amount of OH~ that has been added. The OH~ is expressed as equivalents of total  acetic acid present in the dissociated and undissociated forms. At the midpoint of  this curve, 0.5 equivalents of OH~ have been added, and half of the conjugate acid  has dissociated so that [A~] equals [HA]. This midpoint is expressed in the  Henderson-Hasselbalch equation as the pK a , defined as the pH at which 50% dis-  sociation occurs. As you add more OH~ ions and move to the right on the curve,  more of the conjugate acid molecules (HA) dissociate to generate H + ions, which  combine with the added OH~ ions to form water. Consequently, only a small  increase in pH results. If you add hydrogen ions to the buffer at its pK a (moving to  the left of the midpoint in Fig. 4.7), conjugate base molecules (A~) combine with  the added hydrogen ions to form HA, and almost no decrease in pH occurs.   As can be seen from Figure 4.7, a buffer can only compensate for an influx or  removal of hydrogen ions within approximately 1 pH unit of its pK a . As the pH of a  buffered solution changes from the pK a to one pH unit below the pK a , the ratio of [A~]  to HA changes from 1 : 1 to 1 : 10. If more hydrogen ions were added, the pH would fall  rapidly because relatively little conjugate base remains. Likewise, at 1 pH unit above  the pK a of a buffer, relatively little undissociated acid remains. More concentrated  buffers are more effective simply because they contain a greater total number of buffer  molecules per unit volume that can dissociate or recombine with hydrogen ions.   An average rate of metabolic activity produces roughly 22,000 mEq acid per day. If  all of this acid were dissolved at one time in unbuffered body fluids, their pH would  be less than 1. However, the pH of the blood is normally maintained between 7.36  and 7.44, and intracellular pH at approximately 7.1 (between 6.9 and 7.4). The  widest range of extracellular pH over which the metabolic functions of the liver, the  beating of the heart, and conduction of neural impulses can be maintained is 6.8 to  7.8. Thus, until the acid produced from metabolism can be excreted as C0 2 in  expired air and as ions in the urine, it needs to be buffered in the body fluids. The  major buffer systems in the body are: the bicarbonate-carbonic acid buffer system,  which operates principally in extracellular fluid; the hemoglobin buffer system in  red blood cells; the phosphate buffer system in all types of cells; and the protein  buffer system of cells and plasma.   The pK a for dissociation of bicar-  bonate anion (HC0 3 ") into H + and  carbonate (C0 3 " 2 ) is 9.8; therefore,  only trace amounts of carbonate exist in  body fluids.   where [C0 2 (d)] is the concentration of dis-  solved C0 2 the [HC0 3 _ ] is expressed as  mEq/mL, and Paco 2 as mm Hg. The con-  stant of 0.03 is a solubility constant that also  corrects for the use of different units.   The major source of metabolic acid in the body is the gas C0 2 , produced principally  from fuel oxidation in the TCA cycle. Under normal metabolic conditions, the body  generates more than 13 moles of C0 2 per day (approximately 0.5 — 1 kg). C0 2 dis-  solves in water and reacts with water to produce carbonic acid, H 2 C0 3 , a reaction  accelerated by the enzyme carbonic anhydrase (Fig. 4.8). Carbonic acid is a weak  acid that partially dissociates into H + and bicarbonate anion, HC0 3 ~.   Carbonic acid is both the major acid produced by the body, and its own buffer.  The pK a of carbonic acid itself is only 3.8, so at the blood pH of 7.4 it is almost  completely dissociated and theoretically unable to buffer and generate bicarbonate.  However, carbonic acid can be replenished from C0 2 in body fluids and air because  the concentration of dissolved C0 2 in body fluids is approximately 500 times  greater than that of carbonic acid. As base is added and H + is removed, H 2 C0 3 dis-  sociates into hydrogen and bicarbonate ions, and dissolved C0 2 reacts with H 2 0 to  replenish the H 2 C0 3 (see Fig. 4.8). Dissolved C0 2 is in equilibrium with the C0 2 in  air in the alveoli of the lungs, and thus the availability of C0 2 can be increased or  decreased by an adjustment in the rate of breathing and the amount of C0 2 expired.  The pK a for the bicarbonate buffer system in the body thus combines K h (the hydra-  tion constant for the reaction of water and C0 2 to form H 2 C0 3 ) with the chemical  pK a to obtain the value of 6.1 used in the Henderson-Hasselbalch equation (Equa-  tion 4.6). To use the terms for blood components measured in the emergency room,  the dissolved C0 2 is expressed as a fraction of the partial pressure of C0 2 in arte-  rial blood, Paco 2 .   The bicarbonate buffer system and hemoglobin in red blood cells cooperate in  buffering the blood and transporting C0 2 to the lungs. Most of the C0 2 produced  from tissue metabolism in the TCA cycle diffuses into the interstitial fluid and the  blood plasma and then into red blood cells (Fig. 4.9, circle 1). Although no car-  bonic anhydrase can be found in blood plasma or interstitial fluid, the red blood  cells contain high amounts of this enzyme, and C0 2 is rapidly converted to car-  bonic acid (H 2 C0 3 ) within these cells (circle 2). As the carbonic acid dissociates  (circle 3), the H + released is also buffered by combination with hemoglobin (Hb,  circle 4). The side chain of the amino acid histidine in hemoglobin has a pK a of 6.7  and is thus able to accept a proton. The bicarbonate anion is transported out of the  red blood cell into the blood in exchange for chloride anion, and thus bicarbonate  is relatively high in the plasma (circle 5) (see Table 4.1).   As the red blood cell approaches the lungs, the direction of the equilibrium reverses.  C0 2 is released from the red blood cell, causing more carbonic acid to dissociate into   The partial pressure of C0 2 (Paco 2 ) in Di Abietes' arterial blood was 28 mm Hg  (reference range = 37-43), and her serum bicarbonate level was 8 mEq/L (ref-  erence range = 24-28). Elevated levels of ketone bodies had produced a  ketoacidosis, and Di Abietes was exhaling increased amounts of C0 2 by breathing  deeply and frequently (Kussmaul's breathing) to compensate. Why does this occur?  Ketone bodies are weak acids that partially dissociate, increasing H + levels in the blood  and the interstitial fluid surrounding the "metabolic" respiratory center in the hypothal-  amus that controls the rate of breathing. As a result of the decrease in pH, her respira-  tory rate increased, causing a fall in the partial pressure of arterial C0 2 (Paco 2 ). As bicar-  bonate and increased protons combined to form carbonic acid and produce more C0 2 ,  her bicarbonate concentration decreased (HC0 3 " + H + — > H 2 C0 3 — » C0 2 + H 2 0). As  shown by Di's low arterial blood pH of 7.08, the Kussmaul's breathing was unable to fully  compensate for the high rate of acidic ketone body production.   Fig. 4.9. Buffering systems of the body. C0 2 produced from cellular metabolism is converted to bicarbonate and H + in the red blood cells. Within  the red blood cell, the H + is buffered by hemoglobin (Hb) and phosphate (HP0 4 ~ 2 ). The bicarbonate is transported into the blood to buffer ^gen-  erated by the production of other metabolic acids, such as the ketone body acetoacetic acid. Other proteins (Pr) also serve as intracellular buffers.   C0 2 and water and more hydrogen ions to combine with bicarbonate. Hemoglobin  loses some it of its hydrogen ions, a feature that allows it to bind oxygen more readily  (see Chapter 7). Thus, the bicarbonate buffer system is intimately linked to the deliv-  ery of oxygen to tissues.   The respiratory center within the hypothalamus, which controls the rate of  breathing, is sensitive to changes in pH. As the pH falls, individuals breathe more  rapidly and expire more C0 2 . As the pH rises, they breathe more shallowly. Thus,  the rate of breathing contributes to regulation of pH through its effects on the dis-  solved C0 2 content of the blood.   Bicarbonate and carbonic acid, which diffuse through the capillary wall from the  blood into interstitial fluid, provide a major buffer for both plasma and interstitial  fluid. However, blood differs from interstitial fluid in that the blood contains a high  content of extracellular proteins, such as albumin, which contribute to its buffering  capacity through amino acid side chains that are able to accept and release protons.  The protein content of interstitial fluid is too low to serve as an effective buffer.   were hyperventilating when they  arrived at the emergency room:  Ms. Abietes in response to her primary  metabolic acidosis and Mr. Veere in  response to anxiety. Hyperventilation raised  blood pH in both cases: In Ms. Abietes case  it partially countered the acidosis and in Mr.  Veere's case it produced a respiratory alkalo-  sis (abnormally high arterial blood pH).   Phosphate anions and proteins are the major buffers involved in maintaining a  constant pH of intracellular fluids. The inorganic phosphate anion H 2 P0 4 ~ dis-  sociates to generate H + and the conjugate base, HP0 4 ~ 2 with a pK a of 7.2 (see  Fig. 4.9, circle 6). Thus, phosphate anions play a major role as an intracellular  buffer in the red blood cell and in other types of cells, where their concentration  is much higher than in blood and interstitial fluid (See Table 4.1, extracellular  fluid). Organic phosphate anions, such as glucose 6-phosphate and ATP, also act  as buffers. Intracellular fluid contains a high content of proteins that contain his-  tidine and other amino acids that can accept protons, in a fashion similar to  hemoglobin (see Fig. 4.9, circle 7).   The transport of hydrogen ions out of the cell is also important for mainte-  nance of a constant intracellular pH. Metabolism produces a number of other  acids in addition to C0 2 . For example, the metabolic acids acetoacetic acid and  p -hydroxy butyric acid are produced from fatty acid oxidation to ketone bodies  in the liver, and lactic acid is produced by glycolysis in muscle and other tissues.  The pK a for most metabolic carboxylic acids is below 5, so these acids are com-  pletely dissociated at the pH of blood and cellular fluid. Metabolic anions are  transported out of the cell together with H + (see Fig. 4.9, circle 8). If the cell  becomes too acidic, more H + is transported out in exchange for Na + ions by a  different transporter. If the cell becomes too alkaline, more bicarbonate is trans-  ported out in exchange for CP ions.    Phosphoric acid (H 3 P0 4 ) dissoci-  ates into H + ions and dihydrogen  phosphate (H 2 P0 4 ") with a pK a of  2.15. The dissociation of H 2 P0 4 ~ into H + and  monohydrogen ions (HP0 4 ~ 2 ) occurs with a  pK a of 7.20. Monohydrogen phosphate dis-  sociates into H + and phosphate anions  (P0 4 " 3 ) with a pK a of 12.4.   Ammonium ions are acids that dis-  sociate to form the conjugate base,  ammonia, and hydrogen ions.  What is the form present in blood? In urine?   Diabetes mellitus is diagnosed by  the concentration of plasma glucose  (plasma is blood from which the red  blood cells have been removed by centrifuga-  tion). Because plasma glucose normally  increases after a meal, the normal reference  ranges and the diagnostic level are defined  relative to the time of consumption of food, or  to consumption of a specified amount of glu-  cose during an oral glucose tolerance test.  After an overnight fast, values for the fasting  plasma glucose below 110 mg/dL are consid-  ered normal and above 126 mg/dL define dia-  betes mellitus. Fasting plasma glucose values  greater or equal to 110 and less than 126  mg/dL define an intermediate condition  termed impaired fasting glucose. Normal  casual plasma glucose levels (casual is  defined as any time of day without regard to  the time since a last meal) should not be  above 200 mg/dL. A 2-hour postprandial (after  a meal or after an oral glucose load) plasma  glucose level between 140 and 199 mg/dL  defines a condition known as impaired glu-  cose tolerance. A level above 200 mg/dL  defines "overt" diabetes mellitus.   The nonvolatile acid that is produced from body metabolism cannot be excreted as  expired C0 2 and is excreted in the urine. Most of the nonvolatile acid hydrogen ion  is excreted as undissociated acid that generally buffers the urinary pH between 5.5  and 7.0. A pH of 5.0 is the minimum urinary pH. The acid secretion includes inor-  ganic acids such as phosphate and ammonium ions, as well as uric acid, dicar-  boxylic acids, and tricarboxylic acids such as citric acid (see Table 4.2). One of the  major sources of nonvolatile acid in the body is sulfuric acid (H 2 S0 4 ). Sulfuric acid  is generated from the sulfate-containing compounds ingested in foods and from  metabolism of the sulfur-containing amino acids, cysteine and methionine. It is a  strong acid that is dissociated into H + and sulfate anion (S0 4 -2 ) in the blood and  urine (see Fig. 4.6). Urinary excretion of H 2 P0 4 ~ helps to remove acid. To maintain  metabolic homeostasis, we must excrete the same amount of phosphate in the urine  that we ingest with food as phosphate anions or organic phosphates such as phos-  pholipids. Whether the phosphate is present in the urine as H 2 P0 4 ~ or HP0 4 ~ 2  depends on the urinary pH and the pH of blood.   Ammonium ions are major contributors to buffering urinary pH, but not blood  pH. Ammonia (NH 3 ) is a base that combines with protons to produce ammonium  (NH 4 + ) ions (NH 3 + H + <-> NH 4 + ), a reaction that occurs with a pK a of 9.25.  Ammonia is produced from amino acid catabolism or absorbed through the intes-  tine, and kept at very low concentrations in the blood because it is toxic to neural  tissues. Cells in the kidney generate NH 4 + and excrete it into the urine in propor-  tion to the acidity (proton concentration) of the blood. As the renal tubular cells  transport H + into the urine, they return bicarbonate anions to the blood.   Hydrochloric acid (HC1), also called gastric acid, is secreted by parietal cells of the  stomach into the stomach lumen, where the strong acidity denatures ingested pro-  teins so they can be degraded by digestive enzymes. When the stomach contents are  released into the lumen of the small intestine, gastric acid is neutralized by bicar-  bonate secreted from pancreatic cells and by cells in the intestinal lining.   ^ Dianne Abietes. Di Abietes has type 1 diabetes mellitus (formerly  called juvenile or insulin-dependent diabetes mellitus, IDDM). She main-  tains her insulin level through two daily subcutaneous (under the skin)  injections of insulin. If her blood insulin levels fall too low, free fatty acids leave her  adipocytes (fat cells) and are converted by the liver to the ketone bodies acetoacetic  acid and p -hydroxy butyric acid. As these acids accumulate in the blood, a metabolic  acidosis known as diabetic ketoacidosis (DKA) develops. Until insulin is adminis-  tered to reverse this trend, several compensatory mechanisms operate to minimize  the extent of the acidosis. One of these mechanisms is a stimulation of the respira-  tory center in the hypothalamus induced by the acidosis, which leads to deeper and  more frequent respiration (Kussmaul's respiration). C0 2 is expired more rapidly  than normal, and the blood pH rises. The results of the laboratory studies performed  on Di Abietes in the emergency room were consistent with a moderately severe  DKA. Her arterial blood pH and serum bicarbonate were low, and ketone bodies  were present in her blood and urine (normally, ketone bodies are not present in the  urine). In addition, her serum glucose level was 648 mg/dL (reference range =  80 — 110 fasting and no higher than 200 in a random glucose sample). Her hyper-  glycemia, which induces an osmotic diuresis, contributed to her dehydration and the  hyperosmolality of her body fluids.   Treatment was initiated with intravenous saline solutions to replace fluids lost  with the osmotic diuresis and hyperventilation. The osmotic diuresis resulted from  increased urinary water volume to dilute the large amounts of glucose and ketone  bodies excreted in the urine. Hyperventilation increased the water of respiration lost  with expired air. A loading dose of regular insulin was given as an intravenous bolus  followed by additional insulin each hour as needed. The patient's metabolic  response to the treatment was monitored closely.   The pK a for dissociation of ammo-  •] nium ions is 9.25. Thus, the undis-  sociated conjugate acid form,  NH 4 + , predominates at the physiologic pH of  7.4 and urinary pH of 5.5 to 7.0. Ammonia is  an unusual conjugate base because it is not  an anion.   ^ Dennis Veere. Dennis remained alert in the emergency room. While  j awaiting the report of his initial serum salicylate level, his stomach was  lavaged, and several white tablets were found in the stomach aspirate. He was  examined repeatedly and showed none of the early symptoms of salicylate toxicity,  such as respiratory stimulation, upper abdominal distress, nausea, or headache.   His serum salicylate level was reported as 92 jxg/mL (the usual level in an adult  receiving a therapeutic dosage of 4— 5 g/day is 120—350 jxg/mL, and a level of 800  jxg/mL is considered potentially lethal). He was admitted for overnight observation  and continued to do well. A serum salicylate level the following morning was 24  jxg/mL. He was discharged later that day.   Percy Veere. At the emergency room, Mr. Veere complained of light-  headedness and "pins and needles" (paresthesias) in his hands and  around his lips. These symptoms resulted from an increase in respiratory  drive mediated in this case through the "behavioral" rather than the "metabolic"  central respiratory control system (as seen in Di Abietes when she was in dia-  betic ketoacidosis). The behavioral system was activated in Mr. Veere 's case by  his anxiety over his grandson's potential poisoning. His alveolar hyperventila-  tion caused Mr. Veere 's Paco 2 to decrease below the normal range of 37 to 43  mm Hg. The alkalemia caused his neurologic symptoms. After being reassured  that his grandson would be fine, Mr. Veere was asked to breathe slowly into a  small paper bag placed around his nose and mouth, allowing him to reinhale the  C0 2 being exhaled through hyperventilation. Within 20 minutes, his symptoms  disappeared.   Body water and dehydration. Dehydration, or loss of water, occurs  when salt and water intake is less than the combined rates of renal plus  extrarenal volume loss (Fig. 4.10). In a true hypovolemic state, total body  water, functional ECF volume, and ICF volume are decreased. One of the causes of  hypovolemia is an intake of water that is inadequate to resupply the daily excretion vol-  ume (maintenance of fluid homeostasis). The amount of water lost by the kidneys is  determined by the amount of water necessary to dilute the ions, acids, and other solutes  excreted. Both urinary solute excretion and water loss in expired air, which amount to  almost 400 mL/day, occur during fasting as well as during periods of normal food  intake. Thus, people who are lost in the desert or shipwrecked continue to lose water  in air and urine, in addition to their water loss through the skin and sweat glands.  Comatose patients and patients who are debilitated and unable to swallow also con-  tinue to lose water and become dehydrated. Dysphagia (loss of appetite), such as Percy  Veere experienced during his depression (see Chapters 1 and 3), can result in dehy-  dration because food intake is normally a source of fluid. Cerebral injuries that cause  a loss of thirst also can lead to dehydration.   Dehydration also can occur secondary to excessive water loss through the kid-  neys, lungs, and skin. Excessive renal loss could be the result of impaired kidney  function, or impaired response by the hormones that regulate water balance (e.g.,   Fig. 4.10. Body fluid homeostasis (constant  body water balance). Intake is influenced by  availability of fluids and food, thirst, hunger,  and the ability to swallow. The rates of breath-  ing and evaporation and urinary volume influ-  ence water loss. The body adjusts the volume  of urinary excretion to compensate for varia-  tions in other types of water loss and for varia-  tions in intake. The hormones aldosterone and  antidiuretic hormone (ADH) help to monitor  blood volume and osmolality through mecha-  nisms regulating thirst and sodium and water  balance.   antidiuretic hormone [ADH] and aldosterone). However, in individuals such as Di  Abietes with normal kidney function, dehydration can occur as a response to water  dilution of the high concentrations of ketone bodies, glucose, and other solutes  excreted in the urine (osmotic diuresis). If the urinary water loss is associated with  an acidosis, hyperventilation occurs, increasing the amount of water lost in expired  air. Water loss in expired air (pulmonary water loss) could occur also as a result of  a tracheotomy or cerebral injury. An excessive loss of water and electrolytes  through the skin can result from extensive burns.   Gastrointestinal water losses also can result in dehydration. We secrete approxi-  mately 8 to 10 L fluid per day into our intestinal lumen. Normally, more than 90%  of this fluid is reabsorbed in the intestines. The percentage reabsorbed can be  decreased by vomiting, diarrhea, tube drainage of gastric contents, or loss of water  into tissues around the gut via bowel fistulas.   Many physiology textbooks have good in-depth descriptions of acid-base balance. A very useful refer-  ence for medical students is: Brensilver J, Goldberger, E. A Primer of Water, Electrolyte, and Acid-  Base Syndromes. 8th Ed. Philadelphia: F.A. Davis, 1996 .   For additional information on toxic effects of aspirin, see Roberts LJ II, Morrow JD. Analgesic-  antipyretic and antiinflammatory agents and drugs employed in the treatment of gout. In: Hardman  JG, Limbird LE, Goodman AG, eds. Goodman and Gilman's The Pharmacological Basis of Thera-  peutics. New York: McGraw-Hill, 2001:697-701.   A decrease of blood pH from 7.5 to 6.5 would be accompanied by which of the following changes in ion concentration?   (C) An increase in hydrogen ion concentration by a factor of 7.5/6.5.   (D) A decrease in hydrogen ion concentration by a factor of 6.5/7.5.   (E) A shift in concentration of buffer anions, with no change in hydrogen ion concentration.   (A) Buffers are usually composed of a mixture of strong acids and strong bases.   (B) Buffers work best at the pH at which they are completely dissociated.   (C) Buffers work best at the pH at which they are 50% dissociated.   (D) Buffers work best at one pH unit lower than the pK a   A patient with an enteropathy (intestinal disease) produced large amounts of ammonia (NH 3 ) from bacterial overgrowth  in the intestine. The ammonia was absorbed through the intestine into the portal vein and entered the circulation. Which  of the following is a likely consequence of his ammonia absorption?   4. Which of the following physiologic/pathologic conditions is most likely to result in an alkalosis, provided that the body could  not fully compensate?   (B) Production of ketone bodies by a patient with diabetes mellitus   (D) Diarrhea with loss of the bicarbonate anions secreted into the intestine   5. Laboratory tests on the urine of a patient identified the presence of methylmalonate (~OOC-CH(CH 3 )-COO _ ). Methyl-  malonate   ®CH 3 CH 2 OH Ethanol  CH3OH Methanol  The names of chemical groups are  often incorporated into the common name  of a compound, and denote important differ-  ences in chemical structure. For example, in  the name ethanol, the "eth" denotes the  ethyl group (CH 3 CH 2 -), the "ol" denotes the  alcohol group (OH), and the "an" denotes  the single bonds between the carbon atoms.  Methanol contains a methyl group (CH 3 )  instead of the ethyl group. Methanol (also  called wood alcohol) is much more toxic to  humans than ethanol, the alcohol in alco-  holic beverages. Ingestion of methanol  results in visual disturbances, bradycardia,  coma, and seizures.   ® Hydrophobic molecules are trans-  ported in the blood bound to carrier  proteins or incorporated into  lipoprotein complexes that have a hydropho-  bic lipid core and a more polar surface.   The body contains compounds of great structural diversity, ranging from relatively  simple sugars and amino acids to enormously complex polymers such as proteins  and nucleic acids. Many of these compounds have common structural features  related to their names, their solubility in water, the pathways in which they partic-  ipate, or their physiologic function. Thus, learning the terminology used to  describe individual compounds and classes of compounds can greatly facilitate  learning biochemistry.   In this chapter, we describe the major classes of carbohydrates and lipids and  some of the classes of nitrogen- containing compounds. The structures of amino  acids, proteins, the nucleic acids, and vitamins are covered in more detail in sub-  sequent chapters.   Functional Groups on Molecules, Organic molecules are composed princi-  pally of carbon and hydrogen. However, their unique characteristics are related to  structures termed functional groups involving oxygen, nitrogen, phosphorus, or  sulfur.   Carbohydrates. Carbohydrates, commonly known as sugars, can be classified  by their carbonyl group (aldo- or ketosugars), the number of carbons they contain  (e.g., pentoses, hexoses), or the positions of the hydroxy I groups on their asymmet-  ric carbon atoms (D- or L- sugars, stereoisomers, or epimers). They can also be  categorized according to their substitutents (e.g., amino sugars), or the number of  monosaccharides (such as glucose) joined through glycosidic bonds (disaccha-  rides, oligosaccharides, and polysaccharides ). Glycoproteins and proteoglycans  have sugars attached to their protein components.   Lipids. Lipids are a group of structurally diverse compounds defined by their  hydrophobicity ; they are not very soluble in water. The major lipids of the human  body are the fatty acids, which are esterified to glycerol to form triacylglycerols  (triglycerides) or phosphoacylglycerols (phosphoglycerides). In the sphingolipids ,  a fatty acid is attached to sphingosine, which is derived from serine and another  fatty acid. Glycolipids contain sugars attached to a lipid hydroxyl group. Specific  polyunsaturated fatty acids are precursors of eicosanoids. The lipid cholesterol is  a component of membranes, and the precursor of other compounds that contain  the steroid nucleus, such as the bile salts and steroid hormones. Cholesterol is  one of the compounds synthesized from a 5 -carbon precursor called the isoprene  unit.   Nitrogen-containing compounds. Nitrogen in amino groups or heterocyclic  ring structures often carries a positive charge at neutral pH. Amino acids contain  a carboxyl group, an amino group, and one or more additional carbons. Purines,  pyrimidines, and pyridines have heterocyclic nitrogen- containing ring structures.  Nucleosides comprise one of these ring structures attached to a sugar. The addi-  tion of a phosphate produces a nucleotide.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   • Di Abietes recovered from her bout of diabetic ketoacidosis and was dis-  flP charged from the hospital (see Chapter 4). She has returned for a follow-   " up visit as an outpatient. She reports that she has been compliant with her  recommended diet and that she faithfully gives herself insulin by subcutaneous  injection twice daily. Her serum glucose levels are monitored in the hospital labo-  ratory approximately every 2 weeks, and she self -monitors her blood glucose levels  every other day.   Lotta Topaigne is a 47-year-old woman who came to the physician's  office complaining of a severe throbbing pain in the right great toe that  began 8 hours earlier. The toe has suffered no trauma but appears red and  swollen. It is warmer than the surrounding tissue and is exquisitely tender to even  light pressure. Ms. Topaigne is unable to voluntarily flex or extend the joints of the  digit, and passive motion of the joints causes great pain.   Di Abietes had a metabolic acido-  sis resulting from an increased  hepatic production of ketone bod-  ies. Her response to therapy was followed  with screening tests for ketone bodies in her  urine that employed a paper strip containing  nitroprusside, a compound that reacts with  keto groups. Her blood glucose was meas-  ured with an enzymatic assay that is specific  for the sugar D-glucose and will not react  with other sugars.   The organic molecules of the body consist principally of carbon, hydrogen, oxygen,  nitrogen, sulfur, and phosphorus joined by covalent bonds. The key element is car-  bon, which forms four covalent bonds with other atoms. Carbon atoms are joined  through double or single bonds to form the carbon backbone for structures of vary-  ing size and complexity (Fig. 5.1). Groups containing 1, 2, 3, 4, and 5 carbons plus  hydrogen are referred to as methyl, ethyl, propionyl, butyl, and pentanyl groups,  respectively. If the carbon chain is branched, the prefix "iso" is used. If the com-  pound contains a double bond, "ene" is sometimes incorporated into the name. Car-  bon structures that are straight or branched with single or double bonds, but do not  contain a ring, are called aliphatic.   Carbon-containing rings are found in a number of biologic compounds. One of  the most common is the six-membered carbon-containing benzene ring, sometimes  called a phenyl group (see Fig. 5.1). This ring has three double bonds, but the elec-  trons are shared equally by all six carbons and delocalized in planes above and  below the ring. Compounds containing the benzene ring, or a similar ring structure  with benzene-like properties, are called aromatic.   Biochemical molecules are defined both by their carbon skeleton and by structures  called functional groups that usually involve bonds between carbon and oxygen, car-  bon and nitrogen, carbon and sulfur, and carbon and phosphate groups (Fig. 5.2). In  carbon-carbon and carbon-hydrogen bonds, the electrons are shared equally between  atoms, and the bonds are nonpolar and relatively unreactive. In carbon-oxygen and car-  bon-nitrogen bonds, the electrons are shared unequally, and the bonds are polar and  more reactive. Thus, the properties of the functional groups usually determine the types  of reactions that occur and the physiologic role of the molecule.   Functional group names are often incorporated into the common name of a com-  pound. For example, a ketone might have a name that ends in "one" like acetone,  and the name of a compound that contains a hydroxyl (alcohol or OH group) might  end in "ol" (e.g., ethanol). The acyl group is the portion of the molecule that provides   Fig. 5.1. Examples of aliphatic and aromatic  compounds. A. An isoprene group, which is an  aliphatic group. The "iso" prefix denotes  branching, and the "ene" denotes a double  bond. B. A benzene ring (or phenyl group),  which is an aromatic group.   The ketone bodies synthesized in  the liver are p-hydroxybutyrate and  acetoacetate. A third ketone body,  acetone, is formed by the nonenzymatic  decarboxylation of acetoacetate.   Acetone is volatile and accounts for the  sweet mousy odor in the breath of patients  such as Di Abietes when they have a ketoaci-  dosis. What functional groups are present in  each of these ketone bodies?   - CH 2 - CH 2 - NH 2 ; -CH 2 -i N + - CH 3 ;   Fig. 5.2. Major types of functional groups found in biochemical compounds of the human body.   compound is more oxidized, p-  hydroxybutyrate or acetoacetate? Which is  more reduced?   p-hydroxybutyrate and acetoacetate  | /\^J are carboxylates (dissociated car-  boxylic acids). Acetoacetate and  acetone contain keto/ketone groups. Because  p-hydroxybutyrate contains an alcohol  (hydroxyl) group and not a keto group, the  general name of ketone bodies for these com-  pounds is really a misnomer.   the carbonyl (-C = 0) group in an ester or amide linkage. It is denoted in a name by  an "yl" ending. For example, the fat stores of the body are triacylglycerols. Three  acyl (fatty acid) groups are esterified to glycerol, a compound containing three alco-  hol groups. In the remainder of this chapter, we will bold the portions of names of  compounds that refer to a class of compounds or a structural feature.   The carbon-carbon and carbon-oxygen groups are described as "oxidized" or  "reduced" according to the number of electrons around the carbon atom. Oxidation  is the loss of electrons and results in the loss of hydrogen atoms together with one  or two electrons, or the gain of an oxygen atom or hydroxyl group. Reduction is the  gain of electrons and results in the gain of hydrogen atoms or loss of an oxygen  atom. Thus, the carbon becomes progressively more oxidized (and less reduced) as  we go from an alcohol to an aldehyde or a ketone to a carboxyl group (see Fig. 5.2).  Carbon-carbon double bonds are more oxidized (and less reduced) than  carbon-carbon single bonds.   Acidic groups contain a proton that can dissociate, usually leaving the remainder of  the molecule as an anion with a negative charge (see Chapter 4). In biomolecules,  the major anionic substituents are carboxylate groups, phosphate groups, or sulfate  groups (the "ate" suffix denotes a negative charge) (Fig. 5.3). Phosphate groups  attached to metabolites are often abbreviated as P with a circle around it, or just as  "P", as in glucose-6-P.   Compounds containing nitrogen are usually basic and can acquire a positive  charge (Fig. 5.4). Nitrogen has five electrons in its valence shell. If only three of  these electrons form covalent bonds with other atoms, the nitrogen has no charge.  If the remaining two electrons form a bond with a hydrogen ion or a carbon atom,  the nitrogen carries a positive charge. Amines consist of nitrogen attached through  single bonds to hydrogen atoms and to one or more carbon atoms. Primary amines,   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   such as dopamine, have one carbon-nitrogen bond. These amines are weak acids  with a pK a of approximately 9, so that at pH 7.4 they carry a positive charge. Sec-  ondary, tertiary, and quaternary amines have 2, 3, and 4 nitrogen-carbon bonds,  respectively (see Fig. 5.4).   Polar bonds are covalent bonds in which the electron cloud is denser around one  atom (the atom with the greater electronegativity) than the other. Oxygen is more  electronegative than carbon, and a carbon-oxygen bond is therefore polar, with the  oxygen atom carrying a partial negative charge and the carbon atom carrying a par-  tial positive charge (Fig. 5.5). In nonpolar carbon-carbon bonds and carbon-hydro-  gen bonds, the two electrons in the covalent bond are shared almost equally. Nitro-  gen, when it has only three covalent bonds, also carries a partial negative charge  relative to carbon, and the carbon-nitrogen bond is polarized. Sulfur can carry a  slight partial negative charge.   Water is a dipolar molecule in which the oxygen atom carries a partial negative  charge and the hydrogen atoms carry partial positive charges (see Chapter 4). For  molecules to be soluble in water, they must contain charged or polar groups that can  associate with the partial positive and negative charges of water. Thus, the solubil-  ity of organic molecules in water is determined by both the proportion of polar to  nonpolar groups attached to the carbon-hydrogen skeleton and to their relative posi-  tions in the molecule. Polar groups or molecules are called hydrophilic (water-lov-  ing), and nonpolar groups or molecules are hydrophobic (water-fearing). Sugars  such as glucose 6-phosphate, for example, contain so many polar groups that they  are very hydrophilic and almost infinitely water-soluble (Fig. 5.6). The water mol-  ecules interacting with a polar or ionic compound form a hydration shell around the  compound.   Compounds that have large nonpolar regions are relatively water insoluble. They  tend to cluster together in an aqueous environment and form weak associations  through van der Waals forces, often termed hydrophobic bonds. Hydrophobic com-  pounds are essentially pushed together as the water molecules maximize the num-  ber of energetically favorable hydrogen bonds they can form with each other in the  water lattice. Thus, lipids form droplets or separate layers in an aqueous environ-  ment (e.g., vegetable oils in a salad dressing).   Another consequence of bond polarity is that atoms that carry a partial (or full) neg-  ative charge are attracted to atoms that carry a partial (or full) positive charge and  vice versa. These partial or full charges dictate the course of biochemical reactions —  which follow the same principles of electrophilic and nucleophilic attack character-  istic of organic reactions in general. The partial positive charge on the carboxyl car-  bon attracts more negatively charged groups and accounts for many of the reactions  of carboxylic acids. An ester is formed when a carboxylic acid and an alcohol com-  bine, splitting out water (Fig. 5.7). Similarly, a thioester is formed when an acid com-  bines with a sulfhydryl group, and an amide is formed when an acid combines with  an amine. Similar reactions result in the formation of a phosphoester from phos-  phoric acid and an alcohol and in the formation of an anhydride from two acids.   Biochemists use two systems for the identification of the carbons in a chain. In the  first system, the carbons in a compound are numbered, starting with the carbon in   Fig. 5.3. Examples of anions formed by disso-  ciation of acidic groups. At physiologic pH,  carboxylic acids, phosphoric acid, and sulfuric  acid are dissociated into hydrogen ions and  negatively charged anions.   In medicine and biochemistry, the common  or trivial names of compounds are used  rather than the systematic nomenclature  favored by chemists. Sometimes such  names reflect functional groups, classes of  compounds or the source from which the  compound was first isolated. The compound  testosterone provides an example. It was first  isolated from monkey testis, the "ster" in its  name denotes the steroid ring structure, and  the "one" denotes a ketone group.   "A" contains three alcohol groups  ^^•J and is called glycerol. Compound B  contains an aldehyde group, and is  called glyceraldehyde.   Acetoacetate is more oxidized than  p-hydroxybutyrate. The carbon in  the keto group contains one less  hydrogen than the carbon to which the OH  group is attached. It has lost an electron.   the most oxidized group (e.g., the carboxyl group). In the second system, the car-  bons are given Greek letters, starting with the carbon next to the most oxidized  group. Hence, the compound shown in Figure 5.8 is known as 3 -hydroxy butyrate or  p -hydroxy butyrate .   Simple monosaccharides consist of a linear chain of three or more carbon atoms,  one of which forms a carbonyl group through a double bond with oxygen (Fig. 5.9).  The other carbons of an unmodified monosaccharide contain hydroxyl groups,  resulting in the general formula for an unmodified sugar of C n H 2n O n . The suffix  "ose" is used for the names of sugars. If the carbonyl group is an aldehyde, the sugar  is an aldose; if the carbonyl group is a ketone, the sugar is a ketose. Monosaccha-  rides are also classified according to their number of carbons: Sugars containing 3,  4, 5, 6, and 7 carbons are called trioses, tetroses, pentoses, hexoses, and heptoses,  respectively. Fructose is therefore a ketohexose (see Fig. 5.9), and glucose is an  aldohexose (see Fig. 5.6).   Fig. 5.4. Examples of amines. At physiologic  pH, many amines carry positive charges.   Fig. 5.8. Two systems for identifying the car-  bon atoms in a compound. This compound is  called 3 -hydroxy butyrate or p-hydroxybutyrate.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   The stereospecificity of D-glucose  is still frequently denoted in medi-  cine by the use of its old name,  dextrose. A solution used for intravenous  infusions in patients is a 5% (5 g/100 ml_)  solution of dextrose.   Are D-mannose and D-galactose  stereoisomers? Are they epimers  of each other? (see Fig. 5.12)   Fig. 5.10. D- and L-Glyceraldehyde. The car-  bon in the center contains four different sub-  stituent groups arranged around it in a tetrahe-  dron. A different arrangement creates an  isomer that is a nonsuperimposable mirror  image. If you rotate the mirror image structure  so that groups 1 and 2 align, group 3 will be in  the position of group 4, and group 4 will be in  position 3.   A carbon atom that contains four different chemical groups forms an asymmetric  (or chiral) center (Fig. 5.10). The groups attached to the asymmetric carbon atom  can be arranged to form two different isomers that are mirror images of each other  and not superimposable. Monosaccharide stereoisomers are designated D or L  based on whether the position of the hydroxyl group furthest from the carbonyl  carbon matches D or L glyceraldehyde (Fig. 5.11). Although a more sophisticated  system of nomenclature using the designations of (R) and (S) is generally used to  describe the positions of groups on complex molecules such as drugs, the D and L  designation is still used in medicine for describing sugars and amino acids.  Because glucose (the major sugar in human blood) and most other sugars in human  tissues belong to the D series, sugars are assumed to be D unless L is specifically  added to the name.   Stereoisomers have the same chemical formula but differ in the position of the  hydroxyl group on one or more of their asymmetric carbons (Fig. 5.12). A sugar  with n asymmetric centers has 2 n stereoisomers unless it has a plane of symmetry.  Epimers are stereoisomers that differ in the position of the hydroxyl group at only  one of their asymmetric carbons. D-glucose and D-galactose are epimers of each  other, differing only at position 4, and can be interconverted in human cells by  enzymes called epimerases. D-mannose and D-glucose are also epimers of each  other.   Monosaccharides exist in solution mainly as ring structures in which the carbonyl  (aldehyde or ketone) group has reacted with a hydroxyl group in the same molecule  to form a five- or six-membered ring (Fig. 5.13). The oxygen that was on the  hydroxyl group is now part of the ring, and the original carbonyl carbon, which now  contains a -OH group, has become the anomeric carbon atom. An hydroxyl group  on the anomeric carbon drawn down below the ring is in the a-position; drawn up  above the ring, it is in the p position. In the actual three-dimensional structure, the  ring is not planar but usually takes a "chair" conformation in which the hydroxyl  groups are located at a maximal distance from each other.   In solution, the hydroxyl group on the anomeric carbon spontaneously (non-  enzymatically) changes from the a to the p position through a process called  mutarotation. When the ring opens, the straight chain aldehyde or ketone is formed.  When the ring closes, the hydroxyl group may be in either the a or the p position  (Fig. 5.14). This process occurs more rapidly in the presence of cellular enzymes  called mutarotases. However, if the anomeric carbon forms a bond with another  molecule, that bond is fixed in the a or P position, and the sugar cannot mutarotate.  Enzymes are specific for a or P bonds between sugars and other molecules, and  react with only one type.   Sugars frequently contain phosphate groups, amino groups, sulfate groups or  N- acetyl groups. Most of the free monosaccharides within cells are phosphorylated  at their terminal carbons, which prevents their transport out of the cell (see glucose  6-phosphate in Fig. 5.6). Amino sugars such as galactosamine and glucosamine  contain an amino group instead of a hydroxyl group on one of the carbon atoms,  usually carbon 2 (Fig. 5.15). Frequently this amino group has been acetylated to  form an Af-acetylated sugar. In complex molecules termed proteoglycans, many of   Fig. 5.11. D-Glyceraldehyde and D-glucose.  These sugars have the same configuration at  the asymmetric carbon atom farthest from the  carbonyl group. Both belong to the D series.  Asymmetric carbons are shown in blue.   Fig. 5.12. Examples of stereoisomers. These compounds have the same chemical formula  (C 6 H 12 0 6 ) but differ in the positions of the hydroxyl groups on their asymmetric carbons (in  blue).   They are stereoisomers, but not  •) epimers of each other. They have  the same chemical formula, but  differ in the position of two hydroxyl groups.   Fig. 5.13. Pyranose and furanose rings formed from glucose and fructose. The anomeric car-  bons are highlighted.   Fig. 5.14. Mutarotation of glucose in solution, with percentages of each form at equilibrium.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   the TV-acetylated sugars also contain negatively charged sulfate groups attached to a  hydroxyl group on the sugar.   Sugars can be oxidized at the aldehyde carbon to form an acid. Technically the com-  pound is no longer a sugar, and the ending on its name is changed from "-ose" to  "onic acid" or "onate" (e.g., gluconic acid, Fig. 5.16). If the carbon containing the  terminal hydroxyl group is oxidized, the sugar is called a uronic acid (e.g., glu-  curonic acid).   If the aldehyde of a sugar is reduced, all of the carbon atoms contain alcohol  (hydroxyl) groups, and the sugar is a polyol (e.g., sorbitol)(see Fig. 5.16). If one of   Fig. 5.15. An Af-acetylated amino sugar. The  Af-denotes the amino group to which the acetyl  group is attached, shown in the blue box.   Proteoglycans contain many long  \\ ) unbranched polysaccharide chains  *r attached to a core protein. The  polysaccharide chains, called glycosamino-  glycans, are composed of repeating disac-  charide units containing oxidized acid sugars  (such as glucuronic acid), sulfated sugars,  and N-acetylated amino sugars. The large  number of negative charges causes the gly-  cosaminoglycan chains to radiate out from  the protein so that the overall structure  resembles a bottlebrush. The proteoglycans  are essential parts of the extracellular matrix,  the aqueous humor of the eye, secretions of  mucus-producing cells, and cartilage.    Fig. 5.16. Oxidized and reduced sugars. The affected group is shown in blue. Gluconic acid  (D-gluconate) is formed by oxidation of the glucose aldehyde carbon. Glucuronic acid is  formed by oxidation of the glucose terminal OH group. Sorbitol, a sugar alcohol, is formed  by reduction of the glucose aldehyde group. Deoxyribose is formed by reduction of ribose.   the hydroxyl groups of a sugar is reduced so that the carbon contains only hydro-  gen, the sugar is a deoxysugar, such as the deoxyribose in DNA.   The hydroxyl group on the anomeric carbon of a monosaccharide can react with an  -OH or an -NH group of another compound to form a glycosidic bond. The link-  age may be either a or (3, depending on the position of the atom attached to the  anomeric carbon of the sugar. Af-glycosidic bonds are found in nucleosides and  nucleotides. For example, in the adenosine moiety of ATP, the nitrogenous base  adenine is linked to the sugar ribose through a p-A^-glycosidic bond (Fig. 5.17). In  contrast, O-glycosidic bonds, such as those found in lactose, join sugars to each  other or attach sugars to the hydroxyl group of an amino acid on a protein.   A disaccharide contains two monosaccharides joined by an O -glycosidic bond. Lac-  tose, which is the sugar in milk, consists of galactose and glucose linked through a  (3(1— » 4) bond formed between the p -OH group of the anomeric carbon of galac-  tose and the hydroxyl group on carbon 4 of glucose (see Fig. 5.17). Oligosaccharides  contain from 3 to roughly 12 monosaccharides linked together. They are often found   Fig. 5.17. N- and O-glycosidic bonds. Adenosine triphosphate (ATP) contains a p, Af-glycosidic bond. Lactose contains an O-glycosidic  p(l — >4) bond. Glycogen contains a-1,4 and a-1,6 O-glycosidic bonds.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   attached through N- or (3-glycosidic bonds to proteins to form glycoproteins (see  Chapter 6). Polysaccharides contain tens to thousands of monosaccharides joined by  glycosidic bonds to form linear chains or branched structures. Amylopectin (a form  of starch) and glycogen (the storage form of glucose in human cells) are branched  polymers of glucosyl residues linked through a(l— » 4) and a(l— > 6) bonds.   Fatty acids are usually straight aliphatic chains with a methyl group at one end  (called the to-carbon) and a carboxyl group at the other end (Fig. 5.18). Most fatty  acids in the human have an even number of carbon atoms, usually between 16 and  20. Saturated fatty acids have single bonds between the carbons in the chain, and  unsaturated fatty acids contain one or more double bonds. The most common satu-  rated fatty acids present in the cell are palmitic acid (CI 6) and stearic acid (CI 8).  Although these two fatty acids are generally called by their common names, shorter  fatty acids are often called by the Latin word for the number of carbons, such as  octanoic acid (8 carbons) and decanoic acid (10 carbons).   Monunsaturated fatty acids contain one double bond, and polyunsaturated fatty  acids contain two or more double bonds (see Fig. 5.18). The position of a double  bond is designated by the number of the carbon in the double bond that is closest to  the carboxyl group. For example, oleic acid, which contains 18 carbons and a dou-  ble bond between position 9 and 10, is designated 18:1, A 9 . The number 18 denotes  the number of carbon atoms, 1 (one) denotes the number of double bonds, and A 9   The melting point of a fatty acid  \\ J increases with chain length and  decreases with the degree of satu-  ration. Thus, fatty acids with many double  bonds, such as those in vegetable oils, are  liquid at room temperature and saturated  fatty acids, such as those in butterfat, are  solids. Lipids with lower melting points are  more fluid at body temperature and con-  tribute to the fluidity of our cellular mem-  branes.   Fig. 5.18. Saturated fatty acids and unsaturated fatty acids. In stearic acid, the saturated fatty acid at the top of the figure, all the atoms are shown.  A more common way of depicting the same structure is shown below. The carbons are either numbered starting with the carboxyl group or given  Greek letters starting with the carbon next to the carboxyl group. The methyl (or oo) carbon at the end of the chain is always called the co-carbon  regardless of the chain length. 18:0 refers to the number of carbon atoms (18) and the number of double bonds (0). In the unsaturated fatty acids  shown, not all of the carbons are numbered, but note that the double bonds are cis and spaced at three-carbon intervals. Both co3 and co6 fatty  acids are required in the diet.   The eicosanoids are a group of  \\ ) hormone-like compounds pro-  duced by many cells in the body.  They are derived from polyunsaturated fatty  acids such as arachidonic acid that contain  20 carbons (eicosa) and have 3, 4, or 5 dou-  ble bonds. The prostaglandins, thrombox-  anes, and leukotrienes belong to this group  of compounds.   Palmitoleic acid, oleic acid, and  arachidonic acid are the most com-  mon unsaturated fatty acids in the  cell. Palmitoleic acid is a 16:1, A 9  fatty acid. How would you name it as an w  fatty acid?    Fig. 5.19. Cis and trans double bonds in fatty  acid side chains. Note that the cis double bond  causes the chain to bend.   Fig. 5.20. A triacylglycerol. Note that carbons  1 and 3 of the glycerol moiety are not identi-  cal. The broad end of each arrowhead is closer  to the reader than the narrow, pointed end.   denotes the position of the double bond between the 9th and 10th carbon atoms.  Oleic acid can also be designated 18:1(9), without the A. Fatty acids are also clas-  sified by the distance of the double bond closest to the w end (the methyl group at  the end farthest from the carboxyl group). Thus oleic acid is an oo9 fatty acid, and  linolenic acid is an 0)3 fatty acid. Arachidonic acid, a polyunsaturated fatty acid  with 20 carbons and 4 double bonds, is an 006 fatty acid that is completely described  as 20:4, A 5 ' 8 ' 11 ' 14 .   The double bonds in most naturally occurring fatty acids are in the cis configu-  ration (Fig. 5.19). The designation cis means that the hydrogens are on the same  side of the double bond and the acyl chains on the other side. In trans fatty acids,  the acyl chains are on opposite sides of the double bond. Margarine and the fat used  in preparing French fries are probably the major sources of trans fatty acids found  in humans. Trans fatty acids are produced by the chemical hydrogenation of polyun-  saturated fatty acids in vegetable oils and are not a natural food product.   An acylglycerol comprises glycerol with one or more fatty acids (the acyl group)  attached through ester linkages (Fig. 5.20). Monoacylglycerols, diacylglycerols,  and triacylglycerols contain 1, 2, or 3 fatty acids esterified to glycerol, respectively.  Triacylglycerols rarely contain the same fatty acid at all three positions and are  therefore called mixed triacylglycerols. Unsaturated fatty acids, when present, are  most often esterified to carbon 2. In the three-dimensional configuration of glyc-  erol, carbons 1 and 3 are not identical, and enzymes are specific for one or the  other carbon.   Phosphoacylglycerols contain fatty acids esterified to position 1 and 2 of glycerol  and a phosphate (alone or with a substituent) attached to carbon 3. If only a phos-  phate group is attached to carbon 3, the compound is phosphatidic acid (see Fig.  5.21). Phosphatidic acid is a precursor for the synthesis of the other phosphoacyl-  glycerols.   Phosphatidylcholine is one of the major phosphoacylglycerols found in mem-  branes (see Fig. 5.21). The amine is positively charged at neutral pH, and the phos-  phate negatively charged. Thus, the molecule is amphipathic; it contains large polar  and nonpolar regions. Phosphatidylcholine is also called lecithin. Removal of a fatty  acyl group from a phosphoacyl glycerol leads to a lyso-lipid. For example, remov-  ing the fatty acyl group from lecithin forms lysolecithin.   Sphingolipids do not have a glycerol backbone; they are formed from sphingosine.  (Fig. 5.22). Shingosine is derived from serine and a specific fatty acid, palmitate.  Ceramides are amides formed from sphingosine by attaching a fatty acid to the   Fig. 5.21. Phosphoacylglycerols. Phospholipids found in membranes, such as phosphatidylcholine, have a polar group attached to the phosphate.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   O - P - OCH 2 - CH 2 - N - CH 3 I   Palmitoleic acid is an a>7 fatty acid.  /\ m It has one double bond between  */ the 9th and 10th carbons. It has 16  carbons, like palmitic acid, so the double  bond must be at the 7th carbon from the w  end.   Fig. 5.22. Sphingolipids, derivatives of ceramide. The structure of ceramide is shown at the  bottom of the figure. The portion of ceramide shown in blue is sphingosine. The NH and OH  were contributed by serine. Different groups are added to the hydroxyl group of ceramide to  form sphingomyelin, galactocerebrosides, and gangliosides. NANA = N-acetylneuraminic  acid, also called sialic acid; Glc = glucose; Gal = galactose; GalNAc = N-acetylgalac-  tosamine.   amino group. Various sphingolipids are then formed by attaching different groups  to the hydroxyl group on ceramide. As reflected in the names for cerebrosides and  gangliosides, these sphingolipids contain sugars attached to the hydroxyl group of  ceramide through glycosidic bonds. They are glycolipids (more specifically, gly-  cosphingolipids). Sphingomyelin, which contains a phophorylcholine group  attached to ceramide, is a component of cell membranes and the myelin sheath  around neurons.   Steroids contain a four-ring structure called the steroid nucleus (Fig. 5.23). Choles-  terol is the steroid precursor in human cells from which all of the steroid hormones  are synthesized by modifications to the ring or C20 side chain. Although cholesterol  is not very water soluble, it is converted to amphipathic water-soluble bile salts such  as cholic acid. Bile salts line the surfaces of lipid droplets called micelles in the lumen  of the intestine, where they keep the droplets emulsified in the aqueous environment.   Cholesterol is one of the compounds synthesized in the human from branched 5-  carbon units with one double bond called an isoprenyl unit (see Fig. 5.1 A). Isoprenyl   Fig. 5.23. Cholesterol and its derivatives. The steroid nucleus is shown in blue. The bile salt, cholic acid, and the steroid hormone 17|3-estradiol  are derived from cholesterol and contain the steroid ring structure.   units are combined in long chains to form other structures, such as the side chains of  coenzyme Q in humans and vitamin A in plants.   Nitrogen, as described in Section IB2, is an electronegative atom with two unshared  electrons in its outer valence shell. At neutral pH, the nitrogen in amino groups is  usually bonded to four other atoms and carries a positive charge. However, the pres-  ence of nitrogen atom in an organic compound will increase its solubility in water,  whether the nitrogen is charged or uncharged.   Amino acids are compounds that contain an amino group and a carboxylic acid group.  In proteins, the amino acids are always L-a amino acids (the amino group is attached  to the a carbon in the L-configuration) (Fig. 5.24). These same amino acids also serve  as precursors of nitrogen-containing compounds in the body, such as phosphatidyl-  choline (see Fig. 5.21) and are the basis of most human amino acid metabolism. How-  ever, our metabolic reactions occasionally produce an amino acid that has a p or 7  amino group, such as the neurotransmitter 7-aminobutyric acid (see Fig. 5.24). How-  ever, only a amino acids are incorporated into proteins.   Nitrogen is also a component of ring structures referred to as heterocyclic rings or  nitrogenous bases. The three most common types of nitrogen-containing rings in the   What structural features account  for the differences in the solubility  of cholesterol, estradiol and cholic  acid in the body? (see Fig. 5.23)   Although D-amino acids are not  usually incorporated into proteins  in living organisms, they serve  many other functions in bacteria, such as  synthesis of cross-links in cell walls.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   body are purines (e.g., adenine), pyrimidines (e.g., thymine), and pyridines (e.g.,  the vitamins nicotinic acid, also called niacin, and pyridoxine, also called vitamin  B 6 ) (Fig. 5. 25). The suffix "ine" denotes the presence of nitrogen (amine) in the ring.  The pyrimidine uracil is an exception to this general type of nomenclature. The util-  ity of these nitrogen-containing ring structures lies in the ability of the nitrogen to  form hydrogen bonds and to accept and donate electrons while still part of the ring.  In contrast, the unsubstituted aromatic benzene ring, in which electrons are distrib-  uted equally among all six carbons (see Fig. 5.1), is nonpolar, hydrophobic, and  relatively unreactive.   Nitrogenous bases form nucleosides and nucleotides. A nucleoside consists of a  nitrogenous base joined to a sugar, usually ribose or deoxyribose, through an N-gly-  cosidic bond (see Fig. 5.17). If phosphate groups are attached to the sugar, the com-  pound becomes a nucleotide. In the name of the nucleotide adenosine triphosphate  (ATP), the addition of the ribose is indicated by the name change from adenine to  adenosine (for the glycosidic bond). Monophosphate, diphosphate, or triphosphate  are added to the name to indicate the presence of 1, 2, or 3 phosphate groups in the  nucleotide. The structures of the nucleotides that serve as precursors of DNA and  RNA are discussed in more detail in Section Three, Chapter 12.   In many of the nitrogen-containing rings, the hydrogen can shift to produce a tau-  tomer, a compound in which the hydrogen and double bonds have changed position  (i.e., -N=C-OH — »-NH-C=0) (Fig. 5.26). Tautomers are considered the same  compound, and the structure may be represented either way. Generally one tau-  tomeric form is more reactive than the other. For example, in the two tautomeric  forms of uric acid, a proton can dissociate from the enol form to produce urate.   Radicals are compounds that have a single electron, usually in an outer orbital. Free  radicals are radicals that exist independently in solution or in a lipid environment.    Compounds that cannot be oxi-  dized as fuels in the human are  often excreted in the urine. Chemi-  cal modifications often occur in the liver, kid-  ney, or other tissues that inactivate or detox-  ify the chemicals, make them more  water-soluble, or otherwise target such mol-  ecules for excretion. Uric acid, the basis of  Lotta Topaigne's pain, is excreted in the  urine (see Fig. 5.26). Judging from the simi-  larity in structure, do you think it is derived  from the degradation of purines, pyrim-  idines, or pyridines?   Cholesterol is composed almost  entirely of CH 2 groups and is there-  fore water-insoluble. Estradiol is  likewise relatively water-insoluble. However,  cholic acid contains a hydrophilic carboxyl  group, and three hydroxyl groups. As shown  by the dashed lines, the three hydroxyl  groups all lie on one side of the molecule,  thus creating a hydrophilic surface.   Lotta Topaigne's gout is caused by  depositions of monosodium urate  crystals in the joint of her big toe.  At a blood pH of 7.4, all of the uric acid has  dissociated a proton to form urate, which is  not very water-soluble and forms crystals of  the Na + salt. In the more acidic urine gener-  ated by the kidney, the acidic form, uric acid,  may precipitate to form kidney stones.   I || V = 0 ^ I || \- 0 H I || \-Or   Fig. 5.26. Tautomers of uric acid. The tautomeric form affects the reactivity. The enol form dissociates a proton to form urate.    Free radicals are not just esoteric  reactants; they are the agents of cell  death and destruction. They are  involved in all chronic disease states (e.g.,  coronary artery disease, diabetes mellitus,  arthritis, and emphysema) as well as acute  injury (e.g., radiation, strokes, myocardial  infarction, and spinal cord injury). Through  free radical defense mechanisms in our cells,  we can often restrict the damage attributed to  the "normal" aging process.   Uric acid contains two rings and is  • ) similar to the purines adenine and   guanine (adenine is shown in Fig.  5.25). In fact, it is the urinary excretion prod-  uct formed from the oxidation of these two  purine bases. It is not very soluble in water,  particularly if the pH is near the pK a of its  acidic OH group. If present in excess  amounts, Na + urate tends to precipitate in  joints, causing the severe pain of gout expe-  rienced by Ms. Topaigne.   Although many enzymes generate radicals as intermediates in reactions, these are  not usually released into the cell to become free radicals.   Many of the compounds in the body are capable of being converted to free rad-  icals by natural events that remove one of their electrons, or by radiation. Radiation,  for example, dissociates water into the hydrogen atom and the hydroxyl radical:   In contrast, water normally dissociates into a proton and the negatively charged  hydroxyl ion. The hydroxyl radical forms organic radicals by taking one electron  (as H«) from a compound such as an unsaturated membrane lipid, which then has a  single unpaired electron and is a new radical.   Compounds that are radicals may be written with, or without, the radical show-  ing. For example, nitrogen dioxide, a potent, reactive, toxic radical present in smog  and cigarette smoke, may be designated in medical and lay literature as N0 2 rather  than N0 2 *. Superoxide, a radical produced in the cell and that is the source of much  destruction, is correctly written as the superoxide anion, 0 2 ~. However, to empha-  size its free radical nature, the same compound is sometimes written as 0 2 _# . If a  compound is designated as a radical in the medical literature, you can be certain that  it is a reactive radical, and that its radical nature is important for the pathophysiol-  ogy under discussion. (Reactive oxygen and nitrogen-containing free radicals are  discussed in more detail in Chapter 24).    The reducing sugar test. The reduc-  ing sugar test was used for detection  of sugar in the urine long before spe-  cific enzymatic assays for glucose and galac-  tose became available. In this test, the aldehyde  group of a sugar is oxidized as it donates elec-  trons to copper; the copper becomes reduced  and produces a blue color. In alkaline solution,  keto sugars (e.g., fructose) also react in this test  because they form tautomers that are aldehy-  des. Ring structures of sugars will also react,  but only if the ring can open (i.e., it is not  attached to another compound through a gly-  coside bond). Until a specific test for fructose  becomes available, a congenital disease result-  ing in the presence of fructose in the urine is  indicated by a positive reducing sugar test and  negative results in the specific enzymatic  assays for glucose or galactose.   Dianne Abietes. The severity of clinical signs and symptoms in  patients with diabetic ketoacidosis (DKA), such as Di Abietes, is directly  correlated with the concentration of ketone bodies in the blood. Direct  quantitative methods for measuring acetoacetate and p-hydroxybutyrate are not rou-  tinely available. As a result, clinicians usually rely on semiquantitative reagent strips  (Ketostix, Bayer Diagnostics, Mishawaha, IN) or tablets (Acetest, Bayer Diagnos-  tics, Mishawaha, IN) to estimate the level of acetoacetate in the blood and the urine.  The nitroprusside on the strips and in the tablets reacts with acetoacetate and to a  lesser degree with acetone (both of which have ketone groups), but does not react  with (3-hydroxybutyrate (which does not have a ketone group), p-hydroxybutyrate is  the predominant ketone body present in the blood of a patient in DKA, and its con-  centration could decline at a disproportionately rapid rate compared with that of ace-  toacetate and acetone. Therefore, tests employing the nitroprusside reaction to mon-  itor the success of therapy in such a patient may be misleading.   In contrast to the difficulty of ketone body measurements, diabetic patients can  self-monitor blood glucose levels at home, thereby markedly decreasing the time  and expense of the many blood glucose determinations they need. Blood obtained  from a finger prick is placed on the pad of a plastic strip. The strip has been impreg-  nated with an enzyme (usually the bacterial enzyme glucose oxidase) that specifi-  cally converts the glucose in the blood to a compound (hydrogen peroxide, H 2 0 2 )  that reacts with a dye to produce a color (Fig. 5.27). The intensity of the color,  which is directly proportionate to the concentration of glucose in the patient's  blood, is read on an instrument called a blood glucose monitor.   ^ Lotta Topaigne. Ms. Topaigne has acute gouty arthritis (podagra) involv-  ing her right great toe. Polarized light microscopy of the fluid aspirated from  the joint space showed crystals of monosodium urate phagocytosed by white  blood cells. The presence of the relatively insoluble urate crystals within the joint space  activates an inflammatory cascade leading to the classic components of joint inflam-  mation (pain, redness, warmth, swelling, and limitation of joint motion).   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   f\ Chlorinated Aromatic Hydrocarbon Environmental  Toxins. As a result of human endeavor, toxic compounds containing  chlorinated benzene rings have been widely distributed in the environment.  The pesticide DDT and the class of chemicals called dioxins provide examples of  chlorinated aromatic hydrocarbons and structurally related compounds that are very  hydrophobic and poorly biodegraded (Fig. 5.28). As a consequence of their persist-  ence and lipophilicity, these chemicals are concentrated in the adipose tissue of fish,  fish-eating birds, and carnivorous mammals, including humans.   DDT, a chlorinated biphenyl, was widely used in the United States as an herbi-  cide between the 1940s and 1960s (see Fig. 5.28). Although it has not been used in  this country since 1972, the chlorinated benzene rings are resistant to biodegrada-  tion, and U.S. soil and water are still contaminated with small amounts. DDT is still  used in other parts of the world. Because this highly lipophilic molecule is stored in  the fat of animals, organisms accumulate progressively greater amounts of DDT at  each successive stage of the food chain. Fish-eating birds, one of the organisms at  the top of the food chain, have declined in population because of the effect of DDT  on the thickness of their eggshells. DDT is not nearly as toxic in the human,  although long-term exposure or exposure to high doses may cause reversible  neurologic symptoms, hepatotoxic effects, or cancer.   Dioxins, specifically chlorinated dibenzo-p-dioxins (CDDs), constitute another  class of environmental toxins that are currently of great concern (see Fig. 5.28).  They have been measured at what is termed background levels in the blood, adipose  tissue, and breast milk of all humans tested. CDDs are formed as a byproduct  during the production of other chlorinated compounds and herbicides, and from the  chlorine bleaching process used by pulp and paper mills. They are released during  the incineration of industrial, municipal, and domestic waste, and during the com-  bustion of fossil fuels, and are found in cigarette smoke and the exhaust from gaso-  line and diesel fuels. They can also be formed from the combustion of organic  matter during forest fires. They enter the atmosphere as particulate matter, are  vaporized, and can spread large distances to enter soil and water.   As humans at the top of the food chain, we have acquired our background levels  of dioxins principally through the consumption of food, primarily meat, dairy products,   Most of this chapter has dealt with  Kl J the names and structures of com-  VJ^/ pounds that are nutrients for the  human or metabolites that can be produced  from reactions in the human body. However,  our health is also affected by naturally occur-  ring and man-made xenobiotic compounds  (compounds that have no nutrient value and  are not produced in the human) that we  ingest, inhale, or absorb through our skin.  DDT and dioxins provide examples of chlori-  nated aromatic hydrocarbons, an important  class of manmade chemicals present in the  environment.   The accumulation of DDT in adi-  pose tissue may be protective in  the human because it decreases  the amount of DDT available to pass through  nonpolar lipid membranes to reach neurons  in the brain, or pass through placental mem-  branes to reach the fetus. Eventually we con-  vert DDT to more polar metabolites that are  excreted in the urine. However, some may  pass with lipid into the breast milk of nursing  mothers.   Fig. 5.28. Environmental toxins. Dichlorodiphenyl trichlorethane (DDT ) is a member of a  class of aromatic hydrocarbons that contain two chlorinated benzene (phenyl) rings joined by  a chlorinated ethane molecule. Chlorodibenzo-p-dioxin 's (CDDs) are a related class of more  than 75 chlorinated hydrocarbons that all contain a dibenzo-p-dioxin (DD) molecule com-  prising two benzene rings joined via two oxygen bridges at adjacent carbons on each of the  benzene rings. 2,3,7,8 Tetrachlorodibenzo-p-dioxin, shown above, is one of the most toxic  and the most extensively studied. Chlorinated dibenzofurans (CDF) are structurally and tox-  icologically related.    Most of what is known about the  toxicity of dioxins in the human  comes from individuals exposed  incidentally or chronically to higher levels  (e.g., industrial accidents or presence in  areas sprayed with Agent Orange or other  herbicides contaminated with dioxins.). The  lowest dose effects are probably associated  with thymic atrophy and decreased immune  response, chloracne and related skin lesions,  and neoplasia (cancer). Dioxins can cross  into the placenta to cause developmental  and reproductive effects, decreased prenatal  growth, and prenatal mortality.   and fish. Once in the human body, dioxins are stored in human fat and adipose tis-  sue, and have an average half-life of approximately 5 to 15 years. They are unreac-  tive, poorly degraded, and not readily converted to more water-soluble compounds  that can be excreted in the urine. They are slowly excreted in the bile and feces, and  together with lipids enter the breast milk of nursing mothers.   Nomenclature of chemical compounds. Most undergraduate organic textbooks provide a more detailed  account of the nomenclature used for organic molecules, including the R,S nomenclature for chiral  centers.   Environmental toxins. Good literature reviews for many environmental toxins are published by the U.S.  Department of Health and Human Services: Public Health Service Agency for Toxic Substances and  Disease Registry, including: Toxicological Profile for Chlorinated Dibenzo-p-dioxins, 1998, and the  Toxicological Profile for 4,4'-DDT, 4,4'-DDE, 4,4'-DDD (Update), 1994.   Directions: Select the single best answer for each of the questions below. Base your answers on your knowledge of nomenclature.  You need not recognize any of the structures shown to answer the questions.   1 . Which of the following is a universal characteristic of water-soluble organic compounds?   (B) They must contain a group that has a full negative charge.   (C) They must contain a group that has a full positive charge.   (D) They contain polar groups that can hydrogen bond with water.   A patient was admitted to the hospital emergency room in a coma. Laboratory tests found high levels of the compound shown  above in her blood. On the basis of its structure (and your knowledge of the nomenclature of functional groups), you identify  the compound as   3. A patient was diagnosed with a deficiency of the lysosomal enzyme a-glycosidase. The name of the deficient enzyme su£  gests that it hydrolyzes a glycosidic bond, which is a bond formed   (B) between the anomeric carbon of a sugar and an O-H (or N) of another molecule.   (D) internally between the anomeric carbon of a monosaccharide and its own 5th carbon hydroxyl group.   (E) between the carbon containing the aldol or keto group and the a carbon.   CHAPTER 5 / STRUCTURES OF THE MAJOR COMPOUNDS OF THE BODY   4. A patient was diagnosed with a hypertriglyceridemia. This condition is named for the high blood levels of lipids composed of   5. A patient was diagnosed with a sphingolipidoses, which are congenital diseases involving the inability to degrade sphin-  golipids. All sphingolipids have in common   The genetic code is the sequence of  three bases (nucleotides) in DNA  containing the information for the  linear sequence of amino acids in a polypep-  tide chain (its primary structure). A gene is the  portion of DNA that encodes a functional  product, such as a polypeptide chain. Muta-  tions, which are changes in the nucleotides in  a gene, result in a change in the products of  that gene that may be inherited. The geneti-  cally inherited disease sickle cell anemia is, for  example, caused by a mutation in the gene  encoding one of the subunits of hemoglobin.  Hemoglobin is the protein present in red  blood cells that reversibly binds 0 2 and trans-  ports it to tissues. The adult hemoglobin pro-  tein comprises four polypeptide chains, 2a  and 2p. The a and p subunits differ in primary  structure (i.e., they have different sequences  of amino acids and are encoded by different  genes). Sickle cell anemia is caused by a  mutation in DNA that changes just one amino  acid in the hemoglobin p chains from a glu-  tamic acid to a valine.    Proteins have many functions in the body. They serve as transporters of hydropho-  bic compounds in the blood, as cell adhesion molecules that attach cells to each  other and to the extracellular matrix, as hormones that carry signals from one  group of cells to another, as ion channels through lipid membranes, and as  enzymes that increase the rate of biochemical reactions. The unique characteris-  tics of a protein are dictated by its linear sequence of amino acids, termed its pri-  mary structure. The primary structure of a protein determines how it can fold and  how it interacts with other molecules in the cell to perform its function. The pri-  mary structures of all of the diverse human proteins are synthesized from 20  amino acids arranged in a linear sequence determined by the genetic code.   General properties of amino acids. Each of the amino acids used for protein  synthesis has the same general structure (Fig. 6.1). It contains a carboxylic acid  group, an amino group attached to the a-carbon in an L configuration, a hydro-  gen atom, and a chemical group called a side chain that is different for each  amino acid. In solution, the free amino acids exist as zwitterions, ions in which  the amino group is positively charged and the carboxylate group is negatively  charged. In proteins, these amino acids are joined into linear polymers called  polypeptide chains through peptide bonds between the carboxylic acid group of  one amino acid and the amino group of the next amino acid.   Classification of amino acids according to chemical properties of the side  chains. The chemical properties of the side chain determine the types of bonds and  interactions each amino acid in a polypeptide chain can make with other mole-  cules. Thus, amino acids are often grouped by polarity of the side chain ( charged,  nonpolar hydrophobic, or uncharged polar) or by structural features (aliphatic,  cyclic, or aromatic). The side chains of the nonpolar hydrophobic amino acids  (alanine, valine, leucine, isoleucine, phenylalanine, and methionine) cluster  together to exclude water in the hydrophobic effect. The uncharged polar amino  acids (serine, threonine, tyrosine, asparagine, and glutamine) participate in hydro-  gen bonding. Cysteine, which contains a sulfhydryl group, forms disulfide bonds.  The negatively charged acidic amino acids (aspartate and glutamate) form ionic  (electrostatic) bonds with positively charged molecules, such as the basic amino  acids (lysine, arginine, and histidine). The charge on the amino acid at a particular  pH is determined by the pK a of each group that has a dissociable proton.   Amino acid substitutions in the primary structure. Mutations in the genetic  code result in proteins with an altered primary structure. Mutations resulting in  single amino acid substitutions can affect the functioning of a protein or can con-  fer an advantage specific to a tissue or a set of circumstances. Many proteins such  as hemoglobin exist in the human population as polymorphisms (genetically  determined variations in primary structure.)   Within the same individual, the primary structure of many proteins varies with the  stage of development and is present in fetal and adult isoforms, such as fetal and  adult hemoglobin. The primary structure of some proteins, such as creatine kinase,  can also vary between tissues (tissue-specific isozymes) or between intracellular  locations in the same tissue. Electrophoretic separation of tissue-specific isozymes  has been useful in medicine as a means of identifying the tissue site of injury.   Modified amino acids. In addition to the amino acids encoded by DNA that  form the primary structure of proteins, many proteins contain specific amino acids  that have been modified by phosphorylation, oxidation, carboxylation, or other  reactions. When these reactions are enzyme-catalyzed, they are referred to as  posMranslational modifications .   Will Sichel is a 17 -year-old boy who came to the hospital emergency  room with severe pain in his lower back, abdomen, and legs, which began  after a 2-day history of nausea and vomiting caused by gastroenteritis. He  was diagnosed as having sickle cell disease at age 3 years and has been admitted to  the hospital on numerous occasions for similar vaso-occlusive sickle cell crises.   On admission, the patient's hemoglobin level in peripheral venous blood was 7.8  g/dL (reference range = 12-16 g/dL). The hematocrit or packed cell volume (the  percentage of the total volume of blood made up by red blood cells) was 23.4% (ref-  erence range = 41-53%). His serum total bilirubin level (a pigment derived from  hemoglobin degradation) was 2.3 mg/dL (reference range = 0.2—1.0 mg/dL). An  x-ray of his abdomen showed radiopaque stones in his gallbladder. With chronic  hemolysis (red blood cell destruction), the amount of heme degraded to bilirubin is  increased. These stones are the result of the chronic excretion of excessive amounts  of bilirubin from the liver into the bile, leading to bilirubinate crystal deposition in  the gallbladder lumen.   Cal Kulis is an 18-year-old boy who was brought to the hospital by his  mother because of the sudden onset of severe pain in the left flank radiat-  ing around his left side toward his pubic area. His urine was reddish-brown  in color, and his urinalysis showed the presence of many red blood cells. When his  urine was acidified with acetic acid, clusters of flat hexagonal transparent crystals  of cystine were noted. An x-ray of his abdomen showed radiopaque calculi (stones)  in both kidneys. There was no family history of kidney stone disease.   " disease was first diagnosed (see Chapters 4 and 5). Subsequently, her  physician switched her to synthetic human insulin. At this visit, her physician  changed her insulin therapy and has written a prescription for Humalog mix 75/25  (Eli Lilly, Indianapolis, IN), a mixture of Humalog in protamine suspension and  unbound Humalog insulin (25%).   Ann Jeina is a 54-year-old woman who is 68 inches tall and weighs 198  lb. She has a history of high blood pressure and elevated serum cholesterol  levels. After a heated argument with a neighbor, Mrs. Jeina experienced a  "tight pressure-like band of pain" across her chest, associated with shortness of  breath, sweating, and a sense of light-headedness.   After 5 hours of intermittent chest pain, she went to the hospital emergency  room, where her electrocardiogram showed changes consistent with an acute infarc-  tion of the anterior wall of her heart. She was admitted to the cardiac care unit.  Blood was sent to the laboratory for various tests, including the total creatine kinase  (CK) level and the MB ("muscle-brain") fraction of CK in the blood.   Fig. 6.1. General structure of the amino acids  found in proteins.    The term calculus is used to  describe any abnormal concretion  (concrete-like precipitate) of min-  eral salts. These almost always form within  the cavity of a hollow organ, such as the kid-  ney (kidney or renal stones) or the lumen of  a duct (e.g., common bile duct stones).    The term angina describes a crush-  ing or compressive pain. The term  angina pectoris is used when this  pain is located in the center of the chest,  often radiating to the neck or arms. The most  common mechanism for the latter symptom  is a decreased supply of oxygen to the heart  muscle caused by atherosclerotic coronary  artery disease, which results in obstruction  of the vessels that supply aterial blood to  cardiac muscle.   Fig. 6.2. Dissociation of the a-carboxyl and  a-amino groups of amino acids. At physio-  logic pH (~7), a form in which both the a-car-  boxyl and a-amino groups are charged pre-  dominates. Some amino acids also have  ionizable groups on their side chains.   drugs derived from amino acids  either as "L" or "levo" for the direction in  which L amino acids rotate polarized light.  For example, patients with Parkinson's dis-  ease are treated with L-DOPA (a derivative of  the amino acid L-tyrosine), and hypothyroid  patients are treated with levothyroxine (a  different derivative of L-tyrosine)   Twenty different amino acids are commonly found in proteins. They are all a-amino  acids, amino acids in which the amino group is attached to the a-carbon (the car-  bon atom next to the carboxylate group) (see Fig 6.1). The a-carbon has two addi-  tional substituents, a hydrogen atom and an additional chemical group called a side  chain (-R). The side chain is different for each amino acid.   At a physiologic pH of 7.4, the amino group on these amino acids carries a  positive charge, and the carboxylic acid group is negatively charged (Fig. 6.2).  The pK a of the primary carboxylic acid groups for all of the amino acids is  approximately 2 (1.8-2.4). At pH values much lower than the pK a (higher hydro-  gen ion concentrations), all of the carboxylic acid groups are protonated. At the  pK a , 50% of the molecules are dissociated into carboxylate anions and protons,  and at a pH of 7.4, more than 99% of the molecules are dissociated (see Chapter  4). The pK a for all of the a-amino groups is approximately 9.5 (8.8-1 1.0), so that  at the lower pH of 7.4, most of the amino groups are fully protonated and carry  a positive charge. The form of an amino acid that has both a positive and a neg-  ative charge is called a zwitterion. Because these charged chemical groups can  form hydrogen bonds with water molecules, all of these amino acids are water-  soluble at physiologic pH.   In all of the amino acids but glycine, the a-carbon is an asymmetric carbon atom  that has four different substituents and can exist in either the D or L configuration  (Fig. 6.3). The amino acids in mammalian proteins are all L-amino acids repre-  sented with the amino group to the left if the carboxyl group is at the top of the  structure. These same amino acids serve as precursors of nitrogen-containing com-  pounds synthesized in the body, and thus human amino acid metabolism is also cen-  tered on L-amino acids. The amino acid glycine is neither D nor L because the  a-carbon atom contains two hydrogen atoms.   The chemical properties of the amino acids give each protein its unique charac-  teristics. Proteins are composed of one or more linear polypeptide chains containing   Fig. 6.3. L- and D-amino acids. The L forms are the only ones found in human proteins.  Bonds coming out of the paper are shown by black arrows; those going in, by shaded  arrows. The a-amino groups and H-atoms come toward the reader, and the a-carboxyl and  side chains go away. The L and D forms are mirror images that cannot be superimposed  by rotating the molecule. The reference for the L and D forms are the stereoisomers of  glyceraldehyde.   hundreds of amino acids. The sequence of amino acids, termed the primary structure,  is determined by the genetic code for the protein. In the polypeptide chains, amino  acids are joined through peptide bonds between the carboxylic acid of one amino  acid and the amino group of the adjacent amino acid (Fig. 6.4). Thus, the amino  group, the a-carbon, and the carboxyl groups form the peptide backbone, and the  side chains of the amino acids extend outward from this backbone. The side chains  interact with the peptide backbone of other regions of the chain or with the side  chains of other amino acids in the protein to form hydrophobic regions, electrostatic  bonds, hydrogen bonds, or disulfide bonds. These interactions dictate the folding pat-  tern of the molecule. The three-dimensional folding of the protein forms distinct  regions called binding sites that are lined with amino acid side chains that interact  specifically with another molecule termed a ligand (such as the heme in hemoglo-  bin). Thus, the chemical properties of the side chains determine how the protein  folds, how it binds specific ligands, and how it interacts with its environment (such  as the aqueous medium of the cytoplasm).   The names of the different amino acids have been given three-letter and one-let-  ter abbreviations (Table 6.1). The three-letter abbreviations use the first two letters  in the name plus the third letter of the name or the letter of a characteristic sound,  such as trp for tryptophan. The one-letter abbreviations use the first letter of the  name of the most frequent amino acid in proteins (such as an "A" for alanine). If the  first letter has already been assigned, the letter of a characteristic sound is used  (such as an "R" for arginine). Single-letter abbreviations are usually used to denote  the amino acids in a polypeptide sequence.   Fig. 6.4. Peptide bonds. Amino acids in a  polypeptide chain are joined through peptide  bonds between the carboxyl group of one  amino acid and the amino group of the next  amino acid in the sequence.   According to some biochemists,  "W" stands for the characteristic  sound in "twyptophan" (see Table   In Figure 6.5, the 20 amino acids used for protein synthesis are grouped into dif-  ferent classifications according to the polarity and structural features of the side  chains. These groupings can be helpful in describing common functional roles or  metabolic pathways of the amino acids. However, some amino acid side chains  fit into a number of different classifications and are therefore grouped differently  in different textbooks. Two of the characteristics of the side chain that are useful  for classification are its pK a and its hydropathic index, shown in Table 6.2 The  hydropathic index is a scale used to denote the hydrophobicity of the side chain;  the more positive the hydropathic index, the greater the tendency to cluster with  other nonpolar molecules and exclude water in the hydrophobic effect. The more  negative the hydropathic index of an amino acid, the more hydrophilic is its side  chain.   Glycine is the simplest amino acid, and it really does not fit well into any classifi-  cation because its side chain is only a hydrogen atom. Alanine and the branched  chain amino acids (valine, leucine, and isoleucine) have bulky, nonpolar, aliphatic  side chains. The high degree of hydrophobicitiy of the branched chain amino acid  side chains is denoted by their high hydropathic index (see Table 6.2). Electrons are  shared equally between the carbon and hydrogen atoms in these side chains, so that  they cannot hydrogen bond with water. Within proteins, these amino acid side  chains will cluster together to form hydrophobic cores. Their association is also  promoted by van der Waals forces between the positively charged nucleus of one  atom and the electron cloud of another. This force is effective over short distances  when many atoms pack closely together.   The roles of proline and glycine in amino acid structure differ from those of the  nonpolar amino acids. The amino acid proline contains a ring involving its a-car-  bon and its a-amino group, which are part of the peptide backbone. This rigid ring   *Three-letter abbreviations are generally used. One-  letter abbreviations are used mainly to list the amino  acid sequences of long protein chains.   The proteolytic digestive enzyme  chymotrypsin cleaves the peptide  bonds formed by the carboxyl  groups of large, bulky uncharged amino  acids. Which amino acids fall into this cate-  gory?   Fig. 6.5. The side chains of the amino acids. The side chains are highlighted. The amino acids are grouped by the polarity and structural fea-  tures of their side chains. These groupings are not absolute, however. Tyrosine and tryptophan, often listed with the nonpolar amino acids, are  more polar than other aromatic amino acids because of their phenolic and indole rings, respectively.   *When these amino acids reside in proteins, the pK fl for the side chains may vary to some extent from  the value for the free amino acid, depending on the local environment of the amino acid in the three-  dimensional structure of the protein.   **The hydropathy index is a measure of the hydrophobicity of the amino acid (the higher the number, the  more hydrophobic). Values based on Kyte J, Doolittle RF. J Mol Biol 1 982;1 57: 1 05B1 32.   Chymotrypsin's highest activity is  •] toward peptide bonds formed by  the carboxyl groups of aromatic  amino acids (phenylalanine, tyrosine, trypto-  phan). The side chains of these amino acids  are all large and uncharged. One of the chy-  motrypsin isozymes also exhibits activity  toward leucine and methionine, which are  similar in polarity.   causes a kink in the peptide backbone that prevents it from forming its usual con-  figuration. Because the side chain of glycine is so small compared with that of other  amino acids, it causes the least amount of steric hindrance in a protein (i.e., it does  not significantly impinge on the space occupied by other atoms or chemical  groups). Therefore, glycine is often found in bends or in the tightly packed chains  of fibrous proteins.   The aromatic amino acids have been grouped together because they all contain ring  structures with similar properties, but their polarity differs a great deal. The aro-  matic ring is a six-membered carbon-hydrogen ring with three conjugated double  bonds (the benzene ring or phenyl group). The substituents on this ring determine  whether the amino acid side chain engages in polar or hydrophobic interactions. In  the amino acid phenylalanine, the ring contains no substituents, and the electrons  are shared equally between the carbons in the ring, resulting in a very nonpolar  hydrophobic structure in which the rings can stack on each other (Fig. 6.6). In tyro-  sine, a hydroxyl group on the phenyl ring engages in hydrogen bonds, and the side  chain is therefore more polar and more hydrophilic. The more complex ring struc-  ture in tryptophan is an indole ring with a nitrogen that can engage in hydrogen  bonds. Tryptophan is therefore also more polar than phenylalanine.   Amino acids with side chains that contain an amide group (asparagine and gluta-  mine) or a hydroxyl group (serine and threonine) can be classified as aliphatic,  polar, uncharged amino acids. Asparagine and glutamine are amides of the amino   Fig. 6.6. Hydrophobic and hydrogen bonds.  A. Strong hydrophobic interactions occur with  the stacking of aromatic groups in phenylala-  nine side chains. B. Examples of hydrogen  bonds in which a hydrogen atom is shared by a  nitrogen in the peptide backbone and an oxy-  gen atom in an amino acid side chain or  between an oxygen in the peptide backbone  and an oxygen in an amino acid side chain.    Cal Kulis passed a renal stone  shortly after admission, with  immediate relief of flank pain.  Stone analysis showed its major component  to be cystine. Normally, amino acids are fil-  tered by the renal glomerular capillaries into  the tubular urine but are almost entirely  reabsorbed from this fluid back into the  blood via transport proteins in the proximal  tubular cells of the kidney. Cal Kulis has  cystinuria, a genetically inherited amino acid  substitution in the transport protein that nor-  mally reabsorbs cystine, arginine, and lysine  from the kidney lumen back into the renal  tubular cells. Therefore, his urine contained  high amounts of these amino acids. Cystine,  which is less soluble than other amino acids,  precipitates in the urine to form renal stones  (calculi).   Will Sichel has sickle cell anemia  ■ caused by a point mutation in his  DNA that changes the sixth amino  acid in the (3-globin chain of hemoglobin  from glutamate to valine. What difference  would you expect to find in the chemical  bonds formed by these two amino acids?   acids aspartate and glutamate. The hydroxyl groups and the amide groups in the  side chains allow these amino acids to form hydrogen bonds with water, with each  other and the peptide backbone, or with other polar compounds in the binding sites  of the proteins (see Fig. 6.6). As a consequence of their hydrophilicity, these amino  acids are frequently found on the surface of water-soluble globular proteins. Cysteine,  which is sometimes included in this class of amino acids, has been separated into  the class of sulfur-containing amino acids.   Both cysteine and methionine contain sulfur. The side chain of cysteine contains a  sulfhydryl group that has a pK a of approximately 8.4 for dissociation of its hydro-  gen, so cysteine is predominantly undissociated and uncharged at the physiologic  pH of 7.4. The free cysteine molecule in solution can form a covalent disulfide bond  with another cysteine molecule through spontaneous (nonenzymatic) oxidation of  their sulfhydryl groups. The resultant amino acid, cystine, is present in blood and  tissues, and is not very water-soluble. In proteins, the formation of a cystine disul-  fide bond between two appropriately positioned cysteine sulfhydryl groups often  plays an important role in holding two polypeptide chains or two different regions  of a chain together (Fig. 6.7). Methionine, although it contains a sulfur group, is a  nonpolar amino acid with a large bulky side chain that is hydrophobic. It does not  contain a sulfhydryl group and cannot form disulfide bonds. Its important and cen-  tral role in metabolism is related to its ability to transfer the methyl group attached  to the sulfur atom to other compounds.   The amino acids aspartate and glutamate have carboxylic acid groups that carry a  negative charge at physiologic pH (see Fig. 6.5). The basic amino acids histidine,   Fig. 6.7. A disulfide bond. Covalent disulfide bonds may be formed between two molecules  of cysteine or between two cysteine residues in a protein. The disulfide compound is called  cystine. The hydrogens of the cysteine sulfhydryl groups are removed during oxidation.   lysine, and arginine have side chains containing nitrogen that can be protonated  and positively charged at physiologic and lower pH values. Histidine has a nitro-  gen-containing imidazole ring for a side chain, lysine has a primary amino group  on the 6th or s carbon (from the sequence a, (3, 7, 8, e), and arginine has a guani-  dinium group.   The positive charges on the basic amino acids enables them to form ionic bonds  (electrostatic bonds) with negatively charged groups, such as the side chains of  acidic amino acids or the phosphate groups of coenzymes (Fig. 6.8). In addition,  lysine and arginine side chains often form ionic bonds with negatively charged com-  pounds bound to the protein binding sites, such as the phosphate groups in ATP. The  acidic and basic amino acid side chains also participate in hydrogen bonding and  the formation of salt bridges (such as the binding of an inorganic ion such as Na +  between two partially or fully negatively charged groups).   The charge on these amino acids at physiologic pH is a function of their pK a s for  dissociation of protons from the a-carboxylic acid groups, the a-amino groups, and  the side chains. The titration curve of histidine illlustrates the changes in amino acid  structure occurring as the pH of the solution is changed from less than 1 to 14 by  the addition of hydroxide ions (Fig. 6.9). At low pH, all groups carry protons; amino  groups have a positive charge, and carboxylic acid groups have zero charge. As the  pH is increased by the addition of alkali (OH~), the proton dissociates from the car-  boxylic acid group, and its charge changes from zero to negative with a pK a of  approximately 2, the pH at which 50% of the protons have dissociated.   The histidine side chain is an imidazole ring with a pK a of approximately 6  that changes from a predominantly protonated positively charged ring to an  uncharged ring at this pH. The amino group on the a-carbon titrates at a much  higher pH (between 9 and 10), and the charge changes from positive to zero as  the pH rises. The pH at which the net charge on the molecules in solution is zero  is called the isoelectric point (pi). At this pH, the molecules will not migrate in  an electric field toward either a positive pole (cathode) or a negative pole  (anode), because the number of negative charges on each molecule is equal to the  number of positive charges.   Amino acid side chains change from uncharged to negatively charged, or posi-  tively charged to uncharged as they release protons (Fig. 6.10). The acidic amino  acids lose a proton from their carboxylic acid side chains at a pH of roughly 4, and  are thus negatively charged at pH 7.4. Cysteine and tyrosine lose protons at their  pK a s (~ 8.4 and 10.5, respectively), so their side chains are uncharged at physiologic  pH. Histidine, lysine, and arginine side chains change from positively charged to  neutral at their pK a s. The side chains of the two basic amino acids, arginine and  lysine, have pK a values above 10, so that the positively charged form always pre-  dominates at physiologic pH. The side chain of histidine (pK a ~ 6.0) dissociates  near physiologic pH, so only a portion of the histidine side chains carry a positive  charge (see Fig. 6.9).   In proteins, only the amino acid side chains and the amino group at the amino  terminal and carboxyl group at the carboxyl terminal have dissociable protons. All  of the other carboxylic acid and amino groups on the a-carbons are joined in pep-  tide bonds that have no dissociable protons. The amino acid side chains might have  very different pK a s than those of the free amino acids if they are involved in hydro-  gen or ionic bonds with other amino acid side chains. The pK a of the imidazole  group of histidine, for example, is often shifted to a higher value between 6 and 7  so that it adds and releases a proton in the physiologic pH range.   Glutamate carries a negative  i charge on its side chain at physio-  logic pH and thus can engage in  ionic bonds or hydrogen bonds with water  or other side chains. Valine is a hydrophobic  amino acid and therefore tends to interact  with other hydrophobic side chains to  exclude water. (The effect of this substitution  on hemoglobin structure is described in  more detail in Chapter 7.)   Fig. 6.8. Electrostatic interaction between the  positively charged side chain of lysine and the  negatively charged side chain of aspartate.   Electrophoresis is a technique used  \\ ) to separate proteins on the basis of  \^^/ charge that has been extremely  useful in medicine to identify proteins with  different amino acid composition. The net  charge on a protein at a certain pH is a sum-  mation of all of the positive and negative  charges on all of the ionizable amino acid  side chains plus the N-terminal amino and C-  terminal carboxyl groups. Theoretically, the  net charge of a protein at any pH could be  determined from its amino acid composition  by calculating the concentration of positively  and negatively charged groups from the  Henderson-Hasselbalch equation (see Chap-  ter 4). However, hydrogen bonds and ionic  bonds between amino acid side chains in the  protein make this calculation unrealistic.   Although almost every amino acid in the primary structure of a protein contributes  to its conformation (three-dimensional structure), the primary structure of a protein   + I P K a1 + I P K a2 + I P K a3   Fig. 6.9. Titration curve of histidine. The ionic species that predominates in each region is shown below the graph, pi is the isoelectric point (at  which there is no net charge on the molecule).   Aspartate |- CH 2 - COOH  Glutamate |- CH 2 - CH 2 -COOH   |- CH 2 -CH 2 - COO" + H +  N-   I + 10.5 I  Lysine |— CH 2 -CH 2 -CH 2 -CH 2 -NH 3 < ► |— CH 2 -CH 2 -CH 2 -CH 2 -NH 2 + H +   s // NH 2 12.5 | //NH  Arginine |— CH 2 -CH 2 -CH 2 -NH-C < ► |— CH 2 -CH 2 -CH 2 -NH-C + H +   Fig. 6.10. Dissociation of the side chains of the amino acids. As the pH increases, the charge on the side chain goes from 0 to + or from + to  0. The pK a is the pH at which half the molecules of an amino acid in solution have side chains that are charged. Half are uncharged.   can vary to some degree between species. Even within the human species, the  amino acid sequence of a normal functional protein can vary somewhat among indi-  viduals, tissues of the same individual, and the stage of development. These varia-  tions in the primary structure of a functional protein are tolerated if they are con-  fined to noncritical regions (called variant regions), if they are conservative  substitutions (replace one amino acid with one of similar structure), or if they con-  fer an advantage. If many different amino acid residues are tolerated at a position,  the region is called hypervariable. In contrast, the regions that form binding sites or  are critical for forming a functional three-dimensional structure are usually invari-  ant regions that have exactly the same amino acid sequence from individual to indi-  vidual, tissue to tissue, or species to species.   Within the human population, the primary structure of a protein may vary  slightly among individuals. The variations generally arise from mutations in  DNA that are passed to the next generation. The mutations can result from the  substitution of one base for another in the DNA sequence of nucleotides (a point  mutation), from deletion or insertions of bases into DNA, or from larger changes  (see Chap. 14). For many alleles, the variation has a distinct phenotypic conse-  quence that contributes to our individual characteristics, produces an obvious  dysfunction (a congenital or genetically inherited disease), or increases suscep-  tibility to certain diseases. A defective protein may differ from the most common  allele by as little as a single amino acid that is a nonconservative substitution  (replacement of one amino acid with another of a different polarity or very dif-  ferent size) in an invariant region. Such mutations might affect the ability of the  protein to carry out its function, catalyze a particular reaction, reach the appro-  priate site in a cell, or be degraded. For other proteins, the variations appear to  have no significance.   Variants of an allele that occur with a significant frequency in the population are  referred to as polymorphisms. Thus far in studies of the human genome, almost one  third of the genetic loci appear to be polymorphic. When a particular variation of an  allele, or polymorphism, increases in the general population to a frequency of over   Is the substitution of a glutamate  for a valine in sickle cell hemoglo-  bin a conservative replacement?  What about the substitution of an aspartate  for a glutamate?   For the most part, human chromo-  somes occur as homologous pairs,  with each member of a pair contain-  ing the same genetic information. One mem-  ber of the pair is inherited from the mother  and one from the father. Genes are arranged  linearly along each chromosome. A genetic  locus is a specific position or location on a  chromosome. Alleles are alternate versions of  a gene at a given locus. For each locus (site),  we have two alleles of each gene, one from  our mother and one from our father. If both  alleles of a gene are identical, the individual is  homozygous for this gene; if the alleles are  different, he is heterozygous for this gene. Will  Sichel has two identical alleles for the sickle  variant of the (3-globin gene that results in  substitution of a valine for a glutamate residue  at the sixth position of the p-globin chain. He  is, therefore, homozygous and has sickle cell  anemia. Individuals with one normal gene and  one sickle cell allele are heterozygous. They  are carriers of the disease and have sickle cell  trait.   Will Sichel's hemoglobin, HbS, comprises two normal a chains and 2 (3-globin chains with the sickle cell variant (a 2 £ 2 s ). The  change in amino acid composition from a glutamate to a valine in the (3 chain allows sickle hemoglobin to be separated from  normal adult hemoglobin (HbA, or (a 2 p 2 A )) by electrophoresis. In electrophoresis, an aliquot of blood or other solution contain-  ing proteins is applied to a support, such as paper or a gel. When an electrical field is applied, proteins migrate a distance toward the  anode (negative pole) or cathode (positive pole) that reflects small differences in their net charge. Electrophoresis of Will Sichel's blood  shows that he is homozygous for the sickle cell variant, HbS, and has increased amounts of fetal hemoglobin, HbF. Individuals with sickle  cell trait are heterozygous and have both HbA and HbS, plus small amounts of HbF (a 2 7 2 )-   In heterozygous individuals with sickle cell trait, the sickle cell allele provides some protection against malaria. Malaria is caused by  the parasite Plasmodium falciparum, which spends part of its life cycle in red blood cells. The infected red blood cells of individuals with  normal hemoglobin (HbA) develop protrusions that attach to the lining of capillaries. This attachment occludes the vessels and prevents  oxygen from reaching cells in the affected region, resulting in cell death. In heterozygous individuals, HbS in infected cells aggregates into  long fibers that cause the cell to become distorted. These distorted cells containing the parasite are preferentially recognized by the spleen  and are rapidly destroyed, thus ending the life of the parasite.   In Will Sichel and other homozygous individuals with sickle cell anemia, the red blood cells sickle more frequently, especially under  conditions of low oxygen tension (see Chapter 7). The result is a vaso-occlusive crisis in which the sickled cells clog capillaries and pre-  vent oxygen from reaching cells (ischemia), thereby causing pain. The enhanced destruction of the sickled cells by the spleen results in  anemia. Consequently, the sickle cell allele is of little advantage to homozygous individuals.   Because heterozygous individuals occur more frequently in a population than homozygous individuals, a selective advantage in a het-  erozygous state can outweigh a disadvantage in a homozygous state, causing the mutation to become a stable polymorphism in a popu-  lation. As a consequence, the frequency of sickle cell anemia in parts of equatorial Africa in which malaria was endemic in the past is 1 in  25 births. Migration from Africa accounts for the high frequency of sickle cell anemia among blacks in the United States, which is approx-  imately 1/400 at birth.   The substitution of a glutamate for  a valine is a nonconservative  replacement because a negatively  charged amino acid is substituted for a  hydrophobic branched chain aliphatic amino  acid. However, the substitution of an aspar-  tate for a glutamate is a conservative  replacement because the two amino acids  have the same polarity and nearly the same  size.   Homologous families of proteins  \\ are proteins that have the same   ancestral protein and arose from  the same gene. The term homologs includes  both orthologs and paralogs. Orthologs are  genes from different species that have  evolved from a common ancestral gene as  different species developed (for example,  human and pork insulin). By contrast, par-  alogs are genes related by duplication within  the genome of a single species (e.g., myo-  globin and hemoglobin). Normally, othologs  retain the same function in the course of  evolution, whereas paralogs evolve new  functions that may, or may not, be related to  the original one.   1%, it is considered stable. The sickle cell allele is an example of a point mutation  that is stable in the human population. Its persistence is probably attributable to  selective pressure for the heterozygous mutant phenotype, which confers some pro-  tection against malaria.   A homologous family of proteins is composed of proteins related to the same ances-  tral protein. Groups of proteins with similar, but not identical, structure and func-  tion that have evolved from the same gene after the gene was duplicated are called  paralogs and considered members of the same protein family. Once a gene has  duplicated, one gene can continue to perform original function, and the second copy  can mutate into a protein with another function or another type of regulation. This  process is called divergent evolution. Very large families of homologous proteins  are called a superfamily, which is subdivided by name into families of proteins with  the most similarity in structure.   The paralogs of a protein family are considered different proteins and have  different names because they have different functions. They are all present in  the same individual. Myoglobin and the different chains of hemoglobin, for  example, are paralogs and members of the same globin family that have similar,  but not identical, structures and functions. Myoglobin, an intracellular heme  protein present in most cells that stores and tranports 0 2 to mitochondria, is a  single polypeptide chain containing one heme oxygen-binding site. In contrast,  hemoglobin is composed of four globin chains, each with a heme oxygen-  binding site that is present in red blood cells and transports 0 2 from the lungs  to tissues. The gene for myoglobin is assumed to have evolved from gene dupli-  cation of the a-chain for hemoglobin, which evolved from duplication of the p-  chain. Figure 6.11 compares a region of the structure of myoglobin and the a  and p chains of hemoglobin. Among these three proteins, only 15 invariant  (identical) residues are present, but many of the other amino acid residues are  conservative substitutions.   To compare the primary structure of two homologous polypeptide chains, the  sequences are written left to right from the amino terminal to the carboxyl ter-  minal. The sequences are aligned with computer programs that maximize the  identity of amino acids and minimize the differences caused by segments that are pres-  ent in one protein and not in the other.   Fig 6.11. The primary structures of a region in human globin proteins. Gaps in the structure, indicated with dashes, are introduced to maximize  the alignment between proteins in structure comparisons. They are assumed to coincide with mutations that caused a deletion. Regions of  sequence similarity (identity and conservative substitution) are indicated in blue. Within these regions, there are smaller regions of invariant  residues that are exactly the same from protein to protein. Myoglobin is a single polypeptide chain. The a and p chains are part of hemoglobin  A (a 2 p 2 )- The £ chain is part of embryonic hemoglobin (£2^2) • The 7 chain is part of fetal hemoglobin (HbF), a 2 y 2 -   Within the same individual, different isoforms or isozymes of a protein may be syn-  thesized during different stages of fetal and embryonic development, may be pres-  ent in different tissues, or may reside in different intracellular locations. Isoforms of  a protein all have the same function. If they are isozymes (isoforms of enzymes),  they catalyze the same reactions. However, isoforms have somewhat different prop-  erties and amino acid structure.   The term isozyme was originally  \\ ) defined as meaning enzymes with  V^^y a different primary structure, cat-  alyzing the same reaction, encoded by dif-  ferent genes. However, the term is now used  more broadly. Some isozymes are now  known to arise from alternate splicing of pre-  mRNA to form different mRNAs (mRNA is  the final nucleic acid template used for pro-  tein synthesis.)   Hemoglobin isoforms provide an example of variation during development.  Hemoglobin is expressed as the fetal isozyme HbF during the last trimester of  pregnancy until after birth, when it is replaced with HbA. HbF is composed of  two hemoglobin a and 2 hemoglobin y polypeptide chains, in contrast to the  adult hemoglobin, hemoglobin A, which is 2 a and 2 (3 chains. During the  embryonic stages of development, chains with a different amino acid composi-  tion, the embryonic s and £ chains, are produced (see Fig. 6.11). These differ-  ences are believed to arise evolutionarily from mutation of a duplicated a gene  to produce £, and mutation of a duplicate a gene to produce s. The fetal and  embryonic forms of hemoglobin have a much higher affinity for 0 2 than the adult  forms, and thus confer an advantage at the low 0 2 tensions to which the fetus is  exposed. At different stages of development, the globin genes specific for that  stage are expressed and translated.   Proteins that differ somewhat in primary structure and properties from tissue to tis-  sue, but retain essentially the same function, are called tissue-specific isoforms or  isozymes. The enzyme creatine kinase is an example of a protein that exists as tis-  sue-specific isozymes, each composed of two subunits with 60 to 72% sequence  homology. Of the two creatine kinases that bind to the muscle sarcomere, the M  form is produced in skeletal muscle, and the B polypeptide chains are produced in  the brain. The protein comprises two subunits, and skeletal muscle therefore pro-  duces an MM creatine kinase, and the brain produces a BB form. The heart pro-  duces both types of chains and therefore forms a heterodimer, MB, as well as an  MM dimer. Two more creatine kinase isozymes are found in mitochondria, a heart  mitochondrial creatine kinase, and the "universal" isoform found in other tissues.  (In general, most proteins present in both the mitochondria and cytosol will be pres-  ent as different isoforms.) The advantage conferred on different tissues by having  their own isoform of creatine kinase is unknown. However, tissue- specific isozymes  such as MB creatine kinase are useful in diagnosing sites of tissue injury and cell  death.   The structure of proteins involved in the response to hormones has been studied  in greater depth than many other types of proteins, and most of these proteins are  present as several tissue-specific isoforms that help different tissues respond differ-  ently to the same hormone. One of these proteins present in cell membranes is  adenylyl cyclase, an enzyme that catalyzes the synthesis of intracellular 3', 5' cyclic  adenosine monophosphate (cAMP) (Fig. 6.12). In human tissues, at least nine dif-  ferent isoforms of adenylyl cyclase are coded by different genes in different tissues.  Although they have an overall sequence homology of 50%, the two intracellular  regions involved in the synthesis of cAMP are an invariant consensus sequence with  a 93% identity. The different isoforms help cells respond differently to the same  hormone.    A myocardial infarction (heart  attack) is caused by an atheroma-  tous obstruction or a severe spasm  in a coronary artery that prevents the flow of  blood to an area of heart muscle. Thus, heart  cells in this region suffer from a lack of oxy-  gen and blood-borne fuel. Because the cells  cannot generate ATP, the membranes  become damaged, and enzymes leak from  the cells into the blood.   Creatine kinase (CK or CPK) is one of  these enzymes. The protein is composed of  two subunits, which may be either of the  muscle (M) or the brain (B) type. The MB  form, containing one M and one B subunit, is  found primarily in cardiac muscle. It can be  separated electrophoretically from other CK  isozymes and the amount in the blood used  to determine if a myocardial infarction has  occurred. On admission to the hospital, Ann  Jeina's total CK was 182 units/L (reference  range 5 38-174 U/L). Her MB fraction was  6.8% (reference range 5% or less of the total  CK). Although these values are only slightly  elevated, they are typical of the phase  immediately following a myocardial infarc-  tion. Additional information was provided  by myoglobin and troponin T (Tn-T) meas-  urements.   ® Variations in protein structure  among species have been used to  develop a phylogenetic tree of  ancestral relationships between species  showing the progress of evolution. Accord-  ing to evolutionary theory, related species  evolved from a common ancestor, and pro-  teins all can be grouped into clusters of pro-  teins (called orthologous proteins) that  evolved from a common ancestral protein.  The more similar the amino acid sequence  of an orthologous protein from two different  species, the closer is the relationship  between those species.   Although bovine (beef) insulin is  identical to human insulin in those  amino acid residues essential for  activity, the amino acid residues that are in  the variable regions can act as antigens and  stimulate the formation of antibodies  against bovine insulin. Consequently,  recombinant DNA techniques have been  used for the synthesis of insulin, identical in  structure to native insulin, such as Humulin  (intermediate-acting insulin) (see Chapter 17  for information on recombinant DNA tech-  nology.) Although Di Abietes had not yet  experienced an allergic response to her beef  insulin, the cost of Humulin is now suffi-  ciently low that most patients are being  changed from beef insulin to the synthetic  forms of human insulin.   acids known as the primary struc-  ture of the protein, and determine the fold-  ing pattern for the three-dimensional confor-  mation of the protein. Posttranslational  modifications usually occur once the protein  has already folded into its characteristic pat-  tern. They are like accessories (jewelry, ties,  etc.) that provide additional variations to  your basic wardrobe.   Fig 6.12. Invariant regions in the isoforms of adenylyl cyclase. The invariant regions are on  the cytosolic side of the membrane in the CI and C2 loops shown in blue. These amino acid  residues participate in the catalytic function of the enzyme, synthesis of 3 ',5 '-cyclic AMR  The protein also has several helical regions that span the membrane (Ml helices and M2  helices), represented as tubes. An oligosaccharide chain is attached to an extracellular  domain. N is the amino terminus. (Copyrighted from Taussig R, Gilman AG. J Biol Chem  Mammalian membrane-bound adenylyl cyclases 1995;270:l^k)   Species variations in primary structure are also important in medicine, as illus-  trated by the comparison of human, beef, and pork insulin. Insulin is one of the  hormones that are highly conserved between species, with very few amino acid  substitutions and none in the regions that affect activity. Insulin is a polypeptide  hormone of 51 amino acids that is composed of two polypeptide chains (Fig. 6.13).  It is synthesized as a single polypeptide chain, but is cleaved in three places  before secretion to form the C peptide and the active insulin molecule contain-  ing the A and B chains. The folding of the A and B chains into the correct three-  dimensional structure is promoted by the presence of one intrachain and two  interchain disulfide bonds formed by cysteine residues. The invariant residues  consist of the cysteine residues engaged in disulfide bonds and the residues that  form the surface of the insulin molecule that binds to the insulin receptor. The  amino acid substitutions in bovine and porcine insulin (shown in blue in  Fig. 6.13.) are not in amino acids that affect its activity. Consequently, bovine  and pork insulin were used for many years for the treatment of diabetes mellitus.  However, even with only a few different amino acids, some patients developed  an immune response to these insulins.   After synthesis of a protein has been completed, a few amino acid residues in the  primary sequence may be further modified in enzyme-catalyzed reactions that add  a chemical group, oxidize, or otherwise modify specific amino acids in the protein.  Because protein synthesis occurs by a process known as translation, these changes  are called posttranslational modification. More than 100 different posttranslation-  ally modified amino acid residues have been found in human proteins. These mod-  ifications change the structure of one or more specific amino acids on a protein in  a way that may serve a regulatory function, target or anchor the protein in mem-  branes, enhance a protein's association with other proteins, or target it for degrada-  tion (Fig. 6.14).   Fig. 6.13. The primary structure of human insulin. The substituted amino acids in bovine (beef) and porcine (pork) insulin are shown in blue.  Threonine 30 at the carboxy terminal of the B chain is replaced by alanine in both beef and pork insulin. In beef insulin, threonine 8 on the A  chain is also replaced with alanine, and isoleucine 10 with valine. The cysteine residues, which form the disulfide bonds holding the chains  together, are invariant. In the bioengineered insulin Humalog (lispro insulin), the position of proline at B28 and lysine at B29 is switched. Insulin  is synthesized as a longer precursor molecule, proinsulin, which is one polpeptide chain. Proinsulin is converted to insulin by proteolytic cleav-  age of certain peptide bonds (squiggly lines in the figure). The cleavage removes a few amino acids and the 31-amino acid C-peptide that con-  nects the A and B chains. The active insulin molecule, thus, has two nonidentical chains.   Oligosaccharides (small carbohydrate chains) are bound to proteins by either N-  linkages or O-linkages (see Fig. 6.14). AMinked oligosaccharides are found attached  to cell surface proteins, where they protect the cell from proteolysis or an immune  attack. In contrast, an (9-glycosidic link is a common way of attaching oligosac-  charides to the serine or threonine hydroxyl groups in secreted proteins. The intra-  cellular polysaccharide glycogen is attached to a protein through an (9-glycosidic  linkage to a tyrosine.   Many membrane proteins contain a covalently attached lipid group that interacts  hydrophobically with lipids in the membrane. Palmitoyl groups (CI 6) are often  attached to plasma membrane proteins, and the myristoyl group (C14) is often  attached to proteins in the lipid membranes of intracellular vesicles (see Fig.  6.14). The farnesyl (C15) or geranylgeranyl group (C20) are synthesized from  the five-carbon isoprene unit (isopentenyl pyrophosphate, see Fig. 5.1 A) and are  therefore called isoprenoids. These are attached in ether linkage to a specific  cysteine residue of certain membrane proteins, particularly proteins involved in  regulation.   Adenylyl cyclase is posttranslation-  Kj ) ally modified (see Fig. 6.12). It has  V*^/ an oligosaccharide chain attached  to the external portion of the protein. Some  of the isozymes contain serine residues on  the intracellular portion of the chain that  may be phosphorylated by a protein kinase.   Phosphorylation, acetylation, and adenosine diphosphate (ADP)-ribosylation of  specific amino acid residues in a polypeptide can alter bonding by that residue  and change the activity of the protein (see Fig. 6.14). Phosphorylation of an OH  group on serine, threonine, or tyrosine by a protein kinase (an enzyme that trans-  fers a phosphate group from ATP to a protein) introduces a large, bulky, nega-  tively charged group that can alter the activity of a protein. Reversible acetylation  occurring on lysine residues of histone proteins in the chromosome changes their   O-glycosylation: OH of ser, thr, tyr, /V-glycosylation: NH 2 of asn   H 3 C - (CH 2 )i2 " C - N - CH 2 - C «ww*  |H , ,   CH 3 -C = C-(CH 2 -C = C-CH 2 ) 2 -S   CH 2 -0-P - O-P -0-CH 2  .O v I I I I /O.   Fig. 6.14. Posttranslational modifications of amino acids in proteins. Some of the com-  mon amino acid modifications and the sites of attachment are illustrated. The added group  is shown in blue. Because these modifications are enzyme-catalyzed, only a specific  amino acid in the primary sequence is altered. R-O-hexagon refers to an oligosaccharide.  In Af-glycosylation, the attached sugar is usually N-acetylglucosamine (N-Ac).   interaction with the negatively charged phosphate groups of DNA. ADP-ribosyla-  tion is the transfer of an ADP-ribose from NAD + to an arginine, glutamine, or a  cysteine residue on a target protein in the membrane (primarily in leukocytes,  skeletal muscles, brain, and testes). This modification may regulate the activity of  these proteins   A number of other posttranslational modifications of amino acid side chains alter  the activity of the protein in the cell (see Fig 6.14). Carboxylation of the y carbon  of glutamate (carbon 4) in certain blood clotting proteins is important for attaching  the clot to a surface. Calcium ions mediate this attachment by binding to the two  negatively charged carboxyl groups of 7-glutamate and two additional negatively  charged groups provided by phospholipids in the cell membrane. Collagen, an  abundant fibrous extracellular protein, contains the oxidized amino acid hydrox-  yproline. The addition of the hydroxy 1 group to the proline side chain provides an  extra polar group that can engage in hydrogen bonding between the polypeptide  strands of the fibrous protein.    A number of pathogenic bacteria  produce bacterial toxins that are  ADP-ribosyl transferases (NAD + -  glycohydrolases). These enzymes hydrolyze  the N-glycosidic bond of NAD + and transfer  the ADP-ribose portion to a specific amino  acid residue on a protein in the affected  human cell. Cholera A-B toxin, a pertussis  toxin, and a diptheria toxin are all ADP-ribo-  syl transferases.   The unusual amino acid selenocysteine is found in a few enzymes and is required  for their activity (Fig. 6.15). Its synthesis is not a posttranslational modification,  however, but a modification to serine that occurs while serine is bound to a unique  tRNA. The selenocysteine is then inserted into the protein as it is being synthesized.   Will Sichel. Will Sichel was treated for 3 days with parenteral (intravas-  cular) narcotics, hydration, and nasal inhalation of oxygen for his vaso-  occlusive crisis. The diffuse severe pains of sickle cell crises result from  occlusion of small vessels in a variety of tissues, thereby causing damage to cells  from ischemia (low blood flow) or hypoxia (low levels of oxygen). Vaso-occlusion  occurs when HbS molecules in red blood cells polymerize in the capillaries, where  the partial pressure of 0 2 ( p0 2 ) is low. This polymerization causes the red blood  cells to change from a biconcave disc to a sickle shape that cannot deform to pass  through the narrow capillary lumen. The cells aggregate in the capillaries and  occlude blood flow.   In addition, Will Sichel was treated with hydroxyurea therapy, which  increases the production of red blood cells containing fetal hemoglobin. HbF  molecules cannot participate in sickling. Will Sichel's acute symptoms gradually  subsided. Had his severe pain persisted, partial exchange blood transfusions  would have been considered because no other effective therapy is currently  available. Patients with sickle cell anemia periodically experience sickle cell  crises, and Will's physician urged him to seek medical help whenever symptoms  reappeared.   Cal Kulis. Mr. Kulis has cystinuria, a relatively rare disorder, with a  prevalence that ranges between 1 in 2,500 to 1 in 15,000 births, depending  on the population studied. It is a genetically determined disease with a  complex recessive mode of inheritance resulting from allelic mutations. These  mutations lead to a reduction in the activity of renal tubular cell transport proteins  that normally carry cystine from the tubular lumen into the renal tubular cells. The  transport of the basic amino acids (lysine, arginine, and ornithine, an amino acid   The switch in position of amino  acids in lispro does not affect the  action of this synthetic insulin on  cells because it is not in a critical invariant  region, but it does affect the ability of insulin  to bind zinc. Normally, human insulin is  secreted from the pancreas as a zinc hexa-  mer in which six insulin molecules are  bound to the zinc atom. When zinc insulin is  injected, the binding to zinc slows the  absorption from the subcutaneous (under  the skin) injection site. Lispro cannot bind  zinc to form a hexamer, and thus it is  absorbed much more quickly than other  insulins.   found in the urea cycle but not in proteins) is also often compromised, and they  appear in the urine.   Because cystine is produced by oxidation of cysteine, conservative treatment  of cystinuria includes decreasing the amount of cysteine within the body and,  hence, the amount of cystine eventually filtered by the kidneys. Reduction of  cysteine levels is accomplished by restricting dietary methionine, which con-  tributes its sulfur to the pathway for cysteine formation. To increase the amount  of cystine that remains in solution, the volume of fluid ingested daily is  increased. Crystallization of cystine is further prevented by chronically alkalin-  izing the urine. Finally, drugs may be administered to enhance the conversion of  urinary cystine to more soluble compounds. If these conservative measures fail  to prevent continued cystine stone formation, existing stones may be removed by  a surgical technique that involves sonic fracture. The fragmented stones may  then pass spontaneously or may be more easily extracted surgically because of  their smaller size.   ^ Di Abietes. Di Abietes' treatment was first changed to daily injections  of Humulin instead of beef insulin. Humulin is now mass-produced by  recombinant DNA techniques that insert the human DNA sequences for the  insulin A and B chains into the Escherichia coli or yeast genome (see Chapter 17).  The insulin chains that are produced are then extracted from the media and treated  to form the appropriate disulfide bonds between the chains. As costs have fallen for  production of the synthetic human insulins, they have replaced pork insulin and the  highly antigenic beef insulin.   Di's physician then recommended that she take Humalog, an insulin prepara-  tion containing lispro, an ultra-fast- acting bioengineered insulin analog in which  lysine at position B29 has been moved to B28 and proline at B28 has been moved  to B29 (hence, lispro) (see Fig. 6.13). With lispro, Di will be able to time her  injections minutes before her consumption of carbohydrate -containing meals,  rather than having to remember to give herself an insulin injection 1 hour before  a meal.   Ann Jeina. Mrs. Jeina continued to be monitored in the cardiac care  unit. At admission, her CK levels were elevated (182 units/L compared  with a reference range of 38-174 U/L), and the MB fraction was high at  6.8% of the total (reference = <5% of the total). Her total CK continued to rise  (228 units/L 12 hours after admission and 266 units/L at 24 hours), as did her MB  fraction (8% at 12 hours and 10.8% at 24 hours). Within 2 hours of the onset of an  acute myocardial infarction, the MB form of CK begins leaking from heart cells that  were injured by the ischemic process. These rising serum levels of the MB fraction  (and, therefore, of the total CK) reach their peak 12 to 36 hours later and usually  return to normal within 3 to 5 days from the onset of the infarction (Fig. 6.16). In  addition to the CK measurements, her blood levels were also analyzed for myoglo-  bin and the heart isoform of troponin-T, a protein involved in muscle contraction (see  Chapter 7).   Enzyme and Protein Databases. Large databases of protein  structure have been assembled to collate data from various laboratories  around the world. The National Library of Medicine maintains a  website, PubMed, which catalogues the medical literature, and a search  engine that allows you to search for reference articles by topic or author   Fig. 6.16. Electrophoretic separation of serum creatine kinase enzymes from a normal  healthy adult and from a patient who had a myocardial infarction 24 hours previously. Crea-  tine kinase catalyzes the reversible transfer of a phosphate from ATP to creatine to form phos-  phocreatine and ADR The reaction is an important part of energy metabolism in heart mus-  cle, skeletal muscle, and brain. Three different forms of the dimer exist: BB (or CK-1) found  in brain, MB (or CK-2) found only in heart, and MM (or CK-3), found only in skeletal and  heart muscle (cathode, — ve; anode, +ve).   (http://www.ncbi.nlm.nih.gov/pubmed). From the menu, you can directly enter a  protein database or a structure database. These databases are linked, so that you  can type in the name of a protein, such as human hemoglobin chain A, obtain a  list of contributors to sequence data, and retrieve a complete amino acid  sequence for many proteins. You can link to PubMed to find recent articles about  the protein, or you can link to the structure database. A program called Cn3D can  be downloaded from this site that allows you to view the three-dimensional ver-  sions of the protein structures.   These databases are only a few of more than 500 biologic databases that have  been assembled to collate and exchange biologic information in the areas of DNA,  RNA, genomics, gene mapping, and protein structure. The first issue of the Journal  of Nucleic Acid Research each year provides a description of currently available   biologic databases. Their goal is to provide information that can relate a particular  DNA sequence or mutation to the protein involved, to its function, and to the patho-  logic consequences of a particular amino acid substitution, by comparing proteins  that have similar functional elements.   Will these databases be of any use to the practicing physician or to the prac-  tice-oriented medical student? Very few students will ever want to do protein  modeling. However, students and physicians may wish to use the literature search  in PubMed as part of their approach to evidence-based medicine. They also may  wish to use it to track definitions or fundamental knowledge about particular top-  ics. Thus, a move has begun to link biomedical and basic science textbooks to  PubMed.   Tatusov RL, Koonin EV, Lipman, DJ. A genomic perspective on protein families. Science  1997;278:631-637.   1 . In a polypeptide at physiologic pH, hydrogen bonding may occur between   (A) the side chains of a leucine residue and a lysine residue.   (B) the side chains of an aspartyl residue and a glutamyl residue.   (D) the amide group in the peptide bond and an aspartyl side chain.   2. Which of the following shows the linear sequence of atoms joined by covalent bonds in a peptide backbone?   3. Di Abietes' different preparations of insulin contain some insulin complexed with protamine that is absorbed slowly after  injection. Protamine is a protein preparation from rainbow trout sperm containing arginine-rich peptides that bind insulin.  Which of the following provides the best explanation for complex formation between protamine and insulin?   (A) Arginine is a basic amino acid that binds to negatively charged amino acid side chains in insulin.   (B) Arginine is a basic amino acid that binds to the a-carboxylic acid groups at the N-terminals of insulin chains.   (C) Arginine is a large bulky hydrophobic amino acid that complexes with leucine and phenylalanine in insulin.   (D) Arginine forms disulfide bonds with the cysteine residues that hold the A and B chains together.   (E) Arginine has a side chain that forms peptide bonds with the carboxyl terminals of the insulin chains.   4. Protein kinases phosphorylate proteins only at certain hydroxyl groups on amino acid side chains. Which of the following  groups of amino acids all contain side chain hydroxyl groups?   In a single individual, the primary structures of enzymes catalyzing the same reaction   (A) are exactly the same from cell type to cell type, although the amount of enzyme may differ.   (E) may differ between different cellular compartments of the same cell.   0 © Diseases can be caused by  ( /~oA j changes in protein structure that  y/ affect the protein's ability to bind  other molecules and carry out its function.  They also can be caused by conformational  changes in proteins that affect their solubil-  ity and degradability. In amyloidosis AL,  immunoglobulin chains form an insoluble  protein aggregate called amyloid in organs  and tissues. Alzheimer's disease and familial  amyloid polyneuropathy are neurodegener-  ative diseases characterized by the deposi-  tion of amyloid. Prion diseases result from  misfolding and aggregation of a normal cel-  lular protein. Even in sickle cell anemia, the  mutation in hemoglobin principally affects  the quarternary structure of hemoglobin and  its solubility, and not its ability to bind oxy-  gen.   A multitude of different proteins can be formed from only 20 common amino acids  because these amino acids can be linked together in an enormous variety of  sequences determined by the genetic code. The sequence of amino acids, its primary  structure, determines the way a protein folds into a unique three-dimensional  structure, which is its native conformation. Once folded, the three-dimensional  structure of a protein forms binding sites for other molecules, thereby dictating  the function of the protein in the body. In addition to creating binding sites, a pro-  tein must fold in such a way that it is flexible, stable, able to function in the cor-  rect site in the cell, and capable of being degraded by cellular enzymes.   Levels of protein structure. Protein structure is described in terms of four dif-  ferent levels: primary, secondary, tertiary, and quarternary (Fig. 7.1). The pri-  mary structure of a protein is the linear sequence of amino acids in the polypep-  tide chain. Secondary structure consists of local regions of polypeptide chains  formed into structures generally stabilized by hydrogen bonds, such as the regular  structures called a-helices and ^-sheets. The rigidity of the peptide backbone  determines the types of secondary structure that can occur. The tertiary structure  involves folding of the secondary structural elements into an overall three-dimen-  sional conformation. In globular proteins such as myoglobin, the tertiary struc-  ture generally forms a densely packed hydrophobic core with polar amino acid  side chains on the outside. Some proteins exhibit quaternary structure, the combi-  nation of two or more subunits, each composed of a polypeptide chain.   Domains and folds. The tertiary structure of a globular protein is made up of  structural domains, regions of structure that are recognized as separate and linked  to other domains in a simple way. Within a domain, a combination of secondary   structural elements forms a fold, such as the nucleotide binding fold, or an actin  fold. Folds are defined by their similarity in a number of different proteins.   Quaternary structure. Assembly of globular polypeptide subunits into a multi-  subunit complex can provide the opportunity for cooperative binding ofligands (e.g.,  0 2 binding to hemoglobin), form binding sites for complex molecules (e.g., antigen  binding to immunoglobulin), and increase stability of the protein. The polypeptide  chains of fibrous proteins such as collagen are aligned along an axis, have repeating  elements, and are extensively linked to each other through hydrogen bonds.   Ligand binding. Proteins form binding sites for a specific molecule, called a  ligand (e.g., ATP or 0 2 ) or for another protein. The affinity of a binding site for  its ligand is quantitatively characterized by an association or affinity constant,  K a , (or its dissociation constant, Kj).   Folding of proteins. The primary structure of a protein dictates the way that it  folds into its tertiary structure, which is a stable conformation that is identical to the  shape of other molecules of the same protein (that is, its native conformation.)  Chaperonins act as templates to overcome the kinetic barrier to reaching a stable  conformation. Prion proteins cause neurodegenerative diseases by acting as a tem-  plate for misfolding. Heat, acid, and other agents cause proteins to denature, that is,  to unfold or refold and lose their native three-dimensional conformation.   Will Sichel, who has sickle cell anemia, was readmitted to the hospital  with symptoms indicating that he was experiencing another sickle cell cri-  sis (see Chapter 6).   Anne Jeina is a 54-year-old woman who arrived in the hospital 4 days ago,  approximately 5 hours after she began to feel chest pain (see Chapter 6).  In the emergency room, the physician had drawn blood for the measure-  ment of myoglobin, CK-MB (creatine kinase, muscle-brain fraction) and cTN-T  (cardiac troponin T subunit). The results from these tests had supported the diagno-  sis of an acute MI (myocardial infarction), and Mrs. Jeina was hospitalized.   Amy Lloyd is a 62-year-old woman who presents with weakness, fatigue, an  enlarged tongue (macroglossia), and edema. She has signs and symptoms of  cardiac failure, including electrocardiographic abnormalities. Initial labora-  tory studies showed a serum creatinine of 1.9 mg/dL (reference range [females] =  0.5-1.1) indicating mild renal failure. A urinalysis indicated the presence of a moder-  ate proteinuria and numerous white blood cells in the urinary sediment. She was sub-  sequently diagnosed with amyloidosis/AL secondary to a plasma cell dyscrasia.   Di Abietes returned to her physician's office for a routine visit to monitor her  treatment (see Chapters 4, 5, and 6.) Her physician drew blood for an HbA lc  (pronounced hemoglobin A-l- c) determination. The laboratory reported a  value of 8.5%, compared with to a normal reference range of 5.8 to 7.2%.   The overall conformation of a protein, the particular position of the amino acid side  chains in three-dimensional space, gives a protein its function.   Amyloidosis is a term encompass-  ing many diseases that share a  common feature — the extracellular  deposition of pathologic insoluble fibrillar  proteins called amyloid in organs and tis-  sues. In Amy Lloyd's disease, amyloido-  sis/AL, the amyloid is derived from  immunoglobulin light chains (AL = amyloi-  dosis, light-chain related).   Proteins are generally grouped into major structural classifications: globular proteins,  fibrous proteins, transmembrane proteins, and DNA-binding proteins (Fig. 7.2).  Globular proteins are usually soluble in aqueous medium and resemble irregular balls.  The fibrous proteins are geometrically linear, arranged around a single axis, and have  a repeating unit structure. Another general classification, transmembrane proteins,  consists of proteins that have one or more regions aligned to cross the lipid membrane.  DNA-binding proteins, usually classified separately, are considered in Chapter 16.   The structure of these proteins is often described according to levels called pri-  mary, secondary, tertiary, and quaternary structure (see Fig. 7.1). The primary struc-  ture is the linear sequence of amino acid residues joined through peptide bonds to  form a polypeptide chain. The secondary structure refers to recurring structures  (such as the regular structure of the a-helix) that form in short localized regions of  the polypeptide chain. The overall three-dimensional conformation of a protein is  its tertiary structure. The quaternary structure is the association of polypeptide  subunits in a geometrically specific manner.   The overall three-dimensional structure of a protein must meet certain requirements  to enable the protein to function in the cell or extracellular medium of the body. The  first requirement is the creation of a binding site that is specific for just one mole-  cule, or a group of molecules with similar structural properties. The specific bind-  ing sites of a protein usually define its role. The three-dimensional structure also  must exhibit the degrees of flexibility and rigidity appropriate to its function. Some  rigidity is essential for the creation of binding sites and for a stable structure (i.e., a  protein that just flopped all over the place could not accomplish anything). How-  ever, flexibility and mobility in structure enables the protein to fold as it is synthe-  sized, and to adapt as it binds other proteins and small molecules. The three-dimen-  sional structure must have an external surface appropriate for its environment (e.g.,  cytoplasmic proteins need to keep polar amino acids on the surface to remain solu-  ble in an aqueous environment). In addition, the conformation must also be stable,  with little tendency to undergo refolding into a form that cannot fulfill its function  or that precipitates in the cell. Finally, the protein must have a structure that can de  degraded when it is damaged or no longer needed in the cell. Almost every region  in the sequence of amino acids, the primary structure, participates in fulfilling one  or more of these requirements through the chemical properties of the peptide bonds  and the individual amino acid side chains.   Fig. 7.3. The peptide backbone. Because of the  resonance nature of the peptide bond, the C  and N of the peptide bonds form a series of  rigid planes. Rotation within allowed torsion  angles can occur around the bonds attached to  the a-carbon. The side chains are trans to each  other, and alternate above and below the pep-  tide chain. The actual peptide bond is a hybrid  between the resonance forms shown, resulting  in a partial negative charge on the carbonyl  oxygen, a partial positive charge on the nitro-  gen, and partial double bond character for the  peptide bond itself.   The amino acids in a polypeptide chain are sequentially joined by peptide bonds  between the carboxyl group of one amino acid and amide group of the next amino  acid in the sequence (Fig. 7.3). Usually the peptide bond assumes a trans configu-  ration in which successive a-carbons and their R groups are located on opposite  sides of the peptide bond.   The polypeptide backbone can only bend in a very restricted way. The peptide  bond itself is a hybrid of two resonance structures, one of which has double bond  character, so that the carboxyl and amide groups that form the bond must, therefore,  remain planar (see Fig. 7.3.). As a consequence, the peptide backbone consists of a  sequence of rigid planes formed by the peptide groups (see Fig. 7.3). However, rota-  tion within certain allowed angles (torsion angles) can occur around the bond between  the a-carbon and the a-amino group and around the bond between the a-carbon and  the carbonyl group. This rotation is subject to steric constraints that maximize the   distance between atoms in the different amino acid side chains and forbid torsion  (rotation) angles that place the side chain atoms too close to each other. These folding  constraints, which depend on the specific amino acids present, limit the secondary and  tertiary structures that can be formed from the polypeptide chain.   Regions within polypeptide chains form recurring, localized structures known as  secondary structures. The two regular secondary structures called the a-helix and  the a- sheet contain repeating elements formed by hydrogen bonding between atoms  of the peptide bonds. Other regions of the polypeptide chain form nonregular non-  repetitive secondary structures, such as loops and coils.   The a-helix is a common secondary structural element of globular proteins, mem-  brane-spanning domains, and DNA-binding proteins. It has a stable rigid confor-  mation that maximizes hydrogen bonding while staying within the allowed rotation  angles of the polypeptide backbone. The peptide backbone of the a-helix is formed  by strong hydrogen bonds between each carbonyl oxygen atom and the amide  hydrogen (N-H) of an amino acid residue located four residues further down the  chain (Fig. 7.4). Thus, each peptide bond is connected by hydrogen bonds to the  peptide bond four amino acid residues ahead of it and four amino acid residues  behind it in the amino acid sequence. The core of the helix is tightly packed, thereby  maximizing association energies between atoms. The trans side chains of the amino  acids project backward and outward from the helix, thereby avoiding steric hin-  drance with the polypeptide backbone and with each other (Fig. 7.5).   p-Sheets are a second type of regular secondary structure that maximizes hydro-  gen bonding between the peptide backbones while maintaining the allowed tor-  sion angles. In (3-sheets, the hydrogen bonding usually occurs between regions  of separate neighboring polypeptide strands aligned parallel to each other (Fig. 7.6).  Thus, the carbonyl oxygen of one peptide bond is hydrogen-bonded to the nitro-  gen of a peptide bond on an adjacent strand. (This pattern contrasts with the  a-helix in which the peptide backbone hydrogen bonds are within the same  strand.) Optimal hydrogen bonding occurs when the sheet is bent (pleated) to  form (3 -pleated sheets.    Fig. 7.5. A view down the axis of an a-helix. The side chains (R) jut out from the helix. Steric  hindrance occurs if they come within their van der Waals radii of each other, and a stable  helix cannot form.    Fig. 7.4. The a-helix. Each oxygen of a car-  bonyl group of a peptide bond forms a hydro-  gen bond with the hydrogen atom attached to a  nitrogen atom in a peptide bond four amino  acids further along the chain. The result is a  highly compact and rigid structure.    Fig. 7.6. A |3-pleated sheet. In this case, the chains are oriented in opposite directions (antiparallel). The large arrows show the direction of the  carboxy terminal. The amino acid side chains (R) in one strand are trans to each other, and alternate above and below the plane of the sheet,  which can have a hydrophobic face and a polar face that engages in hydrogen bonding.   A renal biopsy used in the diagno-  sis of Amy Lloyd's disease showed  amorphous deposits in the  glomeruli. When stained with Congo red  dye, these deposits appeared red with ordi-  nary light microscopy and exhibited apple  green fluorescence when viewed in polar-  ized light. This staining is characteristic of  the amyloid fibril structure, which is com-  posed of repeated p-sheets aligned orthogo-  nally (perpendicular) to the axis of the fiber.   A number of diseases involve deposition  of a characteristic amyloid fiber. However, in  each of these diseases, the amyloid is  derived from a different protein that has  changed its conformation (three-dimen-  sional structure) to that of the amyloid  repeated (3-sheet structure. Once amyloid  deposition begins, it seems to proceed rap-  idly, as if the fibril itself were promoting for-  mation and deposition of more fibrils (a phe-  nomenon called "seeding"). The different  clinical presentations in each of these dis-  eases results from differences in the func-  tion of the native protein and the site of amy-  loid deposition.   The p -pleated sheet is described as parallel if the polypeptide strands run in the  same direction (as defined by their amino and carboxy terminals) and anti-parallel  if they run in opposite directions. Antiparallel strands are often the same polypep-  tide chain folded back on itself, with simple hairpin turns or long runs of polypep-  tide chain connecting the strands. The amino acid side chains of each polypeptide  strand alternate between extending above and below the plane of the p-sheet (see  Fig. 7.6). Parallel sheets tend to have hydrophobic residues on both sides of the  sheets; antiparallel sheets usually have a hydrophobic side and a hydrophilic side.  Frequently, sheets twist in one direction.   a-Helices and p -pleated sheets are patterns of regular structure with a repeating ele-  ment, the turn of a helix or a pleat. In contrast, bends, loops, and turns are nonreg-  ular secondary structures that do not have a repeating element. They are character-  ized by an abrupt change of direction and are often found on the protein surface. For  example, p -turns are short regions usually involving four successive amino acid  residues. They often connect strands of antiparallel p- sheets (Fig. 7.7.). The surface  of large globular proteins usually has at least one omega loop, a structure with a  neck like the capital Greek letter omega, fl.    Fig. 7.7. p-turn. The four amino acid residues that form the p-turn (also called a hairpin loop)  are held together by hydrogen bonds, which make this an extremely stable structure.   Figure 7.8 is a three-dimensional drawing of a globular domain in the soluble  enzyme lactate dehydrogenase (LDH). It illustrates the combination of secondary  structural elements to form patterns. This LDH domain is typical of globular pro-  teins, which average approximately 31% a-helical structure and approximately  28% p-pleated sheets (with a wide range of variation.) The helices of globular  domains have an average span of approximately 12 residues, corresponding to  approximately three to four helical turns, although many are much longer. The p-  sheets, represented in diagrams by an arrow for each strand, are an average of six  residues long and six strands wide (2-15 strands). Like the p-sheet in the lactate  dehydrogenase domain, they generally twist to the right, rather than lie flat (see  Fig. 7.8). Most globular domains, such as this LDH domain, also contain motifs.  Motifs are relatively small arrangements of secondary structure recognized in many  different proteins. For example, certain of the p-strands are connected with  a-helices to form the pa Pa P structural motif.   The remaining polypeptide segments connecting the helices and p-sheets are  said to have a coil or loop conformation (see Fig. 7.8). Although some of the con-  necting segments recognized in many proteins have been given names (like the  Il-loops), other segments such as those in this LDH domain appear disordered or  irregular. These nonregular regions, generally called coils, should never be  referred to as "random coils." They are neither truly disordered nor random; they  are stabilized through specific hydrogen bonds dictated by the primary sequence  of the protein and do not vary from one molecule of the protein to another of the  same protein.   The nonregular coils, loops, and other segments are usually more flexible than  the relatively rigid helices and p -pleated sheets. They often form hinge regions that  allow segments of the polypeptide chain to move as a compound binds or to move  as the protein folds around another molecule.   Fig. 7.8. Ribbon drawing showing the arrangement of secondary structures into a three-  dimensional pattern in domain 1 of lactate dehydrogenase. The individual polypeptide  strands in the six-stranded p-sheet are shown with arrows. Different strands are connected by  helices and by nonrepetitive structures (turns, coils and loops), shown in blue. This domain  is the nucleotide binding fold. NAD + is bound to a site created by the helices (upper left of  figure.) (Modified from Richardson JS. Adv Protein Chem. The anatomy and taxonomy of  protein structure 1981;34: 167).   Although it is usually assumed that  proteins can have truly disordered  regions, the more that is learned  about protein structure, the less disordered  these regions seem. Even regions that look  truly disordered may form a specific binding  site for another molecule (e.g., the intracel-  lular C-terminal of transmembrane proteins).  Eventually all regions of all proteins may be  classified as a particular named pattern.   Flexibility is one of the most impor-  \\ ) tant features of protein structure.  VJ^/ Although every portion of every  amino acid in a protein is engaged in bond-  ing to the rest of the protein, to water, or to  a ligand, proteins do not have rigid struc-  tures. The three-dimensional structure is  flexible and dynamic, with rapidly fluctuat-  ing movement in the exact position of amino  acid side chains and domains. These fluctu-  ations are like wiggling or shaking that can  occur without unfolding. They allow ions  and water to diffuse through the structure  and provide alternate conformations for  ligand binding.   The tertiary structure of a protein is the folding pattern of the secondary structural  elements into a three-dimensional conformation, as shown for the LDH domain in  Figure 7.8. As illustrated with examples below, this three-dimensional structure is  designed to serve all aspects of the protein's function. It creates specific and flexi-  ble binding sites for ligands (the compound that binds), illustrated with actin and  myoglobin. The tertiary structure also maintains residues on the surface appropriate  for the protein's cellular location, polar residues for cytosolic proteins, and  hydrophobic residues for transmembrane proteins (illustrated with the p 2 -adrenergic  receptor).   The tertiary structure of large complex proteins is often described in terms of  physically independent regions called structural domains. You can usually iden-  tify domains from visual examination of a three-dimensional figure of a protein,  such as the three-dimensional figure of G-actin shown in Fig. 7.9. Each domain  is formed from a continuous sequence of amino acids in the polypeptide chain  that are folded into a three-dimensional structure independently of the rest of the  protein, and two domains are connected through a simpler structure like a loop  (e.g., the hinge region of Fig. 7.9). The structural features of each domain can be  discussed independently of another domain in the same protein, and the struc-  tural features of one domain may not match that of other domains in the same  protein.   Fig. 7.9. G- Actin. ATP binds in the center of the cleft. The two domains that form the cleft  are further subdivided into subdomains 1-4. The overall structure is found in many ATP-  binding proteins and is called the actin fold. The conformations of the regions shown in blue  are nearly superimposable among the proteins that contain the actin fold. (From Kabsch W,  Holmes KC. The actin fold. FASEB J 1995:9:167-174.)   Folds are relatively large patterns of three-dimensional structure that have been rec-  ognized in many proteins, including proteins from different branches of the phylo-  genetic tree. A characteristic activity is associated with each fold, such as ATP bind-  ing and hydrolysis (the actin fold) or NAD + binding (the nucleotide-binding fold).  These three examples of fold patterns are discussed below.   In the three-dimensional drawing of G-actin shown in Figure 7.9, all four subdo-  mains contribute to a folding pattern called the actin fold, named for the first pro-  tein in which it was described. ATP is bound into the middle of the cleft of the actin  fold by amino acid residues contributed by domains on both sides; thus, ATP bind-  ing promotes a conformational change that closes the cleft. Once bound, ATP is  cleaved to ADP and phosphate.   The actin fold is found in proteins as diverse as actin, which polymerizes to form  the cytoskeleton, heat shock protein 70 (hsp70), which uses ATP energy in chang-  ing the conformation of other proteins and hexokinase, which catalyzes phosphory-  lation of glucose (see Chapter 8 for further discussion of hexokinase.) Although  these proteins have very little sequence identity, three-dimensional drawings of their  actin folds are almost superimposable. The amount of sequence identity they do  have is consistent with their membership in the same fold family and establishes  that they are all homologs of the same ancestral protein. In all of these proteins, ATP  binding results in large conformational changes that contribute to the function of the  protein.   More than 1000 folds have now  \\ ) been recognized, and it is pre-  VJ^/ dieted that there are only a few  thousand different folds for all the proteins  that have ever existed. One of the values of  classifying proteins into fold patterns is that  a specific function, such as ATP binding and  hydrolysis, is associated with each fold. Cur-  rently, we know the primary structure of  many proteins involved in inherited diseases  without knowing the function of the protein.  However, the function of a domain in a pro-  tein can be predicted if its amino acid  sequence can be matched to that of other  proteins in a fold family with a known func-  tion. Classifying proteins into fold families  also may be useful for drug design. The  structure of a bacterial protein targeted by a  drug can be compared with that of human  proteins in the same fold family so that an  antibacterial drug can be designed that more  specifically targets the bacterial protein.   A fold also can be formed by one domain. In the example of secondary structures  provided by lactate dehydrogenase (see Fig. 7.8), domain 1 alone forms the  nucleotide binding fold. This fold is a binding site for NAD + or, in other proteins,  molecules with a generally similar structure (e.g., riboflavin). However, many pro-  teins that bind NAD + or NADP + contain a very different fold from a separate fold  family. These two different NAD + binding folds arise from different ancestral lines  and have different structures, but have similar properties and function. They are  believed to be the product of convergent evolution.   G-actin subunits polymerize to  form F-actin, which forms the  cytoskeleton (see Chapter 10). The  conformational change caused by ATP bind-  ing to the G-actin subunits promotes their  addition to the growing ends of the F-actin  polymers. Hydrolysis of bound ATP to ADP  by the G-actin subunit promotes dissocia-  tion.   Most globular proteins are soluble in the cell. In general, the core of a globular  domain has a high content of amino acids with nonpolar side chains (val, leu, ile,  met, and phe), out of contact with the aqueous medium. This hydrophobic core is  densely packed to maximize attractive van der Waals forces, which exert themselves  over short distances. The charged polar amino acid side chains (arg, his, lys, asp,  and glu) are generally located on the surface of the protein, where they form ion  pairs (salt bridges) or are in contact with aqueous solvent. Charged side chains often  bind inorganic ions (e.g., K + , P0 4 3_ or Cl~) to decrease repulsion between like  charges. When charged amino acids are located on the interior, they are generally  involved in forming specific binding sites. The polar uncharged amino acid side  chains of ser, thr, asn, gin, tyr, and trp are also usually found on the surface of the  protein, but may occur in the interior, hydrogen bonded to other side chains.  Although cystine disulfide bonds (the bond formed by two cysteine sulfhydryl  groups) are sometimes involved in the formation of tertiary structure, they are gen-  erally not needed.   ®Many features of the adrenaline  ((32-adrenergic) receptor are typical  of hormone receptors. Each extra-  cellular loop is actually a structural domain,  but several loops together form the binding  site for the hormone. The binding site is  sometimes referred to as a binding domain  (a functional domain), even though it is not  formed from a continuous segment of the  polypeptide chain. The amino terminus  (residues 1-34) extends out of the membrane  and has branched high mannose oligosac-  charides linked through N-glycosidic bonds  to the amide of asparagine. It is anchored in  the lipid plasma membrane by a palmitoyl  group (shown as a squlggle in Fig. 7.10) that  forms a thioester with the SH residue of a  cysteine. The COOH terminus, which extends  into the cytoplasm, has a number of serine  and threonine phosphorylation sites (shown  as blue circles in Fig. 7.10).   Transmembrane proteins, such as the (3 2 - adrenergic receptor, contain mem-  brane-spanning domains and intracellular and extracellular domains on either  side of the membrane (Fig. 7.10). Many ion channel proteins, transport proteins,  neurotransmitter receptors, and hormone receptors contain similar membrane-  spanning segments that are a-helices with hydrophobic residues exposed to the  lipid bilayer. These rigid helices are connected by loops containing hydrophilic  amino acid side chains that extend into the aqueous medium on both sides of the  membrane. In the p 2 -adrenergic receptor, the helices clump together so that the  extracellular loops form a surface that acts as a binding site for the hormone  adrenaline (epinephrine), our fight or flight hormone. The binding site is some-  times referred to as a binding domain (a functional domain), even though it is  not formed from a continuous segment of the polypeptide chain. Once adrena-  line binds to the receptor, a conformational change in the arrangement of rigid  helical structures is transmitted to the intracellular domains that form a binding  site for another signaling protein, a heterotrimeric G protein (a guanosine  triphosphate [GTP] -binding protein composed of three different subunits).  Thus, receptors require both rigidity and flexibility to transmit signals across  the cell membrane.   As discussed in Chapter 6, transmembrane proteins usually have a number of  posttranslational modifications that provide additional chemical groups to fulfill  requirements of the three-dimensional structure. The amino terminus (residues  1-34) extends out of the membrane and has branched high mannose oligosaccha-  rides linked through TV -glycosidic bonds to the amide of asparagine (see Fig. 7.10).  It is anchored in the lipid plasma membrane by a palmitoyl group that forms a  thioester with the SH residue of a cysteine. The COOH terminus, which extends    Fig. 7.10. p 2 - Adrenergic receptor. The receptor has seven a-helix domains that span the membrane and is therefore a member of the heptaheli-  cal class of receptors. A. The transmembrane domains are drawn in an extended form. B. The seven transmembrane helices (shown as tubes)  form a cylindrical structure. Loops connecting helices form the hormone binding site on the external side of the plasma membrane, and a bind-  ing site for a G-protein is on the intracellular side. The protein also contains oligosaccharide chains, palmityl groups, and phosphorylation sites  (shown in blue; see Chapter 6).   into the cytoplasm, has a number of serine and threonine phosphorylation sites  (shown as blue circles) that regulate receptor activity.   The quaternary structure of a protein refers to the association of individual polypep-  tide chain subunits in a geometrically and stoichiometrically specific manner. Many  proteins function in the cell as dimers, tetramers, or oligomers, proteins in which  two, four, or more subunits, respectively, have combined to make one functional  protein. The subunits of a particular protein always combine in the same number  and in the same way, because the binding between the subunits is dictated by the  tertiary structure, which is dictated by the primary structure, which is determined by  the genetic code.   A number of different terms are used to describe subunit structure. The prefixes  "homo" or "hetero" are used to describe identical or different subunits, respectively,  of 2, 3, or 4 subunit proteins (e.g., heterotrimeric G proteins have three different  subunits). A protomer is a unit structure composed of nonidentical subunits. In con-  trast, F-actin is an oligomer, a multisubunit protein composed of identical G-actin  subunits. "Multimer" is sometimes used as a more generic term to designate a com-  plex with many subunits of more than one type.   The contact regions between the subunits of globular proteins resemble the inte-  rior of a single subunit protein; they contain closely packed nonpolar side chains,  hydrogen bonds involving the polypeptide backbones and their side chains, and  occasional ionic bonds or salt bridges. The subunits of globular proteins are very  rarely held together by interchain disulfide bonds, and never by other covalent  bonds. In contrast, fibrous and other structural proteins may be extensively linked  to other proteins through covalent bonds.   Assembly into a multisubunit structure increases the stability of a protein. The  increase in size increases the number of possible interactions between amino  acid residues and therefore makes it more difficult for a protein to unfold and  refold. As a result, many soluble proteins are composed of two or four identical  or nearly identical subunits with an average size of approximately 200 amino  acids.   A multisubunit structure has many advantages besides increased stability. It may  enable the protein to exhibit cooperativity between subunits in binding ligands  (illustrated later with hemoglobin) or to form binding sites with a high affinity for  large molecules (illustrated with antigen binding to the immunoglobulin molecule.  IgG). An additional advantage of a multisubunit structure is that the different sub-  units can have different activities and cooperate in a common function. Examples  of enzymes that have regulatory subunits or exist as multiprotein complexes are pro-  vided in Chapter 9.   Creatine phosphokinase (CK), one  of the proteins measured to follow  Ann Jeina's myocardial infarction  (see Chapter 6) is present in cells as dimers  (two subunits). The dimers may be homod-  imers (two identical subunits of either the M  [muscle] isozyme or the B [brain] isozyme),  or heterodimers (MB) The MB isozyme is  produced only by the heart and readily  released from injured cardiomyocytes into  the blood (see Chapter 6).   Serum levels of lactate dehydrogenase  (LDH) were formerly used to diagnose an  acute Ml. LDH is present in cells as a  tetramer of four identical, or nearly identical,  subunits. Each subunit is a separate  polypeptide chain with a molecular weight  of 35 kD (approximately 35,000 g/mole).  These subunits are present as two isoforms,  the H isoform (for heart) and the M isoform  (for skeletal muscle). Although the heart pro-  duces principally the H 4 form (four H sub-  units combined into a tetramer) and skeletal  muscles produce principally the M 4 isoform,  the heart, skeletal muscle, and other tissues  produce several intermediate combinations  (e.g., H 3 M, H 2 M 2 ). These tetrameric isoforms  all have similar activities, but the individual  monomeric subunits are inactive. Measure-  ments of LDH isozymes in the serum are no  longer used for diagnosis of a recent Ml  because the enzyme is large, released  slowly, and the isozyme pattern is not as  specific for the heart as is CK.   Insulin is composed of two nonidentical polypeptide chains attached to each  \\ other through disulfide bonds between the chains (see Chapter 6, Fig. 6.13).  *r The subunits of globular proteins are generally not held together by disulfide  bonds, but regions of the same chain may be connected by disulfide bonds that form as  the chain folds. Insulin actually fits this generalization because it is synthesized as a sin-  gle polypeptide chain, which forms the disulfide bonds. Subsequently, a proteolytic  enzyme in secretory vesicles clips the polypeptide chain into two nonidentical subunits.  Generally, each subunit of most protomers and oligomers is synthesized as a separate  polypeptide chain. In fibrous proteins, which have a regular, sometimes repeating  sequence of amino acids, interchain binding serves different functions. In collagen, for  example, extensive interchain binding provides great tensile strength. The structure of  collagen is discussed in Chapter 49 on connective tissue.   Equation 7.1. The association constant,  K a for a binding site on a protein.   Consider a reaction in which a ligand (L)  binds to a protein (P) to form a ligand—  protein complex (LP) with a rate constant  of k q . LP dissociates with a rate constant  of ko.:   The equilibrium constant, K eq , is equal to  the association constant (K a ) or 1/K d , the  dissociation constant. Unless otherwise  given, the concentrations of L, P, and LP  are expressed as mol/L, and K a has the  units of (mol/L) ~\   In the examples of tertiary structure discussed above, the folding of a protein cre-  ated a three-dimensional binding site for a ligand (NAD + for the lactate dehydro-  genase domain 1, ATP for hexokinase, or adrenaline for the p 2 adrenergic receptor).  The binding affinity of a protein for a ligand is quantitatively described by its asso-  ciation constant, K a , which is the equilibrium constant for the binding reaction of a  ligand (L) with a protein (P) (Eq. 7.1).   K a is equal to the rate constant (kj) for association of the ligand with its bind-  ing site divided by the rate constant (k 2 ) for dissociation of the ligand-protein  complex (LP). K d , the dissociation constant for ligand-protein binding, is the  reciprocal of K a . The tighter the binding of the ligand to the protein, the higher  is the K a and the lower is the K d . The K a is useful for comparing proteins pro-  duced by different alleles, or for describing the affinity of a receptor for differ-  ent drugs.   Myoglobin and hemoglobin are two oxygen-binding proteins with a very similar  primary structure (Fig. 7.11). However, myoglobin is a globular protein com-  posed of a single polypeptide chain that has one 0 2 binding site. Hemoglobin is  a tetramer composed of two different types of subunits (2a and 2(3 polypeptide  chains, referred to as two a (3 protomers). Each subunit has a strong sequence  homology to myoglobin and contains an 0 2 binding site. A comparison between  myoglobin and hemoglobin illustrates some of the advantages of a multisubunit  quaternary structure.   The tetrameric structure of hemoglobin facilitates saturation with 0 2 in the lungs  and release of 0 2 as it travels through the capillary beds (Fig. 7.12). When the  amount of oxygen bound to myoglobin or hemoglobin is plotted against the partial  pressure of oxygen (p0 2 ), a hyperbolic curve is obtained for myoglobin, whereas  that for hemoglobin is sigmoidal. These curves show that when the p0 2 is high, as  in the lungs, both myoglobin and hemoglobin are saturated with oxygen. However,   Fig. 7.11. Myoglobin and hemoglobin. Myoglobin consists of a single polypeptide chain, which is similar in structure to the a and |3 subunits  of hemoglobin. In all of the subunits, heme is tightly bound in a hydrophobic binding pocket. The proximal histidine extends down from a  helix to bind to the Fe atom. The oxygen binds between the distal histidine and the heme. Panel C displays the quaternary structure of hemo-  globin (From Frescht A. Structure and Mechanism in Protein Science. New York: WH Freeman and Company, 1999. Used with permission.)   at the lower levels of p0 2 in oxygen-using tissues, hemoglobin cannot bind oxygen  as well as myoglobin (i.e., its percent saturation is much lower). Myoglobin, which  is present in heart and skeletal muscle, can bind the 0 2 released by hemoglobin,  which it stores to meet the demands of contraction. As 0 2 is used in the muscle cell  for generation of ATP during contraction, it is released from myoglobin and picked  up by cytochrome oxidase, a heme-containing enzyme in the electron transport  chain that has an even higher affinity for oxygen than myoglobin.   The tertiary structure of myoglobin consists of eight a-helices connected by short  coils, a structure that is known as the globin fold (see Fig. 7.11). This structure is  unusual for a globular protein in that it has no p- sheets. The helices create a  hydrophobic 0 2 binding pocket containing tightly bound heme with an iron atom  (Fe 2+ ) in its center.   Heme consists of a planar porphyrin ring composed of four pyrrole rings that  lie with their nitrogen atoms in the center, binding an Fe 2+ atom (Fig. 7.13).   Sickle cell anemia is really a disease caused by the wrong quarternary structure.  The painful vasooclusive crises experienced by Will Sichel are caused by the poly-  merization of HbS molecules into long fibers that distort the shape of the red  blood cells into sickle cells. The substitution of a hydrophobic valine for a glutamate in the  p 2 chain creates a knob on the surface of deoxygenated hemoglobin that fits into a  hydrophobic binding pocket on the subunit of a different hemoglobin molecule. A third  hemoglobin molecule, which binds to the first and second hemoglobin molecules through  aligned polar interactions, binds a fourth hemoglobin molecule through its valine knob.  Thus the polymerization continues until long fibers are formed.    Myoglobin is readily released from  skeletal muscle or cardiac tissue  when the cell is damaged. It has a  small molecular weight, 17,000 kDa, and is not  complexed to other proteins in the cell. (Da is  the abbreviation for Dalton, which is a unit of  mass approximately equal to one H atom.  Thus, a molecular weight of 17,000 kDa is  equal to approximately 17,000 g/mole.) Large  injuries to skeletal muscle that result from  physical crushing or lack of ATP production  result in cellular swelling and the release of  myoglobin and other proteins into the blood.  Myoglobin passes into the urine and turns the  urine red because the heme (which is red)  remains covalently attached to the protein.  During an acute Ml, myoglobin is one of the  first proteins released into the blood from  damaged cardiac tissue. (However, the  amount released is not high enough to cause  myoglobinuria.) Laboratory measurements of  serum myoglobin are used for early diagnosis  in patients such as Ann Jeina. Because myo-  globin is not present in skeletal muscle and  the heart as tissue-specific isozymes, and the  amount released from the heart is much  smaller than the amount that can be released  from a large skeletal muscle injury, myoglobin  measurements are not specific for an Ml.   Polymerization of the hemoglobin molecules is highly dependent on the concentration of  HbS and is promoted by the conformation of the deoxygenated molecules. At 100% oxygen  saturation, even high concentrations of HbS will not polymerize. A red blood cell spends the  longest amount of time at the lower oxygen concentrations of the venous capillary bed,  where polymerization is most likely initiated.    10 20 30 40 50 60 70 80 90100  Partial pressure of oxygen (mm Hg)   Fig. 7.12. Oxygen saturation curves for myo-  globin and hemoglobin. Note that the curve for  myoglobin is hyperbolic, whereas that for  hemoglobin is sigmoidal. The effect of the  tetrameric structure is to inhibit 0 2 binding at  low 0 2 concentrations. P 50 is the partial pres-  sure of 0 2 (p0 2 ) at which the protein is half-  saturated with 0 2 . P 50 for myoglobin is 2.8,  and that for hemoglobin is 26.   Fig. 7.13. Heme. The Fe is bound to four nitrogen atoms in the center of the heme porphyrin  ring. Methyl (M, CH 3 ), vinyl (V, -CH = CH 2 ), and propionate (P, CH 2 CH 3 COCr) side chains  extend out from the four pyrrole rings that constitute the porphyrin ring.   Negatively charged propionate groups on the porphyrin ring interact with argi-  nine and histidine side chains from the hemoglobin, and the hydrophobic methyl  and vinyl groups that extend out from the porphyrin ring interact with hydropho-  bic amino acid side chains from hemoglobin. All together, there are approxi-  mately 16 different interactions between myoglobin amino acids and different  groups in the porphyrin ring.   Organic ligands that are tightly bound, such as the heme of myoglobin, are called  prosthetic groups. A protein with its attached prosthetic group is called a holoprotein;  without the prosthetic group, it is called an apoprotein. The tightly bound prosthetic  group is an intrinsic part of the protein and does not dissociate until the protein is  degraded.   Within the binding pocket of myoglobin, 0 2 binds directly to the Fe 2+ atom on  one side of the planar porphyrin ring (Fig. 7.14). The Fe 2+ atom is able to chelate  six different ligands; four of the ligand positions are in a plane and taken by the cen-  tral nitrogens in the planar porphyrin ring. Two ligand positions are perpendicular  to this plane. One of these positions is taken by the nitrogen atom on a histidine,  called the proximal histidine, which extends down from a myoglobin helix. The  other position is taken by 0 2 .   The proximal histidine of myoglobin and hemoglobin is sterically repelled by the  heme porphyrin ring. Thus, when the histidine binds to the Fe 2+ in the middle of the    Fig. 7.14. Oxygen binding to the Fe 2+ of heme in myoglobin. A histidine residue called the proximal histidine binds to the Fe 2+ on one side of  the porphyrin ring; 0 2 binds to Fe 2+ on the other side. 0 2 binding causes a conformational change that pulls the Fe 2+ back into the plane of the  ring. As the proximal histidine moves, it moves the helix that contains it.   ring, it pulls the Fe above the plane of the ring. When oxygen binds on the other side  of the ring, it pulls the Fe 2+ back into the plane of the ring. The pull of 0 2 binding  moves the proximal histidine toward the porphyrin ring, which moves the helix con-  taining the proximal histidine. This conformational change has no effect on the func-  tion of myoglobin. However, in hemoglobin, the movement of one helix leads to the  movement of other helices in that subunit, including one in a comer of the subunit that  is in contact with a different subunit through salt bridges. The loss of these salt bridges  then induces conformational changes in all other subunits, and all four subunits may  change in a concerted manner from their original conformation to a new conformation.   The cooperativity in oxygen binding in hemoglobin comes from conformational  changes in tertiary structure that takes place when 0 2 binds (Fig. 7.15). The con-  formational change of hemoglobin is usually described as changing from a T (tense)  state with low affinity for 0 2 to an R (relaxed) state with a high affinity for 0 2 .  Breaking the salt bridges in the contacts between subunits is an energy-requiring    Fig. 7.15. Equilibria for binding of 0 2 molecules to hemoglobin according to the concerted  model of Monod, Wyman, and Changeux. Hemoglobin exists in two alternate conformations,  the T (tense) state with a low affinity for 0 2 and the R (relaxed) state with a higher affinity.  In the T subunits, the binding sites are hindered, and in the R state the binding sites are open.  Each successive addition of 0 2 shifts the equilibrium further toward the R state. Because the  conformation of all the subunits can change when 0 2 binds to one subunit, oxygen binding  is said to follow the concerted model. Most of the molecules change to the R state when 2 0 2  molecules have bound.   Amy Lloyd's serum protein elec-  trophoresis indicated the presence  of a sharp narrow peak or homoge-  neous "spike" in the characteristic 7-globulin  zone known as an M protein (monoclonal  protein) component. A narrow peak of spike  in electrophoresis, which separates proteins  according to charge distribution of the side  chains, suggests an elevation of proteins  with a similar or identical structure. Subse-  quently, it was shown that Amy Lloyd's  immunoglobulin M-component was com-  posed of a single homogeneous type of  immunoglobulin (just one amino acid  sequence in the N-terminal variable region).  Thus, the M protein was produced by a sin-  gle clone of antibody-secreting cells (cells  that all arose from proliferation of one cell) in  the bone marrow (called plasma cell dyscra-  sia). In amyloidosis, substitutions of particu-  lar amino acids in the light chain variable  region are thought to destabilize its native  conformation, resulting in fibrillogenesis.   In AL amyloidosis, amyloid is formed  from degradation products of the X or k light  chains that deposit most frequently in the  extracellular matrix of the kidney and the  heart but also may deposit in the tongue. In  other types of amyloidosis, the amyloid  arises from other proteins and deposits in a  characteristic organ. For example, the amy-  loid associated with chronic inflammatory  conditions, such as tuberculosis or rheuma-  toid arthritis, is derived from an acute phase  serum protein called serum amyloid A that is  produced by the liver in response to inflam-  mation. It deposits most frequently in the  kidney, and cardiac involvement is rare.   process and, consequently, the binding rate for the first oxygen is very low. When  the next oxygen binds, many of the hemoglobin molecules containing one 0 2 will  already have all four subunits in the R state, and therefore the rate of binding is  much higher. With two 0 2 molecules bound, an even higher percentage of the hemo-  globin molecules will have all four subunits in the R state. This phenomenon,  known as positive cooperativity, is responsible for the sigmoidal oxygen saturation  curve of hemoglobin (see Fig. 7.12).   The immunoglobulins (or antibodies) are one line of defense against invasion of the  body by foreign organisms. In this capacity, they function by binding to ligands  called antigens on the invading organisms, thereby initiating the process by which  these organisms are inactivated or destroyed.   Immunoglobulins all have a similar structure; each antibody molecule contains  two identical small polypeptide chains (the light or L chains) and two identical large  polypeptide chains (the heavy or H chains) (Fig. 7.16). The chains are joined to each  other by disulfide bonds.   The body has five major classes of immunoglobulins. The most abundant  immunoglobulins in human blood are the 7-globulins, which belong to the IgG  class. The 7-globulins have approximately 220 amino acids in their light chains  and 440 in their heavy chains. Like most serum proteins, they have attached  oligosaccharides that participate in targeting the protein for clearance from the  blood. Both the light and heavy chains consist of domains known as the  immunoglobulin fold, which is a collapsed p-barrel made from a number of p-  sheets (Fig. 7.17).   Both the light and heavy chains contain regions termed variable (V) and con-  stant (C) regions. The variable regions of the L and H chains (V L and V H , respec-  tively) interact to produce a single antigen binding site at each branch of the Y-  shaped molecule. Each population (clone) of B cells produces an antibody with a  different amino acid composition in the variable region that is complementary to  the structure of the antigen eliciting the response. The K d of antibodies for their  specific antigens is extremely small and varies from approximately 10~ 7 to 10 -11  M. The antigen thus binds very tightly with almost no tendency to dissociate and  can be removed from circulation as the antigen-antibody complex is ingested by  macrophages. The constant domains that form the Fc part of the antibody are  important for binding of the antigen-antibody complex to phagocytic cells for  clearance and for other aspects of the immune response.   Although the peptide bonds in a protein are rigid, flexibility around the other  bonds in the peptide backbone allow an enormous number of possible confor-  mations for each protein. However, every molecule of the same protein folds into  the same stable three-dimensional structure. This shape is known as the native  conformation.   The tight binding affinity of immunoglobulins for their specific antigen makes them useful for the measurement of small amounts of  other compounds in various radioimmunoassays. The principle of the radioimmunoassay is that the immunoglobulin will specifically  bind the compound being measured, and an additional component of the system that is labeled with a radioactive chemical will bind  the immunoglobulin. The complex is then separated from the solution and the bound radioactivity measured. The different isozymes of CK and  Tn-T used to track Ann Jeina's Ml are measured with a type of radioimmunassay by using antibodies specific for each isozyme. This method  is much faster than the laborious separation of isozymes by electrophoresis. Radioimmunoassays are also useful for measuring the small  amounts of hormones present in the blood for diagnosis of endocrine diseases.   Fig. 7.16. Structure of immunoglobulins. A. Each IgG molecule contains two light (L) and two heavy (H) chains joined by disulfide bonds. Each  light chain contains two domains, a variable domain (V L ) and a region of constant amino acid sequence (C L ). Each heavy chain has four domains:  one variable domain (V H ) and three constant domains (C H ). The conformation of the constant domain contains the |3-sheets that are called the  immunoglobulin fold. The variable domains are specific for the antigen that is bound, whereas the constant regions are the same for all antibody  molecules of a given class. Carbohydrate (CHO) is bound as indicated within the constant region of the heavy chains (CH). The hinge region  allows flexibility when the molecule binds antigen. B. In the space-filled model, the light chains are light in color and the heavy chains are two  different shades of gray. (Modified from Silverton EW, Navia MA, Davies DR, et al. Proc Natl Acad Sci, Three-dimensional structure of an intact  human immunoglobulin USA 1977; 11:5 142)   Very little difference is seen in the  \\ ) energy state of the native confor-   mation and a number of other sta-  ble conformations that a protein might  assume. It appears that the prion protein, the  cause of mad cow disease, is a normal cellu-  lar protein that has refolded into a different  stable conformation with a lower energy  state than its normal functional conforma-  tion (discussed under Biochemical Com-  ments, later). If misfolded proteins do not  precipitate into aggregates, they can be  degraded in the cell by proteolytic reactions,  or even refolded.   The primary structure of a protein determines its three-dimensional conforma-  tion. More specifically, the sequence of amino acid side chains dictates the fold  pattern of the three-dimensional structure and the assembly of subunits into qua-  ternary structure. Under certain conditions, denatured proteins can refold into  their native conformation, regaining their original function. Proteins can be  denatured with organic solvents such as urea that disrupt hydrophobic interac-  tions and convert the protein to a soluble random coil. Many simple single-sub-  unit proteins like ribonuclease that are denatured in this way spontaneously  refold into their native conformation if carefully brought back to physiologic  conditions. Even complex multi-subunit proteins containing bound cof actors can  sometimes spontaneously renature under the right conditions. Thus, the primary  structure essentially specifies the folding pattern.   In the cell, not all proteins fold into their native conformation on their own.  As the protein folds and refolds while it is searching for its native low energy  state, it passes through many high-energy conformations that slow the process  (called kinetic barriers). These kinetic barriers can be overcome by heat shock  proteins, which use energy provided by ATP hydrolysis to assist in the folding  process (Fig. 7.18). Heat shock proteins were named for the fact that their syn-  thesis in bacteria increased when the temperature was suddenly raised. They are   Fig. 7.17. Structure of the V L and C L domains  of IgG. Layers of antiparallel p-sheets are  stacked in these domains, which have been  referred to as collapsed p-barrels. The antigen  binds between the V H and V L immunoglobulin  folds, and NOT in the barrel. The C L domain is  also called the immunoglobulin fold. (Top  modified from Richardson JS. Adv Protein  Chem. The anatomy and taxonomy of protein  structure 1981;34:167; bottom reprinted in part  with permission from Edmundson AB, et al.  Biochemistry 1975;14:3954. © 1975 American  Chemical Society.)   Fig. 7.18. Role of heat shock proteins in folding. A. The Hsp70 family of proteins prevent  folding of the nascent chain and promote unfolding. The ATPase domain of the protein has  the actin fold. B. The Hsp60 class of protein has a barrel shape into which the protein fits. It  acts as a template, binding and rebinding portions of the unfolded protein until folding is  completed. It hydrolyzes many ATP bonds to provide energy for the process.   present in human cells as different families of proteins with different activities.  For example, the hsp70 proteins bind to nascent polypeptide chains as their syn-  thesis is being completed to keep the uncompleted chains from folding prema-  turely. They also unfold proteins prior to their insertion through the membrane  of mitochondria and other organelles. The multi-subunit barrel-shaped hsp60  family of proteins is called chaperonins. The unfolded protein fits into the barrel  cavity that excludes water and serves as a template for the folding process. The  hydrolysis of several ATP molecules is used to overcome the energy barriers to  reaching the native conformation.   A cis-trans isomerase and a disulfide isomerase also participate in folding. The  cis-trans isomerase converts a trans peptide bond preceding a proline into the cis  conformation, which is well suited for making hairpin turns. The disulfide iso-  merase breaks and reforms disulfide bonds between the -SH groups of two cysteine  residues in transient structures formed during the folding process. After the protein  has folded, cysteine-SH groups in close contact in the tertiary structure can react to  form the final disulfide bonds.   Amino acids on proteins can undergo a wide range of chemical modifications  that are not catalyzed by enzymes, such as nonenzymatic glycosylation or oxi-  dation. Such modifications usually lead to a loss of function and denaturation of  the protein, sometimes to a form that cannot be degraded in the cell. In nonen-  zymatic glycosylation, glucose that is present in blood, or in interstitial or intra-  cellular fluid, binds to an exposed amino group on a protein (Fig. 7.19). The two-  step process forms an irreversibly glycosylated protein. Proteins that turn over  very slowly in the body, such as collagen or hemoglobin, exist with a significant  fraction present in the glycosylated form. Because the reaction is nonenzymatic,  the rate of glycosylation is proportionate to the concentration of glucose present,  and individuals with hyperglycemia have much higher levels of glycosylated  proteins than individuals with normal blood glucose levels. Collagen and other  glycosylated proteins in tissues are further modified by nonenzymatic oxidation  and form additional cross-links. The net result is the formation of large protein  aggregates referred to as AGEs (advanced glycosylation end-products). AGE is a  meaningful acronym because AGEs accumulate with age, even in individuals  with normal blood glucose levels.   Proteins can be denatured by changes of pH, temperature, or solvent that disrupt  ionic, hydrogen, and hydrophobic bonds. At a low pH, ionic bonds and hydrogen  bonds formed by carboxylate groups would be disrupted; at a very alkaline pH,  hydrogen and ionic bonds formed by the basic amino acids would be disrupted.  Thus, the pH of the body must be maintained within a range compatible with three-  dimensional structure. Temperature increases vibrational and rotational energies in  the bonds, thereby affecting the energy balance that goes into making a stable three-  dimensional conformation.   Hydrophobic molecules can also denature proteins by disturbing hydrophobic  interactions in the protein. For example, long-chain fatty acids can inhibit many  enzyme-catalyzed reactions by binding nonspecifically to hydrophobic pockets  in proteins and disrupting hydrophobic interactions. Thus, long-chain fatty acids  and other highly hydrophobic molecules have their own binding proteins in the  cell.   Di Abietes' physician used her gly-  cosylated hemoglobin levels, specif-  ically the HbA 1c fraction, to deter-  mine whether she had sustained  hyperglycemia over a long period. The rate of  irreversible nonenzymatic glycosylation of  hemoglobin and other proteins is directly pro-  portional to the glucose concentration to  which they are exposed over the last 4  months. The danger of sustained hyper-  glycemia is that, over time, many proteins  become glycosylated and subsequently oxi-  dized, affecting their solubility and ability to  function. The glycosylation of collagen in the  heart, for example, is believed to result in a  cardiomyopathy in patients with chronic  uncontrolled diabetes mellitus. In contrast,  glycosylation of hemoglobin has little effect  on its function.   ^Proteins are denatured in the gas-  tric juice of the stomach, which has  a pH of 1 to 2. Although this pH  cannot break peptide bonds, disruption of  the native conformation makes the protein a  better substrate for digestive enzymes.   Thermal denaturation is often illus-  trated by the process of cooking an  egg. In the presence of heat, the  protein albumin converts from its native  translucent state to a denatured white pre-  cipitate. Protein precipitates can sometimes  be dissolved by amphipathic agents such as  urea, guanidine HCI, or SDS (sodium dode-  cylsulfate) that form extensive hydrogen  bonds and hydrophobic interactions with  the protein.   Fig. 7.19. Nonenzymatic glycosylation of hemoglobin. Glucose forms a Schiff base with the  N-terminal amino group of the protein, which rearranges to form a stable glycosylated prod-  uct. Similar nonenzymatc glycosylation reactions occur on other proteins.   Fig. 7.20 Sickled red blood cells occlude a  blood vessel, causing hypoxia (low 0 2 in cells)  and necrosis (cell death).   ^ Will Sichel. Will Sichel continues to experience severe low back and  lower extremity pain for many hours after admission. The diffuse pains of  sickle cell crises are believed to result from occlusion of small vessels in a  variety of tissues, thereby depriving cells of oxygen and causing ischemic or anoxic  damage to the tissues. In a sickle cell crisis, long hemoglobin polymers form, caus-  ing the red blood cells to become distorted and change from a biconcave disc to an  irregular shape, such as a sickle (for which the disease was named) or a stellate  structure (Fig. 7. 20). The aggregating Hb polymers damage the cellular membrane  and promote aggregation of membrane proteins, increased permeability of the cell,  and dehydration. Surface charge and antigens of red blood cells are carried on the  transmembrane proteins glycophorin and Band 3 (the erythrocyte anion exchange  channel, see Chapter 10). Hemoglobin S binds tightly to the cytoplasmic portion of  band 3, contributing to further polymer aggregation and uneven distribution of neg-  ative charge on the sickle cell surface. As a result, the affected cells adhere to  endothelial cells in capillaries, occluding the vessel and decreasing blood flow to  the distal tissues. The subsequent hypoxia in these tissues causes cellular damage  and even death.   The sickled cells are sequestered and destroyed mainly by phagocytic cells, par-  ticularly those in the spleen. An anemia results as the number of circulating red  blood cells decreases and bilirubin levels rise in the blood as hemoglobin is  degraded.   After a few days of treatment, Will Sichel's crisis was resolved. In the future,  should Will suffer a cerebrovascular accident as a consequence of vascular occlu-  sion or have recurrent life-threatening episodes, a course of long-term maintenance  blood transfusions to prevent repeated sickle crises may be indicated. Iron chelation  would have to accompany such a program to prevent or delay the development of  iron overload. Although a few individuals with this disease have survived into the  sixth decade, mean survival is probably into the fourth decade. Death usually results  from renal failure or cardiopulmonary disease.   Anne Jeina. Mrs. Jeina's diagnosis of an acute myocardial infarction  (MI) was based partly on measurements of CK-MB, myoglobin, and cTN-  T (the cardiac isozyme of troponin-T, a subunit of the regulatory protein  troponin). Early diagnosis is critical for a decision on the type of therapeutic inter-  vention to be used. Of these proteins, myoglobin appears in the blood most rapidly.  However, its levels are relatively nonspecific for cardiac injury because the amino  acid sequences of cardiac and skeletal muscle myoglobins are identical. Myoglobin  measurements do have a very high negative predictive value within the 2- to 6-hour  period after the onset of symptoms (i.e., if myoglobin is not elevated, a myocardial  infarction did not occur). In contrast, serum cardiac troponin-T is a relatively late,  but highly specific, marker of myocardial injury. It is typically detected in an acute  MI within 3 to 5 hours after onset of symptoms, is positive in most cases within 8  hours, and approaches 100% sensitivity at 10 to 12 hours. It remains elevated for 5  to 10 days.   Mrs. Jeina stayed in the hospital until she had been free of chest pain for 5 days.  She was discharged on a low-fat diet and was asked to participate in the hospital  patient exercise program for patients recovering from a recent heart attack. She was  scheduled for regular examinations by her physician.   Troponin is a heterotrimeric protein  \ \ ) involved in the regulation of striated  V*^/ and cardiac muscle contraction.  Most troponin in the cell is bound to the  actin-tropomyosin complex in the muscle fib-  ril. The three subunits of troponin consist of  troponin-C, troponin-T, and troponin-l, each  with a specific function in the regulatory  process. Troponin-T and troponin-l exist as  different isoforms in cardiac and skeletal mus-  cle (sequences with a different amino acid  composition), thus allowing the development  of specific antibodies against each form. As a  consequence, either cardiac troponin-T or car-  diac troponin-l may be rapidly measured in  blood samples by immunoassay with a good  degree of specificity.   ^ Amy Lloyd. Amy Lloyd has AL amyloidosis, which is characterized by  deposition of amyloid fibers derived principally from the variable region of  X or k immunoglobulin light chains. Increased amounts of the fragments  of the light chains called Bence- Jones proteins appeared in her urine. Fibril deposi-  tion in the extracellular matrix of her kidney glomeruli has resulted in mild renal  failure. Deposition of amyloid in the extracellular matrix of her heart muscle  resulted in the cardiac arrhythmia seen on an electrocardiogram. In addition to other  signs of right-sided heart failure, she had peripheral edema. The loss of weight may  have been caused by infiltrations of amyloid in the gastrointestinal tract or by con-  stipation and diarrhea resulting from involvement of the autonomic nervous system.  Treatment may be directed against the plasma cell proliferation, or against the  symptomatic results of organ dysfunction.   During Amy Lloyd's evaluation, she developed a cardiac arrhythmia that was  refractory to treatment. The extensive amyloid deposits in her heart had disrupted  conduction of electrical impulses in the heart muscle, ultimately resulting in cardiac  arrest. On autopsy, amyloid deposits were found within the heart, tongue, liver, adi-  pose tissue, and every organ examined except the central nervous system, which had  been protected by the blood-brain barrier.   2 Dianne Abietes. Di Abietes' HbA lc of 8.5% was just above the normal  range (5.8-7.2% of total hemoglobin), and her physician decided not to  alter her insulin treatment plan. Glycosylation is a nonenzymatic reaction  that occurs with a rate directly proportionate to the concentration of glucose in the  blood. In the normal range of blood glucose concentrations (approximately 80-140  mg/dL, depending on time after a meal), 6 to 7% of the hemoglobin is glycosylated  to form HbA lc Hemoglobin turns over in the blood as red blood cells are phagocy-  tosed and their hemoglobin degraded and new red blood cells are derived from retic-   Four minor components of adult  KJ ) hemoglobin (HbA) result from  vj^/ posttranslational, nonenzymatic  glycosylation of different amino acid  residues (HbA 1a1 , HbA 1a2 , HbA 1b1 , and HbA 1c ).  In HbA 1c , the fraction that is usually meas-  ured, the glycosylation occurs on an N-  terminal valine.   Prion diseases are categorized as  transmissable spongiform enceph-  alopathies, which are neurodegen-  erative diseases characterized by spongiform  degeneration and astrocytic gliosis in the cen-  tral nervous system. Frequently, protein  aggregates and amyloid plaques are seen.  These aggregates are resistant to proteolytic  degradation.   Familial prion diseases are caused  by point mutations in the gene  encoding the Pr protein (point  mutations are changes in one base in the  DNA nucleotide sequence). The diseases  have a variety of names related to the differ-  ent mutations and the clinical syndrome  (e.g., Gertsmann-Straussler-Scheinker dis-  ease and familial Creutzfeld-Jakob disease).  Familial Creutzfeld-Jakob disease (fCJD)  arises from an inherited mutation and has an  autosomal dominant pedigree. It typically  presents in the fourth decade of life. The  mutation lowers the activation energy for  refolding and the prion proteins fold into the  PrP Sc conformation more readily. It is esti-  mated that the rate of generating prion dis-  ease by refolding of PrP c in the normal cell is  approximately 3,000 to 4,000 years. Lowering  of the activation energy for refolding by  mutation presumably decreases this time to  the observed 30- to 40-year prodromal  period. Sporadic Creutzfeld-Jakob disease  may arise from somatic cell mutation or rare  spontaneous refoldings that initiate a cas-  cade of refolding into the PrP Sc conformation.  The sporadic form of the disease accounts  for 85% of all cases of CJD.   ulocytes. The average lifespan of a red blood cell is 120 days. Thus, the extent of  hemoglobin glycosylation is a direct reflection of the average serum glucose con-  centration to which the cell has been exposed over its 120-day lifespan. Di Abietes'  elevated Hb Alc indicates that her blood glucose levels have been elevated on an aver-  age over the preceding 6 weeks to 4 months. An increase of Di's insulin dosage  would decrease her hyperglycemia but increase her risk of hypoglycemic events.   Protein Misfolding and Prions. Prion proteins are believed to  cause a neurodegenerative disease by acting as a template to misfold other  cellular prion proteins into a form that cannot be degraded. The word prion  stands for proteinaceous infectious agent. The prion diseases can be acquired either  through infection (mad-cow disease), or from sporadic or inherited mutations (e.g.,  Creutzfeldt- Jakob disease). Although the infectious prion diseases represent a small  proportion of human cases, their link to mad cow disease in the United Kingdom  (new variant CJD), to growth hormone inoculations in the United States and France  (iatrogenic or "doctor-induced" CJD), and to ritualistic cannibalism in the Fore  tribespeople (Kuru) have received the most publicity.   The prion protein is normally found in the brain and is encoded by a gene that is  a normal component of the human genome. The disease-causing form of the prion  protein has the same amino acid composition but is folded into a different confor-  mation that aggregates into multimeric protein complexes resistant to proteolytic  degradation (Fig. 7.21). The normal conformation of the prion protein has been des-  ignated PrP c and the disease-causing form PrP Sc (sc for the prion disease scrapies in  sheep). Although PrP Sc and PrP c have the same amino acid composition, the PrP Sc  conformer is substantially enriched in (3-sheet structure compared with the normal  PrP c conformer, which has little or no p- sheet structure and is approximately 40%  a-helix. This difference favors the aggregation of PrP Sc into multimeric complexes.  These two conformations presumably have similar energy levels. Fortunately, spon-  taneous refolding of PrP proteins into the PrP Sc conformation is prevented by a large  activation energy barrier that makes this conversion extremely slow. Thus, very few  molecules of PrP Sc are normally formed during a lifetime.   Fig. 7.21. The conformation of PrP c and PrP sc . The prion proteins have two domains, an N-  terminal region that binds four Cu 2+ per chain, and a C-terminal region. In PrP c , the C-terminal  regions contain three substantial helices and two 3 -residue p strands joined by two to three  hydrogen bonds (approximately 40% a-helix and almost no p-sheet structure). It exists as a  monomer. In PrP sc , the C-terminal region is folded into an extensive p-sheet. The overall  structure is approximately 40 to 50% p-sheet, 20 to 30% a-helices. This conformation pro-  motes aggregation.   The infectious disease occurs with the ingestion of PrP dimers in which the  prion protein is already folded into the high (3 structure. These PrP Sc proteins are  thought to act as a template to lower the activation energy barrier for the conforma-  tional change, causing native proteins to refold into the PrP Sc conformation much  more rapidly (much like the role of chaperonins). The refolding initiates a cascade  as each new PrP Sc formed acts as a template for the refolding of other molecules. As  the number of PrP Sc molecules increase in the cell, they aggregate into a multimeric  assembly that is resistant to proteolytic digestion.   Horwich AL, Weissman JS. Deadly conformations -protein misfolding in prion diseases. Cell  1997;89:499-510.   Raj DSC, Choudhury D, Welbourne TC, Levi M. Advanced glycation end products: a nephrologist's per-  spective. Am J Kidney Dis 2000;35:365-380.    1 . Which of the following characterize a -helix regions of proteins?   (B) They are formed principally by hydrogen bonds between a carbonyl oxygen atom in one peptide bond and the amide  hydrogen from a different peptide bond.   (C) They are formed principally by hydrogen bonds between a carbonyl atom in one peptide bond and the hydrogen atoms  on the side chain of another amino acid.   (D) They are formed by hydrogen bonding between two adjacent amino acids in the primary sequence.   2. Which of the following is a characteristic of globular proteins?   (C) Tertiary structure is formed by hydrophobic and electrostatic interactions between amino acids, and by hydrogen bonds  between amino acids and between amino acids and water.   (D) Secondary structures are formed principally by hydrophobic interactions between amino acids.   (E) Covalent disulfide bonds are necessary to hold the protein in a rigid conformation.   3. A protein has one transmembrane domain composed entirely of a-helical secondary structure. Which of the following amino  acids would you expect to find in the transmembrane domain?   4. Autopsies of patients with Alzheimer's disease show protein aggregates called neurofibrillary tangles and neuritic plaques  in various regions of the brain. These plaques exhibit the characteristic staining of amyloid. Which of the following struc-  tural features is the most likely characteristic of at least one protein in these plaques?   5. While studying a novel pathway in a remote species of bacteria, you discover a new globular protein that phosphorylates a sub-  strate, using ATP as the phosphate donor. This protein most likely contains which of the following structures?   Enzymes are proteins that act as catalysts, compounds that increase the rate of  chemical reactions (Fig. 8.1). Enzyme catalysts bind reactants (substrates), con-  vert them to products, and release the products. Although enzymes may be modi-  fied during their participation in this reaction sequence, they return to their origi-  nal form at the end. In addition to increasing the speed of reactions, enzymes  provide a means for regulating the rate of metabolic pathways in the body. This  chapter describes the properties of enzymes that allow them to function as cata-  lysts. The next chapter explains the mechanisms of enzyme regulation.   Enzyme binding sites. An enzyme binds the substrates of the reaction and con-  verts them to products. The substrates are bound to specific substrate binding  sites on the enzyme through interactions with the amino acid residues of the  enzyme. The spatial geometry required for all the interactions between the sub-  strate and the enzyme makes each enzyme selective for its substrates and ensures  that only specific products are formed.   Active catalytic site. The substrate binding sites overlap in the active catalytic  site of the enzyme, the region of the enzyme where the reaction occurs. Within the  catalytic site, functional groups provided by coenzymes, tightly bound metals,  and, of course, amino acid residues of the enzyme, participate in catalysis.   Activation energy and the transition state. The functional groups in the cat-  alytic site of the enzyme activate the substrates and decrease the energy needed to  form the high-energy intermediate stage of the reaction known as the transition  state complex. Some of the catalytic strategies employed by enzymes, such as gen-  eral acid-base catalysis, formation of covalent intermediates, and stabilization of  the transition state, are illustrated by chymotrypsin.   pH and temperature profiles. Enzymes have a functional pH range determined  by the pK a s of functional groups in the active site and the interactions required for  three-dimensional structure. Non-denaturing increases of temperature increase the  reaction rate.   depends on their ability to inhibit an enzyme. The strongest inhibitors are covalent  inhibitors, compounds that form covalent bonds with a reactive group in the  enzyme active site, or transition state analogues that mimic the transition state  complex.   Enzyme names. Most enzyme names end in "ase." Enzymes usually have both  a common name and a systematic classification that includes a name and an  Enzyme Commission (EC) number.    Fig. 8.1. Catalytic power of enzymes. Many  enzymes increase the rate of a chemical reac-  tion by a factor of 10 11 or higher. To appreciate  an increase in reaction rate by this order of  magnitude, consider a room-sized box of golf  balls that "react" by releasing energy and turn-  ing blue. The 12 ft X 12 ft X 8 ft box contains  380,000 golf balls. If the rate of the reaction in  the absence of enzyme were 100 golf balls per  year, the presence of 1 molecule of enzyme  would turn the entire box of golf balls blue in  1 second (assuming a 10 11 increase in reaction  rate).    Most, if not all, of the tissues and  organs in the body are adversely  affected by chronic ingestion of  excessive amounts of alcohol, including the  liver, pancreas, heart, reproductive organs,  central nervous system, and the fetus. Some  of the effects of alcohol ingestion, such as  the psychotropic effects on the brain or inhi-  bition of vitamin transport, are direct effects  caused by ethanol itself. However, many of  the acute and chronic pathophysiologic  effects of alcohol relate to the pathways of  ethanol metabolism (see Chapter 25).   • A year after recovering from salicylate poisoning (see Chapter 4), Dennis  "the Menace" Veere was playing in his grandfather's basement. Dennis   " drank an unknown amount of the insecticide malathion, which is some-  times used for killing fruit flies and other insects (Fig. 8.2). Sometime later when  he was not feeling well, Dennis told his grandfather what he had done. Mr. Veere  retrieved the bottle and rushed Dennis to the emergency room of the local hospital.  On the way, Dennis vomited repeatedly and complained of abdominal cramps. At  the hospital, he began salivating and had an uncontrollable defecation.   In the emergency room, physicians passed a nasogastric tube for stomach lavage,  started intravenous fluids, and recorded vital signs. Dennis's pulse rate was 48 beats  per minute (slow), and his blood pressure was 78/48 mm Hg (low). The physicians  noted involuntary twitching of the muscles in his extremities.   • Lotta Topaigne was diagnosed with acute gouty arthritis involving her  flP | right great toe (see Chapter 5). The presence of insoluble urate crystals   " within the joint space confirmed the diagnosis. Several weeks after her  acute gout attack subsided, Ms. Topaigne was started on allopurinol therapy in an  oral dose of 150 mg twice per day.   Al Martini, a 44-year-old man who has been an alcoholic for the past 5  years, had a markedly diminished appetite for food. One weekend he  became unusually irritable and confused after drinking two fifths of scotch  and eating very little. His landlady convinced him to visit his doctor. Physical exam-  ination indicated a heart rate of 104 beats/min. His blood pressure was slightly low,  and he was in early congestive heart failure. He was poorly oriented to time, place,  and person.   Fig. 8.2. Organophosphorous compounds.  Malathion and parathion are organophospho-  rous insecticides. Nausea, coma, convulsions,  respiratory failure, and death have resulted  from the use of parathion by farmers who have  gotten it on their skin. Malathion is similar in  structure to parathion, but not nearly as toxic.  The nerve gas Sarin, another organophospho-  rus compound, was used in a terrorist attack in  a Japanese subway.   Enzymes, in general, provide speed, specificity, and regulatory control to reactions  in the body. Enzymes are usually proteins that act as catalysts, compounds that  increase the rate of chemical reactions. Enzyme-catalyzed reactions have three  basic steps:   (2) conversion of bound substrate to bound product: ES <-> EP   An enzyme binds the substrates of the reaction it catalyzes and brings them  together at the right orientation to react. The enzyme then participates in the mak-  ing and breaking of bonds required for product formation, releases the products, and  returns to its original state once the reaction is completed.   Enzymes do not invent new reactions; they simply make reactions occur faster.  The catalytic power of an enzyme (the rate of the catalyzed reaction divided by the  rate of the uncatalyzed reaction) is usually in the range of 10 6 to 10 14 . Without the  catalytic power of enzymes, reactions such as those involved in nerve conduction,  heart contraction, and digestion of food would occur too slowly for life to exist.   Each enzyme usually catalyzes a specific biochemical reaction. The ability of an  enzyme to select just one substrate and distinguish this substrate from a group of  very similar compounds is referred to as specificity (Fig. 8.3). The enzyme converts   this substrate to just one product. The specificity, as well as the speed, of enzyme-  catalyzed reactions result from the unique sequence of specific amino acids that  form the three-dimensional structure of the enzyme.   To catalyze a chemical reaction, the enzyme forms an enzyme-substrate complex in  its active catalytic site (Fig. 8.4). The active site is usually a cleft or crevice in the  enzyme formed by one or more regions of the polypeptide chain. Within the active  site, cofactors and functional groups from the polypeptide chain participate in trans-  forming the bound substrate molecules into products.   Initially, the substrate molecules bind to their substrate binding sites, also called  the substrate recognition sites (see Fig. 8.4B). The three-dimensional arrangement  of binding sites in a crevice of the enzyme allows the reacting portions of the sub-  strates to approach each other from the appropriate angles. The proximity of the  bound substrate molecules and their precise orientation toward each other con-  tribute to the catalytic power of the enzyme.   The active site also contains functional groups that directly participate in the  reaction (see Fig. 8.4C). The functional groups are donated by the polypeptide  chain, or by bound cofactors (metals or complex organic molecules called coen-  zymes). As the substrate binds, it induces conformational changes in the enzyme  that promote further interactions between the substrate molecules and the  enzyme functional groups. (For example, a coenzyme might form a covalent  intermediate with the substrate, or an amino acid side chain might abstract a  proton from the reacting substrate.) The activated substrates and the enzyme  form a transition state complex, an unstable high-energy complex with a strained  electronic configuration that is intermediate between substrate and product.  Additional bonds with the enzyme stabilize the transition state complex and  decrease the energy required for its formation.   Fig. 8.3. Reaction catalyzed by glucokinase,  an example of enzyme reaction specificity.  Glucokinase catalyzes the transfer of a phos-  phate from ATP to carbon 6 of glucose. It can-  not rapidly transfer a phosphate from other  nucleotides to glucose, or from ATP to closely  related sugars such as galactose, or from ATP  to any other carbon on glucose. The only prod-  ucts formed are glucose 6-phosphate and ADP.   Fig. 8.4. Reaction in the enzyme active catalytic site. A. The enzyme contains an active cat-  alytic site, shown in dark blue, with a region or domain where the substrate binds. The active  site also may contain cofactors, nonprotein components that assist in catalysis. B. The sub-  strate forms bonds with amino acid residues in the substrate binding site, shown in light  blue. Substrate binding induces a conformational change in the active site. C. Functional  groups of amino acid residues and cofactors in the active site participate in forming the tran-  sition state complex, which is stabilized by additional noncovalent bonds with the enzyme,  shown in blue. D. As the products of the reaction dissociate, the enzyme returns to its original  conformation.   The transition state complex decomposes to products, which dissociate from the  enzyme (see Fig. 8.4D). The enzyme generally returns to its original form. The free  enzyme then binds another set of substrates, and repeats the process.   Enzyme specificity (the enzyme's ability to react with just one substrate) results from  the three-dimensional arrangement of specific amino acid residues in the enzyme that  form binding sites for the substrates and activate the substrates during the course of  the reaction. The "lock-and-key" and the "induced-fit" models for substrate binding  describe two aspects of the binding interaction between the enzyme and substrate.   The substrate binding site contains amino acid residues arranged in a complemen-  tary three-dimensional surface that "recognizes" the substrate and binds it through  multiple hydrophobic interactions, electrostatic interactions, or hydrogen bonds  (Fig. 8.5). The amino acid residues that bind the substrate can come from very dif-  ferent parts of the linear amino acid sequence of the enzyme, as seen in glucokinase.  The binding of compounds with a structure that differs from the substrate even to a  small degree may be prevented by steric hindrance and charge-repulsion. In the  lock-and-key model, the complementarity between the substrate and its binding site  is compared to that of a key fitting into a rigid lock.   Complementarity between the substrate and the binding site is only part of the picture.  As the substrate binds, enzymes undergo a conformational change ("induced fit") that  repositions the side chains of the amino acids in the active site and increases the  number of binding interactions (see Fig. 8.4). The induced fit model for substrate bind-   Fig. 8.5. Glucose binding site in glucokinase. A. Glucose, shown in blue, is held in its binding site by multiple hydrogen bonds between each  hydroxyl group and polar amino acids from different regions of the enzyme amino acid sequence in the actin fold (see Chapter 7). The position  of the amino acid residue in the linear sequence is given by its number. The multiple interactions enable glucose to induce large conformational  changes in the enzyme. (Modified from Pilkis SJ, Weber IT, Harisson RW, Bell GL J Biol Chem. Glucokinase : structaral analysis of a protein  involved in susceptibility to diabetes 1994;21925-21928.) B. Enzyme specificity is illustrated by the comparison of galactose and glucose. Galac-  tose differs from glucose only in the position of the -OH group shown in blue. However, it is not phosphorylated at a significant rate by the  enzyme. Cells therefore require a separate galactokinase for the metabolism of galactose.   ing recognizes that the substrate binding site is not a rigid "lock" but rather a dynamic  surface created by the flexible overall three-dimensional structure of the enzyme.   The function of the conformational change induced by substrate binding, the  induced fit, is usually to reposition functional groups in the active site in a way that  promotes the reaction, improves the binding site of a cosubstrate, or activates an  adjacent subunit through cooperativity. For example, consider the large conforma-  tional changes that occur in the actin fold of glucokinase when glucose binds. The  induced fit involves changes in the conformation of the whole enzyme that close the  cleft of the fold, thereby improving the binding site for ATP, and excluding water  (which might interfere with the reaction) from the active site (Fig. 8.6). Thus, the  multiple interactions between the substrate and the enzyme in the catalytic site  serve both for substrate recognition and for initiating the next stage of the reaction,  formation of the transition state complex.   For a reaction to occur, the substrates undergoing the reaction need to be activated. If  the energy levels of a substrate are plotted as the substrate is progressively converted  to product, the curve will show a maximum energy level that is higher than that of  either the substrate or the product (Fig. 8.7). This high energy level occurs at the   Fig. 8.6. Conformational change resulting from the binding of glucose to hexokinase. (The  figure is actually yeast hexokinase, which is more similar to human glucokinase than it is to  the other human hexokinase isozymes). The shaded and unshaded areas show the two  domains (four subdomains) that form the actin fold with its ATP-binding cleft. A. Free  enzyme. B. With glucose bound, the cleft closes, forming the ATP binding site. The closure  of the cleft when glucose binds to hexokinase (or human glucokinase) is one of the largest  "induced fits" known. The combination of secondary structures in the actin fold that give  hexokinase the flexibility required for this shift are discussed in Chapter 7, section IV.B.l.,  "The Actin Fold." (From Bennett WS, Steitz TA. J Mol Biol. Structure of a complex between  yeast hexokinase A and glucose II. Detailed comparisons of conformation and active site  configuration with the native hexokinase B. 1980;21 1-230.)   Because the transition state com-  plex binds more tightly to the  enzyme than does the substrate,  compounds that resemble its electronic and  three-dimensional surface (transition state  analogs) are more potent inhibitors of an  enzyme than are substrate analogs. Conse-  quently, a drug developed as a transition  state analog would be highly specific for the  enzyme it is designed to inhibit. However,  transition state analogs are highly unstable  when not bound to the enzyme, and would  have great difficulty making it from the  digestive tract or injection site to the site of  action. Some of the approaches in drug  design that are being used to deal with the  instability problem include: designing drugs  that are almost transition state analogs but  have a stable modification; designing a pro-  drug that is converted to a transition state  analog at the site of action; using the transi-  tion state analog to design a complementary  antibody.   Abzymes (catalytic antibodies) are  \\ ) made as antibodies against analogs  of the transition state complex. They  thus have an arrangement of amino acid side  chains in their variable regions that is similar  to the active site of the enzyme in the transi-  tion state. Consequently, they can act as artifi-  cial enzymes. For example, abzymes have  been developed against analogs of the transi-  tion state complex of cocaine esterase, the  enzyme that degrades cocaine in the body.  These abzymes have esterase activity, and  monthly injections of the abzyme drug can be  used to rapidly destroy cocaine in the blood,  thereby decreasing the dependence of  addicted individuals. (See Chapter 7 for anti-  body structure.)   Hydrolysis is the use of water to  \\ lyse (break) a bond. Proteolysis is   the hydrolysis of a peptide bond in  a protein, a reaction catalyzed by enzymes  called proteases.   Fig. 8.7. Energy diagram showing the energy levels of the substrates as they progress toward  products in the absence of enzyme. The substrates must pass through the high-energy transi-  tion state during the reaction. Although a favorable loss of energy occurs during the reaction,  the rate of the reaction is slowed by the energy barrier to forming the transition state. The  energy barrier is referred to as the activation energy.   transition state. For some enzyme-catalyzed reactions, the transition state is a condi-  tion in which bonds in the substrate are maximally strained. For other enzyme-cat-  alyzed reactions, the electronic configuration of the substrate becomes very strained  and unstable as it enters the transition state. The highest energy level corresponds to  the most unstable substrate configuration, and the condition in which the changing  substrate molecule is most tightly bound to participating functional groups in the  enzyme. The difference in energy between the substrate and the transition state com-  plex is called the activation energy.   According to transition state theory, the overall rate of the reaction is determined by  the number of molecules acquiring the activation energy necessary to form the transi-  tion state complex. Enzymes increase the rate of the reaction by decreasing this acti-  vation energy. They use various catalytic strategies, such as electronic stabilization of  the transition state complex or acid-base catalysis, to obtain this decrease.   Once the transition state complex is formed, it can collapse back to substrates or  decompose to form products. The enzyme does not change the initial energy level  of the substrates or the final energy level of the products.   The enzyme chymotrypsin provides a good example of the strategies and amino acid  side chains used by enzymes to lower the amount of activation energy required. Chy-  motrypsin is a digestive enzyme released into the intestine that catalyzes the hydrol-  ysis of specific peptide bonds in denatured proteins. It is a member of the serine pro-  tease superfamily, enzymes that use a serine in the active site to form a covalent  intermediate during proteolysis. In the overall hydrolysis reaction, an OFT from water  is added to the carbonyl carbon of the peptide bond, and an H + to the N, thereby cleav-  ing the bond (Fig. 8.8). The bond that is cleaved is called the scissile bond.   In the reaction carried out in the absence of enzyme, the negatively charged  hydroxyl group of water attacks the carbonyl carbon, which carries a partial posi-  tive charge. An unstable oxy anion tetrahedral transition state complex is formed in  which the oxygen atom carries a full negative charge. The rate of the chemical  reaction in the absence of chymotrypsin is slow because there are too few OFT   molecules in H 2 0 with enough energy to form the transition state complex and too  few OFT molecules colliding with the substrate at the right orientation.   In the reaction catalyzed by chymotrypsin, the same oxyanion intermediate is  formed by using the hydoxyl group of a serine residue for the attack instead of a  free hydroxyl anion. The rate of the chymotrypsin-catalyzed reaction is faster  because functional groups in the enzyme active site activate the attacking hydroxyl  group, stabilize the oxyanion transition state complexes, form a covalent intermedi-  ate, and destabilize the leaving group. The reaction takes place in two stages: (a)  cleavage of the peptide bond in the denatured substrate protein and formation of a  covalent acyl-enzyme intermediate (Fig. 8.9, steps 1-5), and (b) hydrolysis of the  acyl-enzyme intermediate to release the remaining portion of the substrate protein  (Fig. 8.9, steps 6-9). The names of the catalytic strategies employed in the various  steps are in italics in the following paragraphs.   Chymotrypsin hydrolyzes the peptide bond on the carbonyl side of a phenylalanine,  tyrosine, or tryptophan in a denatured protein. The substrate recognition site consists  of a hydrophobic binding pocket that holds the hydrophobic amino acid contributing  the carbonyl group of the scissile bond (see Fig. 8.9, Step 1). The substrate protein  must be denatured to fit into the pocket and be held rigidly in place by glycines in  the enzyme peptide backbone. Scissile bond specificity is also provided by the sub-  sequent steps of the reaction, such as moving serine 195 into attacking position  (proximity and orientation).    Fig. 8.8. Chymotrypsin hydrolyzes certain  peptide bonds in proteins. The scissile bond is  shown in blue. The carbonyl carbon, which  carries a partial positive charge, is attacked by  a hydroxyl group from water. An unstable  tetrahedral oxyanion intermediate is formed,  which is the transition state complex. As the  electrons return to the carbonyl carbon, it  becomes a carboxylic acid, and the remaining  proton from water adds to the leaving group to  form an amine.   In the first stage of the reaction, the peptide bond of the denatured protein substrate  is cleaved as an active site serine hydroxyl group attacks the carbonyl carbon of the  scissile bond (nucleophilic catalysis-a nucleophile is a chemical group that is  attracted to the positively charged nucleus) (Fig. 8.9, Step 2). Aspartate and histi-  dine cooperate in converting this hydroxyl group (with a partial negative charge on  the oxygen) into a better nucleophilic attacking group by giving it a more negative  charge. An active site histidine acts as a base and abstracts a proton from the serine  hydroxyl (acid-base catalysis). The protonated histidine is stabilized by the nega-  tive charge of a nearby aspartate.   The aspartate-histidine-serine combination, referred to as the catalytic triad, is an  example of cooperative interactions between amino acid residues in the active site.  The strong nucleophilic attacking group created by this charge-relay system has the  same general effect on reaction rate as increasing the concentration of hydroxyl ions  available for collision in the uncatalyzed reaction.   In the next step of the reaction sequence, an oxyanion tetrahedral transition state  complex is formed that is stabilized by hydrogen bonds with -NH groups in the  peptide backbone (Fig. 8.9, Step 3). The original view of the way enzymes form  transition state complexes was that they stretched the bonds or distorted the bond  angles of the reacting substrates. However, most transition state complexes, such as  the oxyanion tetrahedral complex, are better described as showing "electronic  strain," an electrostatic surface that would be highly improbable if it were not sta-  bilized by bonds with functional groups on the enzyme. Stabilization of the transi-  tion state complex lowers its energy level and increases the number of molecules  that reach this energy level.   In the stomach, gastric acid  €B # | decreases the pH to 1 to 2 to dena-  ^/ ture proteins through disruption of  hydrogen bonding. The protease in the stom-  ach, pepsin, is a member of the aspartate pro-  tease superfamily, enzymes that use two  aspartate residues in the active site for acid-  base catalysis of the peptide bond. Why can  they not use histidine like chymotrypsin?   ® Serine proteases in blood coagula-  tion. The use of an active site ser-  ine to cleave a peptide bond is  common in the proteolytic enzymes of blood  coagulation as well as those of digestion.  Blood clots are formed from fibrin, a protein  present in the blood as the inactive precur-  sor, fibrinogen. The serine protease throm-  bin cleaves a peptide bond in fibrinogen to  form active fibrin. Thrombin has the same  aspartate-histidine-serine catalytic triad  found in chymotrypsin and works in essen-  tially the same way. Thrombin is also pres-  ent as an inactive precursor, prothrombin,  which is itself activated through proteolytic  cleavage by another blood coagulation ser-  ine protease.   Subsequently, the serine in the active site forms a full covalent bond with the car-  bon of the carbonyl group as the peptide bond is cleaved (covalent catalysis). The for-  mation of a stable covalent intermediate is a catalytic strategy employed by many  enzymes and often involves serine or cysteine residues. The covalent intermediate is  subsequently hydrolyzed (acid-base catalysis). The dissociating products of an  enzyme-catalyzed reaction are often "destabilized" by some degree of charge repulsion  in the active site. In the case of chymotrypsin, the amino group formed after peptide  bond cleavage is destabilized or "uncomfortable" in the presence of the active site his-  tidine (de stabilization of developing product).   The next sequence of events hydrolyzes the acyl-enzyme intermediate to release the  bound carbonyl-side peptide (Fig. 8.9, Steps 6-9). The active site histidine activates  water to form an OH~ for a nucleophilic attack, resulting in a second oxyanion tran-  sition state complex. When the histidine adds the proton back to serine, the reaction  is complete, and the product dissociates.   The number of steps in real enzymatic reactions results in a multi-bump energy dia-  gram (Fig. 8.10). At the initial stage of the reaction, a dip occurs because energy is  provided by formation of the initial multiple weak bonds between the substrate and  enzyme. As the reaction progresses, the curve rises because additional energy is  required for formation of the transition state complex. This energy is provided by  the subsequent steps in the reaction replacing the initial weak bonds with progres-  sively tighter bonds. Semi-stable covalent intermediates of the reaction have lower  energy levels than do the transition state complexes, and are present in the reaction  diagram as dips in the energy curve. The final transition state complex has the high-  est energy level in the reaction and is therefore the most unstable state. It can col-  lapse back to substrates or decompose to form products.   To participate in general acid-base  J | catalysis, the amino acid side chain  must be able to abstract a proton at  one stage of the reaction, and donate it back  at another. Histidine (pK a 6.3) would be pro-  tonated at this low pH, and could not  abstract a proton from a potential nucle-  ophile. However, aspartic acid, with a (pK a of  about 2) can release protons at a pH of 2.  The two aspartates work together to activate  water through the removal of a proton to  form the hydroxyl nucleophile.   The catalytic strategies employed by chymotrypsin to increase the reaction rate are  common to many enzymes. One of these catalytic strategies, proximity and orien-  tation, is an intrinsic feature of substrate binding and part of the catalytic mecha-  nism of all enzymes. All enzymes also stabilize the transition state by electrostatic  interactions, but not all enzymes form covalent intermediates.   Great variety occurs in the functional groups employed by different enzymes to  carry out these catalytic strategies. Some enzymes, such as chymotrypsin, rely on   Fig. 8.9. Catalytic mechanism of chymotrypsin. The substrate (a dena-  tured protein) is in the shaded area. 1. As the substrate protein binds to  the active site, serine- 195 and a histidine (his57) are moved closer  together and at the right orientation for the nitrogen electrons on  histidine to attract the hydrogen of serine. Without this change of con-  formation on substrate binding, the catalytic triad cannot form. 2.  Histidine serves as a general base catalyst as it abstracts a proton from  the serine, increasing the nucleophilicity of the serine-oxygen, which  attacks the carbonyl carbon. 3. The electrons of the carbonyl group  form the oxyanion tetrahedral intermediate. The oxyanion is stabilized  by the N-H groups of serine- 195 and glycine in the chymotrypsin pep-  tide backbone. 4. The amide nitrogen in the peptide bond is stabilized  by interaction with the histidine proton. Here the histidine acts as a  general acid catalyst. As the electrons of the carbon-nitrogen peptide   bond withdraw into the nitrogen, the electrons of the carboxyanion  return to the substrate carbonyl carbon, resulting in cleavage of the  peptide bond. 5. The cleavage of the peptide bond results in formation  of the covalent acyl-enzyme intermediate, and the amide half of the  cleaved protein dissociates. 6. The nucleophilic attack by H 2 0 on the  carbonyl carbon is activated by histidine, whose nitrogen electrons  attract a proton from water. 7. The second tetrahedral oxyanion inter-  mediate (the transition state complex) is formed. It is again stabilized  by hydrogen bonds with the peptide backbone bonds of glycine and  serine. 8. As the histidine proton is donated to the electrons of the bond  between the serine oxygen and the substrate carbonyl group, the elec-  trons from the oxyanion return to the substrate carbon to form the car-  boxylic acid, and the acyl-enzyme bond is broken. 9. The enzyme, as  it releases substrate, returns to its original state.   Energy required  for removing water  from substrate and  restricting its freedom   Fig. 8.10. A postulated energy diagram for the reaction catalyzed by chymotrypsin. In the presence of enzyme (blue); in the absence of enzyme  (black). The energy barrier to the transition state is lowered in the enzyme-catalyzed reaction by the formation of additional bonds between the  substrate and enzyme in the transition state complex. The energy is provided by substrate binding to the enzyme. The enzyme does not, how-  ever, change the energy levels of the substrate or product.   Because most vitamins function as  \\ coenzymes, the symptoms of vita-  min deficiencies reflect the loss of  specific enzyme activities dependent on the  coenzyme form of the vitamin. Thus, drugs  and toxins that inhibit proteins required for  coenzyme synthesis (e.g., vitamin transport  proteins or biosynthetic enzymes) can cause  the symptoms of a vitamin deficiency. This  type of deficiency is called a functional defi-  ciency, whereas an inadequate intake is called  a dietary deficiency.   Most coenzymes are tightly bound to  their enzymes and do not dissociate during  the course of the reaction. However, a func-  tional or dietary vitamin deficiency that  decreases the level of a coenzyme will result  in the presence of the apoenzyme in cells (an  enzyme devoid of cofactor).   Ethanol is an "antivitamin" that decreases  the cellular content of almost every coenzyme.  For example, ethanol inhibits the absorption  of thiamine, and acetaldehyde produced from  ethanol oxidation displaces pyridoxal phos-  phate from its protein binding sites, thereby  accelerating its degradation.   amino acid residues within the active site. Other enzymes increase their repertoire  by employing cofactors to provide a functional group with the right size, shape, and  properties. Cofactors are nonprotein compounds that participate in the catalytic  process. They are generally divided into three categories: coenzymes, metal ions  (e.g., Fe 2+ , Mg 2+ , or Zn 2+ ), and metallocoenzymes (similar to the Fe 2+ -heme in  hemoglobin, see Chapter 7).   Almost all of the polar amino acids participate directly in catalysis in one or more  enzymes (Table 8.1). Serine, cysteine, lysine, and histidine can participate in covalent   Table 8.1. Some Functional Groups in the Active Site  Function of Amino Acid   Stabilization of anion formed during the reaction  Peptide backbone-NH Chymotrypsin  Arginine-NH Carboxypeptidase A   catalysis. Histidine, because it has a pK a that can donate and accept a proton at neu-  tral pH, often participates in acid-base catalysis. Most of the polar amino acid side  chains are nucleophilic and participate in nucleophilic catalysis by stabilizing more  positively charged groups that develop during the reaction.   Coenzymes are complex nonprotein organic molecules that participate in catalysis  by providing functional groups, much like the amino acid side chains. In the human,  they are usually (but not always) synthesized from vitamins. Each coenzyme is  involved in catalyzing a specific type of reaction for a class of substrates with cer-  tain structural features. Coenzymes can be divided into two general classes: activa-  tion-transfer coenzymes and oxidation-reduction coenzymes.   The activation-transfer coenzymes usually participate directly in catalysis by form-  ing a covalent bond with a portion of the substrate; the tightly held substrate moiety  is then activated for transfer, addition of water, or some other reaction. The portion  of the coenzyme that forms a covalent bond with the substrate is its functional group.  A separate portion of the coenzyme binds tightly to the enzyme.   Thiamine pyrophosphate provides a good illustration of the manner in which  coenzymes participate in catalysis (Fig. 8.11). It is synthesized in human cells  from the vitamin thiamine by the addition of a pyrophosphate. This pyrophos-  phate provides negatively charged oxygen atoms that chelate Mg 2+ , which then  binds tightly to the enzyme. The functional group that extends into the active site  is the reactive carbon atom with a dissociable proton (see Fig. 8.11). In all of the  enzymes that use thiamine pyrophosphate, this reactive thiamine carbon forms a  covalent bond with a substrate keto group while cleaving the adjacent  carbon-carbon bond. However, each thiamine-containing enzyme catalyzes the  cleavage of a different substrate (or group of substrates with very closely related  structures).   Coenzymes have very little activity in the absence of the enzyme and very little  specificity. The enzyme provides specificity, proximity, and orientation in the sub-  strate recognition site, as well as other functional groups for stabilization of the  transition state, acid-base catalysis, etc. For example, thiamine is made into a bet-  ter nucleophilic attacking group by a basic amino acid residue in the enzyme that  removes the dissociable proton (EnzB: in Fig. 8.11), thereby generating a nega-  tively charged thiamine carbon anion. Later in the reaction, the enzyme returns the  proton.   Coenzyme A (Co A), biotin, and pyridoxal phosphate are also activation-transfer  coenzymes synthesized from vitamins. Co A (Co ASH), which is synthesized from  the vitamin pantothenate, contains an adenosine 3', 5'-bisphosphate which binds  reversibly, but tightly, to a site on an enzyme (Fig. 8. 12 A). Its functional group, a  sulfhydryl group at the other end of the molecule, is a nucleophile that always   Most coenzymes, such as functional groups on the enzyme amino acids, are  \\ regenerated during the course of the reaction. However, CoASH and a few of   the oxidation-reduction coenzymes are transformed during the reaction into  products that dissociate from the enzyme at the end of the reaction (e.g., CoASH is con-  verted to an acyl CoA derivative, and NAD + is reduced to NADH). These dissociating  coenzymes are nonetheless classified as coenzymes rather than substrates because they  are common to so many reactions, the original form is regenerated by subsequent reac-  tions in a metabolic pathway, they are synthesized from vitamins, and the amount of  coenzyme in the cell is nearly constant.   atom in the serine -OH) or have a  nitrogen that can act as an electron-donating  group by virtue of its two unpaired elec-  trons. Covalent catalysis and acid-base catal-  ysis are carried out by nucleophilic groups.  Electrophiles carry full or partial positive  charges (e.g., the peptide backbone-NH was  used as an electrophilic group in chy-  motrypsin). In general, nucleophilic and  electrophilic catalysis occur when the  respective nucleophilic or electrophilic  groups on the enzyme stabilize substrate  groups of the opposite polarity that develop  during the reaction.   Although coenzymes look as though  they should be able to catalyze reac-  tions autonomously (on their own),  they have almost no catalytic power when not  bound to the enzyme. Why?    Many alcoholics such as Al Martini  develop thiamine deficiency  because alcohol inhibits the trans-  port of thiamine through the intestinal  mucosal cells. In the body, thiamine is con-  verted to thiamine pyrophosphate (TPP).  TPP acts as a coenzyme in the decarboxyla-  tion of a-keto acids such as pyruvate and a-  ketoglutarate (see Fig. 8.11) and in the uti-  lization of pentose phosphates in the  pentose phosphate pathway. As a result of  thiamine deficiency, the oxidation of a-keto  acids is impaired. Dysfunction occurs in the  central and peripheral nervous system, the  cardiovascular system, and other organs.   For a substrate to react with a  JjL •) coenzyme, it must collide with a   coenzyme at exactly the right  angle. The probability of the substrate and  coenzyme in free solution colliding in  exactly the right place at the exactly right  angle is very small. In addition to providing  this proximity and orientation, enzymes con-  tribute in other ways, such as activating the  coenzyme by abstracting a proton (e.g., thi-  amine-pyrophosphate and coenzyme A) or  polarizing the substrate to make it more sus-  ceptible to nucleophilic attack.   I \\ V=C-CH 2 -CH 2 -0-P-0-P-0"  H 3 C-C^ JJ V II II   Fig. 8.11. The role of the functional group of thiamine pyrophosphate (the reactive carbon  shown in blue) in formation of a covalent intermediate. A. A base on the enzyme (B) abstracts  a proton from thiamine, creating a carbanion (general acid-base catalysis). B. The carbanion  is a strong nucleophile and attacks the partially positively charged keto group on the sub-  strate. C. A covalent intermediate is formed, which is stabilized by resonance forms. The  uncharged intermediate is the stabilized transition state complex.   attacks carbonyl groups and forms acyl thioesters (in fact, the "A" in CoA stands for  the acyl group that becomes attached).   Biotin, which does not contain a phosphate group, is covalently bound to a lysine  in enzymes called carboxylases (see Fig. 8.12B). Its functional group is a nitrogen  atom that covalently binds a C0 2 group in an energy-requiring reaction. This bound  C0 2 group is activated for addition to another molecule. In the human, biotin func-  tions only in carboxylation reactions.   Pyridoxal phosphate is synthesized from the vitamin pyridoxine, which is  also called vitamin B 6 (Fig. 8.13). The reactive aldehyde group usually func-  tions in enzyme-catalyzed reactions by forming a covalent bond with the amino  groups on amino acids. The positively charged ring nitrogen withdraws elec-  trons from a bond in the bound amino acid, resulting in cleavage of that bond.  The enzyme participates by removing protons from the substrate and by keep-  ing the amino acid and the pyridoxal group in a single plane to facilitate shut-  tling of electrons.   Fig. 8.13 Reactive sites of pyridoxal phos-  phate. The functional group of pyridoxal phos-  phate is a reactive aldehyde (shown in blue)  that forms a covalent intermediate with amino  groups of amino acids (a Schiff base). The pos-  itively charged pyridine ring is a strong elec-  tron-withdrawing group that can pull electrons  into it (electrophilic catalysis).   Fig. 8.12. CoA and biotin, activation-transfer coenzymes. A. Coenzyme A (CoA or CoASH)  and phosphopantetheine are synthesized from the vitamin pantothenate (pantothenic acid).  The active sulfhydryl group, shown in blue, binds to acyl groups (e.g., acetyl, succinyl, or  fatty acyl) to form thioesters. B. Biotin activates and transfers C0 2 to compounds in car-  boxylation reactions. The reactive N is shown in blue. Biotin is covalently attached to a lysine  residue in the carboxylase enzyme.   These coenzymes illustrate three features all activation-transfer coenzymes have  in common: (1) a specific chemical group involved in binding to the enzyme, (2) a  separate and different functional or reactive group that participates directly in the  catalysis of one type of reaction by forming a covalent bond with the substrate, and  (3) dependence on the enzyme for additional specificity of substrate and additional  catalytic power.   A large number of coenzymes are involved in oxidation-reduction reactions  catalyzed by enzymes categorized as oxidoreductases. Some coenzymes, such as   In general, coenzymes are nonpro-  \m ) tein organic cofactors that partici-  V^^/ pate in reactions. They may be  covalently bound to enzymes (like biotin),  dissociate during deficiency (thiamine-  pyrophosphate), or freely dissociate at the  end of the reaction (CoASH). Cofactors that  are covalently or very tightly bound to  nonenzyme proteins are usually called pros-  thetic groups. A prosthetic group, such as  the heme in hemoglobin, usually does not  dissociate from a protein until the protein is  degraded.    When a compound is oxidized, it  loses electrons. As a result, the oxi-  dized carbon has fewer H atoms or   gains an O atom. The reduction of a com-  pound is the gain of electrons, which shows  in its structure as the gain of H, or loss of O.  In the oxidation of lactate to pyruvate (see  Fig. 8.14), lactate loses two electrons as a  hydride ion, and a proton (H + ) is released;  NAD + , which accepts the hydride ion, is  reduced to NADH. The carbon atom with the  keto group is now at a higher oxidation state  because both of the electrons in bonds  between carbon and oxygen are counted as  belonging to oxygen, whereas the two elec-  trons in the C-H bond are shared equally  between carbon and hydrogen.   The catalysis of oxidation-reduction reac-  tions is carried out by a class of enzymes  called oxidoreductases. A subclass of oxidore-  ductases is given the common name dehy-  drogenases (such as lactate dehydrogenase),  because they transfer hydrogen (hydrogen  atoms or hydride atoms) from the substrate to  an electron-accepting coenzyme, such as  NAD + .   nicotinamide adenine dinucleotide (NAD + ) and flavin adenine dinucleotide (FAD),  can transfer electrons together with hydrogen and have unique roles in the genera-  tion of ATP from the oxidation of fuels. Other oxidation-reduction coenzymes work  with metals to transfer single electrons to oxygen. Vitamin E and vitamin C (ascor-  bic acid) are oxidation-reduction coenzymes that can act as antioxidants and protect  against oxygen free radical injury. The different functions of oxidation-reduction  coenzymes in metabolic pathways are explained in Chapters 19 through 22.   Oxidation-reduction coenzymes follow the same principles as activation-transfer  coenzymes, except that they do not form covalent bonds with the substrate. Each  coenzyme has a unique functional group that accepts and donates electrons and is  specific for the form of electrons it transfers (e.g., hydride ions, hydrogen atoms,  oxygen). A different portion of the coenzyme binds the enzyme. Like activation-  transfer coenzymes, oxidation-reduction coenzymes are not good catalysts without  participation from amino acid side chains on the enzyme.   The enzyme lactate dehydrogenase, which catalyzes the transfer of electrons  from lactate to NAD + , illustrates these principles (Fig. 8.14). The coenzyme nicoti-  namide adenine dinucleotide (NAD + ) is synthesized from the vitamin niacin (which  forms the nicotinamide ring), and from ATP (which contributes an AMP). The ADP  portion of the molecule binds tightly to the enzyme and causes conformational    AMP provides additional  binding interactions  which induce  conformational changes  in the enzyme   Fig. 8.14 The coenzyme NAD + accepting a hydride ion, shown in blue, from lactate. NAD + -  dependent dehydrogenases catalyze the transfer of a hydride ion (H:) from a carbon to NAD +  in oxidation reactions such as the oxidation of alcohols to ketones or aldehydes to acids. The  positively charged pyridine ring nitrogen of NAD + increases the electrophilicity of the car-  bon opposite it in the ring. This carbon then accepts the negatively charged hydride ion. The  proton from the alcohol group is released into water. NADP functions by the same mecha-  nism, but it is usually involved in pathways of reductive synthesis.   changes in the enzyme. The functional group of NAD + is the carbon on the nicoti-  namide ring opposite the positively charged nitrogen. This carbon atom accepts the  hydride ion (a hydrogen atom that has two electrons) transferred from a specific car-  bon atom on the substrate. The H + from the substrate alcohol (OH) group then dis-  sociates, and a keto group (C = O) is formed. One of the roles of the enzyme is to  contribute a histidine nitrogen that can bind the dissociable proton on lactate,  thereby making it easier for NAD + to pull off the other hydrogen with both elec-  trons. Finally, NADH dissociates.   Metal ions, which have a positive charge, contribute to the catalytic process by act-  ing as electrophiles (electron-attracting groups). They assist in binding of the sub-  strate, or they stabilize developing anions in the reaction. They can also accept and  donate electrons in oxidation-reduction reactions.   The ability of certain metals to bind multiple ligands in their coordination sphere  enables them to participate in binding substrates or coenzymes to enzymes. For  example, Mg 2+ plays a role in the binding of the negatively charged phosphate  groups of thiamine pyrophosphate to anionic or basic amino acids in the enzyme  (see Fig. 8.11). The phosphate groups of ATP are usually bound to enzymes through  Mg 2+ chelation.   The metals of some enzymes bind anionic substrates or intermediates of the reac-  tion to alter their charge distribution, thereby contributing to catalytic power. The  enzyme alcohol dehydrogenase, which transfers electrons from ethanol to NAD + to  generate acetaldehyde and NADH, illustrates this role (Fig. 8.15). In the active site  of alcohol dehydrogenase, an activated serine pulls a proton off the ethanol -OH  group, leaving a negative charge on the oxygen that is stabilized by zinc. This elec-  tronic configuration allows the transfer of a hydride ion to NAD + . Zinc is essen-  tially fulfilling the same function in alcohol dehydrogenase that histidine fulfills in  lactate dehydrogenase.   Cof actors sometimes play a noncatalytic structural role in certain enzymes, binding  different regions of the enzyme together to form the tertiary structure. They also can  serve as substrates that are cleaved during the reaction.   If the activity of most enzymes is plotted as a function of the pH of the reaction,  an increase of reaction rate is usually observed as the pH goes from a very acidic  level to the physiologic range; a decrease of reaction rate occurs as the pH goes  from the physiologic range to a very basic range (Fig. 8.16). The shape of this  curve in the acid region usually reflects the ionization of specific functional groups  in the active site (or in the substrate) by the increase of pH, and the more general  formation of hydrogen bonds important for the overall conformation of the  enzyme. The loss of activity on the basic side usually reflects the inappropriate  ionization of amino acid residues in the enzyme.    In humans, most of ingested  ethanol is oxidized to acetaldehyde  in the liver by alcohol dehydroge-  nase (ADH):   ADH is active as a dimer, with an active  site containing zinc present in each subunit.  The human has at least seven genes that  encode isozymes of ADH, each with a  slightly different range of specificities for the  alcohols it oxidizes.   The acetaldehyde produced from ethanol  is highly reactive, toxic, and immunogenic.  In Al Martini and other patients with chronic  alcoholism, acetaldehyde is responsible for  much of the liver injury associated with  chronic alcoholism   Fig. 8.15. Liver alcohol dehydrogenase cat-  alyzes the oxidation of ethanol (shown in blue)  to acetaldehyde. The active site of liver alcohol  dehydrogenase contains a bound zinc atom,  and a serine side chain -OH and a histidine  nitrogen that participate in the reaction. The  histidine pulls an H + off the active site serine,  which pulls the H + off of the substrate -OH  group, leaving the oxygen with a negative  charge that is stabilized by zinc.   The parietal cells of the stomach secrete HCI into the lumen of the stomach, resulting in a pH between 1 and 2. This strongly  acidic environment is capable of irreversibly denaturing most proteins by protonating amino acids, thereby preventing the  hydrogen bond formation necessary for tertiary structure. Many of the peptide bonds in proteins would not be accessible to  digestive proteases unless the protein was denatured. Pepsin, a digestive protease present in the stomach, is an exceptional enzyme  because its pH optimum is approximately 1.6 and it is active in the acidic environment of the stomach. As denatured dietary proteins pass  into the intestinal lumen, the pH of the gastric juice is raised above 6 by secretion of bicarbonate from the exocrine pancreas. At this higher  pH, chymotrypsin and other proteases from the pancreas can act on the denatured proteins.   Fig. 8.16. pH profile of an enzyme. The rate of the reaction increases as the pH increases  from 6 to 7.4. The exact shape of the curve depends on the protonation state of active site  amino acid residues or on the hydrogen bonding required for maintenance of three-dimen-  sional structure in the enzyme. For the enzyme shown in the figure, the increase of reaction  rate corresponds to deprotonation of the active site histidine. At a pH above 8.5, deprotona-  tion of an amino-terminal -NH 3 + alters the conformation at the active site and the activity  decreases. Other enzymes might have a lower pH maximum, a broader peak, or retain their  activity in the basic side of the curve.   Most human enzymes function optimally at a temperature of approximately  37°C. An increase of temperature from 0°C to 37°C increases the rate of the reac-  tion by increasing the vibrational energy of the substrates. The maximum activity  for most human enzymes occurs near 37°C because denaturation (loss of secondary  and tertiary structure) occurs at higher temperatures.   Inhibitors are compounds that decrease the rate of an enzymatic reaction. Mecha-  nism-based inhibitors mimic or participate in an intermediate step of the catalytic  reaction. The term includes transition state analogs and compounds that can react  irreversibly with functional groups in the active site.   The symptoms experienced by  Dennis Veere resulted from inhibi-  tion of acetylcholinesterase. Acetyl-  cholinesterase cleaves the neurotransmitter  acetylcholine to acetate and choline in the  postsynaptic terminal, thereby terminating  the transmission of the neural signal (see Fig.  8.17). Malathion is metabolized in the liver to  a toxic derivative (malaoxon) that binds to  the active site serine in acetylcholinesterase  and other enzymes, an action similar to that of  diisopropylfluorophosphate. As a result,  acetylcholine accumulates and overstimulates  the autonomic nervous system (the involun-  tary nervous system, including heart, blood  vessels, glands), thereby accounting for Den-  nis's vomiting, abdominal cramps, salivation,  and sweating. Acetylcholine is also a neuro-  transmitter for the somatic motor nervous  system, where its accumulation resulted in  Dennis's involuntary muscle twitching (mus-  cle fasciculations).   Covalent inhibitors form covalent or extremely tight bonds with functional groups  in the active catalytic site. These functional groups are activated by their interac-  tions with other amino acid residues, and are therefore far more likely to be targeted  by drugs and toxins than amino acid residues outside the active site.   The lethal compound diisopropyl phosphofluoridate (DFP, or diisopropylfluo-  rophosphate) is an organophosphorus compound that served as a prototype for the  development of the nerve gas Sarin and other organophosphorus toxins, such as the  insecticides malathion and parathion (Fig. 8.17). DFP exerts its toxic effect by  forming a covalent intermediate in the active site of acetylcholinesterase, thereby  preventing the enzyme from degrading the neurotransmitter acetylcholine. Once the  covalent bond is formed, the inhibition by DFP is essentially irreversible, and activ-  ity can only be recovered as new enzyme is synthesized. DFP also inhibits many  other enzymes that use serine for hydrolytic cleavage, but the inhibition is not as  lethal.   Aspirin (acetylsalicylic acid) provides an example of a pharmacologic drug  that exerts its effect through the covalent acetylation of an active site serine in  the enzyme prostaglandin endoperoxide synthase (cycloxygenase). Aspirin  resembles a portion of the prostaglandin precursor that is a physiologic substrate  for the enzyme.   H 3 C - C r O - CH 2 - CH 2 - N (CH 3 ) 3 HO - CH 2 - CH 2 - N (CH 3 ) 3   Fig. 8.17 A. Acetylcholinesterase normally catalyzes inactivation of the neurotransmitter acetylcholine in a hydrolysis reaction. The active site  serine forms a covalent intermediate with a portion of the substrate during the course of the reaction. B. Diisopropyl phosphofluoridate (DFP),  the ancestor of current organophosphorus nerve gases and pesticides, inactivates acetylcholinesterase by forming a covalent complex with the  active site serine that cannot be hydrolysed by water. The result is that the enzyme cannot carry out its normal reaction, and acetylcholine  accumulates.   B. Transition State Analogs and Compounds that  Resemble Intermediate Stages of the Reaction   Transition state analogs are extremely potent and specific inhibitors of enzymes  because they bind so much more tightly to the enzyme than do substrates or  products. Drugs cannot be designed that precisely mimic the transition state  because of its highly unstable structure. However, substrates undergo progressive  changes in their overall electrostatic structure during the formation of a transi-  tion state complex, and effective drugs often resemble an intermediate stage of  the reaction more closely than they resemble the substrate. Medical literature  often refers to such compounds as substrate analogs, even though they bind more  tightly than substrates.   The antibiotic penicillin is a transition state analog that binds very tightly to gly-  copeptidyl transferase, an enzyme required by bacteria for synthesis of the cell wall  (Fig. 8.18). Glycopeptidyl transferase catalyzes a partial reaction with penicillin  that covalently attaches penicillin to its own active site serine. The reaction is  favored by the strong resemblance between the peptide bond in the p -lactam ring of  penicillin and the transition state complex of the natural transpeptidation reaction.  Active site inhibitors such as penicillin that undergo partial reaction to form irre-  versible inhibitors in the active site are sometimes termed "suicide inhibitors."   Allopurinol, a drug used to treat gout, decreases urate production by inhibiting  xanthine oxidase. This inhibition provides an example of an enzyme that commits  suicide by converting a drug to a transition state analog. The normal physiologic   Fig. 8.18 The antibiotic penicillin inhibits the bacterial enzyme glycopeptide transpeptidase.  The transpeptidase is a serine protease involved in cross-linking components of bacterial cell  walls and is essential for bacterial growth and survival. It normally cleaves the peptide bond  between two D-alanine residues in a polypeptide. Penicillin contains a strained peptide bond  within the p-lactam ring that resembles the transition state of the normal cleavage reaction,  and thus penicillin binds very readily in the enzyme active site. As the bacterial enzyme  attempts to cleave this penicillin peptide bond, penicillin becomes irreversibly covalently  attached to the enzyme's active site serine, thereby inactivating the enzyme.   Lotta Topaigne is being treated  with allopurinol for gout, which is  caused by an accumulation of  sodium urate crystals in joints and joint  fluid, particularly in the ankle and great toe.  Allopurinol is a suicide inhibitor of the  enzyme xanthine oxidase, which is involved  in the degradation of purine nucleotides  AMP and GMP to uric acid (urate). Although  hypoxanthine levels increase in the pres-  ence of allopurinol, hypoxanthine does not  participate in urate crystal formation and  precipitation at this concentration. It is  excreted in the urine.   function of xanthine oxidase is the oxidation of hypoxanthine to xanthine and xan-  thine to uric acid (urate) in the pathway for degradation of purines (Fig. 8.19). The  enzyme contains a molybdenum-sulfide (Mo-S) complex that binds the substrates  and transfers the electrons required for the oxidation reactions. Xanthine oxidase  oxidizes the drug allopurinol to oxypurinol, a compound that binds very tightly to  a molybdenum-sulfide complex in the active site. As a result, the enzyme has  committed suicide and is unable to carry out its normal function, the generation of  uric acid (urate).   Heavy metal toxicity is caused by tight binding of a metal such as mercury (Hg), lead  (Pb), aluminum (Al), or iron (Fe), to a functional group in an enzyme. Heavy metals  are relatively nonspecific for the enzymes they inhibit, particularly if the metal is  associated with high dose toxicity. Mercury, for example, binds to so many enzymes,  often at reactive sulfhydryl groups in the active site, that it has been difficult to deter-  mine which of the inhibited enzymes is responsible for mercury toxicity. Lead pro-  vides an example of a metal that inhibits through replacing the normal functional  metal in an enzyme. Its developmental and neurologic toxicity may be caused by its   Fig. 8.19. Allopurinol is a suicide inhibitor of xanthine oxidase. A. Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine, and  xanthine to uric acid (urate) in the pathway for degradation of purine nucleotides. B. The oxidations are performed by a molybdenum-oxo-sul-  fide coordination complex in the active site that complexes with the group being oxidized. Oxygen is donated from water. The enzyme can  work either as an oxidase (0 2 accepts the 2e" and is reduced to H 2 0 2 ) or as a dehydrogenase (NAD + accepts the 2e" and is reduced to NADH.)  C. Xanthine oxidase is able to perform the first oxidation step and convert allopurinol to alloxanthine (oxypurinol). As a result, the enzyme has  committed suicide; the oxypurinol remains bound in the molybdenum coordination sphere, where it prevents the next step of the reaction.   ability to replace Ca + in two regulatory proteins important in the central nervous  system and other tissues, Ca 2+ -calmodulin and protein kinase C.   ^ Dennis Veere. Dennis Veere survived his malathion intoxication  because he had ingested only a small amount of the chemical, vomited  shortly after the agent was ingested, and was rapidly treated in the emer-  gency room. Lethal doses of oral malathion are estimated at 1 g/kg of body weight  for humans. Emergency room physicians used a drug (oxime) to reactivate the  acetylcholinesterase in Dennis before the aged complex formed. They also used  intravenous atropine, an anticholinergic (antimuscarinic) agent, to antagonize the  action of the excessive amounts of acetylcholine accumulating in cholinergic recep-  tors throughout his body.   After several days of intravenous therapy, the signs and symptoms of acetyl-  choline excess abated, and therapy was slowly withdrawn. Dennis made an unevent-  ful recovery.   0 © Once ingested, the liver converts  /^oA J malathion to the toxic reactive  ^/ compound, malaoxon, by replac-  ing the sulfur with an oxygen. Malaoxon  then binds to the active site of acetyl-  cholinesterase and reacts to form the cova-  lent intermediate. Unlike the complex  formed between diisopropylfluorophos-  phate and acetylcholinesterase, this initial  acylenzyme intermediate is reversible. How-  ever, with time, the enzyme-inhibitor com-  plex "ages" (dealkylation of the inhibitor  and enzyme modification) to form an irre-  versible complex.   a Lotta Topaigne. Within several days of starting allopurinol therapy,  Ms. Topaigne 's serum uric acid level began to decrease. Several weeks  later, the level in her blood was normal. However, while Lotta was adapt-  ing to allopurinol therapy, she experienced a mild gout attack, which was treated  with a low dose of colchicine (see Chapter 10).    At low concentrations of ethanol,  liver alcohol dehydrogenase is the  major route of ethanol oxidation to  acetaldehyde, a highly toxic chemical.  Acetaldehyde not only damages the liver, it  can enter the blood and potentially damage  the heart and other tissues. At low ethanol  intakes, much of the acetaldehyde produced  is safely oxidized to acetate in the liver by  acetaldehyde dehydrogenases.   ^ Al Martini. Al Martini was admitted to the hospital after intravenous  I I thiamine was initiated at a dose of 100 mg/day (compared with an RDA of  1.4 mg/day). His congestive heart failure was believed to be the result, in  part, of the cardiomyopathy (heart muscle dysfunction) of acute thiamine deficiency  known as beriberi heart disease. This nutritional cardiac disorder and the peripheral  nerve dysfunction usually respond to thiamine replacement. However, an alcoholic  cardiomyopathy can also occur in well-nourished patients with adequate thiamine  levels. Exactly how ethanol, or its toxic metabolite acetaldehyde, causes alcoholic  cardiomyopathy in the absence of thiamine deficiency is unknown.   Enzymes have been systematically  \ \ J classified by the Enzyme Commis-  *S sion of the International Union of  Biochemists and Molecular Biologists into  six major groups according to the type of  reaction catalysed. The major groups are  further subdivided so that each enzyme has  an individual Enzyme Commission number.  Most enzymes have systematic names,  reflecting their activities. In addition, most  enzymes have common or trivial names,  which are shorter, reflect the activity of the  enzyme in a less standardized way, and may  not end in "ase". For example, glucokinase  (common name) has the systematic name of  ATP:D-hexose 6-phosphotransferase, and its  enzyme commission number is EC 2.7.1.2.  The "2" is the Enzyme Commission (EC)  number of the general class (transferase),  followed by a period and "1" , the number of  the subclass for transfer of phosphorus-con-  taining groups. The "1" denotes transfer to  an alcohol acceptor, and the final "2" is the  specific number of the enzyme.   Xanthine oxidase uses molybde-  \\ ) num for electron transfer to 0 2 .   This enzyme accounts for the  human dietary requirement for molybde-  num, which is classified as a trace mineral.   Basic Reactions and Classes of Enzymes. In the following  chapters of the text, students will be introduced to a wide variety of reac-  tion pathways and enzyme names. Although it may seem that the number  of reactions is infinite, many of these reactions are similar and occur frequently in  different pathways. Recognition of the type of reaction can aid in remembering the  pathways and enzyme names, thereby reducing the amount of memorization  required. You may wish to use this section for reference as you go through your first  biochemical pathways.   The Enzyme Commission has divided the basic reaction types and the enzymes  catalyzing them into six broad numbered classes: (1) oxidoreductases, (2) trans-  ferases, (3) hydrolases, (4) lyases, (5) isomerases, and (6) ligases. Each broad class  of enzymes includes subsets of enzymes with a systematic name and a common  name (e.g., dehydrogenases and kinases).   Oxidoreductases. Oxidation-reduction reactions are very common  in biochemical pathways and are catalyzed by a broad class of enzymes  called oxidoreductases. Whenever an oxidation-reduction reaction  occurs, at least one substrate gains electrons and becomes reduced, and another  substrate loses electrons and becomes oxidized. One subset of reactions is cat-  alyzed by dehydrogenases, which accept and donate electrons in the form of  hydride ions (H:~) or hydrogen atoms. Usually an electron-transferring coen-  zyme, such as NAD + /NADH, acts as an electron donor or acceptor (e.g., see Fig.  8.14 and Fig. 8.15).   In another subset of reactions, 0 2 donates either one or both of its oxygen atoms  to an acceptor (for example, see xanthine oxidase, Fig. 8.19). When this occurs, 0 2  becomes reduced, and an electron donor is oxidized. Enzymes participating in reac-  tions with 0 2 are called hydroxylases and oxidases when one oxygen atom is incor-  porated into a substrate and the other oxygen atom into water, or both atoms are  incorporated into water. They are called oxygenases when both atoms of oxygen are  incorporated into the acceptor. Most hydroxylases and oxidases require metal ions,  such as Fe 2+ , for electron transfer.   Transferases. Transferases catalyze group transfer reactions — the  transfer of a functional group from one molecule to another. If the trans-  ferred group is a high-energy phosphate (as shown in Fig. 8.3), the enzyme  is a kinase; if the transferred group is a carbohydrate residue, the enzyme is a   gly cosy Itransf erase ; if it is a fatty acyl group, the enzyme is an acyltransf erase. A  common feature of these reactions is that the group being transferred exists as a  good leaving group on the donor molecule.   Another subset of group transfer reactions consists of transaminations (Fig. 8.20).  In this type of reaction, the nitrogen group from an amino acid is donated to an a-  keto acid, forming a new amino acid and the a-keto acid corresponding to the donor  amino acid. Enzymes catalyzing this last type of reaction are called transaminases  or aminotransferases. The coenzyme pyridoxal phosphate is required for all  transaminases (see Fig. 8.13).   When the physiologically important aspect of the reaction is the compound synthe-  sized, the transferase may be called a synthase. For example, the enzyme commonly  called glycogen synthase transfers a glucosyl residue from UDP-glucose to the end of a  glycogen molecule. Its systematic name is UDP-glucose-glycogen glycosyltransferase.   Hydrolases. In hydrolysis reactions, C-O, C-N, or C-S bonds are  cleaved by the addition of H 2 0 in the form of OH~ and H + to the atoms  forming the bond (see, for example, Fig. 8.8). The enzyme class names  specify the group being cleaved (e.g., the enzyme commonly named chy-  motrypsin is a protease, a hydrolase that cleaves peptide bonds in proteins).   fj Lyases. The lyase class of enzymes consists of a diverse group of  ) enzymes cleaving C-C, C-O, and C-N bonds by means other than  hydrolysis or oxidation. Some of the enzymes catalyzing C-C bond  cleavage are called aldolases, decarboxylases (when carbon dioxide is released  from a substrate), and thiolases (when the sulfur-containing nucleophile of cys-  teine or Co ASH is used to break a carbon-carbon bond) (Fig. 8.21). The struc-  tures amenable to carbon-carbon bond cleavage usually require a carbonyl car-  bon that can act as an electron sink to stabilize the carbanion transiently formed  when the carbon-carbon bond breaks.   This broad class of enzymes also includes dehydratases and many synthases.  Dehydratases remove the elements of water from two adjacent carbon-carbon  bonds to form a double bond. Certain enzymes in this group, such as certain  group transferases, are commonly called synthases when the physiologically  important direction of the reaction favors the formation of a carbon-carbon bond  (e.g., citrate synthase).   Fig. 8.20. Transamination reactions. Pyridoxal  phosphate (PLP) on aspartate aminotrans-  ferase transfers an amino group from aspartate  to the a-keto acid (a-ketoglutarate) to form a  new amino acid (glutamate). The enzyme used  to be called glutamate-oxaloacetate transami-  nase.   Fig. 8.21. Aldolases catalyze carbon-carbon cleavage in reactions that are usually reversible.  In glycolysis, the enzyme fructose 1,6-bisphosphate aldolase cleaves a carbon-carbon bond  in fructose 1,6-bisphosphate. Aldolases have a lysine epsilon amino group in the active site  that participates in the reaction.   Fig. 8.22. Isomerases rearrange atoms within a  molecule. In the pathway of glycolysis, triose  phosphate isomerase converts dihydroxyace-  tone phosphate to glyceraldehydes 3-phos-  phate by rearranging hydrogen atoms. No  other substrates or products of the reaction  exist.   ft Isomerases. Many biochemical reactions simply rearrange the existing  atoms of a molecule, that is, create isomers of the starting material (Fig.  8.22). Enzymes rearranging the bond structure of a compound are called   isomerases, whereas enzymes catalyzing movement of a phosphate from one atom   ft Li gases. Ligases synthesize C-C, C-S, C-O, and C-N bonds in reactions  coupled to the cleavage of a high-energy phosphate bond in ATP or another  nucleotide. Carboxylases, for example, add C0 2 to another compound in a  reaction requiring ATP cleavage to provide energy (see Fig. 8.12B). Most carboxy-  lases require the coenzyme biotin. Other ligases are named synthetases (e.g., fatty  acyl Co A synthetase). Synthetases differ from the synthases mentioned under  "lyases" and "group transferases" in that synthetases derive the energy for new bond  formation from cleavage of high-energy phosphate bonds, and synthases use a dif-  ferent source of energy.   Dressier D, Potter H. Discovering enzymes. Scientific American Library. New York: W.H. Freeman, 1991.   Fersht A. Structure and Mechanism in Protein Science. New York: W.H. Freeman, 1999.   EPA has a good website for information on organophosphate compounds at www.epa.gov/pesticides.   The ENZYME database (http://www.expasy.ch/enzyme/) provides basic information about specific  enzymes. It is based on the recommendations of the Nomenclature Committee of the Interna-  tional Union of Biochemistry and Molecular Biology. In this database, you can obtain the EC  number of any enzyme, its recommended name, alternative names, cofactors, and human dis-  eases associated with the enzyme. You can trace the enzyme from its name to the reaction it  catalyses — to the metabolic pathway(s) in which it participates — to the large and intricate  Boehringer Mannheim Biochemical Pathways Wallchart. You also can search the database by  metabolites or pathways.   Questions below cover material from Chapters 6 and 7, as well as Chapter 8 (including Biochemical Comments).   1 . A patient was born with a congenital mutation in an enzyme, severely affecting its ability to bind an activation-transfer coen-  zyme. As a consequence,   (A) the enzyme would be unable to bind the substrate of the reaction.   (B) the enzyme would be unable to form the transition state complex.   (D) the enzyme would normally substitute the functional group of an active site amino acid residue for the coenzyme.   (E) the reaction could be carried out by the free coenzyme, provided the diet carried an adequate amount of its vitamin  precursor.   2. An individual had a congenital mutation in glucokinase in which a proline was substituted for a leucine on a surface helix far  from the active site, but within the hinge region of the actin fold. This mutation would be expected to   (A) have no effect on the rate of the reaction because it is not in the active site.   (B) have no effect on the rate of the reaction because proline and leucine are both nonpolar amino acids.   (C) have no effect on the number of substrate molecules reaching the transition state.   (D) probably affect the binding of ATP or a subsequent step in the reaction sequence.   (E) probably cause the reaction to proceed through an alternate mechanism.   A patient developed a bacterial overgrowth in his intestine that decreased the pH of the luminal contents from their normal  pH of approximately 6.5 down to 5.5. This decrease of pH is likely to   (A) denature proteins reaching the intestine with their native structure intact.   (D) inhibit intestinal enzymes dependent on an active site lysine for binding substrate.   The type of reaction shown above fits into which of the following classifications?   Fig. 9.1. The flux of substrates down a meta-  bolic pathway is analogous to cars traveling  down a highway. The rate-limiting enzyme is  the portion of the highway that is narrowed to  one lane by a highway barrier. This single por-  tion of the highway limits the rate at which  cars can arrive at their final destination miles  later. Cars will back up before the barrier (sim-  ilar to the increase in concentration of a pre-  cursor when a rate-limiting enzyme is inhib-  ited). Some cars may exit and take an alternate  route (similar to precursors entering another  metabolic pathway.) Moving the barrier just a  little to open an additional lane is like activat-  ing a rate-limiting enzyme; it increases flow  through the entire length of the pathway.   In the body, thousands of diverse enzymes are regulated to fulfil their individ-  ual functions without waste of dietary components. Thus, with changes in our  physiologic state, time of eating, environment, diet, or age, the rates of some  enzymes must increase and others decrease. In this chapter, we describe the mech-  anisms for regulation of enzyme activity and the strategies employed to regulate  the metabolic pathways in which they reside.   Regulation matches function. Changes in the rate of a metabolic pathway  occur because at least one enzyme in that pathway, the regulatory enzyme, has  been activated or inhibited, or the amount of enzyme has increased or decreased.  Regulatory enzymes usually catalyze the rate -limiting, or slowest, step in the  pathway, so that increasing or decreasing their rate changes the rate of the entire  pathway (Fig. 9.1). The mechanisms used to regulate the rate-limiting enzyme in a  pathway reflect the function of the pathway.   Substrate concentration. The rate of all enzymes is dependent on substrate con-  centration. Enzymes exhibit saturation kinetics; their rate increases with increas-  ing substrate concentration [S], but reaches a maximum velocity (V max ) when the  enzyme is saturated with substrate. For many enzymes, the Michaelis-Menten  equation describes the relationship between v t (the initial velocity of a reaction),  [S], V max , and the K m (the substrate concentration at which v t = V2 V max ).   Reversible inhibition. Enzymes are reversibly inhibited by structural analogs  and products. These inhibitors are classified as competitive, noncompetitive, or  uncompetitive, depending on their effect on formation of the enzyme-substrate  complex.   Allosteric enzymes. Allosteric activators or inhibitors are compounds that  bind at sites other than the active catalytic site and regulate the enzyme through  conformational changes affecting the catalytic site.   Covalent modification. Enzyme activity also may be regulated by a covalent  modification, such as phosphorylation of a serine, threonine, or tyrosine residue  by a protein kinase.   Protein-protein interactions. Enzyme activity can be modulated through the  reversible binding of a modulator protein, such as Ca 2+ calmodulin. Monomeric G  proteins (GTP -binding proteins) activate target proteins through reversible binding.   Zymogen cleavage. Some enzymes are synthesized as inactive precursors,  called zymogens, that are activated by proteolysis (e.g., the digestive enzyme chy-  motrypsin).   Changes in enzyme concentration. The concentration of an enzyme can be  regulated by changes in the rate of enzyme synthesis (e.g., induction of gene tran-  scription) or the rate of degradation.   Regulation of metabolic pathways. The regulatory mechanisms for the rate-  limiting enzyme of a pathway always reflects the function of the pathway in a  particular tissue. In feedback regulation, the end product of a pathway directly  or indirectly controls its own rate of synthesis; in feedforword regulation, the   substrate controls the rate of the pathway. Biosynthetic and degradative pathways  are controlled through different but complementary regulation. Pathways are also  regulated through compartmentation of enzymes.   Al Martini is a 44-year-old man who has been an alcoholic for the past  5 years. He was recently admitted to the hospital for congestive heart  failure (see Chapter 8). After being released from the hospital, he con-  tinued to drink. One night he arrived at a friend's house at 7:00 P.M. Between his  arrival and 11:00 P.M., he drank four beers and five martinis (for a total ethanol  consumption of 9.5 oz). His friends encouraged him to stay an additional hour  and drink coffee to sober up. Nevertheless, he ran his car off the road on his way  home. He was taken to the emergency room of the local hospital and arrested for  driving under the influence of alcohol. His blood alcohol concentration at the  time of his arrest was 240 mg/dL, compared with the legal limit of ethanol for  driving of 80 mg/dL.   Ann O'Rexia, a 23 -year old woman, 5 feet 7 inches tall, is being treated  for anorexia nervosa (see Chapters 1-3). She has been gaining weight,  and is now back to 99 lb from a low of 85 lb. Her blood glucose is still  below normal (fasting blood glucose of 72 mg/dL, compared with a normal range  of 80-100 mg/dL). She complains to her physician that she feels tired when she  jogs, and she is concerned that the "extra weight" she has gained is slowing her  down.   We will generally be using the  \\ J pathways of fuel oxidation to illus-  trate the role of various mecha-  nisms of enzyme regulation in metabolic  pathways. Chapters 1 through 3 provide  an overview of the names and functions of  these pathways, including the TCA cycle,  glycolysis, glycogen synthesis, glycogenoly-  sis, and fatty acid oxidation.   Al Martini was not able to clear his  blood ethanol rapidly enough to  stay within the legal limit for driv-  ing. Ethanol is cleared from the blood at  about 1 /2 ounce/hr (15 mg/dL per hour). Liver  metabolism accounts for more than 90% of  ethanol clearance from the blood. The major  route of ethanol metabolism in the liver is  the enzyme liver alcohol dehydrogenase  (ADH), which oxidizes ethanol to acetalde-  hyde with generation of NADH.   The multienzyme complex MEOS (microsomal  ethanol oxidizing system), which is also called  cytochrome P450-2E1, provides an additional  route for ethanol oxidation to acetaldehyde in  the liver.   Although the regulation of metabolic pathways is an exceedingly complex sub-  ject, dealt with in most of the subsequent chapters of this text, a number of com-  mon themes are involved. Physiologic regulation of a metabolic pathway  depends on the ability to alter flux through the pathway by activating the enzyme  catalyzing the rate-limiting step in the pathway (see Fig. 9.1). The type of regulation  employed always reflects the function of the pathway and the need for that path-  way in a particular tissue or cell type. Pathways producing a necessary product  are usually feedback-regulated through a mechanism directly or indirectly  involving the concentration of product (e.g., allosteric inhibition or  induction/repression of enzyme synthesis). The concentration of product signals  when enough of the product has been synthesized. Storage and toxic disposal  pathways are usually regulated directly or indirectly through a feedforward  mechanism reflecting the availability of precursor. Regulatory enzymes are often  tissue-specific isozymes whose properties reflect the different functions of a  pathway in particular tissues. Pathways are also regulated through compartmen-  tation, collection of enzymes with a common function within a particular  organelle or at a specific site in the cell.   The mechanisms employed to regulate enzymes have been organized into three  general categories: regulation by compounds that bind reversibly in the active site  (including dependence of velocity on substrate concentration and product levels);  regulation by changing the conformation of the active site (including allosteric reg-  ulators, covalent modification, protein-protein interactions, and zymogen cleav-  age); and regulation by changing the concentration of enzyme (enzyme synthesis  and degradation).   One of the fuels used by Ann O' Rexia's skeletal muscles for jogging is glucose, which is converted to glucose 6-phosphate (glu-  cose 6-P) by the enzymes hexokinase (HK) and glucokinase (GK). Glucose 6-P is metabolized in the pathway of glycolysis to gen-  erate ATP. This pathway is feedback regulated, so that as her muscles use ATP, the rate of glycolysis will increase to generate  more ATP   When she is resting, her muscles and liver will convert glucose 6-phosphate to glycogen (a fuel storage pathway, shown in blue). Glyco-  gen synthesis is feed-forward regulated by the supply of glucose and by insulin and other hormones that signal glucose availability.   Glycogenolysis (glycogen degradation) is activated during exercise to supply additional glucose 6-P for glycolysis. Unless Ann consumes  sufficient calories, her glycogen stores will not be replenished after exercise, and she will tire easily.   The Michaelis-Menten equation  relates the initial velocity of the  reaction (Vj) to the concentration of  enzyme substrate complexes (ES). This  equation is derived for a reaction in which a  single substrate, S, is converted to a single  product, P. The enzyme (E) and S associate  to form ES with the rate constant of k v The  complex dissociates with the rate constant  of k 2 , or is converted to P with the rate con-  stant k 3 . Under conditions in which [S] »  [E], [P] is negligible, and the rate of conver-  sion of ES to an enzyme-product complex is  very fast, Vj = k 3 [ES]. The concentration of  ES is a fraction of E T , the total amount of  enzyme present as ES and E.  Therefore,   Where K m = (k 2 + k 3 )/k 1 . Substitution of V max  for k 3 [ET] gives the Michaelis-Menten equa-  tion (see Equation 9.1)   The velocity of all enzymes is dependent on the concentration of substrate. This  dependence is reflected in conditions such as starvation, in which a number of  pathways are deprived of substrate. In contrast, storage pathways (e.g., glucose  conversion to glycogen in the liver) and toxic waste disposal pathways (e.g., the  urea cycle, which prevents NH 4 + toxicity by converting NH 4 + to urea ) are normally  regulated to speed up when more substrate is available. In the following sections,  we use the Michaelis-Menten equation to describe the response of an enzyme to  changes in substrate concentration and use glucokinase to illustrate the role of sub-  strate supply in regulation of enzyme activity.   The equations of enzyme kinetics provide a quantitative way of describing the depend-  ence of enzyme rate on substrate concentration. The simplest of these equations, the  Michaelis-Menten equation, relates the initial velocity (v { ) to the concentration of sub-  strate [S] and the two parameters K m and V max (Equation 9.1) The V max of the enzyme  is the maximal velocity that can be achieved at an infinite concentration of substrate,  and the K m of the enzyme for a substrate is the concentration of substrate required to  reach l /i V max . The Michaelis-Menten model of enzyme kinetics applies to a simple  reaction in which the enzyme and substrate form an enzyme-substrate complex (ES)  that can dissociate back to the free enzyme and substrate. The initial velocity of prod-  uct formation, v i? is proportionate to the concentration of enzyme-substrate complexes  [ES]. As substrate concentration is increased, the concentration of enzyme-substrate  complexes increases, and the reaction rate increases proportionately.   The graph of the Michaelis-Menten equation (Vi as a function of substrate con-  centration) is a rectangular hyperbola that approaches a finite limit, V max , as the  fraction of total enzyme present as enzyme-substrate complex increases (Fig. 9.2).   where K m = (k 2 + k 3 )/k-|  and V max = k 3 [ET]   At a hypothetical infinitely high substrate concentration, all of the enzyme mole-  cules contain bound substrate, and the reaction rate is at V max The approach to the  finite limit of V max is called saturation kinetics because velocity cannot increase any  further once the enzyme is saturated with substrate. Saturation kinetics is a charac-  teristic property of all rate processes dependent on the binding of a compound to a  protein.   The K m of the enzyme for a substrate is defined as the concentration of sub-  strate at which Vi equals Vi V max . The velocity of an enzyme is most sensitive to  changes in substrate concentration over a concentration range below its K m (see  Fig. 9.2). At substrate concentrations less than Vioth of the K m , a doubling of sub-  strate concentration nearly doubles the velocity of the reaction; at substrate con-  centrations 10 times the K m , doubling the substrate concentration has little effect  on the velocity.   The K m of an enzyme for a substrate is related to the dissociation constant, K d ,  which is the rate of substrate release divided by the rate of substrate binding. For  example, a genetic mutation that decreases the rate of substrate binding to the  enzyme decreases the affinity of the enzyme for the substrate and increases the K d  and K m of the enzyme for that substrate. The higher the K m , the higher is the sub-  strate concentration required to reach x h V max .   Fig. 9.2. A graph of the Michaelis-Menten equa-  tion. V max (solid blue line) is the initial velocity  extrapolated to infinite [S]. K m (dashed blue  line) is the concentration of S at which v { =  V 12    MODY. Patients with maturity onset  diabetes of the young (MODY) have  a rare genetic form of diabetes mel-  litus in which the amount of insulin being  secreted from the pancreas is too low, result-  ing in hyperglycemia. The disease is caused  by mutations in the gene for pancreatic glu-  cokinase (a closely related isozyme of liver  glucokinase) that affect its kinetic properties  (K m or V max ). Glucokinase is part of the mech-  anism controlling release of insulin from the  pancreas. A decreased activity of glucokinase  results in lower insulin secretion for a given  blood glucose level.   A comparison between the isozymes of hexokinase found in red blood cells and in  the liver illustrates the significance of the K m of an enzyme for its substrate. Hexo-  kinase catalyses the first step in glucose metabolism in most cells, the transfer of a  phosphate from ATP to glucose to form glucose 6-phosphate. Glucose 6-phosphate  may then be metabolized in glycolysis, which generates energy in the form of ATP,  or converted to glycogen, a storage polymer of glucose. Hexokinase I, the isozyme  in red blood cells (erythrocytes), has a K m for glucose of approximately 0.05 mM  (Fig. 9.3). The isozyme of hexokinase, called glucokinase, which is found in the liver  and pancreas, has a much higher K m of approximately 5 to 6 mM. The red blood cell  is totally dependent on glucose metabolism to meet its needs for ATP. At the low K m  of the erythrocyte hexokinase, blood glucose could fall drastically below its normal  fasting level of approximately 5 mM, and the red blood cell could still phosphorylate  glucose at rates near V max . The liver, however, stores large amounts of "excess" glu-  cose as glycogen or converts it to fat. Because glucokinase has a K m of approxi-  mately 5 mM, the rate of glucose phosphorylation in the liver will tend to increase  as blood glucose increases after a high-carbohydrate meal, and decrease as blood  glucose levels fall. The high K m of hepatic glucokinase thus promotes the storage of  glucose as liver glycogen or as fat, but only when glucose is in excess supply.    As Ann O'Rexia eats a high carbo-  hydrate meal, her blood glucose  _/ will rise to approximately 20 mM in  the portal vein, and much of the glucose  from her carbohydrate meal will enter the  liver. How will the activity of glucokinase in  the liver change as glucose is increased from  4 mM to 20 mM? (Hint: Calculate Vj as a frac-  tion of V max for both conditions, using a K m  for glucose of 5 mM and the Michaelis-  Menten equation).   Glucokinase, which has a high K m  JjL •) for glucose, phosphorylates glu-  cose to glucose 6-phosphate about  twice as fast after a carbohydrate meal than  during fasting. Substitute the values for S  and K m into the Michaelis-Menten equation.  The initial velocity will be 0.44 times V max  when blood glucose is at 4 mM and about  0.80 times V max when blood glucose is at 20  mM. In the liver, glucose 6-phosphate is a  precursor for both glycogen and fat synthe-  sis. Thus, these storage pathways are par-  tially regulated through a direct effect of  substrate supply. They are also partially reg-  ulated through an increase of insulin and a  decrease of glucagon, two hormones that  signal the supply of dietary fuel.   Fig. 9.3. A comparison between hexokinase I and glucokinase. The initial velocity (vj)  as a fraction of V max is graphed as a function of glucose concentration. The plot for glu-  cokinase (heavy blue line) is slightly sigmoidal (S -shaped), possibly because the rate of  an intermediate step in the reaction is so slow that the enzyme does not follow Michaelis-  Menten kinetics. The dashed blue line has been derived from the Michaelis-Menten  equation fitted to the data for concentrations of glucose above 5 mM. For S-shaped  curves, the concentration of substrate required to reach half V max , or half-saturation, is  sometimes called the S 0 5 or K 0 5 , rather than K m . At v/V max = 0.5, the K m is 5 mM, and  the S 0 5 is 6.7 mM.   The use of V max in the medical liter-  ature to describe the maximal rate  at which a certain amount of tissue  converts substrate to product can be confus-  ing. The best way to describe an increase in  enzyme activity in a tissue is to say that the  maximal capacity of the tissue has  increased. In contrast, the term k cat has been  developed to clearly describe the speed at  which an enzyme can catalyse a reaction  under conditions of saturating substrate  concentration. The rate constant k cat , the  turnover number of the enzyme, has the  units of min" 1 (micromoles of product  formed per minute divided by the micro-  moles of active site).   The rate of a reaction is directly proportional to the concentration of enzyme;  if you double the amount of enzyme, you will double the amount of product  produced per minute, whether you are at low or at saturating concentrations of  substrate. This important relationship between velocity and enzyme concentra-  tion is not immediately apparent in the Michaelis-Menten equation because the  concentration of total enzyme present (E t ) has been incorporated into the term  V max (that is, V max is equal to the rate constant k 3 times E t .) However, V max is  most often expressed as product produced per minute per milligram of enzyme  and is meant to reflect a property of the enzyme that is not dependent on its  concentration.   Most enzymes have more than one substrate, and the substrate binding sites over-  lap in the catalytic (active) site. When an enzyme has more than one substrate, the  sequence of substrate binding and product release affect the rate equation. As a    The liver alcohol dehydrogenase most active in oxidizing ethanol has a very  low K m for ethanol of approximately 0.04 mM, and is at over 99% of its V max  at the legal limit of blood alcohol concentration for driving (80 mg/dL or  about 17 mM). In contrast, the MEOS isozyme most active toward ethanol has a K m of  approximately 1 1 mM. Thus, MEOS makes a greater contribution to ethanol oxidation  and clearance from the blood at higher ethanol levels than lower ones. Liver damage,  such as cirrhosis, results partly from toxic byproducts of ethanol oxidation generated  by MEOS. Al Martini, who has blood alcohol levels of 240 mg/dL (approximately 52  mM), is drinking enough to potentially cause liver damage, as well as his car accident  and arrest for driving under the influence of alcohol. The various isozymes and poly-  morphisms of alcohol dehydrogenase and MEOS are discussed in more detail in  Chapter 25.   consequence, an apparent value of K m (K m app ) depends on the concentration of  cosubstrate or product present.   Equations for the initial velocity of an enzyme-catalyzed reaction, such as the  Michaelis-Menten equation, can provide useful parameters for describing or com-  paring enzymes. However, many multisubstrate enzymes, such as glucokinase, have  kinetic patterns that do not fit the Michaelis-Menten model (or do so under non-  physiologic conditions). The Michaelis-Menten model is also inapplicable to  enzymes present in a higher concentration than their substrates. Nonetheless, the  term "K m " is still used for these enzymes to describe the approximate concentration  of substrate at which velocity equals Vi V max .   One of the ways of altering enzyme activity is through compounds binding in the  active site. If these compounds are not part of the normal reaction, they inhibit  the enzyme. An inhibitor of an enzyme is defined as a compound that decreases  the velocity of the reaction by binding to the enzyme. It is a reversible inhibitor  if it is not covalently bound to the enzyme and can dissociate at a significant rate.  Reversible inhibitors are generally classified as competitive, noncompetitive, or  uncompetitive with respect to their relationship to a substrate of the enzyme. In  most reactions, the products of the reaction are reversible inhibitors of the  enzyme producing them.   A competitive inhibitor "competes" with a substrate for binding at the enzyme's  substrate recognition site and therefore is usually a close structural analog of  the substrate (Fig. 9.4). An increase of substrate concentration can overcome  competitive inhibition; when the substrate concentration is increased to a suffi-  ciently high level, the substrate binding sites are occupied by substrate, and inhibitor  molecules cannot bind. Competitive inhibitors, therefore, increase the apparent K m   Fig. 9.4. Competitive inhibition with respect to substrate A. A and B are substrates for the  reaction forming the enzyme substrate complex (E-AB). The enzyme has separate binding  sites for each substrate, which overlap in the active site. The competitive inhibitor (CI) com-  petes for the binding site of A, the substrate it most closely resembles.   Some of Al Martini's problems  have arisen from product inhibition  of liver alcohol dehydrogenase by  As ethanol is oxidized in liver cells,  is reduced to NADH and the  NADH/NAD+ ratio rises. NADH is an inhibitor  of alcohol dehydrogenase, competitive with  respect to NAD + , so the increased  NADH/NAD + ratio slows the rate of ethanol  oxidation and ethanol clearance from the  blood.   NADH is also a product inhibitor of  enzymes in the pathway that oxidizes fatty  acids. Consequently, these fatty acids accu-  mulate in the liver, eventually contributing to  the alcoholic fatty liver.   of the enzyme (K m app ) because they raise the concentration of substrate necessary to  saturate the enzyme. They have no effect on V max .   If an inhibitor does not compete with a substrate for its binding site, the inhibitor is  either a noncompetitive or uncompetitive inhibitor with respect to that particular  substrate (Fig. 9.5). To illustrate noncompetitive inhibition, consider a multisub-  strate reaction in which substrates A and B react to form a product. An inhibitor (NI)  that is a structural analog of substrate B would fit into substrate B 's binding site, but  the inhibitor would be a noncompetitive inhibitor with regard to the other substrate,  substrate A. An increase of A will not prevent the inhibitor from binding to substrate  B's binding site. The inhibitor, in effect, lowers the concentration of the active  enzyme and therefore changes the V max of the enzyme. If the inhibitor has  absolutely no effect on the binding of substrate A, it will not change the K m for A  (a pure noncompetitive inhibitor).   Some inhibitors, such as metals, might not bind at either substrate recogni-  tion site. In this case, the inhibitor would be noncompetitive with respect to both  substrates.   An inhibitor that is uncompetitive with respect to a substrate will bind only to  enzyme containing that substrate. Suppose, for example, that in Figure 9.5 an  inhibitor that is a structural analog of B and binds to the B site could only bind to  an enzyme that contains A. That inhibitor would be called uncompetitive with  respect to A. It would decrease both the V max of the enzyme and its apparent K m  for A.   All products are reversible inhibitors of the enzymes that produce them and may be  competitive, noncompetitive, or uncompetitive relative to a particular substrate.  Simple product inhibition, a decrease in the rate of an enzyme caused by the accu-  mulation of its own product, plays an important role in metabolic pathways; it pre-  vents one enzyme in a sequence of reactions from generating a product faster than  it can be used by the next enzyme in that sequence.   Product inhibition of hexokinase by  glucose 6-phosphate conserves  blood glucose for tissues needing  it. Tissues take up glucose from the blood  and phosphorylate it to glucose 6-phos-  phate, which can then enter a number of dif-  ferent pathways (including glycolysis and  glycogen synthesis). As these pathways  become more active, glucose 6-phosphate  concentration decreases, and the rate of  hexokinase increases. When these pathways  are less active, glucose 6-phosphate concen-  tration increases, hexokinase is inhibited,  and glucose remains in the blood for other  tissues.   Fig. 9.5. NI is a noncompetitive inhibitor with respect to substrate A. A can still bind to its  binding site in the presence of NI. However, NI is competitive with respect to B because it  binds to the B binding site. In contrast, an inhibitor that is uncompetitive with respect to A  might also resemble B, but it could only bind to the B site after A is bound.   In substrate response and product inhibition, the rate of the enzyme is affected  principally by the binding of a substrate or a product within the catalytic site.  Most rate-limiting enzymes are also controlled through regulatory mechanisms  that change the conformation of the enzyme in a way that affects the catalytic  site. These regulatory mechanisms include: (1) allosteric activation and inhibi-  tion; (2) phosphorylation or other covalent modification; (3) protein-protein  interactions between regulatory and catalytic subunits, or between two proteins;  and (4) proteolytic cleavage. These types of regulation can rapidly change an  enzyme from an inactive form to a fully active conformation.   In the sections below, we describe the general characteristics of these regulatory  mechanisms and illustrate the first three with glycogen phosphorylase, glycogen  phosphorylase kinase, and protein kinase A.   Allosteric activators and inhibitors (allosteric effectors) are compounds that bind to  the allosteric site (a site separate from the catalytic site) and cause a conformational  change that affects the affinity of the enzyme for the substrate. Usually an allosteric  enzyme has multiple interacting subunits that can exist in active and inactive con-  formations, and the allosteric effector promotes or hinders conversion from one  conformation to another.   Allosteric enzymes usually contain two or more subunits and exhibit positive coop-  erativity; the binding of substrate to one subunit facilitates the binding of substrate  to another subunit (Fig. 9.6). The first substrate molecule has difficulty in binding  to the enzyme because all of the subunits are in the conformation with a low affin-  ity for substrate (the taut "T" conformation) (see Chapter 7, section VII.B.). The  first substrate molecule to bind changes its own subunit and at least one adjacent  subunit to the high-affinity conformation (the relaxed "R" state.) In the example of  the tetramer hemoglobin, discussed in Chapter 7, the change in one subunit facili-  tated changes in all four subunits, and the molecule generally changed to the new  conformation in a concerted fashion. However, most allosteric enzymes follow a  more stepwise (sequential) progression through intermediate stages (see Fig. 9.6)   Allosteric enzymes bind activators at the allosteric site, a site physically separate  from the catalytic site. The binding of an allosteric activator changes the conforma-  tion of the catalytic site in a way that increases the affinity of the enzyme for the  substrate.   In general, activators of allosteric enzymes bind more tightly to the high-affinity  R state of the enzyme than the T state (i.e., the allosteric site is open only in the R  enzyme) (Fig. 9.7). Thus, the activators increase the amount of enzyme in the active  state, thereby facilitating substrate binding in their own and other subunits. In contrast,  allosteric inhibitors bind more tightly to the T state, so either substrate concentra-  tion or activator concentration must be increased to overcome the effects of the  allosteric inhibitor. Allosteric inhibitors might have their own binding site on the  enzyme, or they might compete with the substrate at the active site and prevent  cooperativity. Thus, the term "allosteric inhibitor" is more generally applied to any  inhibitor of an allosteric enzyme.    Fig. 9.6. A sequential model for an allosteric  enzyme. The sequential model is actually the  preferred path from the T 0 (taut, with 0 sub-  strate bound) low-affinity conformation to the  R 4 (relaxed, with four substrate molecules  bound) conformation, taken from an array of all  possible equilibrium conformations that differ  by the conformation of only one subunit. The  final result is a stepwise path in which interme-  diate conformations exist, and subunits may  change conformations independently, depend-  ing on their geometric relationship to the sub-  units already containing bound substrate.   Fig. 9.7. Activators and inhibitors of an allosteric enzyme (simplified model). This enzyme  has two identical subunits, each containing three binding sites: one for the substrate (s), one  for the allosteric activator (blue triangle), and one for the allosteric inhibitor (two-pronged  shape). The enzyme has two conformations, a relaxed active conformation (R) and an inac-  tive conformation (T). The activator binds only to its activator site when the enzyme is in the  R configuration. The inhibitor binding site is open only when the enzyme is in the T state.   A plot of velocity (v/V^) versus substrate concentration reveals that binding of the sub-  strate at its binding site stabilizes the active conformation so that the second substrate binds  more readily, resulting in an S (sigmoidal)-shaped curve. The graph of v/V max becomes hyper-  bolic in the presence of activator (which stabilizes the high- affinity R form), and more sig-  moidal with a higher S 0 5 in the presence of inhibitor (which stabilizes the low-affinity form).   Some of the rate-limiting enzymes  \\ ) in the pathways of fuel oxidation  V^^y (e.g., muscle glycogen phosphory-  lase in glycogenosis, phosphofructokinase-  1 in glycolysis and isocitrate dehydrogenase  in the TCA cycle) are allosteric enzymes reg-  ulated by changes in the concentration of  ADP or AMP, which are allosteric activators.  The function of fuel oxidation pathways is  the generation of ATP. When the concentra-  tion of ATP in a muscle cell begins to  decrease, ADP and AMP increase; ADP acti-  vates isocitrate dehydrogenase, and AMP  activates glycogen phosphorylase and phos-  phofructokinase-1. The response is very fast,  and small changes in the concentration of  activator can cause large changes in the rate  of the reaction.   In the absence of activator, a plot of velocity versus substrate concentration for  an allosteric enzyme usually results in a sigmoid or S -shaped curve (rather than the  rectangular hyperbola of Michaelis-Menten enzymes) as the successive binding of  substrate molecules activates additional subunits (see Fig. 9.7). In plots of velocity  versus substrate concentration, the effect of an allosteric activator generally makes  the sigmoidal S-shaped curve more like the rectangular hyperbola, with a substan-  tial decrease in the S 0 5 (K m ) of the enzyme, because the activator changes all of the  subunits to the high-affinity state. Such allosteric effectors are "K effectors"; they  change the K m but not the V max of the enzyme. An allosteric inhibitor makes it more  difficult for substrate or activators to convert the subunits to the most active confor-  mation, and therefore inhibitors generally shift the curve to the right, either increas-  ing the S 0 5 alone, or increasing it together with a decrease in the V max .   Regulation of enzymes by allosteric effectors provides several advantages over  other methods of regulation. Allosteric inhibitors usually have a much stronger   effect on enzyme velocity than competitive, noncompetitive, and uncompetitive  inhibitors in the active catalytic site. Because allosteric effectors do not occupy the  catalytic site, they may function as activators. Thus, allosteric enzymes are not lim-  ited to regulation through inhibition. Furthermore, the allosteric effector need not  bear any resemblance to substrate or product of the enzyme. Finally, the effect of an  allosteric effector is rapid, occurring as soon as its concentration changes in the cell.  These features of allosteric enzymes are often essential for feedback regulation of  metabolic pathways by endproducts of the pathway or by signal molecules that  coordinate multiple pathways.   The activity of many enzymes is regulated through phosphorylation by a protein  kinase or dephosphorylation by a protein phosphatase (Fig. 9.8). Serine/threonine  protein kinases transfer a phosphate from ATP to the hydroxyl group of a specific  serine (and sometimes threonine) on the target enzyme; tyrosine kinases transfer a  phosphate to the hydroxyl group of a specific tyrosine residue. Phosphate is a bulky,  negatively charged residue that interacts with other nearby amino acid residues of  the protein to create a conformational change at the catalytic site. The conforma-  tional change makes certain enzymes more active and other enzymes less active.  The effect is reversed by a specific protein phosphatase that removes the phosphate  by hydrolysis.   Muscle glycogen phosphorylase, the rate-limiting enzyme in the pathway of glyco-  gen degradation, degrades glycogen to glucose 1 -phosphate. It is regulated by the  allosteric activator AMP, which increases in the cell as ATP is used for muscular  contraction (Fig. 9.9). Thus, a rapid increase in the rate of glycogen degradation to  glucose 1 -phosphate is achieved when an increase of AMP signals that more fuel is  needed for ATP generation in the glycolytic pathway.   Glycogen phosphorylase also can be activated through phosphorylation by glyco-  gen phosphorylase kinase. Either phosphorylation or AMP binding can change the  enzyme to the same fully active conformation. The phosphate is removed by protein    When Ann O'Rexia begins to jog,  AMP activates her muscle glycogen  phosphorylase, which degrades  glycogen to glucose 1-phosphate. This com-  pound is converted to glucose 6-phosphate,  which feeds into the glycolytic pathway to  generate ATP for muscle contraction. As she  continues to jog, her adrenaline (epinephrine)  levels rise, producing the signal that activates  glycogen phosphorylase kinase. This enzyme  phosphorylates glycogen phosphorylase,  causing it to become even more active than  with AMP alone (see Fig. 9.9).   Fig. 9.9. Activation of muscle glycogen phosphorylase by AMP and by phosphorylation. Muscle glycogen phosphorylase is composed of two  identical subunits. The substrate binding sites in the active catalytic site are denoted by S. AMP binds to the allosteric site, a site separate from  the active catalytic site. Glycogen phosphorylase kinase can transfer a phosphate from ATP to one serine residue in each subunit. Either phos-  phorylation or binding of AMP causes a change in the active site that increases the activity of the enzyme. The first event at one subunit facili-  tates the subsequent events that convert the enzyme to the fully active form.   Fig. 9.10. Structure of cAMP (3 ',5' -cyclic  AMR) The phosphate group is attached to  hydroxyl groups on both the 3rd (3') and 5th  (5') carbons of ribose, forming a cycle.   Fig. 9.11. Protein kinase A. When the regula-  tory subunits (R) of protein kinase A bind the  allosteric activator, cAMP, they dissociate  from the enzyme, thereby releasing active cat-  alytic subunits (C).   phosphatase- 1. Glycogen phosphorylase kinase links the activation of muscle glyco-  gen phosphorylase to changes in the level of the hormone adrenaline in the blood. It is  regulated through phosphorylation by protein kinase A and by activation of Ca 2+ -  calmodulin (a modulator protein) during contraction.   Some protein kinases, called dedicated protein kinases, are tightly bound to a sin-  gle protein and regulate only the protein to which they are tightly bound. However,  other protein kinases and protein phosphatases will simultaneously regulate a num-  ber of rate-limiting enzymes in a cell to achieve a coordinated response. For exam-  ple, protein kinase A, a serine/threonine protein kinase, phosphorylates a number of  enzymes that regulate different metabolic pathways. One of these enzymes is glyco-  gen phosphorylase kinase (see Fig. 9.9).   Protein kinase A provides a means for hormones to control metabolic pathways.  Adrenaline and many other hormones increase the intracellular concentration of the  allosteric regulator 3', 5' -cyclic AMP (cAMP), which is referred to as a hormonal  second messenger (Fig. 9.10). cAMP binds to regulatory subunits of protein kinase  A, which dissociate and release the activated catalytic subunits (Fig. 9.11). Disso-  ciation of inhibitory regulatory subunits is a common theme in enzyme regulation.  The active catalytic subunits phosphorylate glycogen phosphorylase and other  enzymes at serine residues.   In the example shown in Figure 9.9, adrenaline indirectly increases cAMP, which  activates protein kinase A, which phosphorylates phosphorylase kinase, which  phosphorylates glycogen phosphorylase. The sequence of events in which one  kinase phosphorylates another kinase is called a phosphorylation cascade. Because  each stage of the phosphorylation cascade is associated with one enzyme molecule  activating many enzyme molecules, the initial activating event is greatly amplified.   A number of proteins are covalently modified by the addition of groups such as acetyl,  ADP-ribose, or lipid moieties (see Chapter 6). These modifications may directly acti-  vate or inhibit the enzyme. However, they also may modify the ability of the enzyme  to interact with other proteins or to reach its correct location in the cell.   Changes in the conformation of the active site also can be regulated by direct  protein-protein interaction. This type of regulation is illustrated by Ca 2+ -calmodulin  and small (monomeric) G proteins.   Modulator proteins bind to other proteins and regulate their activity by causing a  conformational change at the catalytic site or by blocking the catalytic site (steric  hindrance). They are protein allosteric effectors that can either activate or inhibit the  enzyme or protein to which they bind.   Ca 2+ -calmodulin is an example of a dissociable modulator protein that binds to  a number of different proteins and regulates their function. It also exists in the  cytosol and functions as a Ca 2+ binding protein (Fig. 9.12). The center of the sym-  metric molecule is a hinge region that bends as Ca 2+ -calmodulin folds over the  protein it is regulating.   One of the enzymes activated by Ca 2+ -calmodulin is muscle glycogen phospho-  rylase kinase, which is also activated by protein kinase A (see Fig. 9.9). When a    Fig. 9.12. Calcium-calmodulin has four binding sites for calcium (shown in blue). Each cal-  cium forms a multiligand coordination sphere by simultaneously binding several amino acid  residues on calmodulin. Thus, it can create large conformational changes in proteins when it  binds. Calmodulin has a flexible region in the middle connecting the two domains.   neural impulse triggers Ca 2+ release from the sarcoplasmic reticulum, Ca 2+ binds  to the calmodulin subunit of muscle glycogen phosphorylase kinase, which under-  goes a conformational change. This activated kinase then phosphorylates glycogen  phosphorylase, ultimately increasing the generation of ATP to supply energy for  muscle contraction. Simultaneously, Ca 2+ binds to troponin-C, a member of the  Ca 2+ -calmodulin superfamily that serves as a nondis sociable regulatory subunit of  troponin, a regulator of muscle contraction. Calcium binding to troponin prepares  the muscle for contraction. Thus, the supply of energy for contraction is activated  simultaneously with the contraction machinery.   The masters of regulation through reversible protein association in the cell are the  monomeric G proteins, small single- subunit proteins that bind and hydrolyze GTP.  GTP (guanosine triphosphate) is a purine nucleotide that, like ATP, contains high-energy  phosphoanhydride bonds that release energy when hydrolyzed. When G proteins bind  GTP, their conformation changes so that they can bind to a target protein, which is  then either activated or inhibited in carrying out its function (Fig. 9.13, step 1).   G proteins are said to possess an internal clock because they are GTPases that  slowly hydrolyze their own bound GTP to GDP and phosphate. As they hydrolyze  GTP, their conformation changes and the complex they have formed with the target  protein disassembles (see Fig. 9.13, step 2). The bound GDP on the inactive G protein  is eventually replaced by GTP, and the process can begin again (see Fig. 9.13, step 3).   The activity of many G proteins is regulated by accessory proteins (GAPs, GEFs,  and GDIs), which may, in turn, be regulated by allosteric effectors. GAPs (GTPase  activating proteins) increase the rate of GTP hydrolysis by the G protein, and there-  fore the rate of dissociation of the G protein-target protein complex (see Fig. 9.13,   Fig. 9.13. Monomeric G proteins. (1) When  GTP is bound, the conformation of the G pro-  tein allows it to bind target proteins, which are  then activated. (2) The G protein hydrolyzes a  phosphate from GTP to form GDP, which  changes the G-protein conformation and  causes it to dissociate from the target protein.  (3) GDP is exchanged for GTP, which reacti-  vates the G protein.   step 2). When a GEF protein (guanine nucleotide exchange factor) binds to a  G-protein, it increases the rate of GTP exchange for a bound GDP, and therefore  activates the G-protein (see Fig. 9.13, step 3). GDI proteins (GDP dissociation  inhibitor) bind to the GDP-G protein complex and inhibit dissociation of GDP,  thereby keeping the G protein inactive.   The Ras superfamily of small G proteins is divided into five families: Ras, Rho,  Arf, Rab, and Ran. These monomeric G proteins play major roles in the regulation  of growth, morphogenesis, cell motility, axonal guidance, cytokinesis, and traffick-  ing through the Golgi, nucleus, and endosomes. They are generally bound to a lipid  membrane through a lipid anchor, such as a myristoyl group or farnesyl group, and  regulate the assembly and activity of protein complexes at these sites. The small G  protein Ras, for example, is involved in regulation of cellular proliferation by a  number of hormones called growth factors (Fig. 9.14). It is attached to the plasma  membrane by a farnesyl group (see Chapter 6, section IV.B.). The activity of Ras is  regulated by a guanine nucleotide exchange protein called SOS (son of sevenless).  When SOS is in its active conformation, it binds to Ras, thereby activating dissoci-  ation of GDP and binding of GTP. When Ras binds GTP, it is activated, allowing it  to bind and activate a protein kinase called Raf . The net effect will be the activation  of transcription of certain genes. (Rho, Arf, Rab, and Ran are illustrated in Chapter  10, and the function of Ras is discussed in greater detail in Chapter 11).   Most of the proteases involved in  \\ ) blood clotting are zymogens, such  V^^x as fibrinogen and prothrombin,  which circulate in blood in the inactive form.  They are cleaved to the active form (fibrin  and thrombin, respectively) by other pro-  teases, which have been activated by their  attachment to the site of injury in a blood  vessel wall. Thus, clots form at the site of  injury and not randomly in circulation.   Although many enzymes undergo some cleavage during synthesis, others enter  lysosomes, secretory vesicles or are secreted as proenzymes, which are precursor  proteins that must undergo proteolytic cleavage to become fully functional. Unlike  most other forms of regulation, proteolytic cleavage is irreversible.   The precursor proteins of proteases (enzymes that cleave specific peptide bonds)  are called zymogens. To denote the inactive zymogen form of an enzyme, the name  is modified by addition of the suffix "ogen" or the prefix "pro." The synthesis of  zymogens as inactive precursors prevents them from cleaving proteins prematurely  at their sites of synthesis or secretion. Chymotrypsinogen, for example, is stored in  vesicles within pancreatic cells until secreted into ducts leading to the intestinal  lumen. In the digestive tract, chymotrypsinogen is converted to chymotrypsin by the  proteolytic enzyme trypsin, which cleaves off a small peptide from the N-terminal  region (and two internal peptides). This cleavage activates chymotrypsin by causing    Fig. 9.14. The monomeric G protein Ras. When SOS is activated, it binds to Ras, a  monomeric G protein anchored to the plasma membrane. SOS is a guanine nucleotide  exchange protein that activates the exchange of GTP for bound GDP on Ras. Activated Ras  containing GTP binds the target enzyme Raf, thereby activating it.   a conformational change in the spacing of amino acid residues around the binding  site for the denatured protein substrate and around the catalytic site.   Tissues continuously adjust the rate at which different proteins are synthesized to  vary the amount of different enzymes present. The expression for V max in the  Michaelis-Menten equation incorporates the concept that the rate of a reaction is pro-  portional to the amount of enzyme present. Thus, the maximal capacity of a tissue  can change with increased protein synthesis, or with increased protein degradation.   Protein synthesis begins with the process of gene transcription, transcribing the  genetic code for that protein from DNA into messenger RNA. The code in messen-  ger RNA is then translated into the primary amino acid sequence of the protein.  Generally the rate of enzyme synthesis is regulated by increasing or decreasing the  rate of gene transcription, processes generally referred to as induction (increase)  and repression (decrease). However, the rate of enzyme synthesis is sometimes reg-  ulated through stabilization of the messenger RNA. (These processes are covered in  Section Three). Compared with the more immediate types of regulation discussed  above, regulation by means of induction/repression of enzyme synthesis is usually  slow in the human, occurring over hours to days.   The content of an enzyme in the cell can be altered through selective regulated  degradation as well as through regulated synthesis. Although all proteins in the cell  can be degraded with a characteristic half-life within lysosomes, protein degrada-  tion via two specialized systems, proteosomes and caspases, is highly selective and  regulated. Protein degradation is dealt with in more detail in Chapter 37.   The different means of regulating enzyme activity described above are used to con-  trol metabolic pathways, cellular events, and physiologic processes to match the  body's requirements. Although many metabolic pathways are present in the body, a  few common themes or principles are involved in their regulation. Of course, the  overriding principle is: Regulation of a pathway matches its function.   Metabolic pathways are a series of sequential reactions in which the product of one  reaction is the substrate of the next reaction (Fig. 9.15). Each step or reaction is usu-  ally catalyzed by a separate enzyme. The enzymes of a pathway have a common  function — conversion of substrate to the final endproducts of the pathway. A path-  way also may have a branchpoint at which an intermediate becomes the precursor  for another pathway.    The maximal capacity of MEOS  (cytochrome P450-2E1) is increased  in the liver with continued ingestion  of ethanol through a mechanism involving  induction of gene transcription. Thus, Al Mar-  tini has a higher capacity to oxidize ethanol  to acetaldehyde than a naive drinker (a per-  son not previously subjected to alcohol).  Nevertheless, the persistance of his elevated  blood alcohol level shows he has saturated  his capacity for ethanol oxidation (V-maxed  out). Once his enzymes are operating near  V max , any additional ethanol he drinks will not  appreciably increase the rate of ethanol  clearance from his blood.   During fasting or infective stress,  protein degradation in skeletal  muscle is activated to increase the  supply of amino acids in the blood for glu-  coneogenesis, or for the synthesis of anti-  bodies and other component of the immune  response. Under these conditions, synthesis  of ubiquitin, a protein that targets proteins  for degradation in proteosomes, is  increased by the steroid hormone Cortisol.   Pathways are principally regulated at one key enzyme, the regulatory enzyme,  which catalyzes the rate-limiting step in the pathway. This is the slowest step and is  usually not readily reversible. Thus, changes in the rate-limiting step can influence  flux through the rest of the pathway (see Fig. 9.1). The rate-limiting step is usually  the first committed step in a pathway, or a reaction that is related to, or influenced   Fig. 9.15. A common pattern for feedback inhibition of metabolic pathways. The letters rep-  resent compounds formed from different enzymes in the reaction pathway. Compound B is  at a metabolic branchpoint: it can go down one pathway to E or down an alternate pathway  to G. The endproduct of the pathway, E, might control its own synthesis by allosterically  inhibiting enzyme 2, the first committed step of the pathway, or inhibiting transcription of the  gene for enzyme 2. As a result of the feedback inhibition, B accumulates and more B enters  the pathway for conversion to G, which could be a storage, or disposal pathway. In this hypo-  thetical pathway, B is a product inhibitor of enzyme 1, competitive with respect to A. Pre-  cursor A might induce the synthesis of enzyme 1, which would allow more A to go to G.   The pathways of energy produc-  tion must be regulated by a mech-  anism that can respond rapidly to  requirements for more ATP, such as the  allosteric regulation of glycogen phosphory-  lase by AMP. However, storage pathways or  biosynthetic pathways can be regulated by a  mechanism that responds more slowly to  changing conditions. For example, choles-  terol partially feedback regulates its own  rate of synthesis by decreasing transcription  of the gene for the rate-limiting enzyme  (HMG-CoA reductase). The enzyme concen-  tration of a tissue may change even more  slowly in response to developmental  changes.   by, the first committed step. Additional regulated enzymes occur after each meta-  bolic branchpoint to direct flow into the branch, (e.g., in Fig. 9.15, feedback inhibi-  tion of enzyme 2 results in accumulation of B, which enzyme 5 then uses for syn-  thesis of compound G). Inhibition of the rate-limiting enzyme in a pathway usually  leads to accumulation of the pathway precursor.   Feedback regulation refers to a situation in which the endproduct of a pathway  controls its own rate of synthesis (see Fig. 9.15). Feedback regulation usually  involves allosteric regulation of the rate-limiting enzyme by the endproduct of a  pathway (or a compound that reflects changes in the concentration of the endprod-  uct). The endproduct of a pathway may also control its own synthesis by inducing  or repressing the gene for transcription of the rate-limiting enzyme in the pathway.  This type of regulation is much slower to respond to changing conditions than  allosteric regulation.   When Ann O'Rexia jogs, the  increased use of ATP for muscle  contraction results in an increase  of AMP, which allosterically activates both  the allosteric enzyme phosphofructokinase-  1, the rate-limiting enzyme of glycolysis, and  glycogen phosphorylase, the rate-limiting  enzyme of glycogenolysis. This is an exam-  ple of feedback regulation by the ATP/AMP  ratio. Unfortunately, her low caloric con-  sumption has not allowed feed-forward acti-  vation of the rate-limiting enzymes in her fuel  storage pathways, and she has very low glyco-  gen stores. Consequently, she has inadequate  fuel stores to supply the increased energy  demands of exercise.   Certain pathways, such as those involved in the disposal of toxic compounds, are  feed-forward regulated. Feed-forward regulation may occur through an increased  supply of substrate to an enzyme with a high K m , allosteric activation of a rate-  limiting enzyme through a compound related to substrate supply, substrate-related  induction of gene transcription (e.g., induction of cytochrome P450-2E1 by  ethanol), or increased concentration of a hormone that stimulates a storage pathway  by controlling enzyme phosphorylation state.   The human body is composed of a number of different cell types that perform  specific functions unique to that cell type and synthesize only the proteins con-  sistent with their functions. Because regulation matches function, regulatory  enzymes of pathways usually exist as tissue-specific isozymes with somewhat  different regulatory properties unique to their function in different cell types. For  example, hexokinase and glucokinase are tissue- specific isozymes with different  kinetic properties.   A pathway for the synthesis of a compound usually has one or more enzymatic  steps that differ from the pathway for degradation of that compound. A biosyn-  thetic pathway can therefore have a different regulatory enzyme than the oppos-  ing degradative pathway, and one pathway can be activated while the other is  inhibited (e.g., glycogen synthesis is activated while glycogen degradation is  inhibited).   In the cell, compartmentation of enzymes into multienzyme complexes or  organelles provides a means of regulation, either because the compartment provides  unique conditions or because it limits or channels access of the enzymes to sub-  strates. Enzymes or pathways with a common function are often assembled into  organelles. For example, enzymes of the TCA cycle are all located within the mito-  chondrion. The enzymes catalyze sequential reactions, and the product of one reac-  tion is the substrate for the next reaction. The concentration of the pathway inter-  mediates remains much higher within the mitochondrion than in the surrounding  cellular cytoplasm.   Another type of compartmentation involves the assembly of enzymes catalyzing  sequential reactions into multi-enzyme complexes so that intermediates of the path-  way can be directly transferred from the active site on one enzyme to the active site  on another enzyme, thereby preventing loss of energy and information.   You may have noticed by now that regulation of metabolic pathways in the human  is exceedingly complex; this might be called the second principle of metabolic reg-  ulation. As you study different pathways in the subsequent chapters of the text, it  may help to develop diagrams such as Fig. 9.15 to keep track of the function and  rationale behind different regulatory interactions.   The different isozymes of hexoki-  \\ nase (e.g., hexokinase I and glu-  VJ^/ cokinase) are tissue-specific  isozymes that arose through gene duplica-  tion. Glucokinase, the low-affinity enzyme  found in liver, is a single polypeptide chain  with a molecular weight of 55 kDa that con-  tains one active catalytic site. The hexoki-  nases found in erythrocytes, skeletal mus-  cles, and most other tissues are 110 kDa and  are essentially two mutated glucokinase  molecules synthesized as one polypeptide  chain. However, only one catalytic site is  functional. All of the tissue-specific hexoki-  nases but glucokinase have K m s for glucose  that are less than 0.2 mM.   An example of a multi-enzyme  complex is provided by MEOS  (microsomal ethanol oxidizing sys-  tem), which is composed of two different  subunits with different enzyme activities.  One subunit transfers electrons from  NADPH to a cytochrome Fe-heme group on  the 2nd subunit, which then transfers the  electrons to 0 2 .   ^ A I Martini. In the Emergency Room, Al Martini was evaluated for  head injuries. From the physical examination and blood alcohol levels,  it was determined that his mental state resulted from his alcohol con-  sumption. Although his chronic ethanol consumption had increased his level of  MEOS (and, therefore, rate of ethanol oxidation in his liver), his excessive drink-  ing resulted in a blood alcohol level greater than the legal limit of 80 mg/dL. He  suffered bruises and contusions but was otherwise uninjured. He left in the cus-  tody of the police officer.   ^ Ann O'Rexia. Ann O'Rexia's physician explained that she had inad-  equate fuel stores for her exercise program. To jog, her muscles require  an increased rate of fuel oxidation to generate the ATP for muscle con-  traction. The fuels used by muscles for exercise include glucose from muscle  glycogen, fatty acids from adipose tissue triacylglycerols, and blood glucose  supplied by liver glycogen. These fuel stores were depleted during her prolonged  bout of starvation. In addition, starvation resulted in the loss of muscle mass as  muscle protein was being degraded to supply amino acids for other processes,  including gluconeogenesis (the synthesis of glucose from amino acids and other  noncarbohydrate precursors). Therefore, Ann will need to increase her caloric  consumption to rebuild her fuel stores. Her physician helped her calculate the    The hormone epinephrine  (released during stress and exer-  cise) and glucagon (released during  fasting) activate the synthesis of cAMP in a  number of tissues. cAMP activates protein  kinase A. Because protein kinase A is able to  phosphorylate key regulatory enzymes in  many pathways, these pathways can be co-  ordinately regulated. In muscle, for example,  glycogen degradation is activated while  glycogen synthesis is inhibited. At the same  time, fatty acid release from adipose tissue is  activated to provide more fuel for muscle.  The regulation of glycolysis, glycogen metab-  olism, and other pathways of metabolism is  much more complex than we have illustrated  here and is discussed in many subsequent  chapters of this text.   additional amount of calories her jogging program will need, and they discussed  which foods she would eat to meet these increased caloric requirements. He also  helped her visualize the increase of weight as an increase in strength.    Fig. 9.16. The Lineweaver-Burk transforma-  tion (shown in blue) for the Michaelis-Menten  equation converts it to a straight line of the  form y = mx + b. When [S] is infinite, 1/[S] =  0, and the line crosses the ordinate (y-axis) at  1/v = 1/Vmax- The slope of the line is K m /V max .  Where the line intersects the abscissa (x-axis),  1/[S] = -1/K m .   ft The Lineweaver-Burk transformation. The K m and V max for an   enzyme can be visually determined from a plot of versus 1/S, called a  ^ Lineweaver-Burk or a double reciprocal plot. The reciprocal of both sides  of the Michaelis-Menten equation generates an equation that has the form of a  straight line, y = mx + b (Fig. 9.16). K m and V max are equal to the reciprocals of the  intercepts on the abscissa and ordinate, respectively. Although double reciprocal  plots are often used to illustrate certain features of enzyme reactions, they are not  directly used for the determination of K m and V max values by researchers.   For the reaction in which an enzyme forms a complex with both substrates, the  K m for one substrate can vary with the concentration of cosubstrate (Fig. 9.17). At  each constant concentration of cosubstrate, the plot of l/v { vs 1/[S] is a straight line.  To obtain V max , the graph must be extrapolated to saturating concentrations of both  substrates, which is equivalent to the intersection point of these lines for different  cosubstrate concentrations.   Lineweaver-Burk plots provide a good illustration of competitive inhibition and  pure noncompetitive inhibition (Fig. 9.18). In competitive inhibition, plots of 1/v vs  1/[S] at a series of inhibitor concentrations intersect on the ordinate. Thus, at infi-  nite substrate concentration, or 1/[S] = 0, there is no effect of the inhibitor. In pure  noncompetitive inhibition, the inhibitor decreases the velocity even when [S] has  been extrapolated to an infinite concentration. However, if the inhibitor has no  effect on the binding of the substrate, the K m is the same for every concentration of  inhibitor, and the lines intersect on the abcissa.   Fig. 9.17. A Lineweaver-Burk plot for a two-substrate reaction in which A and B are con-  verted to products. In the graph, 1/[A] is plotted against 1/v for three different concentra-  tions of the cosubstrate, [B], 2[B], and 3[B]. As the concentration of B is increased, the inter-  section on the abscissa, equal to 1/K m app is increased. The "app" represents "apparent", as the  K m app is the K m at whatever concentration of cosubstrate, inhibitor, or other factor is present  during the experiment.    Fig. 9.18. Lineweaver-Burk plots of competitive and pure noncompetitive inhibition. A. 1/Vj versus 1/[S] in the presence of a competitive inhibitor.  The competitive inhibitor alters the intersection on the abscissa. The new intersection is 1/K m app (also called 1/K' m ). A competitive inhibitor does not  affect V max . B. 1/Vj versus 1/[S] in the presence of a pure noncompetitive inhibitor. The noncompetitive inhibitor alters the intersection on the ordinate,  1/V m ax,app or Wmax> but does not affect 1/K m . A pure noncompetitive inhibitor binds to E and ES with the same affinity. If the inhibitor has different  affinities for E and ES, the lines will intersect above or below the abscissa, and the noncompetitive inhibitor will change both the K' m and the V' m .   1. Which of the following describes a characteristic feature of an enzyme obeying Michaelis-Menten kinetics?   (A) The enzyme velocity is at V2 the maximal rate when 100% of the enzyme molecules contain bound substrate.   (B) The enzyme velocity is at Vi the maximal rate when 50% of the enzyme molecules contain bound substrate.   (C) The enzyme velocity is at its maximal rate when 50% of the enzyme molecules contain bound substrate.   (D) The enzyme velocity is at its maximal rate when all of the substrate molecules in solution are bound by the enzyme.   (E) The velocity of the reaction is independent of the concentration of enzyme.   2. The pancreatic glucokinase of a patient with MODY had a mutation replacing a leucine with a proline. The result was that the  K m for glucose was decreased from a normal value of 6 mM to a value of 2.2 mM, and the V max was changed from 93 units/mg  protein to 0.2 units/mg protein. Which of the following best describes the patient's glucokinase compared with the normal  enzyme?   (A) The patient's enzyme requires a lower concentration of glucose to reach l h V max .   (B) The patient's enzyme is faster than the normal enzyme at concentrations of glucose below 2.2 mM.   (C) The patient's enzyme is faster than the normal enzyme at concentrations of glucose above 2.2 mM.   (D) At near saturating glucose concentration, the patient would need 90 to 100 times more enzyme than normal to achieve  normal rates of glucose phosphorylation.   (E) As blood glucose levels increase after a meal from a fasting value of 5 mM to 10 mM, the rate of the patient's enzyme  will increase more than the rate of the normal enzyme.   3. Methanol (CH 3 OH) is converted by alcohol dehydrogenases to formaldehyde (CHO), a compound that is highly toxic in the  human. Patients who have ingested toxic levels of methanol are sometimes treated with ethanol (CH 3 CH 2 OH) to inhibit methanol  oxidation by alcohol dehydrogenase. Which of the following statements provides the best rationale for this treatment?   (A) Ethanol is a structural analog of methanol, and might therefore be an effective noncompetitive inhibitor.   (B) Ethanol is a structural analog of methanol that would be expected to compete with methanol for its binding site on the  enzyme.   (C) Ethanol would be expected to alter the V max of alcohol dehydrogenase for the oxidation of methanol to formaldehyde.   (D) Ethanol would be an effective inhibitor of methanol oxidation regardless of the concentration of methanol.   (E) Ethanol would be expected to inhibit the enzyme by binding to the formaldehyde binding site on the enzyme, even  though it cannot bind at the substrate binding site for methanol.   4. Which of the following describes a characteristic of most allosteric enzymes?   (B) In the absence of effectors, they generally follow Michaelis-Menten kinetics.   (D) They have allosteric activators that bind in the catalytic site.   (E) They have irreversible allosteric inhibitors that bind at allosteric sites.   5. A rate-limiting enzyme catalyzes the first step in the conversion of a toxic metabolite to a urinary excretion product. Which  of the following mechanisms for regulating this enzyme would provide the most protection to the body?   (A) The product of the pathway should be an allosteric inhibitor of the rate-limiting enzyme.   (B) The product of the pathway should act through gene transcription to decrease synthesis of the enzyme.   (C) The toxin should act through gene transcription to increase synthesis of the enzyme.   (D) The enzyme should have a high K m for the toxin.   (E) The product of the first enzyme should allosterically activate the subsequent enzyme in the pathway.   The basic unit of a living organism is the cell. In the human, each tissue is com-  posed of similar cell types, which differ from those in other tissues. The diversity  of cell types serves the function of the tissue and organs in which they reside, and  each cell type has unique structural features that reflect its role. In spite of their  diversity in structure, human cell types have certain architectural features in com-  mon, such as the plasma membrane, membranes around the nucleus and  organelles, and a cytoskeleton (Fig. 10.1 ). In this chapter, we review some of the  chemical characteristics of these common features, the functions of organelles,  and the transport systems for compounds into cells and between organelles.   Plasma membrane. The cell membrane is a lipid bilayer that serves as a selec-  tive barrier; it restricts the entry and exit of compounds. Within the plasma mem-  brane, different integral proteins facilitate the transport of specific compounds by   The cells of humans and other ani-  mals are eukaryotes (eu, good;  karyon, nucleus) because the  genetic material is organized into a mem-  brane-enclosed nucleus. In contrast, bacteria  are prokaryotes (pro, before; karyon,  nucleus); they do not contain nuclei or other  organelles found in eukaryotic cells.   The cytoplasm of the cell is the  \\ ) portion of the cell between the cell  *r membrane and the nucleus. Mito-  chondria, lysososmes and peroxisomes are  referred to as cytoplasmic organelles. The  Golgi and the endoplasmic reticulum are  referred to as cytoplasmic membrane sys-  tems. The plasma membrane can be gently  disrupted by detergents or shear stress with-  out damage to the other membrane sys-  tems. When a suspension that has been  treated this way is centrifuged for a long  period of time (100,000g for 1 hour), the  organelles and membrane systems will col-  lect at the bottom of the tube. The remaining  clear liquid of soluble enzymes, cofactors,  and metabolites is the cytosol.   energy -requiring active transport, facilitated diffusion, or by forming pores or  gated- channels. The plasma membrane is supported by a membrane skeleton   Organelles and cytoplasmic membrane systems. Most organelles within the  cell are compartments surrounded by a membrane system that restricts exchange  of compounds and information with other compartments (see Fig. 10.1). In gen-  eral, each organelle has unique functions that are served by the enzymes and  other compounds it contains, or the environment it maintains. Lysosomes contain  hydrolytic enzymes that degrade proteins and other large molecules. The nucleus  contains the genetic material and carries out gene replication and transcription  ofDNA, the first step of protein synthesis. The last phase of protein synthesis  occurs on ribosomes. For certain proteins, the ribosomes become attached to the  complex membrane system called the endoplasmic reticulum; for other proteins,  synthesis is completed on ribosomes that remain in the cytoplasm. The endoplas-  mic reticulum is also involved in lipid synthesis and transport of molecules to the  Golgi. The Golgi forms vesicles for transport of molecules to the plasma mem-  brane and other membrane systems, and for secretion. Mitochondria are  organelles of fuel oxidation and ATP generation. Peroxisomes contain many  enzymes that use or produce hydrogen peroxide. The cytosol is the intracellular  compartment free of organelles and membrane systems.   Cytoskeleton. The cytoskeleton is a flexible fibrous protein support system that  maintains the geometry of the cell, fixes the position of organelles, and moves  compounds within the cell or the cell itself. It is composed principally of actin  microfilaments, intermediate filaments , tubulin microtubules, and their attached  proteins.   Al Martini had been drinking heavily when he drove his car off the road and  was taken to the hospital emergency room (see Chapters 8 and 9). Although  he suffered only minor injuries, his driving license was suspended.    V. cholerae epidemics are associ-  ated with unsanitary conditions  affecting the drinking water supply  and are rare in the United States. However,  these bacteria grow well under the alkaline  conditions found in seawater and attach to  chitin in shellfish. Thus, sporadic cases  occur in the southeast United States associ-  ated with the ingestion of contaminated  shellfish.    Two years after Dennis "the Menace" Veere successfully recovered from  his malathion poisoning, he visited his grandfather, Percy Veere. Mr. Veere  took Dennis with him to a picnic at the shore, where they ate steamed  crabs. Later that night, Dennis experienced episodes of vomiting and watery diar-  rhea, and Mr. Veere rushed him to the hospital emergency room. Dennis's hands and  feet were cold, he appeared severely dehydrated, and he was approaching hypov-  olemic shock (a severe drop in blood pressure). He was diagnosed with cholera,  caused by the bacteria Vibrio cholerae.   • Before Lotta Topaigne was treated with allopurinol (see Chapter 8), her  flP j physician administered colchicine (acetyltrimethylcolchicinic acid) for the   " acute attack of gout affecting her great toe. After taking a high dose of  colchicine divided over several-hour intervals, the throbbing pain in her toe had  abated significantly. The redness and swelling also seemed to have lessened slightly.   Cell lysis, the breaking of the cell  membrane and release of cell  contents, occurs when the conti-  nuity of the cell membrane is disrupted.   Membranes are lipid structures that separate the contents of the compartment they  surround from its environment. An outer plasma membrane separates the cell from the   external aqueous environment. Organelles (such as the nucleus, mitochondria, lyso-  somes, and peroxisosmes) are also surrounded by a membrane system that separates  the internal compartment of the organelle from the cytosol. The function of these  membranes is to collect or concentrate enzymes and other molecules serving a com-  mon function into a compartment with a localized environment. The transporters and  receptors in each membrane system control this localized environment and commu-  nication of the cell or organelle with the surrounding milieu.   The following sections describe various organelles and membrane systems  found in most human cells and outline the relationship between their properties  and function. Each organelle has different enzymes and carries out different gen-  eral functions. For example, the nucleus contains the enzymes for DNA and  RNA synthesis.   Not all cells in the human are alike. Different cell types differ quantitatively  in their organelle content, or their organelles may contain vastly different  amounts of a particular enzyme, consistent with the function of the cell. For  example, liver mitochondria contain a key enzyme for synthesizing ketone bod-  ies, but they lack a key enzyme for their use. The reverse is true in muscle mito-  chondria. Thus, the enzymic content of the organelles varies somewhat from cell  type to cell type.   All mammalian cells are enclosed by a plasma membrane composed of a lipid  bilayer (two layers) containing embedded proteins (Fig. 10.2). The membranes  are continuous and sealed so that the hydrophobic lipid bilayer selectively  restricts the exchange of polar compounds between the external fluid and the  intracellular compartment. The membrane is referred to as a fluid mosaic  because it consists of a mosaic of proteins and lipid molecules that can, for the  most part, move laterally in the plane of the membrane. The proteins are classi-  fied as integral proteins, which span the cell membrane, or peripheral proteins,  which are attached to the membrane surface through electrostatic bonds to lipids  or integral proteins. Many of the proteins and lipids on the external leaflet con-  tain covalently bound carbohydrate chains and therefore are glycoproteins and  glycolipids. This layer of carbohydrate on the outer surface of the cell is called  the glycocalyx.   Each layer of the plasma membrane lipid bilayer is formed primarily by phos-  pholipids, which are arranged with their hydrophilic head groups facing the aque-  ous medium and their fatty acyl tails forming a hydrophobic membrane core (see  Fig. 10.2). The principle phospholipids in the membrane are the glycerol lipids  phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine and the  sphingolipid sphingomyelin (Fig. 10.3). The lipid composition varies among dif-  ferent cell types, with phosphatidylcholine being the major plasma membrane lipid  in most cell types and sphingolipids the most variable.   The lipid composition of the bilayer is asymmetric, with a higher content of phos-  phatidylcholine and sphingomyelin in the outer leaflet and a higher content of phos-  phatidylserine and phosphatidylethanolamine in the inner leaflet. Phosphatidylserine  contains a net negative charge that contributes to the membrane potential and might be  important for binding positively charged molecules within the cell. Phosphatidylinosi-  tol, which is found only in the inner membrane, functions in the transfer of informa-  tion from hormones and neurotransmitters across the cell membrane into the cell  (Fig. 10.4).   Bacteria are single cells sur-  rounded by a cell membrane and a  cell wall exterior to the membrane.  They are prokaryotes, which do not contain  nuclei or other organelles (i.e. membrane-  surrounded subcellular structures) found in  eukaryotic cells. Nonetheless, bacteria carry  out many similar metabolic pathways, with  the enzymes located in either the intracellu-  lar compartment or the cell membrane.   The Vibrio cholerae responsible for Dennis  Veere's cholera are gram-negative bacteria.  Their plasma membrane is surrounded by a  thin cell wall composed of a protein-polysac-  charide structure called peptidoglycan and an  outer membrane. In contrast, gram-positive  bacteria have a plasma membrane and a thick  peptidoglycan cell wall that retains the Gram  stain. Vibrio grow best under aerobic condi-  tions, but also can grow under low oxygen  conditions. They possess enzymes similar to  those in human cells for glycolysis, the TCA  cycle, and oxidative phosphorylation. They  have a low tolerance for acid, which partially  accounts for their presence in slightly basic  seawater and shellfish.   The variable carbohydrate compo-  nents of the glycolipids on the cell  surface function as cell recognition  markers. For example, the A, B, or O blood  groups are determined by the carbohydrate  composition of the glycolipids. Cell surface  glycolipids may also serve as binding sites  for viruses and bacterial toxins before pene-  trating the cell. For example, the cholera AB  toxin binds to GM r gangliosides on the sur-  face of the intestinal epithelial cells. The  toxin is then endocytosed in caveolae  (invaginations or "caves" that can form in  specific regions of the membrane).   One of the bacterial toxins secreted  by Clostridium perefringens, the  bacteria that cause gas gangrene,  is a lipase that hydrolyzes phosphocholine  from phosphatidylcholine and from sphin-  gomyelin. The resulting lysis of the cell  membrane releases intracellular contents  that provide the bacteria with nutrients for  rapid growth. These bacteria are strict anaer-  obes and grow only in the absence of oxy-  gen. As their toxins lyse membranes in the  endothelial cells of blood vessels, the capil-  laries are destroyed, and the bacteria are  protected from oxygen transported by the  red blood cells. They are also protected from  antibiotics and components of the immune  system carried in the blood.    Fig. 10.3. Common phospholipids in the mammalian cell membrane. The polar head groups  shown for ethanolamine and serine replace the choline in phosphatidylcholine to form phos-  phatidylethanolamine and phosphatidylserine, respectively. Phosphatidylcholine, phos-  phatidylethanolamine, and phosphatidylserine are phosphoacylglycerols. In contrast, sphin-  gomyelin does not contain the glycerol backbone but has a sphingosine backbone and is a  sphingolipid.   Fig. 10.4. Phosphatidylinositol bisphosphate (PIP 2 ). Rl and R2 are fatty acyl chains. The  portion of PIP 2 that becomes inositol triphosphate, the polar head group extending into the  cytosol, is shown in blue.   Cholesterol, which is interspersed between the phospholipids, maintains  membrane fluidity. In the phosphoacylglycerols, unsaturated fatty acid chains bent  into the cis conformation form a pocket for cholesterol, which binds with its  hydroxyl group in the external hydrophilic region of the membrane and its  hydrophobic steroid nucleus in the hydrophobic membrane core (Fig. 10.5). The  presence of cholesterol and the cis unsaturated fatty acids in the membrane prevent  the hydrophobic chains from packing too closely together. As a consequence, lipid  and protein molecules that are not bound to external or internal structural proteins  can rotate and move laterally in the plane of the leaflet. This movement enables the  plasma membrane to partition between daughter cells during cell division, to    Al Martini is suffering from both  short-term and long-term effects of  ethanol on his central nervous sys-  tem. Data support the theory that the short-  term effects of ethanol on the brain partially  arise from an increase in membrane fluidity  caused when ethanol intercalates between  the membrane lipids. The changes in mem-  brane fluidity may affect proteins that span  the membrane (integral proteins), such as  ion channels and receptors for neurotrans-  mitters involved in conducting the nerve  impulse.   Fig. 10.5. Cholesterol in the plasma membrane. The polar hydroxyl group of cholesterol is  oriented toward the surface. The hydrocarbon tail and the steroid nucleus (blue) lie in the  hydrophobic core. A cis double bond in the fatty acyl chain of a phospholipid bends the chain  to create a hydrophobic binding site for cholesterol.   Two of the prominent integral pro-  teins in the red blood cell mem-  brane are glycophorin, which pro-  vides an external negative charge that repels  other cells, and band 3, which is a channel  for bicarbonate and chloride exchange. The  transport of bicarbonate into the red blood  cell in exchange for chloride helps to carry  the bicarbonate to the lungs, where it is  expired as C0 2 .   All cells contain an inner mem-  brane skeleton of spectrin-like pro-  teins. Red blood cell spectrin was  the first member of the spectrin family  described. The protein dystrophin present in  skeletal muscle cells is a member of the  spectrin family. Genetic defects in the dys-  trophin gene are responsible for Duchenne's  and Becker's muscular dystrophies.   deform as cells pass through capillaries, and to form and fuse with vesicle mem-  branes. The fluidity of the membrane is partially determined by the unsaturated  fatty acid content of the diet.   The composition of the membrane is dynamic. Sections of membrane form buds  that pinch off into vesicles and membrane vesicles formed in the Golgi and else-  where bring new and recycled components back to the membrane. Individual fatty  acyl chains turn over as they are hydrolyzed from the lipids and replaced, and  enzymes called flipases transfer lipids between leaflets.   The integral proteins contain transmembrane domains with hydrophobic amino acid  side chains that interact with the hydrophobic portions of the lipids to seal the mem-  brane (see Fig. 10.2). Hydrophilic regions of the proteins protrude into the aqueous  medium on both sides of the membrane. Many of these proteins function as either  channels or transporters for the movement of compounds across the membrane, as  receptors for the binding of hormones and neurotransmitters, or as structural pro-  teins (Fig. 10.6).   Peripheral membrane proteins, which were originally defined as those proteins  that can be released from the membrane by ionic solvents, are bound through weak  electrostatic interactions with the polar head groups of lipids or with integral pro-  teins. One of the best-characterized classes of peripheral proteins is the spectrin  family of proteins, which are bound to the intracellular membrane surface and pro-  vide mechanical support for the membrane. Spectrin is bound to actin, which  together form a structure that is called the inner membrane skeleton or the cortical  skeleton (see Fig. 10.6).   A third classification of membrane proteins consists of lipid- anchored proteins  bound to the inner or outer surface of the membrane. The glycophosphatidylinosi-  tolglycan (GPI) anchor is a covalently attached lipid that anchors proteins to the    Fig. 10.6. Proteins in the red blood cell membrane. The proteins named Band 3 (the bicar-  bonate-chloride exchange transporter) and glycophorin contain nonpolar a-helical segments  spanning the lipid bilayer. These proteins contain a large number of polar and charged  hydrophilic amino acids in the intracellular and extracellular domains. On the inside of the  cell, they are attached to peripheral proteins constituting the inner membrane skeleton. Band  3 is connected to spectrin filaments via the protein ankyrin. Glycophorin is connected to short  actin filaments and spectrin via protein 4.1.   external surface of the membrane (Fig. 10.7). A number of proteins involved in hor-  monal regulation are anchored to the internal surface of the membrane through  palmityl (CI 6) or myristyl (CI 4) fatty acyl groups or through geranylgeranyl  (C20) or farnesyl (C15) isoprenyl groups (see Ras, Chapter 9, Fig. 9.14, or  Chapter 6, Fig. 6.14). However, many integral proteins also contain attached lipid  groups to increase their stability in the membrane.   Some of the proteins and lipids on the external surface of the membrane contain  short chains of carbohydrates (oligosaccharides) that extend into the aqueous  medium. Carbohydrates therefore constitute 2 to 10% of the weight of plasma mem-  branes. This hydrophilic carbohydrate layer, called the glycocalyx, protects the cell  against digestion and restricts the uptake of hydrophobic compounds.   The glycoproteins generally contain branched oligosaccharide chains of  approximately 15 sugar residues that are attached through N-glycosidic bonds to  the amide nitrogen of an asparagine side chain (N-glycosidic linkage), or through  a glycosidic bond to the oxygen of serine (O-glycoproteins). The membrane gly-  colipids are usually galactosides or cerebrosides. Specific carbohydrate chains on  the glycolipids serve as cell recognition molecules (see Chapter 5 for structures of  classes of compounds).   Membranes form hydrophobic barriers around cells to control the internal environ-  ment by restricting the entry and exit of molecules. As a consequence, cells require  transport systems to permit entry of small polar compounds that they need (e.g.,  glucose) to concentrate compounds inside the cell (e.g., K + ) and to expel other   The prion protein, present in neu-  ronal membranes, provides an  example of a protein attached to  the membrane through a GPI anchor. This is  the protein that develops an altered patho-  genic conformation in both mad cow dis-  ease and Creutzfeldt-Jakob disease (see  Chapter 7, Biochemical Comments).    Fig. 10.7. The glycosylphosphatidylinositol glycan anchor (GPI). The carboxy terminus of  the protein is attached to phosphoethanolamine, which is bound to a branched oligosaccha-  ride that is attached to the inositol portion of phosphatidylinositol. The hydrophobic fatty  acyl chains of the phosphatidylinositol portion are bound in the hydrophobic core of the  membrane.   compounds (e.g., Ca and Na + ). The transport systems for small organic mole-  cules and inorganic ions fall into four categories: simple diffusion through the lipid  bilayer or through a large pore; facilitative diffusion; gated channels; and active  transport pumps (Fig. 10.8). These transport mechanisms are classified as passive if  energy is not required, or active if energy is required. The energy is often provided  by the hydrolysis of ATP.   In addition to these mechanisms for the transport of small individual molecules,  cells engage in endocytosis. The plasma membrane extends or invaginates to sur-  round a particle, a foreign cell, or extracellular fluid, which then closes into a vesi-  cle that is released into the cytoplasm (see Fig. 10.8).   0 o Dennis Veere has become dehy-  / oOA ) drated because he has lost so  y/ much water through vomiting and  diarrhea (see Chapter 4). Cholera toxin  increases the efflux of sodium and chloride  ions from his intestinal mucosal cells into  the intestinal lumen. The increase of water in  his stools results from the passive transfer of  water from inside the cell and body fluids,  where it is in high concentration (i.e., intra-  cellular Na + and CI" concentrations are low),  to the intestinal lumen and bowel, where  water is in lower concentration (relative to  high Na + and CI"). The watery diarrhea is  also high in K + ions and bicarbonate. All of  the signs and symptoms of cholera gener-  ally derive from this fluid loss.   Gases such as 0 2 and C0 2 and lipid-soluble substances (such as steroid hor-  mones) can cross membranes by simple diffusion (see Fig. 10.8). In simple diffu-  sion (free diffusion), molecules move by engaging in random collisions with other  like molecules. There is a net movement from a region of high concentration to a  region of low concentration because molecules keep bumping into each other  where their concentration is highest. Energy is not required for diffusion, and  compounds that are uncharged eventually reach the same concentrations on both  sides of the membrane.   Water is considered to diffuse through membranes by unspecific movement  through ion channels, pores, or around proteins embedded in the lipids. Certain  cells (e.g., renal tubule cells) also contain large protein pores, called aquaporins,  which permit a high rate of water flow from a region of a high water concentra-  tion (low solute concentration) to one of low water concentration (high solute  concentration).   Facilitative diffusion requires that the transported molecule bind to a specific  carrier or transport protein in the membrane (Fig. 10.9). The transporter protein   Fig. 10.8. Common types of transport mechanisms for human cells. The electrochemical gradient consists of the concentration gradient of the  compound and the distribution of charge on the membrane, which affects the transport of charged ions such as Cl~. Both protein amino acid  residues and lipid polar head groups contribute to the net negative charge on the inside of the membrane. Generally, the diffusion of uncharged  molecules (passive transport) is net movement from a region of high concentration to a low concentration, and active transport (energy-requiring)  is net movement from a region of low concentration to one of high concentration.   Fig. 10.9. Facilitative transport. Although the molecule being transported must bind to the  protein transporter, the mechanism is passive diffusion, and the molecule moves from a  region of high concentration to one of low concentration. "Passive" refers to the lack of an  energy requirement for the transport.   All of the cells in the body have  facilitative glucose transporters  that transport glucose across the  plasma membrane down an electrochemical  (concentration) gradient as it is rapidly  metabolized in the cell. In muscle and adi-  pose tissue, insulin increases the content of  facilitative glucose transporters in the cell  membrane, thus increasing the ability of  these tissues to take up glucose. Patients  with type 1 diabetes mellitus, who do not  produce insulin (e.g., Di Abietes, see Chapter  7), have a decreased ability to transport glu-  cose into these tissues, thereby contributing  to hyperglycemia (high blood glucose).   then undergoes a conformational change that allows the transported molecule to  be released on the other side of the membrane. Although the transported mole-  cules are bound to proteins, the transport process is still classified as diffusion  because energy is not required, and the compound equilibrates (achieves a bal-  ance of concentration and charge) on both sides of the membrane.   Transporter proteins, like enzymes, exhibit saturation kinetics; when all the  binding sites on all of the transporter proteins in the membrane are occupied,  the system is saturated and the rate of transport reaches a plateau (the maxi-  mum velocity). By analogy to enzymes, the concentration of a transported  compound required to reach Vi the maximum velocity is often called the K m  (Fig. 10.10). Facilitative transporters are similar to enzymes with respect to  two additional features: they are relatively specific for the compounds they  bind and they can be inhibited by compounds that block their binding sites or  change their conformation.   Fig. 10.10. Saturation kinetics of transporter proteins. When a compound must bind to a  protein to be transported across a membrane, the velocity of transport depends on the  amount of compound bound. It reaches a maximum rate when the compound's concentra-  tion is raised so high that all of the transporter binding sites are occupied. The curve is a rec-  tangular hyperbola that approaches V max at infinite substrate concentration, identical to that  of Michaelis-Menten enzymes. The K m of transport is the concentration of compound  required for l /i V max . In contrast, simple diffusion of a compound does not require its bind-  ing to a protein, and the rate of transport increases linearly with increasing concentration of  the compound.   0 & The cystic fibrosis transmembrane  /^oA J conductance regulator (CFTR) was  named for its role in cystic fibrosis.  A mutation in the gene encoding its trans-  membrane subunits results in dried mucus  accumulation in the airways and pancreatic  ducts.   The CFTR is also involved in the dehydra-  tion experienced by cholera patients such as  Dennis Veere. In intestinal mucosal cells,  cholera A toxin indirectly activates phospho-  rylation of the regulatory domain of CFTR by  protein kinase A. Thus, the channel stays open  and CI" and H 2 0 flow from the cell into the  intestinal lumen, resulting in dehydration.   In gated channels, transmembrane proteins form a pore for ions that is either opened  or closed in response to a stimulus: voltage changes across the membrane (voltage-  gated channels), the binding of a compound (ligand-gated channels), or a regulatory  change in the intracellular domain (phosphorylation-gated and pressure-gated chan-  nels). For example, the conduction of a nerve impulse along the axon depends on  the passive flux of Na + ions through a voltage-gated channel that is opened by  depolarization of the membrane. CFTR (cystic fibrosis transmembrane conduc-  tance regulator) is a CI ~ channel that provides an example of a ligand-gated chan-  nel regulated through phosphorylation (phosphorylation-gated) (Fig. 10.11). CFTR  is a member of the ABC (adenine nucleotide binding cassette, or ATP binding cas-  sette) superfamily of transport proteins. It has two transmembrane domains that  form a closed channel, each connected to an ATP binding site, and a regulatory  domain that sits in front of the channel. When the regulatory domain is phosphory-  lated by a kinase, its conformation changes and it moves away from the ATP bind-  ing domains. As ATP binds and is hydrolyzed, the transmembrane domains change  conformation and open the channel, and chloride ions diffuse through. As the con-  formation reverts back to its original form, the channel closes.   Transport through a ligand-gated channel is considered diffusion, although ATP  is involved, because only a few ATP molecules are being used to open and close the  channel through which many, many chloride ions diffuse. However, the distinction  between ligand-gated channels and facilitative transporters is not always as clear.  Many gated channels show saturation kinetics at very high concentrations of the  compounds being transported.    Protein-mediated transport sys-  tems, whether facilitative or active,  are classified as antiports if they  specifically exchange compounds of similar  charge across a membrane; they are called  symports or cotransporters if they simultane-  ously transport two molecules across the  membrane in the same direction. Band 3 in  the red blood cell membrane, which  exchanges chloride ion for bicarbonate, pro-  vides an example of an antiport.   Both active transport and facilitative transport are mediated by protein transporters  (carriers) in the membrane. However, in facilitative transport, the compound is  transported down an electrochemical gradient (the balance of concentration and  charge across a membrane), usually from a high concentration to a low concentra-  tion, to equilibrate between the two sides of the membrane. In active transport,  energy is used to concentrate the compound on one side of the membrane. If energy  is directly applied to the transporter (e.g., ATP hydrolysis by Na + ,K + -ATPase), the  transport is called primary active transport; if energy is used to establish an ion gra-  dient (e.g., the Na + gradient), and the gradient is used to concentrate another com-  pound, the transport is called secondary active transport.   The Na + ,K + -ATPase spans the plasma membrane, much like a gated pore, with  a binding site for 3 Na + ions open to the intracellular side (Fig. 10.12). Energy from    Fig. 10.11. CFTR, a ligand-gated channel controlled by phosphorylation. Two intracellular binding domains control opening of  the channel, an adenine nucleotide binding domain (ABD) and a regulatory domain (R). 1 Phosphorylation of the regulatory  subunit by protein kinase A causes a conformational change that allows ATP to bind to the adenine nucleotide binding domain  (ABD). 2 Hydrolysis of bound ATP opens the channel so that chloride ions can diffuse through.   Fig. 10.12. Active transport by Na + ,K + -ATPase. Three sodium ions bind to the transporter protein on the cytoplasmic side of the membrane.  When ATP is hydrolyzed to ADP, the carrier protein is phosphorylated and undergoes a change in conformation that causes the sodium ions to  be released into the extracellular fluid. Two potassium ions then bind on the extracellular side. Dephosphorylation of the carrier protein produces  another conformational change, and the potassium ions are released on the inside of the cell membrane. The transporter protein then resumes its  original conformation, ready to bind more sodium ions.   ATP hydrolysis is used to phosphorylate an internal domain and change the trans-  porters' conformation so that bound Na + ions are released to the outside, and two  external K + ions bind. K + binding triggers hydrolysis of the bound phosphate group  and a return to the original conformation, accompanied by release of K + ions inside  the cell. As a consequence, cells are able to maintain a much lower intracellular Na +  concentration and much higher intracellular K + ion concentration than present in  the external fluid.   The Na + gradient, which is maintained by primary active transport, is used to  power the transport of glucose, amino acids, and many other compounds into the cell  through secondary active transport. An example is provided by the transport of glu-  cose into cells of the intestinal epithelium in conjunction with Na + ions (Fig. 10.13).   The Ca 2+ -ATPase, a calcium pump,  \\ ) uses a mechanism similar to that  of Na + ,K + -ATPase to maintain  intracellular Ca 2+ concentration  below 10" 7 M in spite of the high extracellu-  lar concentration of 10 3 M. This transporter  is inhibited by binding of the regulatory pro-  tein calmodulin. When the intracellular Ca 2+  concentration increases, Ca 2+ binds to  calmodulin, which dissociates from the  transporter, thereby activating it to pump  Ca 2+ out of the cell (see Chapter 9 for the  structure of calmodulin). High levels of intra-  cellular Ca 2+ are associated with irreversible  progression from cell injury to cell death.   Fig. 10.13. Secondary active transport of glucose by the Na + -glucose cotransporter. One  sodium ion binds to the carrier protein in the luminal membrane, stimulating the binding of  glucose. After a conformational change, the protein releases Na + and glucose into the cell  and returns to its original conformation. Na + ,K + -ATPase in the basolateral membrane pumps  Na + against its concentration gradient into the extracellular fluid. Thus, the Na + concentra-  tion in the cell is low, and Na + moves from the lumen down its concentration gradient into  the cell and is pumped against its gradient into the extracellular fluid. Glucose, consequently,  moves against its concentration gradient from the lumen into the cell by traveling on the same  carrier as Na + . Glucose then passes down its concentration gradient into the extracellular  fluid on a passive transporter protein.   The dehydration of cholera is often  treated with an oral rehydration  solution containing Na + , K + , and  glucose or a diet of rice (which contains glu-  cose and amino acids). Glucose is absorbed  from the intestinal lumen via the Na + -  dependent glucose cotransporters, which  cotransport Na + into the cells together with  glucose. Many amino acids are also  absorbed by Na + -dependent cotransport.  With the return of Na + to the cytoplasm,  water efflux from the cell into the intestinal  lumen decreases.    The vitamin folate provides an  example of a compound trans-  ported into cells by caveolae,  which form around the occupied folate  receptor. In contrast endocytosis of many  compounds such as membrane hormone  receptors occurs through clathrin-coated  pits. The receptors are targeted for these pits  by adaptor proteins that bind to a specific  amino acid sequence in the receptor.   These cells create a gradient in Na + and then use this gradient to drive the transport  of glucose from the intestinal lumen into the cell against its concentration gradient.   Vesicular transport occurs when a membrane completely surrounds a compound,  particle, or cell and encloses it into a vesicle. When the vesicle fuses with another  membrane system, the entrapped compounds are released. Endocytosis refers to  vesicular transport into the cell, and exocytosis to transport out of the cell. Endocy-  tosis is further classified as phagocytosis if the vesicle forms around particulate  matter (such as whole bacterial cells or metals and dyes from a tattoo), and pinocy-  tosis if the vesicle forms around fluid containing dispersed molecules. Receptor-  mediated endocytosis is the name given to the formation of clathrin-coated vesicles  that mediate the internalization of membrane-bound receptors in vesicles coated on  the intracellular side with subunits of the protein clathrin (Fig. 10.14). Potocytosis  is the name given to endocytosis that occurs via caveolae (small invaginations or  "caves"), which are regions of the cell membrane with a unique lipid and protein  composition (including the protein caveolin-1).   Lysosomes are the intracellular organelles of digestion enclosed by a single  membrane that prevents the release of its digestive enzymes into the cytosol.  They are central to a wide variety of body functions that involve elimination of  unwanted material and recycling their components, including destruction of   Fig. 10.14. Formation of a clathrin-coated vesicle. Ligands entering the cell through recep-  tor-mediated endocytosis bind to receptors that cluster in an area of the membrane. Adaptor  proteins bind to the receptor tails and to the clathrin molecules to enclose the budding mem-  brane in a cage-like clathrin coat.   Molecules of a monomeric G protein called dynamin (from the Rab family) constrict the  neck of the vesicle and pinch it off from the membrane as GTP is hydrolyzed.   infectious bacteria and yeast, recovery from injury, tissue remodeling, involution of  tissues during development, and normal turnover of cells and organelles.   The lysosomal digestive enzymes include nucleases, phosphatases, glycosi-  dases, esterases, and proteases called cathepsins (Fig. 10.15). These enzymes  are all hydrolases, enzymes that cleave amide, ester, and other bonds through  the addition of water. Many of the products of lysosomal digestion, such as the  amino acids, return to the cytosol. Lysosomes are therefore involved in recycling  compounds.   Most of these lysosomal hydrolases have their highest activity near a pH of  approximately 5.5 (the pH optimum). The intralysosmal pH is maintained near 5.5  principally by v-ATPases (vesicular ATPases), which actively pump protons into the  lysosome. The cytosol and other cellular compartments have a pH nearer 7.2 and  are therefore protected from escaped lysosomal hydrolases.   Lysosomes are formed from digestive vesicles called endosomes, which are  involved in receptor-mediated endocytosis. They also participate in digestion of for-  eign cells acquired through phagocytosis and the digestion of internal contents in  the process of autophagocytosis.   Lysosomes are involved in the digestion of compounds brought into the cells in  endocytotic clathrin-coated vesicles formed by the plasma membrane (Fig. 10.16).  These vesicles fuse to form multivesicular bodies called early endosomes. The early  endosomes mature into late endosomes as they recycle clathrin, lipids, and other    Lysosomal storage diseases.  Genetic defects in lysosomal  enzymes, or in proteins such as the  mannose 6-phosphate receptors required for  targeting the enzymes to the lysosome, lead  to an abnormal accumulation of undigested  material in lysosomes that may be con-  verted to residual bodies. The accumulation  may be so extensive that normal cellular  function is compromised, particularly in  neuronal cells. Genetic diseases such as the  Tay-Sachs disease (an accumulation of par-  tially digested gangliosides in lysosomes),  and Pompe's disease (an accumulation of  glycogen particles in lysosomes) are caused  by the absence or deficiency of specific lyso-  somal enzymes.   Fig. 10.15. Lysosomal reactions. Most lysosomal enzymes are hydrolases, which cleave peptide, ester, and glycosidic bonds by adding the  components of water across the bond. These enzymes are active at the acidic pH of the lysosome and inactive if accidentally released into the  cytosol.   The elevated level of uric acid in  Lotta Topaigne's blood led to the  deposition of monosodium urate  crystals in the joint space (synovial fluid) of  her right great toe, resulting in podagra  (painful great toe). Neutrophils, the media-  tors of the acute inflammation that followed,  attempted to phagocytose the urate crystals.  The engulfed urate crystals were deposited  in the late endosomes and lysosomes of the  neutrophil. Because urate crystals are parti-  cles that cannot be degraded by any of the  lysosomal acid hydolases, their accumula-  tion caused lysis of the lysosomal mem-  branes, followed by cell lysis and release of  lysosomal enzymes into the joint space. The  urate crystals also resulted in release of  chemical mediators of inflammation that  recruited other cells into the area. This fur-  ther amplified the acute inflammatory reac-  tion in the tissues of the joint caspsule (syn-  ovitis), leading to the extremely painful  swelling of acute gouty arthritis.   Phagocytosis and autophagy are  part of the normal turnover of body  components, such as degradation  of cells that have a shorter lifespan than the  whole organism and remodeling of tissues  during pregnancy. For example, phagocytes,  located mainly in the spleen and liver,  remove approximately 3 x 10 11 red blood cells  from the circulation each day. During preg-  nancy, breast tissue is remodeled to develop  the capacity for lactation; after weaning of an  infant, the lactating breast returns to its orig-  inal state (involution).    Fig. 10.16. Lysosomes in receptor-mediated endocytosis via clathrin-coated pits. 1 Endo-  cytotic vesicles fuse to form early endosomes. 2 Vesicle contents are sorted, and receptors,  clathrin, and lipids are sent back to the plasma membrane. 3 Transport vesicles from the  trans-Golgi carry lysosomal hydrolases to the late endosome. 4 Lysosomes containing con-  centrated hydrolases digest proteins and other components acquired from endocytotic  vesicles.   membrane components back to the plasma membrane in vesicles called recycling  endosomes. The late endosomes mature into lysosomes as they progressively accu-  mulate newly synthesized acid hydrolases and vesicular proton pumps brought to  them in clathrin-coated vesicles from the Golgi. Thus, lysosomes do not acquire  their full digestive power until after sorting of membrane lipids and proteins for  recycling.   Within the Golgi, enzymes are targeted for endosomes (and eventually lyso-  somes) by addition of mannose 6-phosphate residues that bind to mannose 6-  phosphate receptor proteins in the Golgi membrane. The mannose 6-phosphate  receptors together with their bound acid hydrolases are incorporated into the  clathrin-coated Golgi transport vesicles and released. The transport vesicles lose  their clathrin coat and then fuse with the late endosomal membrane. The acidity of  the endosome releases the acid hydrolases from the receptors into the vesicle lumen.  The receptors are eventually recycled back to the Golgi.   One of the major roles of lysosomes is phagocytosis (Fig. 10.17). Neutrophils and  macrophages, the major phagocytic cells, devour pathogenic microorganisms and  clean up wound debris and dead cells, thus aiding in repair. As bacteria or other par-  ticles are enclosed into clathrin-coated pits in the plasma membrane, these vesicles  bud off to form intracellular phagosomes. The phagosomes fuse with lysosomes,  where the acidity and digestive enzymes destroy the contents. Pinocytotic vescicles  also may fuse with lysosomes.   In autophagy (self -eating), intracellular components such as organelles or glyco-  gen particles are surrounded by a membrane derived from ER vesicles, forming an  autophagosome. The autophagosome fuses with a lysosome, and the contents of the  phagolysosome are digested by lysosomal enzymes. Organelles usually turn over  much more rapidly than the cells in which they reside (e.g., approximately four  mitochondria in each liver cell are degraded per hour). Cells that are damaged but  still viable recover, in part, by using autophagy to eliminate damaged components.   Fig. 10.17. Phagocytosis and autophagy. Cells and large particles are phagocytosed. The  phagosomes fuse with lysosomes to form phagolysosomes. Recyclable amino acids (AA),  fatty acids (FA), and carbohydrates (CHO) are released into the cytosol. Autophagosomes are  formed in the ER as the cell digests mitochondria and its own large particles. These also  merge with lysosomes. Undigested material may remain in the lysosomes to form residual  bodies, which are either extruded or remain in the cell as lipofuscin granules.   If a significant amount of undigestible material remains within the lysosome  after the digestion process is completed, the lysosome is called a residual body.  Depending on the cell type, residual bodies may be expelled (exocytosis) or remain  indefinitely in the cell as lipofuscin granules that accumulate with age.   Fig. 10.18. Mitochondrion. Electron micro-  graph (top); three-dimensional drawing (bot-  tom).   Mitochondria contain most of the enzymes for the pathways of fuel oxidation  and oxidative phosphorylation and thus generate most of the ATP required by  mammalian cells. Each mitochondrion is surrounded by two membranes, an  outer membrane and an inner membrane, separating the mitochondrial matrix  from the cytosol (Fig. 10.18). The inner membrane forms invaginations known  as cristae containing the electron transport chain and ATP synthase. Most of the  enzymes for the TCA cycle and other pathways for oxidation are located in the  mitochondrial matrix, the compartment enclosed by the inner mitochondrial  membrane. (The TCA cycle and electron transport chain are described in more  detail in Chapters 20 and 21.)   The inner mitochondrial membrane is highly impermeable, and the proton gra-  dient that is built up across this membrane during oxidative phosphorylation is  essential for ATP generation from ADP and phosphate. The transport of ions occurs  principally through facilitative transporters in a type of secondary active transport  powered by the proton gradient established by the electron transport chain. The  outer membrane contains pores made from proteins called porins and is permeable  to molecules with a molecular weight up to about 1000 g/mole.   Mitochondria can replicate by division; however, most of their proteins must  be imported from the cytosol. Mitochondria contain a small amount of DNA,  which encodes for only 13 different subunits of proteins involved in oxidative  phosphorylation. Most of the enzymes and proteins in mitochondria are encoded  by nuclear DNA and synthesized on cytoplasmic ribosomes. They are imported    Mitochondial diseases. Mitochon-  dria contain DNA and can repro-  duce by replicating their DNA and  then dividing in half. Although nuclear DNA  encodes most of the enzymes found in mito-  chondria, mitochondrial DNA encodes some  of the subunits of the electron transport  chain proteins and ATP synthase. Mutations  in mitochondrial DNA result in a number of  genetic diseases that affect skeletal muscle,  neuronal, and renal tissues. They are impli-  cated in aging.   through membrane pores by a receptor-mediated process involving members of  the heat shock family of proteins.   Peroxisomes are cytoplasmic organelles, similar in size to lysosomes, that are  involved in oxidative reactions using molecular oxygen (Fig. 10.19). These reac-  tions produce the toxic chemical hydrogen peroxide (H 2 0 2 ), which is subsequently  used or degraded within the peroxisome by catalase and other enzymes. Peroxi-  somes function in the oxidation of very long chain fatty acids (containing 20 or  more carbons) to shorter chain fatty acids, the conversion of cholesterol to bile  acids, and the synthesis of ether lipids called plasmalogens. They are bounded by a  single membrane.   Like mitochondria, peroxisomes can replicate by division. However, they are  dependent on the import of proteins to function. They contain no DNA.    Peroxisomal Diseases. Peroxisomal  diseases are caused by mutations  affecting either the synthesis of  functional peroxisomal enzymes or their  incorporation into peroxisomes. For example,  adrenoleukodystrophy probably involves a  mutation that decreases the content of a  transporter in the peroxisomal membrane.  Zellweger's syndrome is caused by the failure  to complete the synthesis of peroxisomes.   The largest of the subcellular organelles of animal cells is the nucleus (Fig. 10.20).  Most of the genetic material of the cell is located in the chromosomes of the  nucleus, which are composed of DNA, an equal weight of small, positively charged  proteins called histones, and a variable amount of other proteins. This nucleoprotein   Chromatin Outer  (DNA + histones membrane  and other proteins) (continuous  with RER)   complex is called chromatin. The nucleolus, a substructure of the nucleus, is the site  of rRNA transcription and processing, and of ribosome assembly. Replication, tran-  scription, translation, and the regulation of these processes are the major focus of  the molecular biology section of this text (see Section Three).   The nucleus is separated from the rest of the cell (the cytoplasm) by the nuclear  envelope, which consists of two membranes joined at nuclear pores. The outer  nuclear membrane is continuous with the rough endoplasmic reticulum. To convert  the genetic code of the DNA into the primary sequence of a protein, DNA is tran-  scribed into RNA, which is modified and edited into mRNA. The mRNA travels  through the nuclear pores into the cytoplasm, where it is translated into the primary  sequence of a protein on ribosomes (Fig. 10.21). Ribosomes, which are generated  in the nucleolus, also must travel through nuclear pores to the cytoplasm. Conversely,  proteins required for replication, transcription, and other processes pass into the  nucleus through these pores. Thus, transport through the pore is specific for the  molecule and the direction of transport.   Specificity and direction of travel through the nuclear pore (import vs. export) is  dictated by binding proteins (importins vs. exportins), by a small GTP protein  called Ran, and by the location of the regulatory protein, RanGAP (GTPase acti-  vating protein) only on the cytoplasmic side (Fig. 10.22). Proteins transported into  the nucleus have a nuclear localization signal that causes them to bind to one of the  subunits of cytosolic proteins called importins. The other subunit of the importin  molecule binds to cytoplasmic filaments attached to the outer ring of the nuclear  pore. As the importin-protein complex enters the nucleus, the small GTP-binding  protein Ran binds to an importin subunit, causing release of the transported protein  into the nucleus. The Ran-importin complex is returned to the cytosol, where Ran-  GAP (GTPase activating protein) activates hydrolysis of bound GTP to GDP and  phosphate. The energy released by GTP hydrolysis changes the conformation of  Ran and the complex dissociates. The free importin can then bind another protein.   The Ras family of monomeric G  proteins. Ras and Ran belong to a  superfamily of proteins called  small G proteins (also called small GTP-  binding proteins, small GTPases, or  monomeric G proteins: see Chapter 9, sec-  tion III.C.2). These proteins function as tim-  ing regulators for a variety of cell functions.  They are referred to as "small" because they  are composed of a single subunit with a  weight of 20 to 40 kDa, and they are called  GTPases because they slowly hydrolyze  bound GTP. When small G proteins contain  bound GTP, they bind to and activate their  target proteins. As their bound GTP is  hydrolyzed to GDP and Pi, their conforma-  tion changes dramatically, and they dissoci-  ate from the target protein. They thus serve  as "automatic clocks" that shut themselves  off. Many of the monomeric GTP binding  proteins are regulated by GAPs (GTPase  activating proteins), GEFs (GTP exchange  proteins which stimulate GDP dissociation  and GTP binding), or GDIs (GDP-dissociation  inhibitory proteins). (See Chapter 9 for a  more complete discussion of these regula-  tors). The function of each of the five major  classes of Ras monomeric G proteins in cell  biology is summarized in Table 10.1.    Fig. 10.21. The nuclear pore complex. The approximately 100 different polypeptide chains  of the nuclear pore complex form an assembly of 8 spokes attached to two ring structures  (a cytoplasmic ring in the outer nuclear membrane and a nuclear ring through the inner  membrane) with a transporter "plug" in the center. Small molecules, ions, and proteins with  less than a 50-kDa mass passively diffuse through the pore in either direction. However,  RNAs and most proteins are too large to diffuse through, and are actively transported in a  process that requires energy, is selective for the molecule transported, is unidirectional, and  can be regulated.    Fig. 10.22. Nuclear import. Proteins with the nuclear localization signal bind to importins,  which carry them through the nuclear pore into the nucleus. The monomeric G protein Ran  containing bound GTP binds to one of the subunits of importin. This causes dissociation of  the importin subunits and release of the imported protein in the nucleus. The Ran-importin  complex exits a nuclear pore. On the cytoplasmic side, a RanGAP (GTPase activating pro-  tein) activates the hydrolysis of GTP to GDP, which causes dissociation of the complex.  RanGDP is subsequently returned to the nucleus, where an accessory protein activates dis-  sociation of GDP and association of GTP.   RNAs are transported from the nucleus to the cytoplasm as ribonucleoproteins,  which are targeted for export by a specific amino acid sequence called the nuclear  export signal. The nucleoprotein forms a complex with additional proteins called  exportins and with Ran. This complex is transported through the pore to the cyto-  plasm, where RanGAP activates hydrolysis of the bound GTR In the absence of  GTP, the complex dissociates with the release of RNA into the cytoplasm, and the  exportins and Ran are transported back to the nucleus.   The endoplasmic reticulum (ER) is a network of membranous tubules within the cell  consisting of smooth endoplasmic reticulum (SER), which lacks ribosomes, and  rough endoplasmic reticulum (RER), which is studded with ribosomes (Fig. 10.23).  The SER has a number of functions. It contains enzymes for the synthesis of many  lipids, such as triacylglycerols and phospholipids. It also contains the cytochrome  P450 oxidative enzymes involved in metabolism of drugs and toxic chemicals such  as ethanol and the synthesis of hydrophobic molecules such as steroid hormones.  Glycogen is stored in regions of liver cells that are rich in SER.   The RER is involved in the synthesis of certain proteins. Ribosomes attached to  the membranes of the RER give them their "rough" appearance. Proteins produced  on these ribosomes enter the lumen of the RER, travel to the Golgi complex in vesi-  cles, and are subsequently either secreted from the cell, sequestered within  membrane-enclosed organelles such as lysosomes, or embedded in the plasma  membrane. Posttranslational modifications of these proteins, such as the initiation  of N-linked glycosylation and the addition of GPI anchors, occur in the RER. In  contrast, proteins encoded by the nucleus and found in the cytosol, peroxisomes, or  mitochondria are synthesized on free ribosomes in the cytosol and are seldom mod-  ified by the attachment of oligosaccharides.    Chronic ingestion of ethanol has  increased the content of MEOS, the  microsomal ethanol oxidizing sys-  tem, in Al Martini's liver. MEOS is a  cytochrome P450 enzyme that catalyzes the  conversion of ethanol, NADPH and 0 2 to  acetaldehyde, NADP + , and 2 H 2 0 (see Chap-  ter 9). The adjective microsomal is a term  derived from experimental cell biology that  is sometimes used for processes occurring  in the ER. When cells are lysed in the labora-  tory, the ER is fragmented into vesicles  called microsomes, which can be isolated by  centrifugation. Microsomes, as such, are not  actually present in cells.    Fig. 10.23 A. Smooth endoplasmic reticulum. B. Rough endoplasmic reticulum. A and B are  electron micrographs. A three-dimensional drawing is in the middle.   The Golgi complex is involved in modifying proteins produced in the RER and in  sorting and distributing these proteins to the lysosomes, secretory vesicles, or the  plasma membrane. It consists of a curved stack of flattened vesicles in the cyto-  plasm that is generally divided into three compartments: the cis-Golgi network,  which is often convex and faces the nucleus; the medial Golgi stacks; and the trans  Golgi network, which often faces the plasma membrane (Fig. 10.24).   Proteins are transported to and from the Golgi in at least three kinds of vesicles:  coatomer-coated COP I vesicles, coatomer-coated COP II vesicles, and clathrin-  coated vesicles (see Fig. 10.24). Proteins produced on the RER travel in COP II  vesicles to an endoplasmic reticulum-Golgi intermediate compartment (ERGIC),  and then to the cis-Golgi network, where they enter the lumen. Here N-linked  oligosaccharide chains that were added to proteins in the RER are modified, and  O-linked oligosaccharides are added. COP I vesicles recycle material from the  Golgi back to the ER and possibly transfer material from the Golgi to other sites.    Fig. 10.24. Vesicular transport to and from the Golgi complex. COP II vesicles (coatomer-  coated) form in the rough ER and move to the Golgi. COP I vesicles generally go from the  trans to the cis Golgi to the ER. Vesicles that go to late endosomes (eventually lysosomes)  from the Golgi or the plasma membrane are clathrin-coated. Less is known about exocytotic  vesicles. Vesicle transport, as well as transport of organelles and secretory proteins, occurs  along microtubules (structures formed from the protein tubulin).   Vesicles released from the trans face of the Golgi complex travel to endosomes as  clathrin-coated vesicles.   COP vesicles are coated with a complex composed of coatomer proteins  (COP), an Arf family monomeric G protein that mediates vesicle assembly, and  other proteins (Fig. 10.25). COP I vesicles contain the monomeric G protein Arf  (ADP-ribosylating factor), and COP II vesicles contain the monomeric G protein  Sar (another member of the Arf family). In both types of vesicles, hydrolysis of  GTP causes dissociation of the G-protein and disassembly of the vesicle coat. The  vesicle components are then recycled. Glycoproteins or glycolipids once anchored  in the membrane of the vesicle remain in the plasma membrane when the vesicular  and plasma membranes fuse.   Vesicles that have lost their coats are ready to fuse with the target membrane.  The vesicle membranes contain proteins called v-SNARES (vesicle-SNARES)  (see Fig. 10.25). Each type of v-SNARE is able to recognize and bind to its com-  plementary t-SNARE (target SNARE) on the target membrane, thus ensuring that    Fig. 10.25. Transport in COP-coated vesicles. A. Assembly and release. Arf with bound GTP  assembles a region of the trans-Golgi membrane containing receptors for the protein cargo  and coatomers. As GTP is hydrolyzed to GDP, the coat is released. B. Docking. The small G  protein Rab assists in docking. v-Snares in the vesicle membrane recognize complementary  t-Snares in the target membrane. C. Fusion. NSF and SNAP are fusion proteins.   The monomeric G protein Arf was  named for its contribution to the  pathogenesis of cholera and not  for its normal function in the assembly of  COP I vesicles. However, it is also required  for the transport of V. cholerae A-toxin. The  cholera toxin is endocytosed in caveolae  vesicles that subsequently merge with lyso-  somes (or are transformed into lysosomes),  where the acidic pH contributes to activation  of the toxin. As the toxin is transported  through the Golgi and ER, it is further  processed and activated. Arf forms a com-  plex with the A-toxin that promotes its travel  between compartments. The A-toxin is actu-  ally an ADP-ribosylase (an enzyme that  cleaves NAD and attaches the ADP portion  to a protein) (see Chapter 6, Fig. 6.14), and  hence, Arf became known as the ADP-ribo-  sylating factor. The ADP-ribosylation of pro-  teins regulating the CFTR chloride channel  leads to Dennis Veere's dehydration and  diarrhea.   Secretory vesicles. The hormone  insulin is synthesized as a prohor-  mone, proinsulin, which is incorpo-  rated into secretory vesicles. These vesicles  contain a protease that is activated by the  acidic pH of the secretory vesicle. It cleaves  proinsulin into the A, B, and C chains (see  Fig. 6.13).   Lotta Topaigne was given  colchicine, a drug that is frequently  used to treat gout. One of its actions  is to prevent phagocytic activity by binding to  dimers of the a and (3 subunits of tubulin.  When the tubulin dimer-colchicine com-  plexes bind to microtubules, further polymer-  ization of the microtubules is inhibited,  depolymerization predominates, and the  microtubules disassemble. Microtubules are  necessary for vesicular movement of urate  crystals during phagocytosis and release of  mediators that activate the inflammatory  response. Thus, colchicine diminishes the  inflammatory response, swelling and pain  caused by formation of urate crystals.   the vesicle contents are delivered to the right location. Several additional proteins  are required for fusion of the vesicle with the target membrane, including Rab  (another monomeric G protein) and two additional proteins called SNAP (soluble  NSF attachment proteins) and NSF (N-ethylmaleimide sensitive factor).   Exocytotic vesicles release proteins into the extracellular space after fusion  of the vesicular and plasma cell membranes. Exocytotic vesicles containing  hormones also may contain proteases that cleave the prohormone at a specific  site and v-ATPases that acidify the vesicle and activate the protease (similar to  lysosomes).   The structure of the cell, the shape of the cell surface, and the arrangement of  subcellular organelles is organized by three major protein components: micro-  tubules composed of tubulin, which move and position organelles and vesicles;  thin filaments composed of actin, which form a cytoskeleton, and intermediate  filaments composed of different fibrous proteins. Actin and tubulin, which are  involved in cell movement, are dynamic structures composed of continuously  associating and dissociating globular subunits. Intermediate filaments, which  play a structural role, are composed of stable fibrous proteins that turn over more  slowly.   Microtubules, cylindrical tubes composed of tubulin subunits, are present in all  nucleated cells and the platelets in blood (Fig. 10.26). They are responsible for the  positioning of organelles in the cell cytoplasm and the movement of vesicles,  including phagocytic vesicles, exocytotic vesicles, and the transport vesicles  between the ER, Golgi, and endosomes (see Fig. 10.24). They also form the spin-  dle apparatus for cell division. The microtubule network (the minus end) begins in  the nucleus at the centriole and extends outward to the plasma membrane (usually  the plus end). Microtubule-associated proteins (MAPs) attach microtubules to other  cellular components, and can determine cell shape and polarity.   Fig. 10.26. Microtubules composed of ap tubulin heterodimers. MAP, microtubule-associ-  ated protein. These proteins project outward to attach the microtubules to other cellular com-  ponents. The microtubule grows by the addition of ap dimers containing bound GTP to the  plus end of the polymer. Kinesin and dyneins are motor proteins that transport cargo (e.g.,  vesicles) along the microtubule.   Motor proteins called kinesins and cytoplasmic dyneins use ATP energy to move  cargo along the microtubules. Kinesins moves molecules, vesicles, and organelles  toward the plus end of microtubules, usually toward the plasma membrane. Cyto-  plasmic dyneins are huge proteins that move vesicles and organelles to the minus  end, generally toward the nucleus. They are also involved in the positioning of the  Golgi complex and the movement of chromosomes during mitosis.   Microtubules consist of polymerized arrays of a and p tubulin dimers that form  13 protofilaments organized around a hollow core (see Fig. 10.26). Three different  tubulin polypeptides (a, (3, and 7) of similar amino acid composition are encoded  by related genes; a and p dimers polymerize to form most microtubules, and 7-  tubuluin is found only in the centrosome. Tubulin dimers composed of one a and  one p subunit bind GTP, which creates a conformational change in the dimer that  favors addition of dimers to the tubulin polymer. The dimers can add to and disso-  ciate from both ends of the tubulin, but the end to which they add more rapidly (the  plus end) has a net rate of growth, and the end to which they add more slowly (the  minus end) has a net rate of loss. As GTP is hydrolyzed to GDP, the binding of tubu-  lin subunits is weakened, resulting in their dissociation (dynamic instability). Thus,  the net rate and direction of growth is dictated by the fastest growing end of the  microtubule.   Actin filaments form a network controlling the shape of the cell and movement  of the cell surface, thereby allowing cells to move, divide, engulf particles, and  contract. Actin is present in all living cells. The actin polymer, called F-actin, is  composed of a helical arrangement of globular G-actin subunits (Fig. 10.27).  Within the polymer, each G-actin subunit contains a bound ATP or ADP that   Fig. 10.27. Actin filaments. The polymer F-actin is assembled from G-actin subunits con-  taining bound ATP. While bound, the ATP is slowly hydrolyzed to ADR The conformational  change shifts the equilibrium so that dissociation of the G-actin subunits is favorable at the  minus end of the polymer. Once dissociated, the actin subunits exchange ADP for ATP, which  may again associate with the actin polymer. At the plus end of the molecule, association is  favored over dissociation.   A variety of human cells have cilia  and flagella, hairlike projections  from the surface that have a stroke-  like motion. These projections contain a flex-  ible organized array of microtubules. Fluid  or mucus is propelled over the surface of cil-  iated epithelial cells by the coordinated beat-  ing of cilia. A sperm cell swims by means of  a flagellum.   Colchicine has a narrow therapeu-  tic index (i.e., the amount of drug  that produces the desirable thera-  peutic effect is not much lower than the  amount that produces an adverse effect). Its  therapeutic effect depends on inhibiting  tubulin synthesis in neutrophils, but it can  also prevent tubulin synthesis (and, thus,  cell division and other cellular processes) in  other cells. Fortunately, neutrophils concen-  trate colchicine, so they are affected at lower  intakes than other cell types. Neutrophils  lack the transport protein P-glycoprotein, a  member of the ABC cassette family (which  includes the CFTR channel). In most other  cell types, P-glycoprotein exports chemicals  such as colchicine, thus preventing their  accumulation.   1 80 SECTION TWO / CHEMICAL AND BIOLOGICAL FOUNDATIONS OF BIOCHEMISTRY   holds the actin fold into a closed conformation (see Chapter 7). The actin poly-  mer is dynamic. New subunits of G-actin containing ATP continuously combine  with the assembled F-actin polymer at the plus end. As F-actin elongates, bound  ATP is hydrolyzed to ADP, so that most of the polymer contains G-actin- ADP  subunits. The conformation of ADP-actin favors dissociation from the minus end  of the polymer; thus, the polymer is capable of lengthening from the plus end.  This directional growth can account for certain types of cell movement and shape  changes: the formation of pseudopodia that surround other cells during phago-  cytosis, the migration of cells in the developing embryo, or the movement of  white blood cells through tissues.   Actin polymers form the thin filaments (also called microfilaments) in the cell  that are organized into compact ordered bundles or loose network arrays by cross-  linking proteins. Short actin filaments bind to the cross-linking protein spectrin to  form the cortical actin skeleton network (see Fig. 10.6). In muscle cells, long actin  filaments combine with thick filaments, composed of the protein myosin, to pro-  duce muscle contraction. The assembly of G-actin subunits into polymers, bundling  of fibers, and attachments of actin to spectrin and to the plasma membrane proteins  and organelles, are mediated by a number of actin-binding proteins and G-proteins  from the Rho family.   Intermediate filaments (IF) are composed of fibrous protein polymers that provide  structural support to membranes of the cells and scaffolding for attachment of other  cellular components. Each IF subunit is composed of a long rod-like a-helical core  containing globular spacing domains, and globular N- and C-terminal domains. The  a-helical segments of two subunits coil around each other to form a coiled coil, and  then combine with another dimer coil to form a tetramer. Depending on the type of  filament, the dimers may be either hetero- or homo-dimers. The tetramers join end-  to end to form protofilaments and approximately eight proto filaments combine to  form filaments (Fig. 10.28). Filament assembly is partially controlled through phos-  phorylation.   In contrast to actin thin filaments, the 50 or so different types of intermediate fil-  aments are each composed of a different protein having the same general structure  described above (Table 10.2). Some of the intermediate filaments, such as the  nuclear lamins, are common to all cell types. These filaments provide a lattice-like  support network attached to the inner nuclear membrane. Other intermediate fila-  ments are specific for types of cells (e.g., epithelial cells have cytokeratins, and neu-  rons have neurofilaments). These provide an internal network that helps to support  the shape and resilience of the cell.   Fig. 10.28. Formation of a cytokeratin filament. The central rod of the keratin monomer is principally a-helical structure. A  specific acidic keratin monomer combines with a specific basic keratin monomer to form a heterodimer coil (a coiled coil structure). Two dimers  combine in antiparallel fashion to form a tetramer, and the tetramers combine head-to-tail to form protofilaments. Approximately eight protofil-  aments combine to form a filament. The filament is thicker than actin filaments (called thin filaments or microfilaments) and thinner than micro-  tubules (thick tubes) and is therefore called an intermediate filament.   Different epithelial cells express different keratins.  Epithelial cells. Each cell type has a specific acidic  and a specific basic keratin.  Cells of mesenchymal origin  Muscle   lining of cellular nuclear envelopes.  Stem cells of central nervous system   ^ Al Martini. Al Martini has been drinking for 5 years and has begun to  exhibit mental and systemic effects of chronic alcohol consumption. In his  brain, ethanol has altered the fluidity of neuronal lipids, causing changes  in their response to neurotransmitters released from exocytotic vesicles. In his liver,  increased levels of MEOS (CYP2E1) located in the smooth ER increased his rate of  ethanol oxidation to acetaldehyde, a compound that is toxic to the cell. His liver also  continues to oxidize ethanol to acetaldehyde through a cytosolic enzyme, liver alco-  hol dehydrogenase.   One of the toxic effects of acetaldehyde is inhibition of tubulin polymerization.  Tubulin is used in the liver for secretion of very low-density lipoprotein (VLDL)  particles containing newly synthesized triacylglycerols. As a result, these triacyl-  glycerols accumulate in the liver, and he has begun to develop a fatty liver.  Acetaldehyde may also damage protein components of the inner mitochondrial  membrane and affect its ability to pump protons to the cytosol.    Lotta Topaigne had a rapid and gratifying clinical response to the  hourly administration of colchicine. This drug diminishes phagocytosis  and the subsequent release of the lysosomal enzymes that initiate the   The inflammatory response that causes the symptoms of an acute gout attack  begins when neutrophils and macrophages ingest urate crystals. In neutrophils,  urate activates the conversion of the polyunsaturated fatty acid arachidonic acid  (present in membrane phospholipids) to leukotriene B 4 . The release of this  prostaglandin contributes to the pain. Colchicine, through its effect on tubulin,  inhibits phagocytosis, leukotriene B 4 release, and recruitment and cell division of  additional cells involved in inflammation. Colchicine also inhibits the tubulin-  dependent release of histamine from mast cells. As a result, there was a rapid  improvement in the pain and swelling in Lotta 's great toe.   After the gout attack subsided, Ms. Topaigne was placed on allopurinol, a drug  that inhibits urate production (see Chapter 8). During the next 6 months of allop-  urinol therapy, Ms. Topaigne 's blood urate levels decreased. She did not have  another gout attack during this time.   Dennis Veere. Dennis Veere was diagnosed with cholera. He was  placed on intravenous rehydration therapy, followed by oral rehydration  therapy with high glucose and Na + -containing fluids (to be continued in  Chapter 11).   Vibrio cholerae secrete an A toxin that is processed and transported in the cell in  conjunction with the monomeric G protein Arf (ADP-ribosylation factor). The A    In epidermolysis bullosa simplex,  the skin blisters in response to a  very slight mechanical stress. The  familial form of the disease is generally  caused by mutations in either of the two  forms of keratin that constitute the keratin  heterodimers of the basal layer of the epider-  mis. The weakened keratin cytoskeleton  results in cytolysis when stress is applied.  The disease also can be caused by mutations  in plektin, the protein that attaches keratin to  the membrane protein integrin in  hemidesmosomes (a structure involved in  attaching cells to the extracellular matrix).   toxin ADP-ribosylates the G a subunit of a heterotrimeric G protein (a process  discussed in Chapter 11.) The net result is activation of protein kinase A, which then  phosphorylates the CFTR (cystic fibrosis transmembrane conductance regulator)  chloride channel so that it remains permanently open. The subsequent efflux of  chloride, sodium, and water into the bowel lumen is responsible for Dennis Veere's  diarrhea and subsequent dehydration.   The mitochondria of eukaryotic  \\ ) cells have many features in com-  VJ^/ mon with prokaryotic cells and, in  fact, may have originated when primordial  anaerobic eukaryotes engulfed ancient aero-  bic prokaryotes, establishing a symbiotic  relationship. These prokaryotes provided  eukaryotes with a more efficient mechanism  for oxidizing fuels to obtain energy.   f\ Differences between bacteria and human cells. Different  species of bacteria have some common structural features that distinguish  them from animal cells. They are single-cell organisms that are prokary-  otes ("before nucleus"). Their genetic material (DNA) is concentrated in the central  region of the cell called a nucleoid, rather than a nucleus, because it is not separated  from the rest of the cellular contents by a membrane. Likewise, bacteria contain no  cytoplasmic organelles defined by membranes. They do have a plasma membrane  that encloses the cytoplasm. External to this membrane is a peptidoglycan cell wall  composed of extensively cross-linked polysaccharides that form a protective shield  on the surface of the cell.   Bacterial cells obtain nutrients from the medium on which they grow. Many  of their metabolic pathways for fuel oxidation are similar to those in eukaryotes  and generate NADH and ATP; however, individual steps in these pathways may  use different coenzymes or very different enzymes for catalysis than do human  cells. Like human cells, bacteria use intermediates of glycolysis and other basic  degradative pathways to serve as precurors for biosynthetic pathways, and  energy acquired from catabolic pathways is used in anabolic pathways. Aerobic  bacteria, such as Escherichia coli, contain enzymes of the tricarboxylic acid  (TCA) cycle and the components of the electron transport chain, which are  located in the cell membrane. However, many bacteria are anaerobes and can  function in the absence of oxygen.   Many of the metabolic differences between human cells and bacteria are related  to their interactions with their environment. Some bacteria, such as E. coli, can  adapt to adverse or changing conditions (high versus low 0 2 tension or a single sup-  ply of nutrients from which to synthesize everything) by dramatic shifts in the genes  that are transcribed. Other bacteria find a unique environmental niche where they do  not have to compete with other bacteria for nutrients (e.g., Lactobaccili in yogurt  are adapted to an acidic pH). In contrast, the human cells are adapted to interacting  with blood and interstitial fluid, which provides a well-controlled pH, a constant  nutrient supply, and a medium for communication between very distant cells. As a  consequence of their constant environment, adult human cells seldom need to adapt  (or can adapt) to widely fluctuating conditions through large variations in the genes  transcribed. As a consequence of being organized into a multicellular organism,  human cell types have been able to specialize in function, structure, and enzyme  content.   Emmerson BT. The management of gout. N Engl J Med. 1996;334:445-451.   Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001;81:153-208. Although this is  a lengthy review for medical students, it is very useful in identifying the roles of different G proteins.   A number of extremely good cell and molecular biology textbooks have recently been published. Some  of the most recent textbooks that we found helpful include:   Karp G. Cell and Molecular Biology: Concepts and Experiments. 3rd Ed. New York: John Wiley and  Sons, 2002.   Cooper GM. The Cell: A Molecular Approach. 2nd Ed. Washington, DC: ASM Press, Sinauer Assoc., 2000.  Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J. Molecular Cell Biology. 4th  Ed. New York: W.H. Freeman, 2000. The section on cell structure is particularly good.  Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th Ed.  New York: Garland Science, 2002.   1 . Which of the following is a characteristic of the plasma membrane?   (C) It contains phospholipids with their acyl groups extending into the cytosol.   (D) It contains more phosphatidylserine in the inner than the outer leaflet.   (E) It contains oligosaccharides sandwiched between the inner and outer leaflets.   (A) can usually be dissociated from membranes without disrupting the lipid bilayer.   (D) contain hydrophilic amino acid residues extending into the lipid bilayer.   A patient had a sudden heart attack caused by inadequate blood flow through the vessels of the heart. As a consequence,  there was an inadequate supply of oxygen to generate ATP in his cardiomyocytes.   3. The compartment of the cardiomyocyte most directly involved in ATP generation is the   4. The transport process most directly affected by his decreased rate of ATP generation is   A patient in a nursing home who developed severe diarrhea was diagnosed with a Clostridium difficile infection. The severe  diarrhea associated with C. difficile is caused principally by two toxins that are UDP-glucosyltransf erases. These toxins  modify a monomeric G protein, thereby disrupting cellular attachments associated with the actin skeleton.   5. On the basis of its effect, which of the following G protein families is the most likely target of this modification?   Within a complex organism such as the human, different organs, tissues, and indi-  vidual cell types have developed specialized functions. Yet each cell must contribute  in an integrated way as the body grows, differentiates, and adapts to changing con-  ditions. Such integration requires communication that is carried out by chemical  messengers traveling from one cell to another or by direct contact of cells with the  extracellular matrix or with each other. The eventual goal of such signals is to  change actions carried out in target cells by intracellular proteins ( metabolic  enzymes, gene regulatory proteins, ion channels, or cytoskeletal proteins). In this  chapter, we present an overview of signaling by chemical messengers.   Chemical messengers. Chemical messengers (also called signaling molecules)  transmit messages between cells. They are secreted from one cell in response to a  specific stimulus and travel to a target cell, where they bind to a specific receptor  and elicit a response (Fig. 11.1). In the nervous system, these chemical messen-  gers are called neurotransmitters; in the endocrine system, they are hormones,  and in the immune system, they are called cytokines. Additional chemical mes-  sengers include retinoids, eicosanoids, and growth factors. Depending on the dis-  tance between the secreting and target cells, chemical messengers can be classi-  fied as endocrine (travel in the blood), paracrine (travel between nearby cells), or  autocrine (act on the same cell or on nearby cells of the same type).   Receptors and Signal Transduction, Receptors are proteins containing a bind-  ing site specific for a single chemical messenger and another binding site involved  in transmitting the message (see Fig. 11.1). The second binding site may interact  with another protein or with DNA. They may be either plasma membrane receptors  (which span the plasma membrane and contain an extracellular binding domain for  the messenger) or intracellular binding proteins (for messengers able to diffuse into  the cell) (see Fig. 11.1). Most plasma membrane receptors fall into the categories of  ion channel receptors, tyrosine kinase receptors, tyrosine-kinase associated recep-  tors (JAK-STAT receptors), serine-threonine kinase receptors, or heptahelical  receptors (proteins with seven a-helices spanning the membrane). When a chemical  messenger binds to a receptor, the signal it is carrying must be converted into an  intracellular response. This conversion is called signal transduction.   Signal Transduction for Intracellular Receptors. Most intracellular receptors  are gene-specific transcription factors, proteins that bind to DNA and regulate  the transcription of certain genes ( Gene transcription is the process of copying  the genetic code from DNA to RNA.).   Signal Transduction for Plasma Membrane Receptors. Mechanisms of signal  transduction that follow the binding of signaling molecules to plasma membrane  receptors include phosphorylation of receptors at tyrosine residues ( receptor tyro-  sine kinase activity), conformational changes in signal transducer proteins (e.g.,  proteins with SH2 domains, the monomeric G protein Ras, heterotrimeric G pro-  teins) or increases in the levels of intracellular second messengers. Second mes-  sengers are nonprotein molecules generated inside the cell in response to   hormone binding that continue transmission of the message. Examples include  3' ,5' -cyclic AMP (cAMP), inositol trisphosphate (IP 3 ), and diacy I glycerol (DAG).   Signaling often requires a rapid response and rapid termination of the mes-  sage, which may be achieved by degradation of the messenger or second messen-  ger, the automatic G protein clock, deactivation of signal transduction kinases by  phosphatases, or other means.   • Mya Sthenia is a 37-year-old woman who complains of increasing mus-  flP cle fatigue in her lower extremities with walking. If she rests for 5 to 10   " minutes, her leg strength returns to normal. She also notes that if she talks  on the phone, her ability to form words gradually decreases. By evening, her upper  eyelids droop to the point that she has to pull her upper lids back in order to see nor-  mally. These symptoms are becoming increasingly severe. When Mya is asked to  sustain an upward gaze, her upper eyelids eventually drift downward involuntarily.  When she is asked to hold both arms straight out in front of her for as long as she  is able, both arms begin to drift downward within minutes. Her physician suspects  that Mya Sthenia has myasthenia gravis and orders a test to determine whether she  has antibodies in her blood directed against the acetylcholine receptor.   Ann O'Rexia, who suffers from anorexia nervosa, has increased her  weight to 102 lb from a low of 85 lb (see Chapter 9). On the advice of her  physician, she has been eating more to prevent fatigue during her daily  jogging regimen. She runs about 10 miles before breakfast every second day and  forces herself to drink a high-energy supplement immediately afterward.   • Dennis Veere was hospitalized for dehydration resulting from cholera toxin  (see Chapter 10). In his intestinal mucosal cells, cholera A toxin indirectly   " activated the CFTR channel, resulting in secretion of chloride ion and Na +  ion into the intestinal lumen. Ion secretion was followed by loss of water, resulting in  vomiting and watery diarrhea. Dennis is being treated for hypovolemic shock.   Certain universal characteristics to chemical messenger systems are illustrated in  Figure 11.1. Signaling generally follows the sequence: (1) the chemical messenger  is secreted from a specific cell in response to a stimulus; (2) the messenger diffuses  or is transported through blood or other extracellular fluid to the target cell; (3) a  receptor in the target cell (a plasma membrane receptor or intracellular receptor)  specifically binds the messenger; (4) binding of the messenger to the receptor elic-  its a response; (5) the signal ceases and is terminated. Chemical messengers elicit  their response in the target cell without being metabolized by the cell.   Another general feature of chemical messenger systems is that the specificity of  the response is dictated by the type of receptor and its location. Generally, each  receptor binds only one specific chemical messenger, and each receptor initiates a  characteristic signal transduction pathway that will ultimately activate or inhibit  certain processes in the cell. Only certain cells, the target cells, carry receptors for  that messenger and are capable of responding to its message.   The means of signal termination is an exceedingly important aspect of cell signaling,  and failure to terminate a message contributes to a number of diseases, such as cancer.    Acetylcholine is released by neu-  rons and acts on acetylcholine  receptors at neuromuscular junc-  tions to stimulate muscular contraction.  Myasthenia gravis is an acquired autoim-  mune disease in which the patient has devel-  oped pathogenic antibodies against these  receptors. Mya Sthenia's decreasing ability to  form words and her other symptoms of mus-  cle weakness are being caused by the inabil-  ity of acetylcholine to stimulate repeated  muscle contraction when the numbers of  effective acetylcholine receptors at neuro-  muscular junctions are greatly reduced.   Endocrine hormones enable Ann  O'Rexia to mobilize fuels from her  adipose tissue during her periods of  fasting and during jogging. While she fasts  overnight, a-cells of her pancreas increase  secretion of the polypeptide hormone  glucagon. The stress of prolonged fasting and  chronic exercise stimulates release of Corti-  sol, a steroid hormone, from her adrenal cor-  tex. The exercise of jogging also increases  secretion of the hormones epinephrine and  norepinephrine from the adrenal medulla.  Each of these hormones is being released in  response to a specific signal and causes a  characteristic response in a target tissue,  enabling her to exercise. However, each of  these hormones binds to a different type of  receptor and works in a different way.   Ann O'Rexia's fasting is accompa-  nied by high levels of the  endocrine hormone glucagon,  which is secreted in response to low blood  glucose levels. It enters the blood and acts  on the liver to stimulate a number of path-  ways, including the release of glucose from  glycogen stores (glycogenolysis) (see Chap-  ter 3). The specificity of its action is deter-  mined by the location of receptors. Although  liver parenchymal cells have glucagon  receptors, skeletal muscle and many other  tissues do not. Therefore, glucagon cannot  stimulate glycogenolysis in these tissues.   and endocrine hormones) are con-  tained in vesicles that fuse with a region of  the cell membrane when the cell receives a  stimulus to release the messenger. Most  secretory cells use a similar set of proteins to  enable vesicle fusion, and fusion is usually  triggered by Ca 2+ influx, as seen with the  release of acetylcholine.   Myasthenia gravis is a disease of  autoimmunity caused by the pro-  duction of an antibody directed  against the acetylcholine receptor in skeletal  muscle. In this disease, B and T lympho-  cytes cooperate in producing a variety of  antibodies against the nicotinic acetyl-  choline receptor. The antibodies then bind  to various locations in the receptor and  cross-link the receptors, forming a multi-  receptor antibody complex. The complex is  endocytosed and incorporated into lyso-  somes, where it is degraded. Mya Sthenia,  therefore, has fewer functional receptors for  acetylcholine to activate.   A. General Features of Chemical Messenger Systems  Applied to the Nicotinic Acetylcholine Receptor   The individual steps involved in cell signaling by chemical messengers are illus-  trated with acetylcholine, a neurotransmitter that acts on nicotinic acetylcholine  receptors on the plasma membrane of certain muscle cells. This system exhibits the  classic features of chemical messenger release and specificity of response.   Neurotransmitters are secreted from neurons in response to an electrical stimu-  lus called the action potential (a voltage difference across the plasma membrane,  caused by changes in Na + and K + gradients, that is propagated along a nerve). The  neurotransmitters diffuse across a synapse to another excitable cell, where they  elicit a response (Fig. 1 1.2). Acetylcholine is the neurotransmitter at neuromuscular  junctions, where it transmits a signal from a motor nerve to a muscle fiber that elic-  its contraction of the fiber. Before release, acetylcholine is sequestered in vesicles  clustered near an active zone in the presynaptic membrane. This membrane also has  voltage-gated Ca 2+ channels that open when the action potential reaches them,  resulting in an influx of Ca 2+ . Ca 2+ triggers fusion of the vesicles with the plasma  membrane, and acetylcholine is released into the synaptic cleft. Thus, the chemical  messenger is released from a specific cell in response to a specific stimulus.   Acetylcholine diffuses across the synaptic cleft to bind to plasma membrane  receptors on the muscle cells called nicotinic acetylcholine receptors (Fig. 11.3).  The subunits are assembled around a channel, which has a funnel-shaped opening  in the center. As acetylcholine binds to the receptor, a conformational change opens  the narrow portion of the channel (the gate), allowing Na + to diffuse in and K + to  diffuse out (A uniform property of all receptors is that signal transduction begins  with conformational changes in the receptor.). The change in ion concentration    Fig. 11.3. The nicotinic acetylcholine recep-  tor. Each receptor is composed of five sub-  units, and each subunit has four membrane-  spanning helical regions. The two a subunits  are identical and contain binding sites for  acetylcholine. When two acetylcholine mole-  cules are bound, the subunits change their con-  formation so that the channel in the center of  the receptor is open, allowing K + ions to dif-  fuse out and Na + ions to diffuse in.   Fig. 11.2. Acetylcholine receptors at the neuromuscular junction. A motor nerve terminates  in several branches; each branch terminates in a bulb-shaped structure called the presynaptic  bouton. Each bouton synapses with a region of the muscle fiber containing junctional folds.  At the crest of each fold, there is a high concentration of acetylcholine receptors, which are  gated ion channels.   activates a sequence of events that eventually triggers the cellular response —  contraction of the fiber.   Once acetylcholine secretion stops, the message is rapidly terminated by acetyl-  cholinesterase, an enzyme located on the postsynaptic membrane that cleaves  acetylcholine. It is also terminated by diffusion of acetylcholine away from the  synapse. Rapid termination of message is a characteristic of systems requiring a  rapid response from the target cell.   The actions of chemical messengers are often classified as endocrine, paracrine, or  autocrine (Fig. 11.4). Each endocrine hormone is secreted by a specific cell type   Mya Sthenia was tested with an  inhibitor of acetylcholinesterase,  edrophonium chloride, adminis-  intravenously (see Chapter 8, Fig.  After this drug inactivates acetyl-  cholinesterase, acetylcholine that is released  from the nerve terminal accumulates in the  synaptic cleft. Even though Mya expresses  fewer acetylcholine receptors on her muscle  cells (due to the auto-antibody-induced  degradation of receptors), by increasing the  local concentration of acetylcholine, these  receptors have a higher probability of being  occupied and activated. Therefore, acute  intravenous administration of this short-  acting drug briefly improves muscular weak-  ness in patients with myasthenia gravis.   Fig. 11.4. Endocrine, autocrine, and paracrine actions of hormones and other chemical mes-  sengers.   (generally in an endocrine gland), enters the blood, and exerts its actions on specific  target cells, which may be some distance away. In contrast to endocrine hormones,  paracrine actions are those performed on nearby cells, and the location of the cells  plays a role in specificity of the response. Synaptic transmission by acetylcholine  and other neurotransmitters (sometimes called neurocrine signaling) is an example  of paracrine signaling. Acetylcholine activates only those acetylcholine receptors  located across the synaptic cleft from the signaling nerve and not all muscles with  acetylcholine receptors. Paracrine actions are also very important in limiting the  immune response to a specific location in the body, a feature that helps prevent the  development of autoimmune disease. Autocrine actions involve a messenger acting  on the cell from which it is secreted, or on nearby cells that are the same type as the  secreting cells.   H 3 C-C-j-0-CH 2 -CH 2 - N(CH 3 ) 3  Acetylcholine   -OOC-CH 2 -CH 2 -CH 2 - NH 3  y-aminobutyrate (GAB A)   Three major signaling systems in the body employ chemical messengers: the nerv-  ous system, the endocrine system, and the immune system. Some messengers are  difficult to place in just one such category.   The nervous system secretes two types of messengers: small molecule neurotrans-  mitters, often called biogenic amines, and neuropeptides. Small molecule neuro-  transmitters are nitrogen-containing molecules, which can be amino acids or are  derivatives of amino acids (e.g., acetylcholine and 7-aminobutyrate, Fig. 11.5).  Neuropeptides are usually small peptides (between 4 and 35 amino acids), secreted  by neurons, that act as neurotransmitters at synaptic junctions or are secreted into  the blood to act as neurohormones.   Q o The catecholamine hormone epi-  ( / \vi 5 nephrine (also called adrenaline) is  ^ the fight, fright, and flight hor-  mone. Epinephrine and the structurally sim-  ilar hormone norepinephrine are released  from the adrenal medulla in response to a  variety of immediate stresses, including  pain, hemorrhage, exercise, hypoglycemia,  and hypoxia. Thus, as Ann O'Rexia begins to  jog, there is a rapid release of epinephrine  and norepinephrine into the blood.   Endocrine hormones are defined as compounds, secreted from specific endocrine  cells in endocrine glands, that reach their target cells by transport through the  blood. Insulin, for example, is an endocrine hormone secreted from the (3 cells  of the pancreas. Classic hormones are generally divided into the structural cate-  gories of polypeptide hormones (e.g., insulin -see Chapter 6, Fig. 6.15 for the  structure of insulin ), catecholamines such as epinephrine (which is also a neu-  rotransmitter), steroid hormones (which are derived from cholesterol), and thy-  roid hormone (which is derived from tyrosine). Many of these endocrine hor-  mones also exert paracrine or autocrine actions. The hormones that regulate  metabolism are discussed throughout this chapter and in subsequent chapters of  this text.   Some compounds normally considered hormones are more difficult to catego-  rize. For example, retinoids, which are derivatives of vitamin A (also called retinol)  and vitamin D (which is also derived from cholesterol) are usually classified as hor-  mones, although they are not synthesized in endocrine cells.   The messengers of the immune system, called cytokines, are small proteins with a   factors) are secreted by cells of the immune system and usually alter the behavior  of other cells in the immune system by activating the transcription of genes for pro-  teins involved in the immune response.   Selection and Proliferation of B Cells Producing the Desired Antibody. Interleukins, a class of cytokine, illustrate some of the sig-  naling involved in the immune response. Interleukins are polypeptide factors with molecular weights ranging from 15,000 to  25,000 Daltons. They participate in a part of the immune response called humoral immunity, which is carried out by a popula-  tion of lymphoid B cells producing just one antibody against one particular antigen. The proliferation of cells producing that particular  antibody is mediated by receptors and by certain interleukins.   Bacteria phagocytized by macrophages are digested by lysosomes. (1) A partially digested fragment of the bacterial protein (the blue  antigen) is presented on the extracellular surface of the macrophage by a membrane protein called an MHC (major histocompatibility com-  plex). (2) Certain lymphoid cells called T-helper cells contain receptors that can bind to the displayed antigen-MHC complex, a process  that activates the T-cell (direct cell-to-cell signaling, requiring recognition molecules). (3) The activated T-helper cell then finds and binds  to a B cell whose antigen receptor binds a soluble fragment of that same bacterially derived antigen molecule (again, direct cell-to-cell sig-  naling). The bound T cell secretes interleukins, which act on the B cell (a paracrine signal). The interleukins thus stimulate proliferation of  only those B cells capable of synthesizing and secreting the desirable antibody. Furthermore, the interleukins determine which class of  antibody is produced.   The eicosanoids (including prostaglandins [PG], thromboxanes, and leukotrienes)  control cellular function in response to injury (Fig. 11.6) These compounds are all  derived from arachidonic acid, a 20-carbon polyunsaturated fatty acid that is usu-  ally present in cells as part of the membrane lipid phosphatidylcholine (see Chap-  ter 5, Fig. 5.21). Although almost every cell in the body produces an eicosanoid in  response to tissue injury, different cells produce different eicosanoids. The  eicosanoids act principally in paracrine and autocrine functions, affecting the cells  that produce them or their neighboring cells. For example, vascular endothelial cells  (cells lining the vessel wall) secrete the prostaglandin PGI 2 (prostacyclin), which  acts on nearby smooth muscle cells to cause vasodilation (expansion of the blood  vessel).   Growth factors are polypeptides that function through stimulation of cellular prolifer-  ation. For example, platelets aggregating at the site of injury to a blood vessel secrete   Lotta Topaigne suffered enormously painful gout attacks affecting her great toe  (see Clinical Comments, Chapter 8). The extreme pain was caused by the release  of a leukotriene that stimulated pain receptors. The precipitated urate crystals in  her big toe stimulated recruited inflammatory cells to release the leukotriene.    Fig. 11.6. Eicosanoids are derived from  arachidonic acid and retain its original 20 car-  bons (thus the name eicosanoids). All  prostaglandins, such as prostacyclin, also have  an internal ring.   PDGF (platelet-derived growth factor). PDGF stimulates the proliferation of nearby  smooth muscle cells, which eventually form a plaque covering the injured site. Some  growth factors are considered hormones, and some have been called cytokines.   Each of the hundreds of chemical messengers has its own specific receptor,  which will usually bind no other messenger.   Fig. 11.7. Intracellular vs. plasma membrane  receptors. Plasma membrane receptors have  extracellular binding domains. Intracellular  receptors bind steroid hormones or other mes-  sengers able to diffuse through the plasma  membrane. Their receptors may reside in the  cytoplasm and translocate to the nucleus,  reside in the nucleus bound to DNA, or reside  in the nucleus bound to other proteins.   The structural properties of a messenger determine, to some extent, the type of  receptor it binds. Most receptors fall into two broad categories: intracellular recep-  tors or plasma membrane receptors (Fig. 11.7) Messengers using intracellular  receptors must be hydrophobic molecules able to diffuse through the plasma mem-  brane into cells. In contrast, polar molecules such as peptide hormones, cytokines,  and catecholamines cannot rapidly cross the plasma membrane and must bind to a  plasma membrane receptor.   Most of the intracellular receptors for lipophilic messengers are gene-specific  transcription factors. A transcription factor is a protein that binds to a specific site  on DNA and regulates the rate of transcription of a gene (i.e., synthesis of the  mRNA). External signaling molecules bind to transcription factors that bind to a  specific sequence on DNA and regulate the expression of only certain genes; they  are called gene- specific or site- specific transcription factors.   Lipophilic hormones that use intracellular gene- specific transcription factors  include the steroid hormones, thyroid hormone, retinoic acid (active form of vita-  min A), and vitamin D (Fig. 11.8). Because these compounds are water-insoluble,  they are transported in the blood bound to serum albumin, which has a hydrophobic  binding pocket, or to a more specific transport protein, such as steroid hormone-  binding globulin (SHBG) and thyroid hormone-binding globulin (TBG). The intra-  cellular receptors for these hormones are structurally similar and are referred to as  the steroid hormone/thyroid hormone superfamily of receptors.   The steroid hormone/thyroid hormone superfamily of receptors reside primarily  in the nucleus, although some are found in the cytoplasm. The glucocorticoid recep-  tor, for example, exists as cytoplasmic multimeric complexes associated with heat  shock proteins. When the hormone Cortisol (a glucocorticoid) binds, the receptor  undergoes a conformational change and dissociates from the heat shock proteins,  exposing a nuclear translocation signal (see Chapter 10, Section VI.) The receptors  dimerize, and the complex (including bound hormone) translocates to the nucleus,  where it binds to a portion of the DNA called the hormone response element (e.g.,   The steroid hormone Cortisol is synthesized and released from the adrenal cor-  tex in response to the polypeptide hormone ACTH (adrenal corticotrophic hor-  mone). Chronic stress (pain, hypoglycemia, hemorrhage, and exercise) signals  are passed from the brain cortex to the hypothalamus to the anterior pituitary, which  releases ACTH. Cortisol acts on tissues to change enzyme levels and redistribute nutrients  in preparation for acute stress. For example, it increases transcription of the genes for  regulatory enzymes in the pathway of gluconeogenesis, thereby increasing the content of  these enzymes (called gene-specific activation of transcription, or induction of protein  synthesis). Induction of gluconeogenic enzymes prepares the liver to respond rapidly to  hypoglycemia with increased synthesis of glucose. Ann O'Rexia, who has been frequently  fasting and exercising, has an increased capacity for gluconeogenesis in her liver.   H-C-CH 2 -CH 2 -CH 2 -C-OH  H 3 C | CHo   Fig. 11.8. Steroid hormone/thyroid hormone superfamily. A. Cortisol (a glucocorticoid). B. Aldosterone (an androgen). C. Thyroid hormone D.  Vitamin D 3 . E. Retinoids.   the glucocorticoid receptor binds to the glucocorticoid response element, GRE).  Most of the intracellular receptors reside principally in the nucleus, and some of  these are constitutively bound, as dimers, to their response element in DNA (e.g.,  the thyroid hormone receptor). Binding of the hormone changes its activity and its  ability to associate with, or disassociate from, DNA. Regulation of gene transcrip-  tion by these receptors is described in Chapter 16.   Recently several nuclear receptors have been identified that play important  \ \ ) roles in intermediary metabolism, and they have become the target of lipid-  *S lowering drugs. These include the peroxisome proliferator activated receptors  (PPAR a, p and 7), the liver X-activated receptor (LXR), the farnesoid X-activated recep-  tors (FXR), and the pregnane X receptor (PXR). These receptors form heterodimers with  the 9-c/s retinoic acid receptor (RXR) and bind to their appropriate response elements in  DNA in an inactive state. When the activating ligand binds to the receptor (oxysterols for  LXR, bile salts for FXR, secondary bile salts for PXR, and fatty acids and their derivatives  for the PPARs), the complex is activated, and gene expression is altered. Unlike the Cor-  tisol receptor, these receptors reside in the nucleus and are activated once their ligands  enter the nucleus and bind to them.   Signal transduction pathways, like  a river, run in one direction. From a  ^t<^ given point in a signal transduction  pathway, events closer to the receptor are  referred to as "upstream," and events closer  to the response are referred to as "down-  stream."   /^5V Protein kinases transfer a phos-  \\ phate group from ATP to the  \^^/ hydroxyl group of a specific  amino acid residue in the protein. Tyrosine  kinases transfer the phosphate group to  the hydroxyl group of a specific tyrosine  residue and serine/threonine protein  kinases to the hydroxyl of a specific serine  or threonine residue (serine is more often  phosphorylated than threonine in target  proteins). Different protein kinases have  specificity for distinct amino acid  sequences (containing a tyrosine, serine,  or threonine). Thus, two different protein  kinases target distinct sequences (and  usually different proteins) for phosphory-  lation. A protein containing both target  sequences could be a substrate for both  protein kinases.   All plasma membrane receptors are proteins with certain features in common: an  extracellular domain that binds the chemical messenger, one or more membrane-  spanning domains that are a-helices, and an intracellular domain that initiates sig-  nal transduction. As the ligand binds to the extracellular domain of its receptor, it  causes a conformational change that is communicated to the intracellular domain  through the rigid a-helix of the transmembrane domain. The activated intracellular  domain initiates a characteristic signal transduction pathway that usually involves  the binding of a specific intracellular signal transduction protein.   The pathways of signal transduction for plasma membrane receptors have two  major types of effects on the cell: (1) rapid and immediate effects on cellular ion  levels or activation/inhibition of enzymes and/or (2) slower changes in the rate of  gene expression for a specific set of proteins. Often, a signal transduction pathway  will diverge to produce both kinds of effects.   Individual plasma membrane receptors are grouped into the categories of ion chan-  nel receptors, receptors that are kinases or bind kinases, and receptors that work  through second messengers. This classification is based on the receptor's general  structure and means of signal transduction.   The ion channel receptors are similar in structure to the nicotinic acetylcholine  receptor (see Fig. 11.3). Signal transduction consists of the conformational change  when ligand binds. Most small molecule neurotransmitters and some neuropeptides  use ion channel receptors.   Several types of receptors that are kinases or bind kinases are illustrated in Figure  11.9. Their common feature is that the intracellular domain of the receptor (or an  associated protein) is a kinase that is activated when the messenger binds to the  extracellular domain. The receptor kinase phosphorylates an amino acid residue on  the receptor (autophosphorylation) or an associated protein. The message is propa-  gated through signal transducer proteins that bind to the activated messenger-  receptor complex (e.g., Grb2, STAT, or Smad).   Fig. 11.9. Receptors that are kinases or bind kinases. The kinase domains are shown in blue, and the phosphorylation sites are indicated with  blue arrows. A. Tyrosine kinase receptors. B. JAK-STAT receptors. C. Serine/threonine kinase receptors.   Heptahelical receptors (which contain 7-membrane spanning a-helices) are the  most common type of plasma membrane receptor. They work through second mes-  sengers, which are small nonprotein compounds, such as cAMP, generated inside  the cell in response to messenger binding to the receptor (Fig. 11.10). They continue  intracellular transmission of the message from the hormone/cytokine/neurotrans-  mitter, which is the "first" messenger. Second messengers are present in low con-  centrations so that their concentration, and hence the message, can be rapidly  initiated and terminated.   The tyrosine kinase receptors are summarized in Figure 1 1.9A. They generally exist  in the membrane as monomers with a single membrane-spanning helix. One mole-  cule of the growth factor generally binds two molecules of the receptor and pro-  motes their dimerization (Fig. 11.11). Once the receptor dimer has formed, the  intracellular tyrosine kinase domains of the receptor phosphorylate each other on  certain tyrosine residues (autophosphorylation). The phosphotyrosine residues form  specific binding sites for signal transducer proteins.   One of the domains of the receptor containing a phosphotyrosine residue forms a  binding site for intracellular proteins with a specific three-dimensional structure  known as the SH2 domain (the Src homology 2 domain, named for the first protein  in which it was found, the src protein of the Rous sarcoma virus). The adaptor   Fig. 11.10. Heptahelical Receptors and Second  Messengers. The secreted chemical messenger  (hormone, cytokine, or neurotransmitter) is the  first messenger, which binds to a plasma mem-  brane receptor such as the heptahelical recep-  tors. The activated hormone-receptor complex  activates a heterotrimeric G protein and via  stimulation of membrane-bound enzymes, dif-  ferent G-proteins lead to generation of one or  more intracellular second messengers, such as  cAMP, diacylglycerol (DAG), or inositol  trisphosphate (IP 3 ).   Although many different signal transducer proteins have SH2 domains, and  many receptors have phosphotyrosine residues, each signal transducer protein  is specific for one type of receptor. This specificity of binding results from the  fact that each phosphotyrosine residue has a different amino acid sequence around it  that forms the binding domain. Likewise, the SH2 domain of the transducer protein is  only part of its binding domain.    2. Auto-crossphosphorylation 3. Binding of adaptor proteins 6. Ras binds raf and initiates   Fig. 11.11. Signal transduction by tyrosine kinase receptors. (1) Binding and dimerizaion. (2) Autophosphorylation. (3) Binding of Grb2 and SOS.  (4) SOS is a GEF (guanine nucleotide exchange protein) that binds Ras, a monomeric G protein anchored to the plasma membrane. (5) GEF acti-  vates the exchange of GTP for bound GDP on Ras. (6) Activated Ras containing GTP binds the target enzyme Raf, thereby activating it.   Fig. 11.12. Major route for generation of the  phosphatidyl inositide signal molecules, inosi-  tol 1,4,5-trisphosphate (IP 3 ) and phos-  phatidylinositol 3,4,5 trisphosphate (PI-3,4,5-  trisP). PI 3-kinase phosphorylates PI-4,5-bisP  and PI-4P at the 3 position. Prime symbols are  sometimes used in these names to denote the  inositol ring. DAG is also a second messenger.   protein Grb2, which is bound to a membrane phosphoinositide, is one of the  proteins with an SH2 domain that binds to phosphotyrosine residues on growth fac-  tor receptors. Binding to the receptor causes a conformational change in Grb2 that  activates another binding site called an SH3 domain. These activated SH3 domains  bind the protein SOS (SOS is an acronym for "son of sevenless," a name unrelated  to the function or structure of the compound). SOS is a guanine nucleotide  exchange factor (GEF) for Ras, a monomeric G protein located in the plasma mem-  brane (see Chapter 9, Section III.C.2.) SOS activates exchange of guanosine  triphosphate (GTP) for guanosine diphosphate (GDP) on Ras, causing a conforma-  tional change in Ras that promotes binding of the protein Raf. Raf is a serine pro-  tein kinase that is also called MAPKKK (mitogen activated protein kinase kinase  kinase.) Raf begins a sequence of successive phosphorylation steps called a phos-  phorylation cascade (When a kinase in a cascade is phosphorylated, it binds and  phosphorylates the next enzyme in the cascade.). The MAP kinase cascade termi-  nates at a gene transcription factor, thereby regulating transcription of certain genes  involved in cell survival and proliferation.   Many tyrosine kinase receptors (as well as heptahelical receptors) also have  additional signaling pathways involving phosphatidylinositol phosphates.   Phosphatidylinositol phosphates serve two different functions in signal transduction:  (1) Phosphatidylinositol 4', 5' bisphosphate (PI-4,5-bisP) can be cleaved to generate the  two intracellular second messengers, diacylglycerol (DAG) and inositol trisphosphate  (IP 3 ); and (2) Phosphatidylinositol 3 ',4', 5' trisphosphate (PI-3,4,5-trisP) can serve as a  plasma membrane docking site for signal transduction proteins.   Phosphatidyl inositol, which is present in the inner leaflet of the plasma membrane,  is converted to PI-4,5-bisP by kinases that phosphorylate the inositol ring at the 4' and  5' positions (Fig. 11.12). PI-4,5-bisP, which has three phosphate groups, is cleaved by  a phospholipase C-isozyme to generate IP 3 and DAG. The phospholipase isozyme C 7  (PLC 7 ) is activated by tyrosine kinase growth factor receptors, and phospholipase Cp  is activated by a heptahelical receptor-G protein signal transduction pathway.   PI-4,5-bisP can also be phosphorylated at the 3' position of inositol by the enzyme  phosphatidylinositol 3' kinase (PI 3-kinase) to form PI -3,4,5- trisP (see Fig. 11.12).  PI-3,4,5- tris P (and PI -3,4 bis P) form membrane docking sites for proteins contain-  ing a certain sequence of amino acids called the pleckstrin homology (PH) domain. PI  3- kinase contains an SH2 domain and is activated by binding to a specific phosphoty-  rosine site on a tyrosine kinase receptor or receptor-associated protein.   The insulin receptor, a member of the tyrosine kinase family of receptors, provides  a good example of divergence in the pathway of signal transduction. Unlike other  growth factor receptors, the insulin receptor exists in the membrane as a preformed  dimer, with each half containing an a and a p subunit (Fig. 11.13). The p subunits   Insulin is a growth factor that is essential for cell viability and growth. It  increases general protein synthesis, which strongly affects muscle mass. How-  ever, it also regulates immediate nutrient availability and storage, including  glucose transport into skeletal muscle and glycogen synthesis. Thus, Di Abietes and  other patients with type I diabetes mellitus who lack insulin rapidly develop hyper-  glycemia once insulin levels drop too low. They also exhibit muscle "wasting." To medi-  ate the diverse regulatory roles of insulin, the signal transduction pathway diverges after  activation of the receptor and phosphorylation of IRS, which has multiple binding sites  for different signal mediator proteins.    Fig. 11.13. Insulin receptor signaling. The insulin receptor is a dimer of two membrane-spanning  a-(3 pairs. The tyrosine kinase domains are shown in blue, and arrows indicate auto-crosspho-  sphorylation. The activated receptor binds IRS molecules (insulin receptor substrates) and phos-  phorylates IRS at multiple sites, thereby forming binding sites for proteins with SH2 domains:  Grb2, phospholipase C^CPLC^), and PI 3 -kinase. These proteins are associated with various  phosphatidylinositol phosphates (all designated with PIP) in the plasma membrane.   autophosphorylate each other when insulin binds, thereby activating the receptor.  The activated phosphorylated receptor binds a protein called IRS (insulin receptor  substrate). The activated receptor kinase phosphorylates IRS at multiple sites, cre-  ating multiple binding sites for different proteins with SH2 domains. One of the  sites binds Grb2, leading to activation of Ras and the MAP kinase pathway. Grb2 is  anchored to PI-3,4,5-trisP in the plasma membrane through its PH (pleckstrin  homology) domain. At another phosphotyrosine site, PI 3 -kinase binds and is acti-  vated. At a third site, phospholipase C 7 (PLC 7 ) binds and is activated. The insulin  receptor can also transmit signals through a direct docking with other signal trans-  duction intermediates.   The signal pathway initiated by the insulin receptor complex involving PI 3-  kinase leads to activation of protein kinase B, a serine-threonine kinase that medi-  ates many of the downstream effects of insulin (Fig. 11.14). PI 3- kinase binds and  phosphorylates PI-4,5- bis P in the membrane to form PI-3,4,5- trisP. Protein kinase   Protein kinase B is a serine-  \ \ ) threonine kinase, also known as  Akt. One of the signal transduction  pathways from protein kinase B (Akt) leads to  the effects of insulin on glucose metabolism.  Other pathways, long associated with Akt,  result in the phosphorylation of a host of  other proteins that affect cell growth, cell  cycle entry, and cell survival. In general, phos-  phorylation of these proteins by Akt inhibits  their action and promotes cell survival.    Fig. 11.14. The insulin receptor-protein kinase B signaling pathway. Abbreviations: Ins, insulin; IRS, insulin receptor substrate; PH domains,  pleckstrin homology domains; PDK1, phosphoinositide-dependent protein kinase 1; PKB, protein kinase B. The final phosphorylation step that  activates PKB is shown in blue.   B and PDK1 (phosphoinositide-dependent kinase- 1) are recruited to the membrane  by their PH domains, where PDK1 phosphorylates and activates protein kinase B.  Many other signal transducer proteins have PH domains and are docked at the mem-  brane, where they can find and bind each other. Thus, the insulin signal diverges  again and again. Insulin is covered in more detail in Chapters 26, 36 and 43.   Although Jak is an acronym for  \ \ ) janus kinase, it has been sug-  gested that it stands for "just  another kinase". It was named for Janus, a  two-headed god of the Romans.   Tyrosine kinase-associated receptors called Jak- STAT receptors are often used by  cytokines to regulate the proliferation of certain cells involved in the immune  response (see Fig. 11. 9B). The receptor itself has no intrinsic kinase activity but  binds (associates with) the tyrosine kinase Jak (janus kinase). Their signal trans-  ducer proteins, called STATs (signal transducer and activator of transcription), are  themselves gene- specific transcription factors. Thus, Jak- STAT receptors have a  more direct route for propagation of the signal to the nucleus than tyrosine kinase  receptors.   Each receptor monomer has an extracellular domain, a membrane-spanning  region, and an intracellular domain. As the cytokine binds to these receptors, they  form dimers (either homodimers or heterodimers, between two distinct receptor mol-  ecules) and may cluster (Fig. 11.15). The activated Jaks phosphorylate each other  and intracellular tyrosine residues on the receptor, forming phosphotyrosine-binding  sites for the SH2 domain of a STAT. STATs are inactive in the cytoplasm until they  bind to the receptor complex, where they are also phosphorylated by the bound JAK.  Phosphorylation changes the conformation of the STAT, causing it to dissociate from  the receptor and dimerize with another phosphorylated STAT, thereby forming an  activated transcription factor. The STAT dimer translocates to the nucleus and binds  to a response element on DNA, thereby regulating gene transcription.   There are many different STAT proteins, each with a slightly different amino  acid sequence. Receptors for different cytokines bind different STATs, which then  form heterodimers in various combinations. This microheterogeneity allows differ-  ent cytokines to target different genes.   Proteins in the transforming growth factor superfamily use receptors that have ser-  ine/threonine kinase activity and associate with proteins from the Smad family,  which are gene-specific transcription factors (see Fig. 11. 9C). This superfamily  includes transforming growth factor (3 (TGF-|3), a cytokine/hormone involved in  tissue repair, immune regulation, and cell proliferation, and bone morphogenetic  proteins (BMPs), which control proliferation, differentiation, and cell death during  development.   Fig. 11.16. Serine/threonine receptors and Smad proteins. TGF-p (transforming growth factor p), which is composed of two identical subunits,  communicates through a receptor dimer of type I and type II subunits that have serine kinase domains. The type I receptor phosphorylates an  R-Smad (receptor- specific Smad), which binds a Co-Smad (common Smad, also called Smad 4).   A simplified version of TGF-P 1 binding to its receptor complex and activating  Smads is illustrated in Fig. 11.16 The TGF-P receptor complex is composed of two  different single membrane- spanning receptor subunits (type I and type II), which  have different functions even though they both have serine kinase domains. TGF-P  binds to a type II receptor. The activated type II receptor recruits a type I receptor,  which it phosphorylates at a serine residue, forming an activated receptor complex.  The type I receptor then binds a receptor- specific Smad protein (called R-Smads),  which it phosphorylates at serine residues. The phosphorylated R-Smad undergoes  a conformational change and dissociates from the receptor. It then forms a complex  with another member of the Smad family, Smad 4 (Smad 4 is known as the com-  mon Smad, Co-Smad, and is not phosphorylated). The Smad complex, which may  contain several Smads, translocates to the nucleus, where it activates or inhibits the  transcription of target genes. Receptors for different ligands bind different Smads,  which bind to different sites on DNA and regulate the transcription of different  genes.   The heptahelical receptors are named for their 7 -membrane spanning domains,  which are a-helices (see Fig. 11.10; see also Chapter 7, Fig. 7.10). Although hun-  dreds of hormones and neurotransmitters work through heptahelical receptors, the  extracellular binding domain of each receptor is specific for just one polypeptide  hormone, catecholamine, or neurotransmitter (or its close structural analog).  Heptahelical receptors have no intrinsic kinase activity but initiate signal transduc-  tion through heterotrimeric G proteins composed of a, P and 7 subunits. However,  different types of heptahelical receptors bind different G proteins, and different G  proteins exert different effects on their target proteins.   The function of heterotrimeric G proteins is illustrated in Figure 11.17 using a hor-  mone that activates adenylyl cyclase (e.g., glucagon or epinephrine). While the a  subunit contains bound GDP, it remains associated with the p and 7 subunits, either  free in the membrane or bound to an unoccupied receptor (see Fig. 11.17, part 1).  When the hormone binds, it causes a conformational change in the receptor that acti-  vates GDP dissociation and GTP binding. The exchange of GTP for bound GDP  causes dissociation of the a subunit from the receptor and from the P7 subunits (see  Fig. 11.17, part 2). The a and p subunits are tethered to the intracellular side of the    Fig. 11.17. Heptahelical receptors and heterotrimeric G proteins. (1) The intracellular domains of the receptor form a binding site for a G pro-  tein containing GDP bound to the a-subunit. (2) Hormone binding to the receptor promotes the exchange of GTP for GDP. As a result, the com-  plex disassembles, releasing the G protein a-subunit from the p7 complex. (3) The Gs a-subunit binds to a target enzyme, thereby changing its  activity. The p7 complex may simultaneously target another protein and change its activity. (4) Over time, bound GTP is hydrolysed to GDP,  causing dissociation of the a-subunit from adenylyl cyclase. The GDP-a-subunit reassociates with the p7 subunit and the receptor.   0 o The importance of signal termina-  ( /~oA ) tion is illustrated by the "internal  ™ clock" of G proteins, which is the  rate of spontaneous hydrolysis of GTP to GDP.  Mutations in ras (the gene encoding Ras) that  decrease the rate of GTP hydrolysis are found  in about 20 to 30% of all human cancers,  including approximately 25% of lung cancers,  50% of colon cancers, and more than 90% of  pancreatic cancers. In these mutations of Ras,  GTP hydrolysis is decreased and Ras remains  locked in the active GTP-bound form, rather  than alternating normally between inactive  and active state in response to extracellular  signals. Consequently, MAP kinase pathways  are continuously stimulated and drive cell pro-  liferation, even in the absence of growth fac-  tors that would be required for ras activation  in normal cells.   plasma membrane through lipid anchors, but can still move around on the membrane  surface. The GTP-a subunit binds its target enzyme in the membrane, thereby chang-  ing its activity. In this example, the a-subunit binds and activates adenylyl cyclase,  thereby increasing synthesis of cAMP (see Fig. 11.17, part 3).   With time, the Ga subunit inactivates itself by hydrolyzing its own bound GTP  to GDP and Pj. This action is unrelated to the number of cAMP molecules formed.  Like the monomeric G proteins, the GDP-a subunit then dissociates from its target  protein, adenylyl cyclase (see Fig. 11.16, part 4). It reforms the trimeric G protein  complex, which may return to bind the empty hormone receptor. As a result of this  GTPase "internal clock," sustained elevations of hormone levels are necessary for  continued signal transduction and elevation of cAMP.   A large number of different heterotrimeric G protein complexes are generally  categorized according to the activity of the a subunit (Table 11.1). The 20 or more dif-  ferent isoforms of Ga fall into four broad categories: Ga s , Ga i/0 , Ga q/11 , and  Gal2/13 13 . Ga s refers to a subunits, which, like the one in Figure 11.17, stimulate  adenylyl cyclase (hence the s). Ga subunits that inhibit adenylyl cyclase are called Ga L  The p7 subunits likewise exist as different isoforms, which also transmit messages.   Acetylcholine has two types of receptors: nicotinic ion channel receptors, the receptors inhibited by antibodies in myasthenia  \\ gravis, and muscarinic receptors, which exist as a variety of subtypes. The M2 muscarinic receptors activate a Ga i/0 het-  erotrimeric G protein in which release of the p7 subunit controls K + channels and pacemaker activity in the heart. Epinephrine  has several types and subtypes of heptahelical receptors: (3 receptors work through a Ga s and stimulate adenylyl cyclase; a 2  receptors in other cells work through a Ga, protein and inhibit adenylyl cyclase; a-, receptors work through Ga q subunits and activate phos-  pholipase Cp. This variety in receptor types allows a messenger to have different actions in different cells.   Glucagon and epinephrine to  regulate metabolic enzymes, regulatory polypeptide hormones to control steroid hor-  mone and thyroid hormone synthesis, and by some neurotransmitters (e.g., dopamine)  to control ion channels   Epinephrine, many neurotransmitters including acetylcholine, dopamine, serotonin.  Has a role in the transducin pathway, which mediates detection of light  in the eye.   Epinephrine, acetylcholine, histamine, thyroid-stimulating hormone (TSH),  interleukin 8 , somatostatin, angiotensin  Thromboxane A2, lysophosphatidic acid   *There is a growing tendency to designate the heterotrimeric G protein subunits without using subscripts so that they are actually visible to the naked eye.   cAMP is referred to as a second messenger because changes in its concentration  reflect changes in the concentration of the hormone (the first messenger). When  a hormone binds and adenylyl cyclase is activated, it synthesizes cAMP from  adenosine triphosphate (ATP). cAMP is hydrolyzed to AMP by cAMP phospho-  diesterase, which also resides in the plasma membrane (Fig. 11.18). The  concentration of cAMP and other second messengers is kept at very low levels  in cells by balancing the activity of these two enzymes so that cAMP levels can  change rapidly when hormone levels change. Some hormones change the con-  centration of cAMP by targeting the phosphodiesterase enzyme rather than  adenylyl cyclase. For example, insulin lowers cAMP levels by causing phospho-  diesterase activation.   cAMP exerts diverse effects in cells. It is an allosteric activator of protein kinase  A (see Chapter 9, section III.B.3), which is a serine/threonine protein kinase that  phosphorylates a large number of metabolic enzymes, thereby providing a rapid  response to hormones such as glucagon and epinephrine. The catalytic subunits of  protein kinase A also enter the nucleus and phosphorylate a gene-specific tran-  scription factor called CREB (cyclic AMP response element-binding protein). Thus,  cAMP also activates a slower response pathway, gene transcription. In other cell  types, cAMP directly activates ligand-gated channels.   Certain heptahelical receptors bind the q isoform of the Ga subunit (Ga q ), which  activates the target enzyme phospholipase C p (see Fig. 11.12). When activated,  phospholipase C p hydrolyzes the membrane lipid phosphatidyl inositol bis phos-  phate (PI-4,5-bisP) into two second messengers, diacylglycerol (DAG) and   I, 4,5-inositol trisphosphate (IP 3 ). IP 3 has a binding site in the sarcoplasmic  reticulum and the endoplasmic reticulum that stimulates the release of Ca 2+ (Fig.   II. 19). Ca 2+ activates enzymes containing the calcium-calmodulin subunit,  including a protein kinase. Diacylglycerol, which remains in the membrane, acti-  vates protein kinase C, which then propagates the response by phosphorylating  target proteins.   Dennis Veere was hospitalized for  dehydration resulting from cholera  toxin (see Chapter 10). Cholera A  toxin was absorbed into the intestinal  mucosal cells, where it was processed and  complexed with Arf (ADP-ribosylation fac-  tor), a small G protein normally involved in  vesicular transport. Cholera A toxin is an  NAD-glycohydrolase, which cleaves NAD  and transfers the ADP ribose portion to other  proteins. It ADP-ribosylates the Ga s subunit  of heterotrimeric G proteins, thereby inhibit-  ing their GTPase activity. As a consequence,  they remain actively bound to adenylyl  cyclase, resulting in increased production of  cAMP. The CFTR channel is activated, result-  ing in secretion of chloride ion and Na + ion  into the intestinal lumen. The ion secretion is  followed by loss of water, resulting in vomit-  ing and watery diarrhea.   Some signaling pathways cross  \\ J from the MAP kinase pathway to   phosphorylate CREB, and all het-  erotrimeric G protein pathways diverge to  include a route to the MAP kinase pathway.  These types of complex interconnections in  signaling pathways are sometimes called  hormone cross-talk.   Tissues vary in their ability to respond to a message through changes in receptor  activity or number. Many receptors contain intracellular phosphorylation sites  that alter their ability to transmit signals. Receptor number is also varied through   Fig. 11.18. Formation and cleavage of the cyclic phosphodiester bond in cAMR When activated by Gas, adenyl cyclase converts ATP to 3 ',5'-  cyclic AMP + PPi . cAMP phosphodiesterase hydrolyzes cAMP to AMR   0 o In myasthenia gravis, increased  I r\A ) endocytosis and degradation of  ™ acetylcholine receptors lead to a  signal transduction pathway that decreases  synthesis of new receptors. Thus, downreg-  ulation of acetylcholine receptors is part of  this disease.   down-regulation. After a hormone binds to the receptor, the hormone-receptor  complex may be taken into the cell by the process of endocytosis in clathrin-  coated pits (see Chapter 10, Section III.B.l.) The receptors may be degraded or  recycled back to the cell surface. This internalization of receptors decreases the  number available on the surface under conditions of constant high hormone lev-  els when more of the receptors are occupied by hormones and results in decreased  synthesis of new receptors. Hence, it is called down-regulation.   Fig. 11.19. IP 3 signaling calcium release from  the endoplasmic reticulum.   Some signals, such as those that modify the metabolic responses of cells or trans-  mit neural impulses, need to turn off rapidly when the hormone is no longer being  produced. Other signals, such as those that stimulate proliferation, turn off more  slowly. In contrast, signals regulating differentiation may persist throughout our  lifetime. Many chronic diseases are caused by failure to terminate a response at the  appropriate time.   The first level of termination is the chemical messenger itself (Fig. 11.20). When  the stimulus is no longer applied to the secreting cell, the messenger is no longer  secreted, and existing messenger is catabolized. For example, many polypeptide  hormones such as insulin are taken up into the liver and degraded. Termination of  the acetylcholine signal by acetylcholinesterase has already been mentioned.   Within each pathway of signal transduction, the signal may be turned off at spe-  cific steps. The receptor might be desensitized to the messenger by phosphorylation.  G proteins, both monomeric and heterotrimeric, automatically terminate messages  as they hydrolyze GTP. Termination also can be achieved through degradation of the  second messenger (e.g., phosphodiesterase cleavage of cAMP). Each of these ter-  minating processes is also highly regulated.   Another important pathway for reversing the message is through protein phos-  phatases, enzymes that reverse the action of kinases by removing phosphate groups   from proteins. Specific tyrosine or serine/threonine phosphatases (enzymes that  remove the phosphate group from specific proteins) exist for all of the sites phos-  phorylated by signal transduction kinases. Some receptors are even protein phos-  phatases.   ^ Mya Sthenia. Mya Sthenia has myasthenia gravis, an autoimmune dis-  ) ease caused by the production of antibodies directed against the nicotinic  acetylcholine receptor in skeletal muscles. The diagnosis is made by his-  tory (presence of typical muscular symptoms), physical examination (presence of  inability to do specific repetitive muscular activity over time), and tests such as the  inhibition of acetylcholinesterase activity. The diagnosis can be further confirmed  with an electromyogram (EMG) showing a partial blockade of ion flux across mus-  cular membranes and a diagnostic procedure involving repetitive electrical nerve  stimulation.   Ann O'Rexia. Anorexia nervosa presents as a distorted visual self-  image often associated with compulsive exercise. Although Ann has been  gaining weight, she is still relatively low on stored fuels needed to sustain  the metabolic requirements of exercise. Her prolonged starvation has resulted in  release of the steroid hormone Cortisol and the polypeptide hormone glucagon,  whereas levels of the polypeptide hormone insulin have decreased. Cortisol acti-  vates transcription of genes for some of the enzymes of gluconeogenesis (the syn-  thesis of glucose from amino acids and other precursors; see Chapter 3.) Glucagon  binds to heptahelical receptors in liver and adipose tissue and, working through  cAMP and protein kinase A, activates many enzymes involved in fasting fuel  metabolism. Insulin, which is released when she drinks her high-energy supple-  ment, works through a specialized tyrosine kinase receptor to promote fuel stor-  age. Epinephrine, a catecholamine released when she exercises, promotes fuel  mobilization.   Dennis Veere. In the emergency room, Dennis received intravenous  rehydration therapy (normal saline [0.9% NaCl]) and oral hydration ther-  apy with a glucose-electrolyte solution to increase his glucose-dependent  Na + uptake from the intestinal lumen (see Chapter 10). Dennis quickly recovered  from his bout of cholera. Cholera is self -limiting, possibly because the bacteria  remain in the intestine, where they are washed out of the system by the diffuse  watery diarrhea. Over the past three years, Percy Veere has persevered through the  death of his wife and the subsequent calamities of his grandson Dennis "the  Menace" Veere, including salicylate poisoning, suspected malathion poisoning, and  now cholera. Mr. Veere decided to send his grandson home for the remainder of the  summer.   Fig. 11.20. Sites of signal termination.  Processes that terminate signals are shown in  blue.   Death domain receptors. The cytokine TNF (tumor necrosis fac-  tor) uses a type of receptor called the death domain receptor (Fig. 11.21).  These receptors function as a trimer when they bind TNF (which is also a  trimer). On TNF binding, an inhibitory protein called the "silencer of death" is  released from the receptor. The receptor then binds and activates several adaptor  proteins. One adaptor protein, FADD (fas-associated death domain), recruits and  activates the zymogen form of a proteolytic enzyme called caspase. Caspases    Fig. 11.21. Death domain receptors. The portion of the receptor shown in blue is called the  death domain because it binds adaptor proteins that initiate different signaling pathways lead-  ing to cell death. The adaptor protein FADD forms a scaffold on which proteolytic procas-  pases cleave each other, thereby initiating a death pathway. The adaptor protein TRADD  binds a protein that binds a serine-threonine kinase (Ser-T kinase) that initiates another  signaling pathway leading to activation of the transcription factor NF-kB.   Caspases are proteolytic enzymes  fjl ] that have a critical role in pro-  *S grammed cell death (also called  apoptosis) (see Chapter 16). Caspases are  present as latent zymogens until their auto-  proteolysis (self-cleavage) is activated by  "death signals" to the receptor complex.  Once activated, they work systematically to  dismantle a cell through degrading a wide  variety of proteins, such as DNA repair  enzymes and cellular structural proteins.   initiate a signal transduction pathway leading to apoptosis (programmed cell death)  (see Chapter 18). Another adaptor protein, TRADD (TNF receptor-associated  death domain), initiates signaling pathways that lead to activation of the gene-spe-  cific transcription factors Jun and NF-kB (nuclear factor- kB). Through these path-  ways, TNF mediates cell- specific responses, such as cell growth and death, the  inflammatory response, and immune function.   Guanylyl cyclase receptors. Guanylyl cyclase receptors convert  GTP to the second messenger 3 ',5' cyclic GMP (cGMP), which is analo-  gous to cAMP (Fig. 11.22). Like cAMP, cGMP is degraded by a mem-  brane-bound phosphodiesterase. Elevated cGMP activates protein kinase G, which  then phosphorylates target proteins to propagate the response.   One type of guanylyl cyclase exists in the cytoplasm and is a receptor for nitric  oxide (NO), a neurotransmitter/neurohormone. NO is a lipophilic gas that is able to  diffuse into the cell. This receptor thus is an exception to the rule that intracellular  receptors are gene transcription factors. The other type of guanyl cyclase receptor  is a membrane-spanning receptor in the plasma membrane with an external binding  domain for a signal molecule (e.g., natriuretic peptide).   Signaling Pathways. The May 31, 2002 issue of Science, Vol 296, provides a 2-3-  page synopsis of many signaling pathways. The articles most relevant to the path-  ways discussed in this chapter are:   Aaronson DA, Horvath CM. A road map for those who don't know JAK-STAT. Science   2002;296:1653-1655.  Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655-1657.  Attisano L, Wrana, JL. Signal transduction by the TGF-(3 superfamily. Science 2002;296:1646-1647.  Neves SR, Ram PT, Iyengar R. G protein pathways. Science 2002;296:1636-1639.  Chen Q, Goeddel DV. TNF-R1 signaling: A beautiful pathway. Science 2002;296:1634-1635.   1. Which of the following is a general characteristic of all chemical messengers?   (A) They are secreted by one cell, enter the blood, and act on a distant target cell.   (B) To achieve a coordinated response, each messenger is secreted by several types of cells.   (C) Each messenger binds to a specific protein receptor in a target cell.   (E) Chemical messengers are metabolized to intracellular second messengers to transmit their message.   2. Which of the following is a characteristic of chemical messengers that bind to intracellular transcription factor receptors?   (E) They are always present in high concentrations in the blood.   Use the following case history for questions 3 and 4. To answer this question, you do not need to know more about parathy-  roid hormone or pseudophypoparathyroidism than the information given.   Pseudohypoparathyroidism is a heritable disorder caused by target organ unresponsiveness to parathyroid hormone (a  polypeptide hormone secreted by the parathyroid gland). One of the mutations causing this disease occurs in the gene encod-  ing G s a in certain cells.   (C) a receptor that must be endocytosed in clathrin-coated pits to transmit its signal.   (D) decreases generation of IP 3 in response to parathyroid hormone.   (E) decreases synthesis of phosphatidylinositol 3,4,5-trisphosphate in response to parathyroid hormone.   5. SH2 domains on proteins are specific for which of the following sites?   In the middle of the 20th century, DNA was identified as the genetic material,  and its structure was determined. Using this knowledge, researchers then dis-  covered the mechanisms by which genetic information is inherited and  expressed. During the last quarter of the 20th century, our understanding of  this critical area of science, known as molecular biology, grew at an increas-  ingly rapid pace. We now have techniques to probe the human genome that  will completely revolutionize the way medicine is practiced in the 21st century.   The genome of a cell consists of all its genetic information, encoded in DNA  (deoxyribonucleic acid). In eukaryotes, DNA is located mainly in nuclei, but small  amounts are also found in mitochondria. Nuclear genes are packaged in chromo-  somes that contain DNA and protein in tightly coiled structures (Chapter 12).   The molecular mechanism of inheritance involves a process known as replication,  in which the strands of parental DNA serve as templates for the synthesis of DNA  copies (Fig. 1) (Chapter 13). After DNA replication, cells divide, and these DNA  copies are passed to daughter cells. Alterations in genetic material occur by recombi-  nation (the exchange of genetic material between chromosomes) and by mutation (the  result of chemical changes that alter DNA). DNA repair mechanisms correct much of  this damage, but, nevertheless, many gene alterations are passed to daughter cells.   The expression of genes within cells requires two processes: transcription and  translation (see Fig. 1) (Chapters 14 and 15). DNA is transcribed to produce ribonu-  cleic acid (RNA). Three major types of RNA are transcribed from DNA and subse-  quently participate in the process of translation (The synthesis of proteins). Mes-  senger RNA (mRNA) carries the genetic information from the nucleus to the  cytoplasm, where translation occurs on ribosomes, structures containing proteins  complexed with ribosomal RNA (rRNA). Transfer RNA (tRNA) carries individual  amino acids to the ribosomes, where they are joined in peptide linkage to form pro-  teins. During translation, the sequence of nucleic acid bases in mRNA is read in sets  of three (each set of three bases constitutes a codon). The sequence of codons in the  mRNA dictates the sequence of amino acids in the protein. Proteins function in cell  structure, signaling, and catalysis and, therefore, determine the appearance and  behavior of cells and the organism as a whole. The regulation of gene expression  (Chapter 16) determines which proteins are synthesized and the amount synthesized  at any time, thus allowing cells to undergo development and differentiation and to  respond to changing environmental conditions.   Research in molecular biology has produced a host of techniques, known col-  lectively as recombinant DNA technology, biotechnology, or genetic engineering,  that can be used for the diagnosis and treatment of disease (Chapter 17). These  techniques can detect a number of genetic diseases that previously could only be  diagnosed after symptoms appeared. Diagnosis of these diseases can now be made  with considerable accuracy even before birth, and carriers of these diseases also can  be identified.   Fig. 1. Replication, transcription, and transla-  tion. Replication: DNA serves as a template  for producing DNA copies. Transcription:  DNA serves as a template for the synthesis of  RNA. Translation: RNA provides the informa-  tion for the synthesis of proteins.   geted to interfere with their ability  to synthesize RNA and proteins. Thus, med-  ical students need to know the basics of bac-  terial DNA replication, RNA synthesis, and  protein synthesis.   Ethical dilemmas have come along  with technological advances in  moleuclar biology. Consider the  case of a patient with a mild case of  ornithine transcarbamoylase deficiency, a  urea cycle defect that, if untreated, leads to  elevated ammonia levels and nervous sys-  tem dysfunction. The patient was being  effectively treated by dietary restriction of  protein. However, in 1999, he was treated  with a common virus carrying the normal  gene for ornithine transcarbamoylase. The  patient developed a severe immune  response to the virus and died as a result of  the treatment. This case history raises the  issues of appropriate patient consent, appro-  priate criteria to be included in this type of  study, and the types of diseases for which  gene therapy is appropriate. These are  issues that you, the student, will be facing as  you enter your practice of medicine.   Tumors may be benign or malig-  nant. A tumor is malignant if it  invades locally or if cells break  away from the tumor and travel to other  parts of the body, where they establish new  growths (a process called metastasis), result-  ing in destruction of the tissues they invade.  Many of the drugs used to treat malignant  tumors are directed toward inhibition of DNA  replication. These chemotherapeutic drugs  are more toxic to cancer cells than normal  cells, because the cancer cells divide more  rapidly. However, such drugs also may  inhibit normal rapidly dividing cells, such as  the cells of the bone marrow (causing a  decrease in white blood cell count) or cells in  the hair follicles (resulting in hair loss during  chemotherapy).    With recent developments in the field of gene therapy, diseases that for centuries  have been considered hopeless are now potentially curable. Much of the therapy for  these diseases is currently experimental. However, during the 21st century, physi-  cians may be using genetic engineering techniques routinely for both the diagnosis  and treatment of their patients.   Replication and cell division are highly regulated processes in the human. Can-  cer is a group of diseases in which a cell in the body has been transformed and  begins to grow and divide out of control (Chapter 18). It results from multiple muta-  tions or changes in DNA structure in the genes that activate cell growth, called  proto-oncogenes, and those that ensure that DNA replication and repair are normal,  called growth suppressor or tumor suppressor genes. Mutations that activate proto-  oncogenes to oncogenes disturb the regulation of the cell cycle and the rate of cell  proliferation. Mutations disrupting tumor suppressor genes lead to an increased  incidence of these proto-oncogene-activating mutations. Such mutations may be  inherited, causing a predisposition to a type of cancer. They also may arise from  DNA replication or copying errors that remain uncorrected, from chemicals or radi-  ation that damages DNA, from translocation of pieces of chromosomes from one  chromosome to another during replication, or from incorporation of viral encoded  DNA into the genome.   Nucleotides in DNA and RNA. Nucleotides are the monomeric units of the  nucleic acids, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Each  nucleotide consists of a heterocyclic nitrogenous base, a sugar, and phosphate.  DNA contains the purine bases adenine (A) and guanine (G) and the pyrimidine  bases cytosine (C) and thymine (T). RNA contains A, G, and C, but it has uracil  (U) instead of thymine. In DNA, the sugar is deoxyribose, whereas in RNA it is  ribose.   Polynucleotides such as DNA and RNA are linear sequences of nucleotides  linked by 3' - to 5' -phosphodiester bonds between the sugars (Fig. 12.1). The  bases of the nucleotides can interact with other bases or with proteins.   DNA Structure, Genetic information is encoded by the sequence of different  nucleotide bases in DNA. DNA is double -stranded; it contains two antiparallel  polynucleotide strands. The two strands are joined by hydrogen bonding between  their bases to form base-pairs. Adenine pairs with thymine, and guanine pairs  with cytosine. The two DNA strands run in opposite directions. One strand runs  5 9 to 3' , and the other strand runs 3' to 5' . The two DNA strands wind around  each other, forming a double helix.   Transcription of a gene generates a single -stranded RNA that is identical in  nucleotide sequence to one of the strands of the duplex DNA. The three major  types of RNA are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer  RNA (tRNA).   RNA Structures, mRNAs contain the nucleotide sequence that is converted  into the amino acid sequence of a protein in the process of translation. Eukaryotic  mRNA has a structure known as a cap at the 5' -end, a sequence of adenine  nucleotides (a poly(A) tail) at the 3' -end, and a coding region in the center con-  taining codons that dictate the sequence of amino acids in a protein or relay a  signal. Each codon in the genetic code is a different sequence of three nucleotides.   rRNAs and tRNAs are part of the apparatus for protein synthesis, but do not  encode proteins, rRNA has extensive internal base pairing and complexes with  proteins to form ribonucleoprotein particles called ribosomes. The ribosomes  bind mRNA and tRNAs during translation. Each tRNA binds and activates a spe-  cific amino acid for insertion into the polypeptide chain and therefore has a  somewhat different nucleotide sequence than other tRNAs. A unique trinucleotide  sequence on each tRNA called an anticodon binds to a complementary codon on  the mRNA, thereby ensuring insertion of the correct amino acid. In spite of their  differences, all tRNAs contain a number of unusual nucleotides and assume a  similar cloverleaf structure.    Fig. 12.1. Structure of a polynucleotide. The  5 '-carbon of the top sugar and the 3 '-carbon of  the bottom sugar are indicated.   • Ivy Sharer is a 26-year-old intravenous (IV) drug abuser who admitted to  flP ) sharing unsterile needles with another addict for several years. Five months   " before presenting to the hospital emergency department with soaking night  sweats, she experienced a 3 -week course of a flu-like syndrome with fever, malaise,  and muscle aches. Four months ago, she noted generalized lymph node enlargement  associated with chills, anorexia, and diarrhea, which led to a 22-lb weight loss. Tests  were positive for human immunodeficiency virus (HIV). Because her symptoms  indicated that she now had the acquired immunodeficiency syndrome (AIDS), a  multidrug regimen including zidovudine (ZDV), formerly called azidothymidine  (AZT), was initiated.   0 o An adenoma is a mass of rapidly  ( /~OA J proliferating cells, called a neoplasm  ™ (neo = new; plasm = growth), that  is formed from epithelial cells growing into a  glandlike structure. The cells lining all the  external and internal organs are epithelial  cells, and most human tumors are adenocar-  cinomas. Adenematous polyps are adenomas  that grow into the lumen of the colon or rec-  tum. The term malignant applied to a neo-  plasm refers to invasive unregulated growth.  Colin Tuma has an adenocarcinoma, which is  a malignant adenoma that has started to grow  through the wall of the colon into surrounding  tissues. Cells from adenocarcinomas can  break away and spread through the blood or  lymph to other parts of the body, where they  form "colony" tumors. This process is called  metastasis.   • Colin Tuma had intestinal polyps at age 45, which were removed via a  1|1 ) colonoscope. However, he did not return for annual colonoscopic exami-   " nations as instructed. At age 56, he reappeared, complaining of tar-colored  stools (melena), which are caused by intestinal bleeding. The source of the blood  loss was an adenocarcinoma growing from a colonic polyp of the large intestine. At  surgery, it was found that the tumor had invaded the gut wall and perforated the vis-  ceral peritoneum. Several pericolic lymph nodes contained cancer cells, and several  small nodules of metastatic cancer were found in the liver. After resection of the  tumor, the oncologist began treatment with 5-fluorouracil (5-FU) combined with  other chemotherapeutic agents.   Agneu ("neu") Moania complains to his physician of a fever and cough.  His cough produces thick yellow-brown sputum. A stain of his sputum  shows many Gram-positive, bullet-shaped diplococci. A sputum culture  confirms that he has pneumonia, a respiratory infection caused by Streptococcus  pneumoniae, which is sensitive to penicillin, erythromycin, tetracycline, and other  antibiotics. Because of a history of penicillin allergy, he is started on oral erythro-  mycin therapy.   DNA is a double-stranded mole-  \ \ ) cule that forms base pairs (bp)   between strands. The bp designa-  tion is often used to indicate the size of a  DNA molecule. For example, in a stretch of  DNA 200 bp long, both strands are included  with 200 bases in each strand, for a total of  400 bases.   DNA and RNA serve as the genetic material for prokaryotic and eukaryotic cells,  for viruses, and for plasmids, each of which stores it in a different arrangement or  location. In prokaryotes, DNA is not separated from the rest of the cellular contents.  In eukaryotes, however, DNA is located in the nucleus, where it is separated from  the rest of the cell by the nuclear envelope (see Fig. 10.20). Eukaryotic DNA is  bound to proteins, forming a complex called chromatin. During interphase (when  cells are not dividing), some of the chromatin is diffuse (euchromatin) and some is  dense (heterochromatin), but no distinct structures can be observed. However,  before mitosis (when cells divide), the DNA is replicated, resulting in two identical  chromosomes called sister chromatids. During metaphase (a period in mitosis),  these condense into discrete, visible chromosomes.   Less than 0.1% of the total DNA in a cell is present in mitochondria. The genetic  information in a mitochondrion is encoded in less than 20,000 base pairs of DNA;  the information in a human haploid nucleus (i.e., an egg or a sperm cell) is encoded  in approximately 3 X 10 9 (3 billion) base pairs. The DNA and protein synthesizing  systems in mitochondria more closely resemble the systems in bacteria, which do   not have membrane-enclosed organelles, than those in the eukaryotic nucleus and  cytoplasm. It has been suggested that mitochondria were derived from ancient bac-  terial invaders of primordial eukaryotic cells.   Viruses are small infectious particles consisting of a DNA or RNA genome (but  not both), proteins required for pathogenesis or replication, and a protein coat. They  lack, however, complete systems for replication, transcription, and translation and,  consequently, viruses must invade other cells and commandeer their DNA, RNA,  and protein- synthesizing machinery to reproduce. Both eukaryotes and prokaryotes  can be infected by viruses. Viruses that infect bacteria are known as bacteriophage  (or more simply as phage).   Plasmids are small, circular DNA molecules that can enter bacteria and repli-  cate autonomously, that is, outside the host genome. In contrast to viruses, plas-  mids are not infectious; they do not convert their host cells into factories devoted  to plasmid production. Genetic engineers use plasmids as tools for transfer of  foreign genes into bacteria because segments of DNA can readily be incorpo-  rated into plasmids.   In 1865, Frederick Meischer first isolated DNA, obtaining it from pus scraped  from surgical bandages. Initially, scientists speculated that DNA was a cellular  storage form for inorganic phosphate, an important but unexciting function that  did not spark widespread interest in determining its structure. In fact, the details  of DNA structure were not fully determined until 1953, almost 90 years after it  had first been isolated, but only 9 years after it had been identified as the genetic  material.   Early in the 20th century, the bases of DNA were identified as the purines adenine  (A) and guanine (G), and the pyrimidines cytosine (C) and thymine (T) (Fig. 12.2).  The sugar was found to be deoxyribose, a derivative of ribose, lacking a hydroxyl  group on carbon 2 (Fig. 12.3).   Nucleotides, composed of a base, a sugar, and phosphate, were found to be the  monomeric units of the nucleic acids (Table 12.1). In nucleosides, the nitrogenous  base is linked by an Af-glycosidic bond to the anomeric carbon of the sugar, either  ribose or deoxyribose. A nucleotide is a nucleoside with an inorganic phosphate  attached to a 5' -hydroxyl group of the sugar in ester linkage (Fig. 12.4). The names  and abbreviations of nucleotides specify the base, the sugar, and the number of  phosphates attached (MP, monophosphate; DP, diphosphate; TP, triphosphate). In  deoxynucleotides, the prefix "d" precedes the abbreviation. For example, GDP is  guanosine diphosphate (the base guanine attached to a ribose that has two phos-  phate groups) and dATP is deoxy adenosine triphosphate (the base adenine attached  to a deoxyribose with three phosphate groups).   Adenine (A)  Guanine (G)  Cytosine (C)  Thymine (T)  Uracil (U)  Hypoxanthine (I)   a If the sugar is deoxyribose rather than ribose, the nucleoside has "deoxy" as a prefix (e.g., deoxyadeno-  sine). Nucleotides are given the name of the nucleoside plus mono, di, or triphosphate (e.g., adenosine  triphosphate or deoxyadenosine triphosphate).   b The base hypoxanthine is not found in DNA but is produced during degradation of the purine bases. It is  found in certain tRNA molecules. Its nucleoside, inosine, is produced during synthesis of the purine  nucleotides (see Chapter 41).   Fig. 12.3. Deoxyribose and ribose, the sugars  of DNA and RNA. The carbon atoms are num-  bered from 1 to 5. When the sugar is attached  to a base, the carbon atoms are numbered from  1' to 5' to distinguish it from the base. In  deoxyribose the X = H; in ribose the X = OH.   Fig. 12.4. Nucleoside and nucleotide structures. Shown with ribose as the sugar. The  corresponding deoxyribonucleotides are abbreviated dNMP, dNDP, and dNTP. N = any base  (A, G, C, U, or T).   After Ivy Sharer was diagnosed  with AIDS, she was treated with a  mixture of drugs including zidovu-  dine (ZDV), formerly called AZT. This drug is  an analog of the thymine nucleotide found in  DNA (the modified group is shown in the  dashed box). ZDV is phosphorylated in the  body by the kinases that normally phospho-  rylate nucleosides and nucleotides. As the  viral DNA chain is being synthesized in a  human cell, ZDV is then added to the grow-  ing 3'-end by viral reverse transcriptase.  However, ZDV lacks a 3' OH group and,  therefore, no additional nucleotides can be  attached through a 5'— »3' bond. Thus, chain  elongation of the DNA is terminated.  Reverse transcriptase has a higher affinity  for ZDV than does normal human cellular  DNA polymerases, enabling the drug to tar-  get viral replication more specifically than  cellular replication.   In 1944, after Oswald Avery's experiments establishing DNA as the genetic mate-  rial were published, interest in determining the structure of DNA intensified. Diges-  tion with enzymes of known specificity proved that inorganic phosphate joined the  nucleotide monomers, forming a phosphodiester bond between the 3' -carbon of one  sugar and the 5 '-carbon of the next sugar along the polynucleotide chain (Fig. 12.5).  Another key to DNA structure was provided by Erwin Chargaff. He analyzed the  base composition of DNA from various sources and concluded that, on a molar basis,  the amount of adenine was always equal to the amount of thymine, and the amount  of guanine was equal to the amount of cytosine.   During this era, James Watson and Francis Crick joined forces and, using the  x-ray diffraction data of Maurice Wilkins and Rosalind Franklin, incorporated the  available information into a model for DNA structure. In 1953, they published a  brief paper, describing DNA as a double helix consisting of two polynucleotide  strands joined by pairing between the bases (adenine with thymine and guanine  with cytosine). The model of base-pairing they proposed is the basis of modern  molecular biology.   As proposed by Watson and Crick, each DNA molecule consists of two polynu-  cleotide chains joined by hydrogen bonds between the bases. In each base pair, a  purine on one strand forms hydrogen bonds with a pyrimidine on the other strand.  In one type of base pair, adenine on one strand pairs with thymine on the other  strand (Fig. 12.6). This base pair is stabilized by two hydrogen bonds. The other  base pair, formed between guanine and cytosine, is stabilized by three hydrogen  bonds. As a consequence of base-pairing, the two strands of DNA are complemen-  tary, that is, adenine on one strand corresponds to thymine on the other strand, and  guanine corresponds to cytosine.   The concept of base-pairing proved to be essential for determining the mecha-  nism of DNA replication (in which the copies of DNA are produced that are dis-  tributed to daughter cells) and the mechanisms of transcription and translation (in   Watson and Crick's one-page paper, published in 1953, contained little more  \ \ ) than 900 words. However it triggered a major revolution in the biologic  sciences and produced the conceptual foundation for the discipline of molecu-  lar biology.   Fig. 12.5. A segment of a polynucleotide chain of DNA. The dashes at the 5'- and 3 '-ends  indicate that the molecule contains more nucleotides than are shown.   Fig. 12.6. Base pairs of DNA. Note that the pyrimidine bases are "flipped over" from the positions in which they are usually shown (see Fig.  12.5). The bases must be in this orientation to form base pairs. The dotted lines indicate hydrogen bonds between the bases. Although the hydro-  gen bonds hold the bases and thus the two DNA strands together, they are weaker than covalent bonds and allow the DNA strands to separate  during replication and transcription.   "It has not escaped our notice that  \\ ) the specific pairing we have postu-  *S lated immediately suggests a pos-  sible copying mechanism for the genetic  material." J.D. Watson and F.H.C. Crick,  Nat ure, April 25, 1953.   Fig. 12.7. DNA strands serve as templates.  During replication, the strands of the helix sep-  arate in a localized region. Each parental  strand serves as a template for the synthesis of  a new DNA strand.   Multi-drug regimens used to treat  cancers (e.g., lymphomas) some-  times include the drug doxorubicin  (adriamycin). It is a natural product with a  complex multi-ring structure that interca-  lates or slips in between the stacked base  pairs of DNA and inhibits replication and  transcription.   which mRNA is produced from genes and used to direct the process of protein syn-  thesis). Obviously, as Watson and Crick suggested, base-pairing allows one strand  of DNA to serve as a template for the synthesis of the other strand (Fig. 12.7). Base-  pairing also allows a strand of DNA to serve as a template for the synthesis of a  complementary strand of RNA.   As concluded by Watson and Crick, the two complementary strands of DNA run in  opposite directions. On one strand, the 5' -carbon of the sugar is above the 3' -car-  bon (Fig. 12.8). This strand is said to run in a 5' to 3' direction. On the other strand,  the 3' -carbon is above the 5' -carbon. This strand is said to run in a 3' to 5' direc-  tion. Thus, the strands are antiparallel (that is, they run in opposite directions.) This  concept of directionality of nucleic acid strands is essential for understanding the  mechanisms of replication and transcription.   Because each base pair contains a purine bonded to a pyrimidine, the strands are  equidistant from each other throughout. If two strands that are equidistant from each  other are twisted at the top and the bottom, they form a double helix (Fig. 12.9). In  the double helix of DNA, the base pairs that join the two strands are stacked like a  spiral staircase along the central axis of the molecule. The electrons of the adjacent  base pairs interact, generating stacking forces that, in addition to the hydrogen  bonding of the base pairs, help to stabilize the helix.   The phosphate groups of the sugar-phosphate backbones are on the outside of the  helix (see Fig. 12.9). Each phosphate has two oxygen atoms forming the phospho-  diester bonds that link adjacent sugars. However, the third -OH group on the phos-  phate is free and dissociates a hydrogen ion at physiologic pH. Therefore, each  DNA helix has negative charges coating its surface that facilitate the binding of spe-  cific proteins.   The helix contains grooves of alternating size, known as the major and minor  grooves (see Fig. 12.9). The bases in these grooves are exposed and therefore can  interact with proteins or other molecules.   Fig. 12.8. Antiparallel strands of DNA. For the strand on the left, the 5 '-carbon of each sugar  is above the 3 '-carbon, so it runs 5' to 3'. For the strand on the right, the 3 '-carbon of each  sugar is above the 5 '-carbon, so it runs 3' to 5'.   Watson and Crick described the B form of DNA, a right-handed helix, contain-  ing 3.4 A between base pairs and 10.4 base pairs per turn. Although this form pre-  dominates in vivo, other forms also occur (Fig. 12.10). The A form, which pre-  dominates in DNA-RNA hybrids, is similar to the B form, but is more compact (2.3  A between base pairs and 1 1 base pairs per turn). In the Z form, the bases of the two  DNA strands are positioned toward the periphery of a left-handed helix . There are  3.8 A between base pairs and 12 base pairs per turn in Z DNA. This form of the  helix was designated "Z" because, in each strand, a line connecting the phosphates  "zigs" and "zags."   Both alkali and heat cause the two strands of the DNA helix to separate (denature).  Many techniques employed to study DNA or to produce recombinant DNA mole-  cules make use of this property. Although alkali causes the two strands of DNA to   If you look up through the bottom of a helix along the central axis and the helix spirals  away from you in a clockwise direction (toward the arrowhead in the drawing), it is a  right-handed helix. If it spirals away from you in a counterclockwise direction, it is a left-  handed helix.   Fig. 12.9. Two DNA strands twist to form a  double helix. The distance between the two  phosphodiester backbones, shown with a rib-  bon, is about 1 1 A. The hydrogen-bonded base  pairs, shown in blue, create stacking forces  with adjacent base pairs. Each phosphate  group contains one negatively charged oxygen  atom that provides the phosphodiester back-  bone with a negative charge. Because of the  twisting of the helix, grooves are formed along  the surface, the larger one being the major  groove, and the smaller one the minor groove.   Fig. 12.10. Z, B, and A forms of DNA. The solid black lines connect one phosphate group  to the next. Modified from Saenger W. Principles of nucleic acid structure. New York:  Springer Verlag, 1984:257-286.   Heating and cooling cycles are  fjl J used to separate and reanneal   DNA strands in the polymerase  chain reaction (PCR), a technique for obtain-  ing large quantities of DNA from very small  samples for research or for clinical and  forensic testing (see Chapter 17).   Fig. 12.11. Effect of alkali on DNA and RNA.  DNA strands stay intact, but they separate.  RNA strands are degraded to nucleotides.   If histones contain large amounts  of arginine and lysine, will their net  charge be positive or negative?   separate, it does not break the phosphodiester bonds (Fig. 12.11). In contrast, the  phosphodiester bonds of RNA are cleaved by alkali. Therefore, alkali is used to  remove RNA from DNA and to separate DNA strands before, or after, elec-  trophoresis on polyacrylamide or agarose gels.   Heat alone converts double- stranded DNA to single- stranded DNA. The separa-  tion of strands is called melting, and the temperature at which 50% of the DNA is  separated is called the T m . If the temperature is slowly decreased, complementary  single strands can realign and base-pair, re-forming a double helix essentially iden-  tical to the original DNA. This process is known as renaturation, reannealing, or  hybridization. The process by which a single-stranded DNA anneals with comple-  mentary strands of RNA is also called hybridization (Fig. 12.12). Hybridization is  used extensively in research and clinical testing.   A prokaryotic cell generally contains a single chromosome composed of double-  stranded DNA that forms a circle. These circular DNA molecules are extremely  large. The entire chromosome of the bacterium Escherichia coli, composed of a sin-  gle, circular double-stranded DNA molecule, contains over 4 X 10 6 base pairs. Its  molecular weight is over 2,500 X 10 6 g/mol (compared to the molecular weight for  a glucose molecule of 180 g/mol). If this molecule were linear, its length would  measure almost 2 mm.   DNA from eukaryotic cells is approximately 1,000 times larger than that from  bacterial cells. In eukaryotes, each chromosome contains one continuous, linear  DNA helix. The DNA of the longest human chromosome is over 7 cm in length. In  fact, if the DNA from all 46 chromosomes in a diploid human cell were placed end  to end, our total DNA would span a distance of about 2 m (over 6 feet). Our total  DNA contains about 6 X 10 9 base pairs.   DNA molecules require special packaging to enable them to reside within cells  because the molecules are so large. In E. coli, the circular DNA is supercoiled and  attached to an RNA-protein core. Packaging of eukaryotic DNA is much more com-  plex because it is larger and must be contained within the nucleus of the eukaryotic  cell. Eukaryotic DNA binds to an equal weight of histones, which are small basic  proteins containing large amounts of arginine and lysine. The complex of DNA and  proteins is called chromatin. The organization of eukaryotic DNA into chromatin is  essential for controlling transcription, as well as for packaging. When chromatin is  extracted from cells, it has the appearance of beads on a string (Fig. 12.13). The  beads with DNA protruding from each end are known as nucleosomes, and the  beads themselves are known as nucleosome cores (Fig. 12.14). Two molecules of  each of four histone classes (histones H2A, H2B, H3, and H4) form the center of  the core around which approximately 140 base pairs of double- stranded DNA are  wound. The DNA wrapped around the nucleosome core is continuous and joins one   DNA consists of a double helix, with the two strands of DNA wrapping around  each other to form a helical structure. To compact, the DNA molecule coils  about itself to form a structure called a supercoil. A telephone cord, which con-  nects the handpiece to the phone, displays supercoiling when the coiled cord wraps  about itself. When the strands of a DNA molecule separate and unwind over a small local  region (which occurs during DNA replication), supercoils are introduced into the remain-  ing portion of the molecule, thereby increasing stress on this portion. Enzymes known  as topoisomerases relieve this stress so that unwinding of the DNA strands can occur.   nucleosome core to the next. The DNA joining the cores is complexed with the fifth  type of histone, HI. Further compaction of chromatin occurs as the strings of nucle-  osomes wind into helical, tubular coils called solenoid structures.   Although complexes of DNA and histones form the nucleosomal substructures  of chromatin, other types of proteins are also associated with DNA in the nucleus.  These proteins were given the unimaginative name of "non-histone chromosomal  proteins." The cells of different tissues contain different amounts and types of these  proteins, which include enzymes that act on DNA and factors that regulate  transcription.   The genome, or total genetic content, of a human haploid cell (a sperm or an egg)  is distributed in 23 chromosomes. Haploid cells contain one copy of each chromo-  some. The haploid egg and haploid sperm cells combine to form the diploid zygote,  which continues to divide to form our other cells (mitosis), which are diploid.  Diploid cells thus contain 22 pairs of autosomal chromosomes, with each pair com-  posed of two homologous chromosomes containing a similar series of genes (Fig.  12.15). In addition to the autosomal chromosomes, each diploid cell has two sex  chromosomes, designated X and Y. A female has two X chromosomes, and a male  has one X and one Y chromosome. The total number of chromosomes per diploid  cell is 46.   Genes are arranged linearly along each chromosome. A gene, in genetic  terms, is the fundamental unit of heredity. In structural terms, a gene encom-  passes the DNA sequence encoding the structural components of the gene prod-  uct (whether it be a polypeptide chain or RNA molecule) along with the DNA  sequences adjacent to the 5" end of the gene which regulates its expression. A  genetic locus is a specific position or location on a chromosome. Each gene on  a chromosome in a diploid cell is matched by an alternate version of the gene at  the same genetic locus on the homologous chromosome (Fig. 12.16). These  alternate versions of a gene are called alleles. We thus have two alleles of each    Fig. 12.14. A polynucleosome, indicating the histone cores and linker DNA.    At physiologic pH, arginine and  ) lysine carry positive charges on  their side chains; therefore, his-  tones have a net positive charge. The argi-  nine and lysine residues are clustered in  regions of the histone molecules. These pos-  itively charged regions of the histones inter-  act with the negatively charged DNA phos-  phate groups.   Fig. 12.16. Homologous chromosomes and  their protein products. A set of homologous  chromosomes is shown diagrammatically. (Of  course, during interphase when they are pro-  ducing their protein products, they cannot be  visualized as discrete entities.) Four genes are  shown as examples on each homologue. The  genes of the homologues are alleles (e.g., AA,  Bb, CC, dD). They may be identical (e.g., AA,  CC), or they may differ (e.g., Bb, dD) in DNA  sequence. Thus the corresponding protein  products may be identical or they may differ in  amino acid sequence.   Fig. 12.15. Human chromosomes from a male dipolid cell. Each diploid cell contains 22  pairs of autosomes (the numbered chromosomes 1-22) plus one X and one Y. Each female  diploid cell contains two X chromosomes. Each haploid cell contains chromosomes 1  through 22 plus either an X or a Y. From Gelehrter TD, Collins FS, Ginsburg D. Principles  of Human Genetics, 2nd Ed. Baltimore: Williams & Wilkins, 1998.   0 o Will Sichel has sickle cell anemia  / /~oA j (see Chapters 6 and 7). He has two  ™ alleles for the p-globin gene that  both generate the mutated form of hemo-  globin, HbS. His younger sister Amanda, a  carrier for sickle cell trait, has one normal  allele (that produces HbA) and one that pro-  duces HbS. A carrier would theoretically be  expected to produce HbA:HbS in a 50:50  ratio. However, what is generally seen in  electrophoresis is 60:40 ratio of HbA:HbS.  Dramatic deviations from this ratio imply the  occurrence of an additional hemoglobin  mutation (e.g., thalassemia).   Fig. 12.17. Comparison of the structures of  uracil and thymine. They differ in structure  only by a methyl group, outlined in blue.   gene, one from our mother and one from our father. If the alleles are identical in  base sequence, we are homozygous for this gene. If the alleles differ, we are het-  erozygous for this gene and may produce two versions of the encoded protein  that differ somewhat in primary structure.   The genomes of prokaryotic and eukaryotic cells differ in size. The genome of  the bacterium E. coli contains approximately 3,000 genes. All of this bacterial DNA  has a function; it either codes for proteins, rRNA, and tRNA, or it serves to regulate  the synthesis of these gene products. In contrast, the genome of the human haploid  cell contains between 30,000 and 50,000 genes, 10 to 15 times the number in  E. coli. The function of most of this extra DNA has not been determined (an issue  considered in more detail in Chapter 15).   RNA is similar to DNA. Like DNA, it is composed of nucleotides joined by 3'- to  5'-phosphodiester bonds, the purine bases adenine and guanine, and the pyrimidine  base cytosine. However, its other pyrimidine base is uracil rather than thymine.  Uracil and thymine are identical bases except that thymine has a methyl group at  position 5 of the ring (Fig. 12.17). In RNA, the sugar is ribose, which contains a  hydroxyl group on the 2 "-carbon (see Fig 12.3. The prime refers to the position on  the ribose ring). (The presence of this hydroxyl group allows RNA to be cleaved to  its constituent nucleotides in alkaline solutions.)   RNA chains are usually single- stranded and lack the continuous helical structure  of double- stranded DNA. However, RNA still has considerable secondary and ter-  tiary structure because base pairs can form in regions where the strand loops back  on itself. As in DNA, pairing between the bases is complementary and antiparallel.  But in RNA, adenine pairs with uracil rather than thymine (Fig. 12.18). Base-  pairing in RNA can be extensive, and the irregular looped structures generated are   important for the binding of molecules, such as enzymes, that interact with specific  regions of the RNA.   The three major types of RNA (mRNA, rRNA, and tRNA) participate directly in  the process of protein synthesis. Other less abundant RNAs are involved in replica-  tion or in the processing of RNA, that is, in the conversion of RNA precursors to  their mature forms.   Some RNA molecules are capable of catalyzing reactions. Thus, RNA, as well  as protein, can have enzymatic activity. Certain rRNA precursors can remove inter-  nal segments of themselves, splicing the remaining fragments together. Because this  RNA is changed by the reaction that it catalyzes, it is not truly an enzyme and there-  fore has been termed a "ribozyme." Other RNAs act as true catalysts, serving as  ribonucleases that cleave other RNA molecules or as a peptidyl transferase, the  enzyme in protein synthesis that catalyzes the formation of peptide bonds.   Colin Tuma is being treated with 5-  fluorouracil (5-FU), a pyrimidine  base similar to uracil and thymine.  5-FU inhibits the synthesis of the thymine  nucleotides required for DNA replication.  Thymine is normally produced by a reaction  catalyzed by thymidylate synthase, an  enzyme that converts deoxyuridine  monophosphate (dUMP) to deoxythymidine  monophosphate (dTMP). 5-FU is converted  in the body to F-dUMP, which binds tightly to  thymidylate synthase in a transition state  complex and inhibits the reaction (recall that  thymine is 5-methyl uracil). Thus, thymine  nucleotides cannot be generated for DNA  synthesis, and the rate of cell proliferation  decreases.   Each mRNA molecule contains a nucleotide sequence that is converted into the  amino acid sequence of a polypeptide chain in the process of translation. In eukary-  otes, messenger RNA (mRNA) is transcribed from protein-coding genes as a long  primary transcript that is processed in the nucleus to form mRNA. The various pro-  cessing intermediates, which are mRNA precursors, are called pre-mRNA or  hnRNA (heterogenous nuclear RNA). mRNA travels through nuclear pores to the  cytoplasm, where it binds to ribosomes and tRNAs and directs the sequential inser-  tion of the appropriate amino acids into a polypeptide chain.   Eukaryotic mRNA consists of a leader sequence at the 5 ' end, a coding region,  and a trailer sequence at the 3' end (Fig 12.19). The leader sequence begins with a  guanosine cap structure at its 5 ' end. The coding region begins with a trinucleotide  start codon that signals the beginning of translation, followed by the trinucleotide  codons for amino acids, and ends at a termination signal. The trailer terminates at  its 5' end with a poly (A) tail that may be up to 200 nucleotides long. Most of the  leader sequence, all of the coding region, and most of the trailer are formed by tran-  scription of the complementary nucleotide sequence in DNA. However, the termi-  nal guanosine in the cap structure and the poly(A) tail do not have complementary  sequences; they are added posttranscriptionally.   Ribosomes are subcellular ribonucleoprotein complexes on which protein synthesis  occurs. Different types of ribosomes are found in prokaryotes and in the cytoplasm  and mitochondria of eukaryotic cells (Fig. 12.20). Prokaryotic ribosomes contain  three types of rRNA molecules with sedimentation coefficients of 16, 23, and 5S.  The 30S ribosomal subunit contains the 16S rRNA complexed with proteins, and   Fig. 12.19. The regions of eukaryotic mRNA.  The wavy line indicates the polynucleotide  chain of the mRNA and the As constituting the  poly(A) tail. The 5 '-cap consists of a guano-  sine residue linked at its 5 ' hydroxyl group to  three phosphates, which are linked to the 5'-  hydroxyl group of the next nucleotide in the  RNA chain. The start and stop codons repre-  sent where protein synthesis is initiated and  terminated from this mRNA.   Fig. 12.20. Comparison of prokaryotic and  eukaryotic ribosomes. The cytoplasmic ribo-  somes of eukaryotes are shown. Mitochondrial  ribosomes are similar to prokaryotic ribosomes,  but they are smaller (55S rather than 70S).   0 0 Erythromycin, the antibiotic used  I S\A ) to treat Neu Moania, inhibits pro-  ™ tein synthesis on prokaryotic ribo-  somes, but not on eukaryotic ribosomes. It  binds to the 50S ribosomal subunit, which is  absent in eukaryotes. Therefore, it will selec-  tively inhibit bacterial growth. However,  because mitochondrial ribosomes are simi-  lar to those of bacteria, mitochondrial pro-  tein synthesis can also be inhibited. This fact  is important in understanding some of the  side effects of antibiotics that work by  inhibiting bacterial protein synthesis.   A sedimentation coefficient is a measure of the rate of sedimentation of a  macromolecule in a high-speed centrifuge (an ultracentrifuge). It is expressed  in Svedberg units (S). Although larger macromolecules generally have higher  sedimentation coefficients than do smaller macromolecules, sedimentation coefficients  are not additive. Because frictional forces acting on the surface of a macromolecule slow  its migration through the solvent, the rate of sedimentation depends not only on the  density of the macromolecule, but also on its shape.   the 50S ribosomal subunit contains the 23 S and 5S rRNAs complexed with proteins.  The 30S and 50S ribosomal subunits join to form the 70S ribosome, which partici-  pates in protein synthesis.   Cytoplasmic ribosomes in eukaryotes contain four types of rRNA molecules of  18, 28, 5, and 5.8S. The 40S ribosomal subunit contains the 18S rRNA complexed  with proteins, and the 60S ribosomal subunit contains the 28, 5, and 5.8S rRNAs  complexed with proteins. In the cytoplasm, the 40S and 60S ribosomal subunits  combine to form the 80S ribosomes that participate in protein synthesis.   Mitochondrial ribosomes, with a sedimentation coefficient of 55S, are smaller  than cytoplasmic ribosomes. Their properties are similar to those of the 70S ribo-  somes of bacteria.   rRNAs contain many loops and exhibit extensive base-pairing in the regions  between the loops (Fig. 12.21). The sequences of the rRNAs of the smaller riboso-  mal subunits exhibit secondary structures that are common to many different genera.   During protein synthesis, tRNA molecules carry amino acids to ribosomes and  ensure that they are incorporated into the appropriate positions in the growing  polypeptide chain (Fig. 12.22). This is done through base-pairing of three bases of  the tRNA (the anticodon) with the three base codons within the coding region of the  mRNA. Therefore, cells contain at least 20 different tRNA molecules that differ  somewhat in nucleotide sequence, one for each of the amino acids found in proteins.  Many amino acids have more than one tRNA.   tRNA molecules contain not only the usual nucleotides, but also derivatives of  these nucleotides that are produced by posttranscriptional modifications. In  eukaryotic cells, 10 to 20% of the nucleotides of tRNA are modified. Most tRNA  molecules contain ribothymidine (T), in which a methyl group is added to uridine  to form ribothymidine. They also contain dihydrouridine (D), in which one of the  double bonds of the base is reduced; and pseudouridine OP), in which uracil is  attached to ribose by a carbon-carbon bond rather than a nitrogen-carbon bond (see  Chapter 14). The base at the 5 '-end of the anticodon of tRNA is frequently modified.   tRNA molecules are rather small compared with both mRNA and the large rRNA  molecules. On average, tRNA molecules contain approximately 80 nucleotides and  have a sedimentation coefficient of 4S. Because of their small size and high content  of modified nucleotides, tRNAs were the first nucleic acids to be sequenced. Since  1965 when Robert Holley deduced the structure of the first tRNA, the nucleotide  sequences of many different tRNAs have been determined. Although their primary  sequences differ, all tRNA molecules can form a structure resembling a cloverleaf  (discussed in more detail in Chapter 14).   In addition to the three major types of RNA described above, other RNAs are pres-  ent in cells. These RNAs include the oligonucleotides that serve as primers for DNA  replication and the RNAs in the small nuclear ribonucleoproteins (snRNPs or  snurps) that are involved in the splicing and modification reactions that occur dur-  ing the maturation of RNA precursors (see Chapter 14).   a Ivy Sharer. Ivy Sharer's clinical course was typical for the development  ) of full-blown AIDS, in this case caused by the use of needles contaminated  with HIV. The progressive immunologic deterioration that accompanies  this disease ultimately results in life-threatening opportunistic infections with fungi  (e.g., Candida, cryptococcus), other viruses (e.g., cytomegalovirus, herpes sim-  plex), and bacteria (e.g., Mycobacterium, Pneumocystis carinii, Salmonella). The  immunologic incompetence also frequently results in the development of certain  neoplasms (e.g., Kaposi's sarcoma, non-Hodgkin's lymphoma) as well as meningi-  tis, neuropathies, and neuropsychiatric disorders causing cognitive dysfunction.  Although recent advances in drug therapy can slow the course of the disease, no  cure is yet available.   a Colin Tuma. Colin Tuma's original benign adenomatous polyp was  ) located in the ascending colon, where 10% of large bowel cancers eventu-  ally arise. Because Mr. Tuma's father died of a cancer of the colon, his  physician had warned him that his risk for developing colon cancer was three times  higher than for the general population. Unfortunately, Mr. Tuma neglected to have  his annual colonoscopic examinations as prescribed, and he developed an adeno-  carcinoma that metastasized.   The most malignant characteristic of neoplasms is their ability to metastasize,  that is, form a new neoplasm at a noncontiguous site. The initial site of metas-  tases for a tumor is usually at the first capillary bed encountered by the malig-  nant cells once they are released. Thus, cells from tumors of the gastrointestinal  tract often pass through the portal vein to the liver, which is Colin Tuma's site of  metastasis. Because his adenocarcinoma has metastasized, there is little hope of  eradicating it, and his therapy with 5-FU is palliative (directed toward reducing  the severity of the disease and alleviation of the symptoms without actually cur-  ing the disease.)   S protein synthesis in eukaryotic cells, it has the potential to alter host cell  function, leading to such side effects as epigastric distress, diarrhea, and, infre-  quently, cholestatic jaundice.   ©Retroviruses. RNA also serves as the genome for certain types of  viruses, including retroviruses (e.g., the human immunodeficiency virus  [HIV] that causes AIDS). Viruses must invade host cells to reproduce. They  are not capable of reproducing independently. Some viruses that are pathogenic to    Fig. 12.21. Secondary structure of the portion  of 16S-type ribosomal RNA that is common to  many species. Darkened areas are base-paired.  Circles are unpaired loops. Reproduced with  permission, from Annu Rev Biochem.  1984;53:137. © 1984, by Annual Reviews, Inc.   Colin Tuma completed his first course of intravenous 5-fluorouracil (5-FU) in the hospital. He tolerated the therapy with only mild  anorexia and diarrhea and with only a mild leukopenia (a decreased white blood cell count; leuko = white). Thirty days after the  completion of the initial course, these symptoms abated and he started his second course of chemotherapy with 5-FU as an out-  patient.   Because 5-FU inhibits synthesis of thymine, DNA synthesis is affected in all cells in the human body that are rapidly dividing, such as  the cells in the bone marrow that produce leukocytes and the mucosal cells lining the intestines. Inhibition of DNA synthesis in rapidly  dividing cells contributes to the side effects of 5-FU and many other chemotherapeutic drugs.   Fig. 12.23. The life cycle of a retrovirus. The virus contains two identical RNA strands, only one of which is shown for clarity. After penetrat-  ing the plasma membrane, the single- stranded viral RNA genome is reverse-transcribed to a double- stranded DNA form. The viral DNA migrates  to the nucleus and integrates into the chromosomal DNA, where it is transcribed to form a viral RNA transcript. The viral transcript can form  the viral RNA genome for progeny viruses, or can be translated to generate viral structural proteins.   humans contain DNA as their genetic material. Others contain RNA as their genetic  material.   Some viruses that contain an RNA genome are known as retroviruses. HIV, the  human immunodeficiency virus, is the retrovirus that causes AIDS (Fig 12.23). It  invades cells of the immune system and prevents the affected individual from  mounting an adequate immune response to combat infections.   According to the "central dogma" proposed by Francis Crick, information flows  from DNA to RNA to proteins. For the most part, this concept holds true. However,  retroviruses provide one violation of this rule. When retroviruses invade cells, their  RNA genome is transcribed to produce a DNA copy. The enzyme that catalyzes this  process is encoded in the viral RNA and is known as reverse transcriptase. This  DNA copy integrates into the genome of the infected cell, and enzymes of the host  cell are used to produce many copies of the viral RNA, as well as viral proteins,  which can be packaged into new viral particles.   Watson JD, Crick FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid.   Nature 1953;171:737-738.  American Cancer Society Web site (http://www.cancer.org): Colorectal cancer. This web site provides   information on the cause, treatment and prevention of a number of different cancers, and links to   The Human Genome Project Web site (http://www.nhgri.nih.gov/About_NHGRI/Der/Elsi/): This site  discusses the ethical issues involved in gene therapy protocols.   1. For the following DNA sequence, determine the sequence and direction of the complementary strand.  5 ' - ATCGATCGATCGATCG - 3 '   2. If the DNA strand shown below serves as a template for the synthesis of RNA, which of the following choices gives the  sequence and direction of the RNA?   3. In DNA, the bond between the deoxyribose sugar and the phosphate is which of the following?   4. How many double- stranded DNA molecules 8 base pairs long are theoretically possible?   5. The backbone of a DNA strand is composed of which of the following?   Fig. 13.1. A replicating DNA helix. The  parental strands separate at the replication  fork. Each parental strand serves as a template  for the synthesis of a new strand.   DNA synthesis occurs by the process of replication. During replication, each of  the two parental strands of DNA serves as a template for the synthesis of a com-  plementary strand. Thus, each DNA molecule generated by the replication process  contains one intact parental strand and one newly synthesized strand (Fig. 13.1).  In eukaryotes, DNA replication occurs during the S phase of the cell cycle, which  is followed by the G 2 phase. The cell divides during the next phase (M), and each  daughter cell receives an exact copy of the DNA of the parent cell.   The Replication Fork. In both prokaryotes and eukaryotes, the site at which  replication is occurring at any given moment is called the replication fork. As  replication proceeds, the two parental strands separate in front of the fork. Behind  the fork, each newly synthesized strand of DNA base-pairs with its complementary  parental template strand. A complex of proteins is involved in replication.  Helicases and topoisomerases unwind the parental strands, and single-strand  binding proteins prevent them from reannealing.   The major enzyme involved in replication is a DNA polymerase that copies  each parental template strand in the 3' to 5' direction, producing new strands in a  5' to 3' direction. Deoxyribonucleoside triphosphates serve as the precursors.  One strand of newly synthesized DNA grows continuously, whereas the other  strand is synthesized dis continuously in short segments known as Okazaki frag-  ments. These fragments are subsequently joined by DNA ligase.   Initiation. DNA polymerase cannot initiate the synthesis of new  strands. Therefore, a short primer is produced, which contains ribonucleotides  (RNA). DNA polymerase can add deoxy ribonucleotides to the 3' end of this  primer. This RNA primer is subsequently removed and replaced by  deoxy ribonucleotides .   Telomeres. The ends of linear chromosomes are called telomeres. The enzyme  telomerase, an RNA-dependant DNA polymerase that carries its own RNA tem-  plate, is required for their replication.   Errors and Repair. Errors occurring during replication could lead to deleteri-  ous mutations. However, many errors are corrected by enzyme activities associ-  ated with the complex at the replication fork. The error rate is thus kept at a very  low level.   Damage to DNA molecules also causes mutations. Repair mechanisms correct  DNA damage, usually by removing and replacing the damaged region. The intact,  undamaged strand serves as a template for the DNA polymerase involved in the  repair process.   Recombination. Although cells have mechanisms to correct replication  errors and to repair DNA damage, some genetic change is desirable. It pro-  duces new proteins or variations of proteins that may increase the survival rate  of the species. Genetic change is produced by unrepaired mutations and by a  mechanism known as recombination in which portions of chromosomes are  exchanged.   Ivy Sharer is having difficulty complying with her multidrug regimen.  She often forgets to take her pills. When she returns for a checkup, she asks  whether such a large number of pills are really necessary for treatment of  acquired immunodeficiency syndrome (AIDS).   Di Abietes responded to treatment for her diabetes mellitus but subse-  quently developed a low-grade fever, an increase in urinary urgency and  frequency, and burning at the urethral opening with urination (dysuria). A  urinalysis showed a large number of white blood cells and many Gram-negative  bacilli. A urine culture indicated many colonies of Escherichia coli, which is sensi-  tive to several antibiotics, including the quinolone norfloxacin.   Melvin (Mel) Anoma is a 46-year-old man who noted a superficial, brown-  ish-black, 5 -mm nodule with irregular borders in the skin on his chest. He  was scheduled for outpatient surgery, at which time a wide excision biopsy  was performed. (In an excision biopsy, the complete mole is removed and biopsy  performed). Examination of the nodule indicated histologic changes characteristic of  a malignant melanoma reaching a thickness of only 0.7 mm from the skin surface  (Stage I).   Nick O'Tyne is a 62-year-old electrician who has smoked two packs of  cigarettes a day for 40 years. He recently noted that his chronic cough had  gotten worse. His physician ordered a chest radiograph, which showed a  2-cm nodule in the upper lobe of the right lung. Cytologic study of the sputum by  Papanicolaou technique showed cells consistent with the presence of a well-  differentiated adenocarcinoma of the lung.   The basic features of the mechanism of DNA replication are illustrated by the  processes occurring in the bacterium E. coli. This bacillus grows symbiotically in the  human colon. It has been extensively studied and serves as a model for the more com-  plex and, consequently, less well-understood processes that occur in eukaryotic cells.   Replication of the circular, double- stranded DNA of the chromosome of E. coli  begins with the binding of approximately 30 molecules of the protein DnaA at a sin-  gle point of origin, designated oriC, where the DNA coils around the DnaA core  (Fig. 13.2). With the assistance of other proteins (e.g., a helicase, gyrase, and sin-  gle-stranded binding protein), the two parental strands separate within this region,  and both strands are copied simultaneously. Synthesis begins at the origin and  occurs at two replication forks that move away from the origin bidirectionally (in  both directions at the same time). Replication ends on the other side of the chro-  mosome at a termination point. One round of synthesis, involving the incorporation  of over 4 million nucleotides in each new strand of DNA, is completed in approxi-  mately 40 minutes. However, a second round of synthesis can begin at the origin  before the first round is finished. These multiple initiations of replication allow bac-  terial multiplication to occur much more quickly than the time it takes to complete  a single round of replication.    Fig. 13.2. Bidirectional replication of a circu-  lar chromosome. Replication begins at the  point of origin (oriC) and proceeds in both  directions at the same time.   Fig. 13.3. Proteins involved in separating and  unwinding parental DNA strands at the repli-  cation fork in prokaryotes.   Di Abietes' urinary tract infection  was treated with norfloxacin, a fluo-  rinated member of the quinolone  family. This group of drugs inhibits bacterial  DNA gyrase, a topoisomerase that unwinds  the closed circular bacterial DNA helix ahead  of the replication fork, and thus inhibits bacte-  rial DNA synthesis. Because eukaryotic cells  have linear DNA and do not contain DNA  gyrase, they are not affected by quinolones.   One of the drugs used to treat Ivy  Sharer was didanosine (ddl). It is a  dideoxynucleoside, an example of  which is shown below.   The dideoxynucleosides do not have a  hydroxyl group on either the 2'- or 3'-car-  bon. They can be converted to dideoxynu-  cleoside triphosphates in cells and, like ZDV,  terminate chain growth when incorporated  into DNA. In the case of the dideoxynucleo-  sides, chain termination results from the  absence of a hydroxyl group on the 3' car-  bon. The HIV virus mutates very rapidly  (mostly because reverse transcriptase lacks  3' to 5' exonuclease activity, the proofread-  ing activity) and frequently develops resist-  ance to one or more of these drugs. There-  fore, it is recommended that AIDS patients  take a number of drugs, including more than  one reverse transcriptase inhibitor.   Each daughter chromosome contains one of the parental DNA strands and one  newly synthesized, complementary strand. Therefore, replication is said to be semi-  conservative; i.e., the parental strands are conserved but are no longer together.  Each one is paired with a newly synthesized strand (see Figs. 13.1 and 13.2).   Replication requires separation of the parental DNA strands and unwinding of the  helix ahead of the replication fork. Helicases (DnaB) separate the DNA strands and  unwind the parental duplex. Single-strand binding proteins prevent the strands from  reassociating and protect them from enzymes that cleave single- stranded DNA  (Fig. 13.3). Topoisomerases, enzymes that can break phosphodiester bonds and  rejoin them, relieve the supercoiling of the parental duplex caused by unwinding.  DNA gyrase is a major topoisomerase in bacterial cells.   Enzymes that catalyze the synthesis of DNA are known as DNA polymerases.   E. coli has three DNA polymerases, Pol I, Pol II, and Pol III. Pol III is the major  replicative enzyme (Table 13.1). All DNA polymerases that have been studied copy  a DNA template strand in its 3 ' to 5 ' direction, producing a new strand in the 5 ' to  3' direction (Fig. 13.4). Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP,  and dTTP) serve as substrates for the addition of nucleotides to the growing chain.   The incoming nucleotide forms a base pair with its complementary nucleotide on  the template strand. Then an ester bond is formed between the first 5 '-phosphate of  the incoming nucleotide and the free 3' -hydroxyl group at the end of the growing  chain. Pyrophosphate is released. The release of pyrophosphate and its subsequent  cleavage by a pyrophosphatase provide the energy that drives the polymerization  process.   DNA polymerases that catalyze the synthesis of new strands during replication  exhibit a feature called processivity. They remain bound to the parental template  strand while continuing to "process" down the chain, rather than dissociating and  reassociating as each nucleotide is added. Consequently, synthesis is much more  rapid than it would be with an enzyme that was not processive.   In E. coli, the replicative enzyme Pol III also performs a proofreading or editing  function. This enzyme has 3 '-5' -exonuclease activity in addition to its polymerase  activity (see Table 13.1). If the nucleotide at the end of the growing chain is incor-  rectly base-paired with the template strand, Pol III removes this nucleotide before  continuing to lengthen the growing chain. This proofreading activity eliminates   Pol I Filling of gap after removal 5' to 3' and 3' to 5'   conjunction with RNAse H  Pol II DNA repair 3' to 5'   a Synthesis of new DNA strands always occurs 5' to 3'   b Exonucleases remove nucleotides from DNA strands and act at the 5' end (cleaving 5' to 3') or at the 3'  end (cleaving 3' to 5').   Fig. 13.4. Action of DNA polymerase. Deoxyribonucleoside triphosphates serve as precur-  sors (substrates) used by DNA polymerase to lengthen the DNA chain. DNA polymerase  copies the DNA template strand in the 3' to 5' direction. The new strand grows 5' to 3'.   most base-pairing errors as they occur. Only about one base pair in a million is mis-  matched in the final DNA product; the error rate is about 10 -6 . If this proofreading  activity is experimentally removed from the enzyme, the error rate increases to  about 10~ 3 .   After replication, other mechanisms replace mismatched bases that escaped  proofreading so that the fidelity of DNA replication is very high. The two processes  of proofreading and postreplication mismatch repair result in an overall error rate of  about 1CT 10 , that is, less than one mismatched base pair in 10 billion.   DNA polymerase cannot initiate the synthesis of new strands; it requires the pres-  ence of a free 3' -OH group to function. Therefore, a primer is required to supply the  free 3' -OH group. This primer is an RNA oligonucleotide. It is synthesized in a 5'  to 3' direction by an RNA polymerase (primase) that copies the DNA template  strand. DNA polymerase initially adds a deoxyribonucleotide to the 3'-hydroxyl  group of the primer and then continues adding deoxyribonucleotides to the 3 '-end  of the growing strand (Fig. 13.5).   Both parental strands are copied at the same time in the direction of the replication  fork, an observation difficult to reconcile with the known activity of DNA poly-  merase, which can produce chains only in a 5 ' to 3 ' direction. Because the parental  strands run in opposite directions relative to each other, synthesis should occur in a  5 ' to 3 ' direction toward the fork on one template strand and in a 5 ' to 3 ' direction  away from the fork on the other template strand.   Okazaki resolved this dilemma by showing that synthesis on one strand, called  the leading strand, is continuous in the 5' to 3' direction toward the fork. The other    Nucleases cleave phosphodiester  bonds. Endonucleases cleave  bonds within polynucleotide  chains. Exonucleases cleave bonds at the  ends of polynucleotide chains, removing  one nucleotide at a time. 5'-Exonucleases  start at the 5'-end of the chain and cleave 5'  to 3'. 3'-Exonucleases start at the 3'-end and  cleave 3' to 5'.   Fig. 13.5. Synthesis of DNA at the replication  fork. (See Fig. 13.6 for the ligation reaction.)   RNase H is a ribonuclease that  \\ ) specifically degrades RNA from an  V/ RNA-DNA hybrid. The HIV virus  (see Chapter 12) converts an RNA genome to  a double-stranded DNA copy using the  enzyme reverse transcriptase. An intermedi-  ate in the conversion of the single-stranded  RNA genome to double-stranded DNA is an  RNA-DNA hybrid. To remove the RNA so a  double-stranded DNA molecule can be  made, reverse transcriptase contains RNase  H activity. Scientists are currently attempting  to target the RNase H activity of reverse tran-  scriptase with inhibitors as a means of con-  trolling spread of the virus.   strand, called the lagging strand, is synthesized discontinuously in short fragments  (see Fig. 13.5). These fragments, named for Okazaki, are produced in a 5' to 3'  direction (away from the fork), but then joined together so that, overall, synthesis  proceeds toward the replication fork.   As replication progresses, the RNA primers are removed from Okazaki fragments,  probably by the combined action of DNA polymerase I (Pol I, using its 5 ' — » 3 '  exonuclease activity) and RNase H. Pol I fills in the gaps produced by removal of  the primers. Because DNA polymerases cannot join two polynucleotide chains  together, an additional enzyme, DNA ligase, is required to perform this function.  The 3' -hydroxy 1 group at the end of one fragment is ligated to the phosphate group  at the 5' -end of the next fragment (Fig. 13.6).   Fig. 13.6. Action of DNA ligase. Two polynu-  cleotide chains, one with a free 3' -OH group  and one with a free 5 '-phosphate group, are  joined by DNA ligase, which forms a phos-  phodiester bond.   The process of replication in eukaryotes is similar to that in prokaryotes. Differ-  ences in the processes are related mainly to the vastly larger amount of DNA in  eukaryotic cells (over 1,000 times the amount in E. coli) and the association of  eukaryotic DNA with histones in nucleosomes. Enzymes with DNA polymerase,  primase, ligase, helicase, and topoisomerase activity are all present in eukaryotes,  although these enzymes differ in some respects from those of prokaryotes.   The cell cycle of eukaryotes consists of four phases (Fig. 13.7). The first three  phases (G 1? S, and G 2 ) constitute interphase. Cells spend most of their time in these  three phases, carrying out their normal metabolic activities. The fourth phase is  mitosis, the process of cell division. This phase is very brief.   The first phase of the cell cycle, Q x (the first "gap" phase), is the most variable  in length. Late in G 1? the cells prepare to duplicate their chromosomes (e.g., by pro-  ducing nucleotide precursors). In the second or S phase, DNA replicates. Nucleo-  somes disassemble as the replication forks advance. Throughout S phase, the syn-  thesis of histones and other proteins associated with DNA is markedly increased.   Fig. 13.7. Eukaryotic cell cycle. The times given for the length of each phase are for cells  growing in culture.   The amount of DNA and histones both double, and chromosomes are duplicated.  Histones complex with DNA, and nucleosomes are formed very rapidly behind the  advancing replication forks.   During the third phase of the cell cycle, G 2 (the second "gap" phase), the cells  prepare to divide and synthesize tubulin for construction of the microtubules of the  spindle apparatus. Finally, division occurs in the brief mitotic or M phase.   After mitosis, some cells reenter G 1? repeatedly going through the phases of the  cell cycle and dividing. Other cells leave the cycle after mitosis, never to divide  again, or they enter an extended G T phase (sometimes called G 0 ), in which they  remain for long periods. On the appropriate signal, cells in G 0 are stimulated to  reenter the cycle and divide.   In contrast to bacterial chromosomes (see section LA. of this chapter), eukaryotic  chromosomes have multiple points of origin at which replication begins. "Bubbles"  appear at these points on the chromosomes. At each end of a bubble, a replication  fork forms; thus, each bubble has two forks. DNA synthesis occurs at each of these  forks, as illustrated in Figure 13.8. As the bubbles enlarge, they eventually merge,  and replication is completed. Because eukaryotic chromosomes contain multiple  points of origin of replication (and, thus, multiple replicons-units of replication),  duplication of such large chromosomes can occur within a few hours.   At least nine DNA polymerases exist in eukaryotic cells (a, (3, 7, 8, e, £, k, t|, and 1)  (Table 13.2). Polymerase 8 (pol 8) is the major replicative enzyme. Pol a and pol e  are also involved in relication. Polymerases p and e, as well as pol a, appear to be  involved in DNA repair. Pol 7 is located in mitochondria and replicates the DNA of  this organelle. Polymerases £, k, t\ and 1, which lack 3' —> 5' exonuclease activity,  are used when DNA is damaged.   HeLa cells, derived from a human  \\ ) cervical carcinoma, are rapidly   dividing cells that can be grown in  culture flasks. Their cell cycle is approxi-  mately 20 hours. Only 1 hour of this time is  spent in mitosis.   Cells within the body that divide less fre-  quently, such as liver cells, spend days,  weeks, or months in interphase before going  through a brief mitotic phase.   Although Prometheus was chained  to a rock as punishment for his  theft of fire from the gods, and a  vulture pecked at his liver each day, he sur-  vived. Can you guess why?   In the human body, many cells  cycle frequently, e.g., hair follicles,  skin cells, and cells of the duodenal  crypts. Other cells, such as the precursors of  red blood cells, divide a number of times,  then lose their nuclei and leave the cell cycle  to form mature red blood cells. These cells  transport oxygen and carbon dioxide  between the lungs and other tissues for  about 120 days, then die. Other cells are nor-  mally quiescent (in G 0 ). However, they can  be stimulated to divide. In many instances,  the stimuli are growth factors or hormones  (e.g., mammary aveolar cells and uterine  cells). In the case of liver cells, the stimulus  is produced by death of some of the cells.   Many proteins bind at or near the replication fork and participate in the process of  duplicating DNA (Fig. 13.9)(Table 13.3). Polymerase 8 (pol 8) is the major replica-   a Synthesis of new DNA strands always occurs 5' to 3'.   b Bypass polymerase are able to "bypass" areas of DNA damage and continue DNA replication. Some  enzymes are error-free and insert the correct bases; other enzymes are error prone and insert random  bases.   Fig. 13.8. Replication of a eukaryotic chro-  mosome. Synthesis is bidirectional from each  point of origin (O) and semiconservative, each  daughter DNA helix contains one intact  parental strand (solid line) and one newly syn-  thesized strand (dashed line).   Okazaki fragments are much  smaller in eukaryotes than in  prokaryotes (about 200 nucleotides  vs. 1,000 to 2,000). Because the size of  eukaryotic Okazaki fragments are equivalent  to the size of the DNA found in nucleosomes,  it seems likely that one nucleosome at a time  may release its DNA for replication.   Fig. 13.9. Replication complex in eukaryotes. The lagging strand is shown looped around  the replication complex. Single- strand binding proteins (not shown) are bound to the  unpaired, single- stranded DNA. Other proteins also participate in this complex (see text).   tive enzyme. However, before it acts, a primase associated with polymerase a (pol  8) produces an RNA primer (approximately 10 nucleotides in length). Then pol a  adds about 20 deoxyribonucleotides to this RNA and dissociates from the template,  because of the low processivity of pol a. On the leading strand, pol 8 adds deoxyri-  bonucleotides to this RNA-DNA primer, continuously producing this strand. Pol 8  is a highly processive enzyme.   The lagging strand is produced from a series of Okazaki fragments (see Fig.  13.5). Synthesis of each Okazaki fragment is initiated by pol a and its associated  primase, as described above. After pol a dissociates, pol 8 adds deoxyribonu-  cleotides to the primer, producing an Okazaki fragment. Pol 8 stops synthesizing  one fragment when it reaches the start of the previously synthesized Okazaki frag-  ment (see Fig. 13.5). The primer of the previously synthesized Okazaki fragment is   ® Liver cells are in G 0 . Up to 90% of  the human liver can be removed.  The remaining liver cells are stimu-  lated to re-enter the cell cycle and divide,  regenerating a mass equivalent to the origi-  nal mass of the liver within a few weeks. The  myth of Prometheus indicates that the  capacity of the liver to regenerate was rec-  ognized even in ancient times.   5' — > 3', copying a DNA template 3' — > 5'   - Flap endonucleases 1 (FEN1) -  recognizes "flap"  (Unannealed portion of RNA) near  5'-end of primer and cleaves downstream  in DNA region of primer   - Joins, by forming a phosphodiester  bond, two adjacent DNA strands that are  bound to the same template   removed by flap endonuclease 1 (FEN1) and Rnase H. The gap left by the primer  is filled by a DNA polymerase that uses the parental DNA strand as its template and  the newly synthesized Okazaki fragment as its primer. DNA ligase subsequently  joins the Okazaki fragments together (see Fig. 13.6).   Obviously, eukaryotic replication requires many proteins. The complexity of the  fork and the fact that it is not completely understood limits the detail shown in Fig-  ure 13.9. One protein not shown is proliferating cell nuclear antigen (PCNA), which  is involved in organizing and orchestrating the replication process.   Additional activities that occur during replication include proofreading and  DNA repair. Pol 8, which is part of the replication complex, has the 3' — > 5'-  exonuclease activity required for proofreading. Enzymes that catalyze repair of  mismatched bases are also present (see section III.B.3 of this chapter). Conse-  quently, eukaryotic replication occurs with high fidelity; approximately one mis-  pairing occurs for every 10 9 to 10 12 nucleotides incorporated into growing DNA  chains.   Proliferating cell nuclear antigen  (PCNA) is used clinically as a diag-  nostic marker for proliferating   Eukaryotic chromosomes are linear, and the ends of the chromosomes are called  telomeres. As DNA replication approaches the end of the chromosome, a problem  develops in the lagging strand (Fig. 13.10). Either primase cannot lay down a primer  at the very end of the chromosome, or, after DNA replication is complete, the RNA  at the end of the chromosome is degraded. Consequently, the newly synthesized  strand is shorter at the 5' end, and there is a 3' -overhang in the DNA strand being  replicated. If the chromosome became shorter with each successive replication,  genes would be lost. How is this problem solved?   The 3 ' overhang is lengthened by the addition of telomeres so that primase can  bind and synthesize the complementary strand. Telomeres consist of a repeating  sequence of bases (TTAGGG for humans), which may be repeated thousands of  times. The enzyme telomerase contains both proteins and RNA and acts as an RNA-  dependent DNA polymerase (just like reverse transcriptase). The RNA within  telomerase contains the complementary copy of the repeating sequence in the  telomeres and can base pair with the existing 3 '-overhang (Fig. 13.11). The poly-  merase activity of telomerase then uses the existing 3'- hydroxyl group of the over-  hang as a primer, and its own RNA as a template, and synthesizes new DNA that  lengthens the 3'- end of the DNA strand. The telomerase moves down the DNA  toward the new 3'- end and repeats the process a number of times. When the 3'-  overhang is sufficiently long, primase binds, and synthesis of the complementary  strand is completed. The 3' overhang can also form a complicated structure with  telomere binding proteins to protect the ends of the chromosomes from damage and  nuclease attack once they have been lengthened   Fig. 13.10. The "end-replication" problem in  linear chromosomes. After replication, the  telomeres have 3 '-overhangs. When these mol-  ecules are replicated, chromosome shortening  will result. The figure depicts a linear chromo-  some with one origin of replication. At the ori-  gin, two replication forks are generated, each  moving in the opposite direction, labeled as  Fork I and Fork II. As Fork I moves to the  right, the bottom strand is read in the 3' to 5'  direction, which means it is the template for  the leading strand. The newly synthesized  DNA complementary to the upper strand at  Fork 1 will be the lagging strand. Now con-  sider Fork 2. As this replication fork moves to  the left, the upper strand is read in the 3' to 5'  direction, so the newly synthesized DNA com-  plementary to this strand will be the leading  strand. For this fork, the newly synthesized  DNA complementary to the bottom strand will  be the lagging strand.    Fig. 13.11. Telomerase action. The RNA present in telomerase base-pairs with the over-  hanging 3 '-end of telomeres and extends it by acting both as a template and a reverse tran-  scriptase. After copying a small number of repeats, the complex moves down to the 3 '-end  of the overhang and repeats the process.   An inability to replicate telomeres  \\ ) has been linked to cell aging and  *S death. Many somatic cells do not  express telomerase; when placed in culture  they survive a fixed number of population  doublings, enter senescence, and then die.  Analysis has shown significant telomere  shortening in those cells. In contrast, stem  cells do express telomerase and appear to  have an infinite lifetime in culture. Research  is underway to understand the role of telom-  eres in cell aging, growth, and cancer.   Nick O'Tyne has been smoking for  40 years in spite of the warnings on  cigarette packs that this habit can  be dangerous and even deadly. The burning  of tobacco, and, for that matter, the burning  of any organic material, produces many dif-  ferent carcinogens, such as benzo[a]pyrene.  These carcinogens coat the airways and  lungs. They can cross cell membranes and  interact with DNA, causing damage to bases  that interferes with normal base pairing. If  these DNA lesions cannot be repaired or if  they are not repaired rapidly enough, a per-  manent mutation can be produced when the  cells replicate. Some mutations are silent,  whereas other mutations can lead to abnor-  mal cell growth, and cancer results.   Melanomas develop from exposure  of the skin to the ultraviolet rays of  the sun. The ultraviolet radiation  causes pyrimidine dimers to form in DNA.  Mutations may result that produce  melanomas, appearing as dark brown  growths on the skin.   Fortunately, Mel Anoma's malignant skin  lesion was discovered at an early stage.  Because there was no evidence of cancer in  the margins of the resected mass, full recov-  ery was expected. However, lifelong surveil-  lance for return of the melanoma was  planned.   Pyrimidine dimers, most com-  \ \ ) monly thymine dimers, can be  *r repaired by photoreactivating  enzymes that cleave the bonds between the  bases by using energy from visible light. In  this process, nucleotides are not removed  from the damaged DNA. This repair process  is used by bacteria and might serve as a very  minor repair mechanism in human cells.   Despite proofreading and mismatch repair during replication, some mismatched  bases do persist. Additional problems may arise from DNA damaged by mutagens,  chemicals produced in cells, inhaled, or absorbed from the environment that cause  mutations. Mutagens that cause normal cells to become cancer cells are known as  carcinogens. Unfortunately, mismatching of bases and DNA damage produce thou-  sands of potentially mutagenic lesions in each cell every day. Without repair mech-  anisms, we could not survive these assaults on our genes.   DNA damage can be caused by radiation and by chemicals (Fig. 13.12). These  agents can directly affect the DNA or they can act indirectly. For example, x-rays,  a type of ionizing radiation, act indirectly to damage DNA by exciting water in the  cell and generating the hydroxyl radical, which reacts with DNA, thereby altering  the structure of the bases or cleaving the DNA strands.   While exposure to x-rays is infrequent, it is more difficult to avoid exposure to  cigarette smoke and virtually impossible to avoid exposure to sunlight. Cigarette  smoke contains carcinogens such as the aromatic polycyclic hydrocarbon  benzo[<2]pyrene (see Fig. 13.12). When this compound is oxidized by cellular  enzymes, which normally act to make foreign compounds more water soluble and  easy to excrete, it becomes capable of forming bulky adducts with guanine residues  in DNA. Ultraviolet rays from the sun, which also produce distortions in the DNA  helix, excite adjacent pyrimidine bases on DNA strands, causing them to form cova-  lent dimers (Fig. 13.13).   The mechanisms used for the repair of DNA have many similarities (Fig. 13.14).  First, a distortion in the DNA helix is recognized, and the region containing the dis-  tortion is removed. The gap in the damaged strand is replaced by the action of a  DNA polymerase that uses the intact, undamaged strand as a template. Finally, a lig-  ase seals the nick in the strand that has undergone repair.    Fig. 13.12. Oxidation of benzo[a]pyrene and covalent binding to DNA. Benzo[«]pyrene is  not carcinogenic until it is oxidized within cells. Then it can covalently bind to guanine  residues in DNA, interrupting hydrogen bonding in G-C base pairs and producing distortions  of the helix.   Nucleotide excision repair involves local distortions of the DNA helix, such as  mismatched bases or bulky adducts (e.g., oxidized benzoyl pyrene) (Fig. 13.15, see  also Fig. 13.14). Endonucleases cleave the abnormal chain and remove the distorted  region. The gap is then filled by a DNA polymerase that adds deoxyribonucleotides,  one at a time, to the 3' -end of the cleaved DNA, using the intact, complementary  DNA strand as a template. The newly synthesized segment is joined to the 5 '-end  of the remainder of the original DNA strand by a DNA ligase.   DNA glycosylases recognize small distortions in DNA involving lesions caused by  damage to a single base (e.g., the conversion of cytosine to uracil). A glycosylase  cleaves the TV-glycosidic bond that joins the damaged base to deoxyribose (see Fig.  13.15). The sugar-phosphate backbone of the DNA now lacks a base at this site  (known as an apurinic or apyrimidinic site, or an AP site). Then an AP endonucle-  ase cleaves the sugar-phosphate strand at this site. Subsequently, the same types of  enzymes involved in other types of repair mechanisms restore this region to normal.   Mismatched bases (bases that do not form normal Watson-Crick base pairs) are rec-  ognized by enzymes of the mismatch repair system. Because neither of the bases in  a mismatch is damaged, these repair enzymes must be able to determine which base  of the mispair to correct.   The mismatch repair enzyme complex acts during replication when an incorrect, but  normal base (i.e., A, G, C, or T) is incorporated into the growing chain (Fig. 13.16). In  bacteria, parental DNA strands contain methyl groups on adenine bases in specific  sequences. During replication, the newly synthesized strands are not immediately  methylated. Before methylation occurs, the proteins involved in mismatch repair can  distinguish parental from newly synthesized strands. A region of the new, unmethy-  lated strand, containing the mismatched base, is removed and replaced.   Human enzymes also can distinguish parental from newly synthesized strands  and repair mismatches. However, the mechanisms have not yet been as clearly  defined as those in bacteria.   Pyrimidine dinners occur frequently in the skin. Usually repair mechanisms cor-  rect this damage, and cancer rarely occurs. However, in individuals with xero-  derma pigmentosum, cancers are extremely common. These individuals have  defects in their DNA repair systems. The first defect to be identified was a deficiency of  the endonuclease involved in removal of pyrimidine dimers from DNA. Because of the  inability to repair DNA, the frequency of mutation increases. A cancer develops once  proto-oncogenes or tumor suppressor genes mutate. By scrupulously avoiding light,  these individuals can reduce the number of skin cancers that develop.   Spontaneous deamination occurs frequently in human DNA and converts cyto-  KJ ] sine bases to uracil. This base is not normally found in DNA and is potentially   harmful because U pairs with A, forming U-A base pairs instead of the normal  C-G pairs. To prevent this change from occurring, a uracil A/-glycosylase removes uracil,  and it is replaced by a cytosine via base excision repair.   Hereditary nonpolyposis colorectal cancer (a human cancer that does not arise  from intestinal polyps) is caused by mutations in genes for proteins involved in  mismatch repair (hMSH1, hMSH2, hPMS1, or hPMS2). The inability to repair  mismatches increases the mutation frequency, resulting in cancers from mutations in  growth regulatory genes.   Fig. 13.13. A thymine dimer in a DNA strand.  Ultraviolet light can cause two adjacent pyrim-  idines to form a covalent dimer.   Fig. 13.15. Types of damage and various  repair mechanisms. In base excision repair, the  glycosylase cleaves the glycosidic bond  between the altered base (shown with an X)  and ribose. In nucleotide excision repair, the  entire nucleotide is removed at once. The gap  formed by the incision (cut) and excision  (removal) endonucleases is usually several  nucleotides wider than the one shown.   Fig. 13.16. Mismatch repair. Normal, undamaged but mismatched bases bind proteins of the  mismatch repair system. In bacteria, these proteins recognize the older, parental strand  because it is methylated and replace a segment of newly synthesized (and unmethylated)  DNA containing the mismatched base. The mechanism for distinguishing between parental  and newly synthesized strands in humans is not as well understood.   Genes that are actively transcribed to produce mRNA are preferentially repaired.  The RNA polymerase that is transcribing a gene (see Chapter 14 for a description  of the process) stalls when it encounters a damaged region of the DNA template.  Excision repair proteins are attracted to this site and repair the damaged region.  Subsequently, RNA polymerase can resume transcription.   The exchange of segments between DNA molecules occurs quite frequently and is  responsible for genetic alterations that can have beneficial or devastating conse-  quences for the affected individuals and, in some instances, for their offspring. The  DNA segments that are exchanged may be homologous (that is, of very similar  sequence) or they may be totally unrelated. The size of these segments can range  from a few nucleotides to tens of thousands and can include many different genes  or portions of genes. Many of the enzymes involved in these exchanges are the same  as or similar to those used for replication and repair and include endonucleases,  exonucleases, unwinding enzymes, topoisomerases, DNA polymerases, and ligases.   One type of genetic rearrangement that has been observed for many years is  "crossing-over" between homologous chromosomes during meiosis. Another type  occurs in stem cells as they differentiate into lymphocytes. Segments of the genes  of stem cells are rearranged so that the mature cell is capable of producing only a  single type of antibody (see Chapter 16). Other types of genetic exchanges involve  transposable elements (transposons) that can move from one site in the genome to  another or produce copies that can be inserted into new sites. Translocations occur  when chromosomes break and portions randomly become joined to other chromo-  somes, producing gross changes that can be observed under the light microscope.  Genetic exchanges can even occur between species, for example, when foreign  DNA is inserted into the human genome as a result of viral infection.   Various models, supported by experimental evidence, have been proposed for the  mechanism of recombination between homologous DNA sequences. Although   these mechanisms are complex, a simplified scheme for one type of recombination  is presented in Figure 13.17.   Initially, two homologous chromosomes or segments of double-helical (duplex)  DNA that have very similar, but not necessarily identical, sequences become  aligned (see Fig. 13.17). One strand of one duplex is nicked by an enzyme and  invades the other DNA duplex, base-pairing with a region of complementary  sequence. The match between the sequences does not have to be perfect, but a sig-  nificant number of bases must pair so that the strand displaced from its partner can  form a displacement (D) loop. This D loop is nicked, and the displaced strand now  base-pairs with the former partner of the invading strand. Ligation occurs, and a  Holliday structure is generated (see Fig. 13.17). The branch point of the Holliday  structure can migrate and may move many thousands of nucleotides from its origi-  nal position. The Holliday structure, named for the scientist who discovered it, is  finally cleaved and then religated, forming two chromosomes that have exchanged  segments. In addition to enzymes similar to those used in DNA replication,  enzymes for strand invasion, branch migration, and cleavage of the Holliday struc-  ture are required.   Breaks in chromosomes, caused by agents such as x-rays or chemical carcino-  gens, can result in gross chromosomal rearrangements (Fig. 13.18). If the free  ends of the DNA at the break point reseal with the free ends of a different bro-  ken chromosome, a translocation is produced. These exchanges of large portions  of chromosomes can have deleterious effects and are frequently observed in  cancer cells.   Movable (or transposable) genetic elements, "jumping genes," were first observed  by Barbara McClintock in the 1940s. Her work, initially greeted with skepticism,  was ultimately accepted, and she was awarded the Nobel Prize in 1983.   Transposons are segments of DNA that can move from their original position in  the genome to a new location (Fig. 13.19). They are found in all organisms. Trans-  posons contain the gene for an enzyme called a transposase, which is involved in  cleaving the transposon from the genome and moving it from one location to  another.   Retroposons are similar to transposons except that they involve an RNA inter-  mediate. Reverse transcriptase (see below) makes a single-stranded DNA copy of  the RNA. A double- stranded DNA is then produced that is inserted into the genome  at a new location.   Reverse transcriptase is an enzyme that uses a single- stranded RNA template and  makes a DNA copy (Fig. 13.20). The RNA template can be transcribed from DNA  by RNA polymerase or obtained from another source, such as an RNA virus. The  DNA copy of the RNA produced by reverse transcriptase is known as complemen-  tary DNA (because it is complementary to the RNA template), or cDNA. Retro-  viruses (RNA viruses) contain a reverse transcriptase, which copies the viral RNA  genome. A double-stranded cDNA is produced, which can become integrated into  the human genome (see Fig. 12.23). After integration, the viral genes may be inac-  tive, or they may be transcribed, sometimes causing diseases such as AIDS or can-  cer (see Chapter 18).   Fig. 13.18. A chromosomal translocation. A  portion of the long arm of chromosome 8 is  exchanged for a portion of the long arm of  chromosome 14. This chromosomal transloca-  tion occurs in Burkitt's lymphoma.   Fig. 13.20. Action of reverse transcriptase.  This enzyme catalyzes the production of a  DNA copy from an RNA template. The RNA  of a DNA-RNA hybrid is degraded, and the  single DNA strand is used as a template to  make double- stranded DNA. This figure is a  simplified version of a more complex process.   Fig. 13.19. Transposons. The steps involved in transposition are shown. Direct repeats are  regions of DNA that have the same base sequence in the 5 ' — > 3 ' direction.   ^ Ivy Sharer. Ivy Sharer contracted AIDS when she used needles con-  ) taminated with HIV to inject drugs intravenously. Intravenous drug abusers  account for 15 to 20% of new AIDS cases in the United States. HIV  mutates rapidly, and, therefore, current treatment involves a combination of drugs  that affect different aspects of its life cycle. This multidrug therapy lowers the viral  titer (the number of viral particles found in a given volume of blood), sometimes to  undetectable levels. However, if treatment is not followed carefully (i.e., if the  patient is not "compliant"), the titer increases rapidly. Therefore, Ivy's physician  emphasized that she must carefully follow her drug regimen.   Di Abietes. Di Abietes' poorly controlled diabetes mellitus predisposed  her to a urinary tract infection because glucose in the urine serves as a "cul-  ture medium" for bacterial growth. The kidney glomerulotubular unit reab-  sorbs filtered glucose so that normally, the urine is glucose-free. However, when  serum blood glucose levels exceed 175 to 185 mg/dL (the tubular threshold for glu-  cose), the capacity for reabsorption is exceeded. In Ms. Abietes' case, blood glucose  levels frequently exceed this threshold.   ^ Mel Anoma. The average person has 20 moles on the body surface;  however, only seven people of every 100,000 develop a malignant  melanoma. The incidence of malignant melanoma, however, is rising rap-  idly. Because 35 to 40% of patients with malignant melanoma die as a result of this  cancer, the physician's decision to perform a biopsy on a pigmented mole with an  irregular border and variation of color probably saved Mel Anoma's life.   Nick O'Tyne. Lung cancer currently accounts for one fifth of all cancers  in men and one tenth in women. The overall 5 -year survival rate is still less  than 15%. For those who smoke two or more packs of cigarettes daily, as does   Nick O'Tyne, the death rate is 265 per 100,000 population. Thankfully, cigarette  smoking has declined in the United States. Whereas 50% of men and 32% of women  smoked in 1965, these figures have currently fallen to 26% and 24%, respectively.   Chemical carcinogens and tumor promoters. Once it was  realized that a number of chemicals react with DNA, leading to muta-  tions, and that mutations may lead to tumor formation, toxicologists  searched for chemicals with the ability to cause skin tumors in rats. To test a  chemical, it was applied to an area of shaved skin on the back of a rat to see  whether a tumor would develop. The chemicals fell into two groups: Group I, the  initiators, caused mutations in DNA and group II, the promoters, greatly  enhanced the probability that cells that had been previously initiated would  develop a tumor. Compounds falling into group II activate a protein kinase (pro-  tein kinase C), which is usually only transiently activated when normal cells are  stimulated to grow. Tumor promoters, however, are long-lived, and lead to an  enhanced activation of protein kinase C.   Is there an easy way to determine whether a compound is an initiator? Bruce  Ames developed a rapid and simple test to determine whether chemicals are muta-  gens. The basic test uses bacteria that have a mutation in a gene necessary for his-  tidine biosynthesis and require histidine for growth. The bacteria are treated with  the test chemicals and the number that can grow in the absence of histidine meas-  ured. Bacteria that no longer require histidine for growth must have acquired a sec-  ond, chemically induced mutation that opposed the inactivating, original mutation.  In this fashion, the ability of chemicals to alter DNA can be determined. Because  many chemicals (such as benz[o]pyrene) do not become carcinogenic in humans  until they are metabolized by the liver, the Ames test also involves incubating the  chemical with a liver extract, and then testing the metabolized extract for DNA-  modifying activity.   Hanawalt P. Transcription-coupled repair and human disease. Science 1994;266:1957-1958.  Kunkel T. DNA replication fidelity. J Biol Chem 1992;267:18251-18254.  Modrich P. Mismatch repair, genetic stability, and cancer. Science 1994;266:1959-1960.  Sancar A. Mechanisms of DNA excision repair. Science 1994;266:1954-1956.   Waga S, Stillman B. The DNA replication fork in eukaryotic cells. Annu Rev Biochem 1998;67:721-751.    1 . Reverse transcriptase, an RNA-dependant DNA polymerase, differs from DNA polymerase 8 by which of the following?    236 SECTION THREE / GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS   2. If a 1 ,000-kilobase fragment of DNA has 10 evenly spaced and symmetric replication origins and DNA polymerase moves at  1 kilobase per second, how many seconds will it take to produce two daughter molecules (ignore potential problems at the  ends of this linear piece of DNA)? Assume that the 10 origins are evenly spaced from each other, but not from the ends of the  chromosome.   3. Primase is not required during DNA repair processes because of which of the following?   (E) DNA polyemerase I or III can use any 3 ' -OH for elongation.   4. Which of the following enzymes is required to actively enhance the separation of DNA strands during replication?   5. The key mechanistic failure in patients with xeroderma pigmentosum involves which of the following?   Synthesis of RNA from a DNA template is called transcription. Genes are tran-  scribed by enzymes called RNA polymerases that generate a single -stranded  RNA identical in sequence ( with the exception of U in place of T) to one of the  strands of the double-stranded DNA. The DNA strand that directs the sequence  of nucleotides in the RNA by complementary base-pairing is the template  strand. The RNA strand that is initially generated is the primary transcript.  The DNA template is copied in the 3' to 5' direction, and the RNA transcript is  synthesized in the 5' to 3' direction. RNA polymerases differ from DNA poly-  merases in that they can initiate the synthesis of new strands in the absence of  a primer.   In addition to catalyzing the polymerization of ribonucleotides, RNA poly-  merases must be able to recognize the appropriate gene to transcribe, the appro-  priate strand of the double -stranded DNA to copy, and the startpoint of transcrip-  tion (Fig. 14.1 ). Specific sequences on DNA, called promoters, determine where  the RNA polymerase binds and how frequently it initiates transcription. Other reg-  ulatory sequences, such as promoter-proximal elements and enhancers, also  affect the frequency of transcription.   In bacteria, a single RNA polymerase produces the primary transcript precur-  sors for all three major classes of RNA : messenger RNA (mRNA), ribosomal RNA  (rRNA), and transfer RNA (tRNA). Because bacteria do not contain nuclei, ribo-  somes bind to mRNA as it is being transcribed, and protein synthesis occurs  simultaneously with transcription.   Eukaryotic genes are transcribed in the nucleus by three different RNA  polymerases, each principally responsible for one of the major classes of RNA.  The primary transcripts are modified and trimmed to produce the mature  RNAs. The precursors of mRNA (called pre-mRNA) have a guanosine "cap"  added at the 5' -end and a poly (A) "tail" at the 3 '-end. Exons, which contain  the coding sequences for the proteins, are separated in pre-mRNA by introns,  regions that have no coding function. During splicing reactions, introns are  removed and the exons connected to form the mature mRNA. In eukaryotes,  tRNA and rRNA precursors are also modified and trimmed, although not as  extensively as pre-mRNA.   Fig. 14.1. Regions of a gene. A gene is a seg-  ment of DNA that functions as a unit to gener-  ate an RNA product or, through the processes  of transcription and translation, a polypeptide  chain. The transcribed region of a gene con-  tains the template for synthesis of an RNA,  which begins at the startpoint. A gene also  includes regions of DNA that regulate produc-  tion of the encoded product, such as a pro-  moter region. In a structural gene, the tran-  scribed region contains the coding sequences  that dictate the amino acid sequence of a  polypeptide chain.   Anne Niemick is a 4-year-old girl of Mediterranean ancestry whose  height and body weight are below the 20th percentile for girls of her age.  She is listless, tires easily, and complains of loss of appetite and shortness  of breath on exertion. A dull pain has been present in her right upper quadrant for   The thalassemias are a heteroge-  nous group of hereditary anemias  that constitute the most common  gene disorder in the world, with a carrier  rate of almost 7%. The disease was first dis-  covered in countries around the Mediter-  ranean Sea and was named for the Greek  word "thalassa" meaning "sea". However, it  is also present in areas extending into India  and China that are near the equator.   The thalassemia syndromes are caused  by mutations that decrease or abolish the  synthesis of the a or p chains in the adult  hemoglobin A tetramer. Individual syn-  dromes are named according to the chain  whose synthesis is affected and the severity  of the deficiency. Thus, in p° thalassemia,  the superscript 0 denotes none of the (3 chain  is present; in (3 + thalassemia, the + denotes  a partial reduction in the synthesis of the p  chain. More than 170 different mutations  have been identified that cause p tha-  lassemia; most of these interfere with the  transcription of (3-globin mRNA or its pro-  cessing or translation.   the last 3 months. Her complexion is slate-gray and she appears pale. Initial labo-  ratory studies indicate a severe anemia (decreased red blood cell count) with a  hemoglobin of 6.2 g/dL (reference range, 12-16). A battery of additional hemato-  logic tests shows that Anne has p + -thalassemia, intermediate type.   Ivy Sharer, a patient with AIDS (see Chapters 12 and 13), has developed  a cough that produces a gray, slightly blood-tinged sputum. A chest radi-  ograph indicates infiltrates in the cavities of both upper lung fields (cavi-  tary infiltrates). A stain of sputum shows the presence of acid-fast bacilli, suggest-  ing a diagnosis of pulmonary tuberculosis caused by Mycobacterium tuberculosis.   Amanda Tin picked mushrooms in a wooded area near her home. A few  hours after eating one small mushroom, she experienced mild nausea and  diarrhea. She brought a mushroom with her to the hospital emergency  room. A poison expert identified it as Amanita phalloides (the "death cap"). These  mushrooms contain the toxin a-amanitin.   Sis Lupus, a 28 -year-old computer programmer, notes increasing fatigue,  pleuritic chest pain, and a nonproductive cough. In addition, she com-  plains of joint pains, especially in her hands. A rash on both cheeks and  the bridge of her nose ("butterfly rash") has been present for the last 6 months. Ini-  tial laboratory studies indicate a subnormal white blood cell count and a mild reduc-  tion in hemoglobin. Tests result in a diagnosis of systemic lupus erythematous  (SLE) (frequently called lupus).   Transcription, the synthesis of RNA from a DNA template, is carried out by RNA poly-  merases (Fig. 14.2). Like DNA polymerases, RNA polymerases catalyze the formation  of ester bonds between nucleotides that base-pair with the complementary nucleotides  on the DNA template. Unlike DNA polymerases, RNA polymerases can initiate the  synthesis of new chains in the absence of primers. They also lack the 3' to 5' exonu-  clease activity found in DNA polymerases. A strand of DNA serves as the template for  RNA synthesis and is copied in the 3' to 5' direction. Synthesis of the new RNA mol-  ecule occurs in the 5' to 3' direction. The ribonucleoside triphosphates ATP, GTP, CTP,  and UTP serve as the precursors. Each nucleotide base sequentially pairs with the com-  plementary deoxyribonucleotide base on the DNA template (A, G, C, and U pair with  T, C, G and A, respectively). The polymerase forms an ester bond between the a-phos-  phate on the ribose 5' -hydroxy 1 of the nucleotide precursor and the ribose 3'-hydroxyl  at the end of the growing RNA chain. The cleavage of a high-energy phosphate bond  in the nucleotide triphosphate and release of pyrophosphate (from the p and 7 phos-  phates) provides the energy for this polymerization reaction. Subsequent cleavage of  the pyrophosphate by a pyrophosphatase also helps to drive the polymerization reac-  tion forward by removing a product.   RNA polymerases must be able to recognize the startpoint for transcription of each  gene and the appropriate strand of DNA to use as a template. They also must be sensi-  tive to signals that reflect the need for the gene product and control the frequency of  transcription. A region of regulatory sequences called the promoter, usually contiguous  with the transcribed region, controls the binding of RNA polymerase to DNA and iden-  tifies the startpoint (see Fig. 14.1). The frequency of transcription is controlled by reg-  ulatory sequences within the promoter, nearby the promoter (promoter-proximal ele-  ments), and by other regulatory sequences, such as enhancers, that may be located at  considerable distances, sometimes thousands of nucleotides, from the startpoint. Both  the promoter-proximal elements, and the enhancers interact with proteins that stabilize  RNA polymerase binding to the promoter.    Fig. 14.2. RNA synthesis. The a-phosphate from the added nucleotide (shown in black) con-  nects the ribosyl groups.   Bacterial cells have a single RNA polymerase that transcribes DNA to generate all  of the different types of RNA (mRNA, rRNA, and tRNA). The RNA polymerase of  Escherichia coli contains four subunits (a 2 pp'), which form the core enzyme.  Another protein called a a (sigma) factor binds the core enzyme and directs bind-  ing of RNA polymerase to specific promoter regions of the DNA template. The a  factor dissociates shortly after transcription begins. E. coli has a number of differ-  ent a factors that recognize the promoter regions of different groups of genes. The  major a factor is a 70 , a designation related to its molecular weight of 70,000  Daltons.   In contrast to prokaryotes, eukaryotic cells have three RNA polymerases (Table  14.1). Polymerase I produces most of the rRNAs, polymerase II produces mRNA,  and polymerase III produces small RNAs, such as tRNA and 5S rRNA. All of these  RNA polymerases have the same mechanism of action. However, they recognize  different types of promoters.   Double- stranded DNA consists of a coding strand and a template strand (Fig.  14.3) The DNA template strand is the strand that is actually used by RNA poly-  merase during the process of transcription. It is complementary and antiparallel  both to the coding (nontemplate) strand of the DNA and to the RNA transcript  produced from the template. Thus, the coding strand of the DNA is identical in  base sequence and direction to the RNA transcript, except, of course, that wherever  this DNA strand contains a T, the RNA transcript contains a U. By convention, the   Patients with acquired immune  deficiency syndrome (AIDS) fre-  quently develop tuberculosis. After  Ivy Sharer's sputum stain suggested that  she had tuberculosis, a multidrug antituber-  culous regimen, which includes an antibiotic  of the rifamycin family (rifampin), was  begun. A culture of her sputum was taken to  confirm the diagnosis.   Rifampin inhibits bacterial RNA poly-  merase, selectively killing the bacteria that  cause the infection. The nuclear RNA poly-  merase from eukaryotic cells is not affected.  Although rifampin can inhibit the synthesis  of mitochondrial RNA, the concentration  required is considerably higher than that  used for treatment of tuberculosis.    RNA polymerase I: RNA  RNA polymerase II: mRNA  RNA polymerase III: tRNA +    The mushrooms picked by Amanda Tin contained a-amanitin, an inhibitor of  eukaryotic RNA polymerases. It is particularly effective at blocking the action of  RNA polymerase II. This toxin initially causes gastrointestinal disturbances,  then electrolyte imbalance and fever, followed by liver and kidney dysfunction. Between  40 and 90% of the individuals who ingest a-amanitin die within a few days.   nucleotide sequence of a gene is represented by the letters of the nitrogenous  bases of the coding strand of the DNA duplex. It is written from left to right in  the 5' to 3' direction.   During translation, mRNA is read 5' to 3' in sets of three bases, called codons,  that determine the amino acid sequence of the protein (see Fig. 14.3) Thus, the base  sequence of the coding strand of the DNA can be used to determine the amino acid  sequence of the protein. For this reason, when gene sequences are given, they refer  to the coding strand.   A gene consists of the transcribed region and the regions that regulate transcrip-  tion of the gene (e.g., promoter and enhancer regions)(Fig. 14.4). The base in the  coding strand of the gene serving as the startpoint for transcription is numbered + 1 .  This nucleotide corresponds to the first nucleotide incorporated into the RNA at the  5 '-end of the transcript. Subsequent nucleotides within the transcribed region of the  gene are numbered +2, +3, etc., toward the 3' -end of the gene. Untranscribed  sequences to the left of the startpoint, known as the 5 '-flanking region of the gene,  are numbered -1, -2, -3, etc., starting with the nucleotide (-1) immediately to the  left of the startpoint (+1) and moving from right to left. By analogy to a river, the  sequences to the left of the startpoint are said to be upstream from the startpoint and  those to the right are said to be downstream.   For genes to be expressed, RNA polymerase must recognize the appropriate point  to start transcription and the strand of the DNA to transcribe (the template strand).   The two strands of DNA are  Ki ) antiparallel, with complementary   nucleotides at each position. Thus,  each strand would produce a different  mRNA, resulting in different codons for  amino acids and a different protein product.  Therefore, it is critical that RNA polymerase  transcribe the correct strand.   L T JL T J L T JL T J  Protein N -M et -P ro -Va I -Gly-His -Leu-Ser -C   Fig. 14.3. Relationship between the coding strand of DNA, the DNA template strand, the  mRNA transcript, and the protein produced from the gene. The bases in mRNA are used in  sets of three (called codons) to specify the order of the amino acids inserted into the growing  polypeptide chain during the process of translation (see Chapter 15).   Fig. 14.4. A schematic view of a eukaryotic gene, and steps required to produce a protein product. The gene consists of promoter and transcribed  regions. The transcribed region contains introns, which do not contain coding sequence for proteins, and exons, which do carry the coding  sequences for proteins. The first RNA form produced is heterogenous nuclear RNA (hn RNA), which contains both intronic and exonic  sequences. The hnRNA is modified such that a cap is added at the 5' end (cap site), and a poly-A tail added to the 3' end. The introns are removed  (a process called splicing) to produce the mature mRNA, which leaves the nucleus to direct protein synthesis in the cytoplasm. Py is pyrimidine  (C or T).   RNA polymerase also must recognize which genes to transcribe because transcribed  genes are only a small fraction of the total DNA. The genes that are transcribed dif-  fer from one type of cell to another and change with changes in physiologic condi-  tions. The signals in DNA that RNA polymerase recognizes are called promoters.  Promoters are sequences in DNA (often composed of smaller sequences called  boxes or elements) that determine the startpoint and the frequency of transcription.  Because they are located on the same molecule of DNA and near the gene they reg-  ulate, they are said to be cis acting (i.e., "cis" refers to acting on the same side).  Proteins that bind to these DNA sequences and facilitate or prevent the binding of  RNA polymerase are said to be trans acting.   The binding of RNA polymerase and the subsequent initiation of gene transcription  involves a number of consensus sequences in the promoter regions of the gene (Fig.  14.5). A consensus sequence is the sequence most commonly found in a given  region when many genes are examined. In both prokaryotes and eukaryotes, an ade-  nine- and thymine-rich consensus sequence in the promoter determines the start-  point of transcription by binding proteins that facilitate the binding of RNA poly-  merase. In the prokaryote E. coli, this consensus sequence is TATA AT, which is  known as the TATA or Pribnow box. It is centered about -10 and is recognized by  the sigma factor a 70 . A similar sequence in the -25 region of eukaryotic genes has  a consensus sequence of TATA(A/T) A. (The (A/T) in the fifth position indicates that  either A or T occurs with equal frequency.) This eukaryotic sequence is also known  as a TATA box, but is sometimes named the Hogness or Hogness-Goldberg box  after its discoverers. Other consensus sequences involved in binding of RNA poly-  merase are found further upstream in the promoter region (see Fig. 14.5). Bacterial  promoters contain a sequence TTGACA in the -35 region. Eukaryotes frequently  have CAAT boxes and GC-rich sequences in the region between -40 and -110.  Eukaryotic genes also contain promoter-proximal elements (in the region of -100    Anne Niemick has a p + tha-  lassemia classified clinically as p-  thalassemia intermedia. She pro-  duces an intermediate amount of functional  (3 globin chains (her hemoglobin is 6.2 g/dL;  normal is 12-16). ^-thalassemia intermedia  is usually the result of two different muta-  tions (one that mildly affects the rate of syn-  thesis of p-globin and one severely affecting  its rate of synthesis), or, less frequently,  homozygosity for a mild mutation in the rate  of synthesis, or a complex combination of  mutations. For example, mutations within  the promoter region of the p-globin gene  could result in a significantly decreased rate  of p-globin synthesis in an individual who is  homozygous for the allele, without com-  pletely abolishing synthesis of the protein.   Two of the point mutations that result in  a p + phenotype are within the TATA box (A— »  G or A— » C in the -28 to -31 region). These  mutations reduce the accuracy of the start-  point of transcription so that only 20 to 25%  of the normal amount of (3-globin is synthe-  sized. Other mutations that also reduce the  frequency of (3-globin transcription have  been observed further upstream in the pro-  moter region (-87 G and -88 C^T ).   Fig. 14.5. Prokaryotic and eukaryotic promoters. The promoter-proximal region contains  binding sites for transcription factors which that can accelerate the rate at which RNA poly-  merase binds to the promoter. Pu = purine; Py = pyrimidine.   What property of an AT-rich region  of a DNA double helix makes it  suitable to serve as a recognition   Fig. 14.6. Bacterial operon. A cistron encodes  a single polypeptide chain. In bacteria, a single  promoter may control transcription of an  operon containing many cistrons. A single  polycistronic mRNA is transcribed. Its transla-  tion produces a number of polypeptide chains.   to -200), which are sites that bind other gene regulatory proteins. Genes vary in the  number of such sequences present.   In bacteria, a number of protein-producing genes may be linked together and  controlled by a single promoter. This genetic unit is called an operon (Fig. 14.6).  Proteins bind to the promoter and either inhibit or facilitate transcription of the  operon. Repressors are proteins that bind to a region in the promoter known as the  operator and inhibit transcription by preventing the binding of RNA polymerase to  DNA. Activators are proteins that stimulate transcription by binding within the -35  region or upstream from it, facilitating the binding of RNA polymerase. (Operons  are described in more detail in Chapter 16.)   In eukaryotes, proteins known as general transcription factors (or basal fac-  tors) bind to the TATA box and facilitate the binding of RNA polymerase II, the  polymerase that transcribes mRNA (Fig. 14.7). This binding process involves at  least six basal transcription factors (labeled as TFIIs, transcription factors for  RNA polymerase II). The TATA-binding protein (TBP), which is a component  of TFIID, initially binds to the TATA box. TFIID consists of both the TBP and  a number of transcriptional coactivators. TFIIA and TFIIB interact with TBP.  RNA polymerase II binds to the complex of transcription factors and to DNA,  and is aligned at the startpoint for transcription. TFIIE, TFIIF, and TFIIH sub-  sequently bind, cleaving adenosine triphosphate (ATP), and transcription of the  gene is initiated.   With only these transcription (or basal) factors and RNA polymerase II attached  (the basal transcription complex), the gene is transcribed at a low or basal rate.   TFIIH plays a number of roles in both transcription and DNA repair. In both  processes, it acts as an ATP-dependent DNA helicase, unwinding DNA for  either transcription or repair to occur. Two of the forms of xeroderma pigmen-  tosum (XPB and XPD; see Chapter 13) arise from mutations within two different helicase  subunits of TFIIH. TFIIH also contains a kinase activity, and RNA polymerase II is phos-  phorylated by this factor during certain phases of transcription.   Fig. 14.7. Transcription apparatus. The TATA-binding protein (TBP), a component of TFIID, binds to the TATA box. Transcription factors TFII  A and B bind to TBP. RNA polymerase binds, then TFII E, F, and H bind. This complex can transcribe at a basal level. Some coactivator pro-  teins are present as a component of TFIID, and these can bind to other regulatory DNA binding proteins (called specific transcription factors or  transcriptional activators).   The rate of transcription can be further increased by binding of other regulatory  DNA binding proteins to additional gene regulatory sequences (such as the pro-  moter proximal or enhancer regions). These regulatory DNA binding proteins are  called gene- specific transcription factors (or transactivators) because they are spe-  cific to the gene involved (see Chapter 16). They interact with coactivators in the  basal transcription complex.   In bacteria, binding of RNA polymerase with a a factor to the promoter region of  DNA causes the two DNA strands to unwind and separate within a region approxi-  mately 10 to 20 nucleotides in length. As the polymerase transcribes the DNA, the  untranscribed region of the helix continues to separate, whereas the transcribed  region of the DNA template rejoins its DNA partner (Fig. 14.8). The sigma factor  is released when the growing RNA chain is approximately 10 nucleotides long. The  elongation reactions continue until the RNA polymerase encounters a transcription  termination signal. One type of termination signal involves the formation of a hair-  pin loop in the transcript, preceding a number of U residues. The second type of   ^"■"V In regions in which DNA is being  ^\ •) transcribed, the two strands of the  DNA must be separated. AT base  pairs in DNA are joined by only two hydro-  gen bonds, whereas GC pairs have three  hydrogen bonds. Therefore, in AT-rich  regions of DNA, the two strands can be sep-  arated more readily than in regions that con-  tain GC base pairs.   Although each eukaryotic mRNA  only codes for one polypeptide  chain, a number of proteins can  contain different polypeptide chains or have  multiple active sites, allowing the protein to  catalyze more than one reaction.   The terms hnRNA (heterogeneous  nuclear RNA) and pre-mRNA are  both used to denote mRNA precur-  sors. The term hnRNA was originally applied  to a pool of RNA molecules in the nucleus  that were rapidly synthesized and varied  greatly in size. These RNA molecules are  now known to be the mRNA precursors that  vary greatly in size because they contain  exons that encode different sizes of polypep-  tide chains and introns that vary in amount  and size.    Fig. 14.9. Bacterial rRNA and tRNA transcripts. One large precursor is cleaved (at arrows)  to produce 16S, 23S, and 5S rRNA and some tRNAs.   mechanism for termination involves the binding of a protein, the rho factor, which  causes release of the RNA transcript from the template. The signal for both termi-  nation processes is the sequence of bases in the newly synthesized RNA.   A cistron is a region of DNA that encodes a single polypeptide chain. In bacte-  ria, mRNA is usually generated from an operon as a polycistronic transcript (one  that contains the information to produce a number of different proteins). The poly-  cistronic transcript is translated as it is being transcribed. This transcript is not mod-  ified and trimmed, and it does not contain introns (regions within the coding  sequence of a transcript that are removed before translation occurs). Several differ-  ent proteins are produced during translation of the polycistronic transcript, one from  each cistron (see Fig. 14.6).   In prokaryotes, rRNA is produced as a single, long transcript that is cleaved to  produce the 16S, 23 S, and 5S ribosomal RNAs. tRNA is also cleaved from larger  transcripts (Fig. 14.9). One of the cleavage enzymes, RNase P, is a protein contain-  ing an RNA molecule. This RNA actually catalyzes the cleavage reaction.   The process of transcription in eukaryotes is similar to that in prokaryotes. RNA  polymerase binds to the transcription factor complex in the promoter region and to  the DNA, the helix unwinds within a region near the startpoint of transcription,  DNA strand separation occurs, synthesis of the RNA transcript is initiated, and the  RNA transcript is elongated, copying the DNA template. The DNA strands separate  as the polymerase approaches and rejoin as the polymerase passes.   One of the major differences between eukaryotes and prokaryotes is that eukary-  otes have more elaborate mechanisms for processing the transcripts, particularly the  precursors of mRNA (pre-mRNA). Eukaryotes also have three polymerases, rather  than just the one present in prokaryotes. Other differences include the facts that  eukaryotic mRNA usually contains the coding information for only one polypeptide  chain and that eukaryotic RNA is transcribed in the nucleus and migrates to the  cytoplasm where translation occurs.   In eukaryotes, extensive processing of the primary transcript occurs before the  mature mRNA is formed and can migrate to the cytosol, where it is translated into  a protein product. RNA polymerase II synthesizes a large primary transcript from  the template strand that is capped at the 5' end as it is transcribed (Fig. 14.10). The  transcript also rapidly acquires a poly (A) tail at the 3' end. Pre-mRNAs thus con-  tain untranslated regions at both the 5' and 3' ends (the leader and trailing  sequences, respectively). These untranslated regions are retained in the mature   Fig. 14.10. Overview of mRNA synthesis. Transcription produces hnRNA from the DNA  template. hnRNA processing involves addition of a 5 '-cap and a poly (A) tail and splicing to  join exons and remove introns. The product, mRNA, migrates to the cytoplasm, where it will  direct protein synthesis.   mRNA. The coding region of the pre-mRNA, which begins with the start codon  for protein synthesis and ends with the stop codon, contains both exons and  introns. Exons consist of the nucleotide codons that dictate the amino acid  sequence of the eventual protein product. Between the exons, interspersing  regions called introns contain nucleotide sequences that are removed by splicing  reactions to form the mature RNA. The mature RNA thus contains a leader  sequence (that includes the cap), a coding region comprising exons, and a tailing  sequence that includes the poly(A) tail.   This mature mRNA complexes with the poly(A) binding protein and other  proteins. It travels through pores in the nuclear envelope into the cytoplasm.  There it combines with ribosomes and directs the incorporation of amino acids  into proteins.   "Capping" of the primary transcript synthesized by RNA polymerase II occurs at  its 5' -end as it is being transcribed (Fig. 14. 11). The 5'- terminal, the initial  nucleotide of the transcript, is a pyrimidine with three phosphate groups attached  to the 5'- hydroxyl of the ribose. To form the cap, the terminal triphosphate loses  one phosphate, forming a 5' -diphosphate. The p-phosphate of the diphosphate  then attacks the a -phosphate of GTP, liberating pyrophosphate, and forming an  unusual 5' to 5' triphosphate linkage. A methyl group is transferred from S-  adenosylmethionine (SAM), a universal methyl donor, to position 7 of the added  guanine ring. Methylation also occurs on the ribose 2' -hydroxyl group in the ter-  minal nucleotide to which the cap is attached, and sometimes the 2 '-hydroxyl  group of the adjacent nucleotide ribose. This cap "seals" the 5' end of the pri-  mary transcript and decreases the rate of degradation. It also serves as a recog-  nition site for the binding of the mature mRNA to a ribosome at the initiation of  protein synthesis.   Once SAM (S-adenosylmethion-  \m ) ine) donates its methyl group, it   must be regenerated by reactions  that require the vitamins folate and B 12 . Thus  formation of mRNA is also one of the  processes affected by a deficiency of these  vitamins.   There are three different types of  methyl caps, shown in blue: CAPO  refers to the methylated guanosine  (on the nitrogen at the seven position, N 7 )  added in the 5' to 5' linkage to the mRNA;  CAP1 refers to CAPO with the addition of a  methyl to the 22' carbon of ribose on the  nucleotide (N-,) at the 5' end of the chain; and  CAP2 refers to CAP1 with the addition of  another 2' methyl group to the next  nucleotide (N 2 ). The methyl groups are  donated by S-adenosylmethionine (SAM).   CH 3 (n 7 )  G-5'-PPP-5 l -N 1 N 2 N 3 -   CH 3 CH 3  G-5'-PPP-5'-N r N 2 -N 3 -   Fig. 14.11. The cap structure in eukaryotic mRNA. The phosphates in blue originated from the original RNA transcript; the phosphate in black  comes from GTR   Fig. 14.12. Synthesis of the poly(A) tail. As  RNA polymerase continues to transcribe the  DNA, enzymes cleave the transcript (hnRNA)  at a point 10-20 nucleotides beyond an  AAUAAA sequence, just before a run of Us  (or Gs). Approximately 250 adenine  nucleotides are then added to the 3 '-end of the  hnRNA, one at a time, by poly(A) polymerase.   After the RNA polymerase transcribes the stop codon for protein translation, it  passes a sequence called the polyadenylation signal (AAUAAA) (Fig. 14.12). It  continues past the polyadenylation signal until it reaches an unknown, and possibly  unspecific, termination signal many nucleotides later. However, as the primary tran-  script is released from the RNA polymerase elongation complex, an enzyme com-  plex binds to the polyadenylation signal and cleaves the primary transcript approx-  imately 10 to 20 nucleotides downstream, thereby forming the 3' end. After this  cleavage, a poly(A) tail that can be over 200 nucleotides in length is added to the  3 '-end. Thus, there is no poly(dT) sequence in the DNA template that corresponds  to this tail; it is added posttranscriptionally. ATP serves as the precursor for the  sequential addition of the adenine nucleotides. They are added one at a time, with  poly(A) polymerase catalyzing each addition. The poly(A) tail is a protein binding  site that protects the mRNA from degradation.   Within a few days of initiation of treatment for tuberculosis, laboratory staining  results of Ivy Sharer's sputum confirmed the diagnosis of pulmonary tuberculo-  sis caused by M. tuberculosis. Therefore, the multidrug therapy, which included  the antibiotic rifampin, was continued. Rifampin binds to the RNA polymerases of several  bacteria. M. tuberculosis rapidly develops resistance to rifampin through mutations that  result in an RNA polymerase that cannot bind the complex structure. Simultaneous treat-  ment with a drug that works through a different mechanism decreases the selective  advantage of the mutation and the rate at which resistance develops.   The presence of a poly (A) tail on eukaryotic mRNA allows this form of RNA to  be easily separated from the more abundant rRNA. After extracting all of the  RNA from a cell, the total RNA is applied to a column of beads to which oligo-  dT has been covalently attached. As the mRNA flows through the column, its poly (A) tail  will base pair with the oligo-dT, and the mRNA will become bound to the column. All  other types of RNA will flow through the column and not bind to the beads. The bound  mRNA can then be eluted from the column by changing the ionic strength of the buffer.   Eukaryotic pre-mRNA transcripts contain regions known as exons and introns.  Exons appear in the mature mRNA; introns are removed from the transcript and are  not found in the mature mRNA (see Fig. 14. 10). Introns, therefore, do not contribute  to the amino acid sequence of the protein. Some genes contain 50 or more introns.  These introns are carefully removed from the pre-mRNA transcript and the exons  spliced together, so that the appropriate protein is produced from the gene.   The consensus sequences at the intron/exon boundaries of the pre-mRNA are  AGGU (AGGT in the DNA). The sequences vary to some extent on the exon side  of the boundaries, but almost all introns begin with a 5' GU and end with a 3' AG  (Fig. 14.13). These intron sequences at the left splice site and the right splice site  are, therefore, invariant. Because every 5' GU and 3' AG combination does not  result in a functional splice site, clearly other features within the exon or intron help  to define the appropriate splice sites. These features, which are currently being  identified, involve the presence of positive- and negative-acting ds-regulatory  sequences within the intron.   A complex structure known as a spliceosome ensures that exons are spliced  together with great accuracy (Fig. 14.14). Small nuclear ribonucleoproteins   Fig. 14.13. Splice junctions in hnRNA. The  intron sequences shown in blue dashed boxes  are invariant. They always appear at this posi-  tion in introns. The sequences on the exon side  of the splice sites are more variable.   Anne Niemick has p + -thalassemia  (enough of the (3-chain is produced  to maintain blood hemoglobin lev-  els above 6.0 g/dL). One mutation resulting  in p + -thalassemia is a point mutation  (AATAAA -> AACAAA) that changes the  sequence in hnRNA at the polyadenylation  signal site from AAUAAA to AACAAA.  Homozygous individuals with this mutation  produce only one-tenth the amount of nor-  mal p-globin mRNA.   Fig. 14.14. Splicing process. Nuclear ribonucleoproteins (snurps U l to U 6 ) bind to the intron,  causing it to form a loop. The complex is called a splicesosome. The Ul snurp binds near the  first exon/intron junction, and U2 binds within the intron in a region containing an adenine  nucleotide residue. Another group of snurps, U4, U5, and U6, binds to the complex, and the  loop is formed. The phosphate attached to the G residue at the 5 '-end of the intron forms a  2 '-5' linkage with the 2' -hydroxy 1 group of the adenine nucleotide residue. Cleavage occurs  at the end of the first exon, between the AG residues at the 3' end of the exon and the GU  residues at the 5' end of the intron. The complex continues to be held in place by the spliceo-  some. A second cleavage occurs at the 3 '-end of the intron after the AG sequence. The exons  are joined together. The intron, shaped like a lariat, is released and degraded to nucleotides.   0 © Some types of (^-thalassemia (little  | /~oA J or none of the hemoglobin p chain  ™ produced) are caused by homozy-  gous mutations in the splice junction  sequences at intron/exon boundaries. In  some individuals, an AT replaces a GT in the  gene at the 5' end of the first or second  intron. Mutations also occur within the  splice junction sequences at the 3'-end of  introns (GT at the donor site 5'-end and AG  at the acceptor site 3'-end). Mutations at  either site totally abolish normal splicing  and result in p° thalassemia.   (snRNPs), called "snurps," are involved in formation of the spliceosome. Because  snurps are rich in uracil, they are identified by numbers preceded by a U.   Exons frequently code for separate functional or structural domains of proteins.  Proteins with similar functional regions (e.g., ATP or NAD binding regions) fre-  quently have similar domains, although their overall structure and amino acid  sequence is quite different. A process known as exon shuffling has probably  occurred throughout evolution, allowing new proteins to develop with functions  similar to those of other proteins.   Ribosomal RNAs (rRNAs) form the ribonucleoprotein complexes on which protein  synthesis occurs. In eukaryotes, the rRNA gene exists as many copies in the nucleolar  organizer region of the nucleus (Fig. 14.15, circle 1). Each gene produces a large, 45S  transcript that is cleaved to produce the 18S, 28S, and 5.8S rRNAs. Approximately  1,000 copies of this gene are present in the human genome. The genes are linked in tan-   Systemic lupus erythematous is an autoimmune disease characterized by a  particular spectrum of autoantibodies against many cellular components,  including chromatin, ribonucleoprotein, and cell membrane phospholipids. In  this disorder, the body makes these antibodies against its own components. snRNPs are  one of the targets of these antibodies. In fact, snRNPs were discovered as a result of  studies using antibodies obtained from patients with SLE.   Tests were performed on Sis Lupus's blood to detect elevated levels of antinuclear anti-  bodies (including antibodies to DNA, antibodies to histone, antibodies to ribonucleopro-  teins, and antibodies to nuclear antigens). The tests were strongly positive and, in conjunc-  tion with her symptoms, led to a diagnosis of systemic lupus erythematosus (SLE).    Fig. 14.15. rRNA and ribosome synthesis. The 5S rRNA is transcribed in the nucleoplasm and moves into the nucleolus. The other rRNAs are  transcribed from DNA and mature in the nucleolus, forming the 40S and 60S ribosomal subunits, which migrate to the cytoplasm.   dem, separated by spacer regions that contain the termination signal for one gene and  the promoter for the next. Promoters for rRNA genes are located in the 5' -flanking  region of the genes and extend into the region surrounding the startpoint. rRNA genes  caught in the act of transcription by electron micrographs show that many RNA poly-  merase I molecules can be attached to a gene at any given time, all moving toward the  3' end as the 45 S rRNA precursors are synthesized.   As the 45 S rRNA precursors are released from the DNA, they complex with pro-  teins, forming ribonucleoprotein particles that generate the granular regions of the  nucleolus (see Fig. 14.15, circle 2). Processing of the transcript occurs in the gran-  ular regions. 5S rRNA, produced by RNA polymerase III from genes located out-  side the nucleolus in the nucleoplasm, migrates into the nucleolus and joins the  ribonucleoprotein particles.   One to two percent of the nucleotides of the 45 S precursor become methylated,  primarily on the 2'-hydroxyl groups of ribose moieties (see Fig. 14.15, circle 3).  These methyl groups may serve as markers for cleavage of the 45 S precursors and  are conserved in the mature rRNA. A series of cleavages in the 45 S transcripts occur  to produce the mature rRNAs (Fig. 14.16).   In the production of cytoplasmic ribosomes in human cells, one portion of the  45S rRNA precursor becomes the 18S rRNA that, complexed with proteins, forms  the small 40S ribosomal subunit (Fig. 14.15, circle 4). Another segment of the pre-  cursor folds back on itself and is cleaved, forming 28S rRNA, hydrogen-bonded to  the 5.8 S rRNA. The 5S rRNA, transcribed from nonnucleolar genes, and a number  of proteins complex with the 28S and 5.8S rRNAs to form the 60S ribosomal sub-  unit (Fig. 14.15, circle 5). The ribosomal subunits migrate through the nuclear  pores. In the cytoplasm, the 40S and 60S ribosomal subunits interact with mRNA,  forming the 80S ribosomes on which protein synthesis occurs.   A transfer RNA has one binding site for a specific sequence of three nucleotides in  mRNA (the anticodon site) and another binding site for the encoded amino acid.  tRNAs thus ensure that the genetic code is translated into the correct sequence of   Fig. 14.16 Maturation of the 45S rRNA pre-  cursor. The clear regions are removed, and the  shaded regions become the mature rRNAs.  (The 5S rRNA is not produced from this pre-  cursor.)   become the mature rRNAs. These  introns are removed by the rRNA precursors  themselves, which undergo self-splicing  reactions. The rRNA precursors that catalyze  their own splicing are known as ribozymes  (see Chapter 12, III.A).   Fig. 14.17. The tRNA cloverleaf. Bases that commonly occur in a particular position are indicated by letters. Base-pairing in stem regions is indicated  by lines between the strands. The locations of the modified bases dihydrouridine (D), ribothymidine (T), and pseudouridine (VP) are indicated.   Fig. 14.18. The three-dimensional folding of  tRNA. (Reprinted with permission from Kim  SH, et al. Science 1974; 185:436. Copyright  1974 American Association for the Advance-  ment of Science.)   Fig. 14.19. Promoter for tRNA transcription. The segments of the genes from which the  mature tRNA is produced are indicated by the light blue color. The two regions of the pro-  moter lie within these segments, and are indicated by the dark blue color.   amino acids. At least 20 types of tRNAs occur in cells, one for every amino acid that  is incorporated into growing polypeptide chains during the synthesis of proteins.  tRNAs have a cloverleaf structure that folds into a three-dimensional L shape, and  contains a number of bases that are modified posttranscriptionally (Fig. 14.17). The  loop closest to the 5' -end is known as the D-loop because it contains dihydrouridine  (D). The second, or anticodon, loop contains the trinucleotide anticodon that base-  pairs with the codon on mRNA. The third loop (the T^C loop) contains both riboth-  ymidine (T) and pseudouridine OF). A fourth loop, known as the variable loop  because it varies in size, is frequently found between the anticodon and T^C loops.  Base-pairing occurs in the stem regions of tRNA, and a three-nucleotide sequence  (e.g., CCA) at the 3' -end is the attachment site for the specific amino acid carried  by each tRNA. Different tRNAs bind different amino acids. The three-dimensional  structure of tRNA has been determined and is shown in Figure 14.18. tRNA is pro-  duced by RNA polymerase III, which recognizes a split promoter within the tran-  scribed region of the gene (Fig. 14.19). One segment of the promoter is located  between +8 and +19. A second segment is 30 to 60 base pairs downstream from  the first.   tRNA precursors of approximately 100 nucleotides in length are generated.  (Fig. 14.20, circle 1). The pre-tRNA assumes a cloverleaf shape and is subsequently  cleaved at the 5'- and 3 '-ends (see Fig. 14.20, circle 2). The enzyme that acts at the   Fig. 14.20. Overview of tRNA synthesis. D, T, "VP, and ■ indicate modified bases.  D = dihydrouracil, T = ribothymine, \\f = pseudouridine, ■ = other modified bases   5 '-end is RNase P, similar to the RNase P of bacteria. Both enzymes contain a small  RNA (Ml) that has catalytic activity and serves as an endonuclease. Some tRNA  precursors contain introns that are removed by endonucleases. To close the opening,  a 2'- or 3 '-phosphate group from one end is ligated to a 5'-hydroxyl on the other  end by an RNA ligase.   The bases are modified at the same time the endonucleolytic cleavage reactions are  occurring (see Fig. 14.20, circle 3). Three modifications occur in most tRNAs: (1)  Uracil is methylated by S-adenosylmethionine (SAM) to form thymine; (2) one of the  double bonds of uracil is reduced to form dihydrouracil;, and (3) a uracil residue  (attached to ribose by an TV-glycosidic bond) is rotated to form pseudouridine, which  contains uracil linked to ribose by a carbon-carbon bond, (see Fig. 14.17). Other, less  common but more complex, modifications also occur and involve bases other than  uracil. Of particular note is the deamination of adenosine to form the base inosine.   The final step in forming the mature tRNA is the addition of a CCA sequence at  its 3 '-end (see Fig 14.20, circle 4). These nucleotides are added one at a time by  nucleotidyltransferase. The tRNA then migrates to the cytoplasm. The terminal  adenosine at the 3 '-end is the site at which the specific amino acid for each tRNA  is bound and activated for incorporation into a protein.   Except for the germ cells, most normal human cells are diploid. Therefore, they  contain two copies of each chromosome, and each chromosome contains genes that  are alleles of the genes on the homologous chromosome. Because one chromosome  in each set of homologous chromosomes is obtained from each parent, the alleles  can be identical, containing the same DNA sequence, or they can differ. A diploid  human cell contains 2,000 times more DNA than the genome of the bacterium in  the haploid E. coli cell (approximately 4 X 10 6 base pairs).   Eukaryotic introns contribute to the DNA size difference between bacteria and human  cells. In eukaryotic genes, introns (noncoding regions) occur within sequences that  code for proteins. Consequently, the primary transcript (heterogeneous nuclear RNA or  hnRNA) averages roughly 10 times longer than the mature mRNA produced by  removal of the introns. In contrast, bacterial genes do not contain introns.   Although diploidy and introns account for some of the difference between the DNA  content of humans and bacteria, a large difference remains that is related to the  greater complexity of the human organism. Bacterial cells have a single copy of  each gene, called unique DNA, and they contain very little DNA that does not pro-  duce functional products. Eukaryotic cells contain substantial amounts of DNA that  does not code for functional products (i.e., proteins or rRNA and tRNA). In addi-  tion, some genes that encode functional products are present in multiple copies,   The removal of introns in pre-tRNA  \m ) has been most extensively studied  in yeast. The intron, less than 20  nucleotides long, is located within the anti-  codon loop at the 3'-end. Removal of the  intron by endonucleases leaves the two  halves of the tRNA, which remain held  together by hydrogen bonds between base  pairs in the stem regions. The opening is  closed by an RNA ligase.   Calculate the number of different  proteins, 300 amino acids in  length, which could be produced  from the E. coli genome (4 X 10 6 base pairs  of DNA).    A number of differences between eukaryotes and prokaryotes affect the processes of replication, transcription, and translation,  in addition to the content of their DNA. Eukaryotic DNA is complexed with histones, and prokaryotic DNA is not. In eukaryotic  cells, the process of transcription, which occurs in the nucleus, is separated by the nuclear envelope from the process of trans-  lation (protein synthesis from the mRNA template), which occurs in the cytoplasm. Because prokaryotes lack nuclei, the processes of tran-  scription and translation occur simultaneously. Transcription of bacterial DNA requires only one promoter per operon. In contrast, human  DNA requires one promoter for each gene.   Complexity may explain some of  \ \ ) the differences between the DNA   content of bacteria and humans.  But an extension of this line of reasoning  would lead to the conclusion that frogs are  more complex than humans, because frogs  have 8 feet of DNA per diploid nucleus com-  pared to the 6 feet in a human cell. Logic, or  perhaps vanity, suggests that the amount of  DNA per cell does not necessarily reflect the  complexity of the organism. One of the fea-  tures of frog DNA that may explain its length  is that frogs have more repetitive DNA than  humans. More than 75% of the frog genome  is in the moderately and highly repetitive  category, whereas only about 35% of the  human genome is repetitive.   ®Four million base pairs contain 4 X  10 6 /3 or 1.33 million codons. If each  protein contained approximately  300 amino acids, E. coli could produce about  4,000 different proteins (1.33 X 10 6 /300).   called highly repetitive or moderately repetitive DNA. Approximately 64% of the  DNA in the human genome is unique, consisting of DNA sequences present in one  or a very few copies in the genome (Fig. 14.21). Unique DNA sequences are tran-  scribed to generate mRNA, which is translated to produce proteins.   Highly repetitive DNA consists of sequences approximately 6 to- 100 base pairs in  length that are present in hundreds of thousands to millions of copies, clustered within  a few locations in the genome (see Fig. 14.21). It occurs in centromeres (which join  sister chromatids during mitosis) and in telomeres (the ends of chromosomes). This  DNA represents approximately 10% of the human genome. It is not transcribed.   Moderately repetitive DNA is present in a few to tens of thousands of copies in the  genome (see Fig. 14.21). This fraction constitutes approximately 25% of the human  genome. It contains DNA that is functional and transcribed to produce rRNA, tRNA,  and also some mRNA. The histone genes, present in a few hundred copies in the  genome, belong to this class. Moderately repetitive DNA also includes some gene  sequences that are functional but not transcribed. Promoters and enhancers (which are  involved in regulating gene expression) are examples of gene sequences in this cate-  gory. Other groups of moderately repetitive gene sequences that have been found in the  human are called the Alu sequences (approximately 300 base pairs in length). Alu   0 o Alu sequences in DNA were named for the enzyme Alu (obtained from  j Arthrobacter luteus), that which is able to cleave them. Alu sequences make up  ™ 6 to 8% of the human genome. In some cases of familial hypercholesterolemia,  homologous recombination is believed to have occurred between two Alu repeats,  resulting in a large deletion in the low- density lipoprotein (LDL) receptor gene. The LDL  receptor mediates uptake of the cholesterol-containing LDL particle into many cell types  and, in the absence of functional LDL receptors, blood cholesterol levels are elevated.  Patients who are homozygous for this mutation may die from of cardiac disease as early  as in their second or third decade of life.   Fig. 14.21. Distribution of unique, moderately repetitive, and highly repetitive sequences in a hypothetical human chromosome. Unique genes encode  mRNA. These genes occur in single copies. The genes for the large rRNA and the tRNA precursors occur in multiple copies that are clustered in the  genome. The large rRNA genes form the nucleolar organizer. Moderately repetitive sequences are dispersed throughout the genome, and highly repet-  itive sequences are clustered around the centromere and at the ends of the chromosome (the telomeres). From Wolfe SL. Mol Cell Biol 1993:761.   sequences are also examples of SINEs (Short INterspersed Elements). The LINE  sequences (Long INterspersed Elements) are 6,000 to 7,000 base pairs in length. The  function of the Alu and LINE sequences has not been determined.   Major differences between prokaryotic and eukaryotic DNA and RNA are sum-  marized in Table 14.2.   ^ Anne Niemick. Patients with (3 + -thalassemia who maintain their  hemoglobin levels above 6.0 to 7.0 g/dL are usually classified as having  thalassemia intermedia. In the p -thalassemias, the a chains of adult hemo-  globin A (a 2 p 2 ) continue to be synthesized at a normal rate. These chains accumu-  late in the bone marrow, where the red blood cells are synthesized in erythropoiesis  (generation of red blood cells). The accumulation of a chains diminishes ery-  thopoiesis, resulting in an anemia. Individuals who are homozygous for a severe  mutation require constant transfusions.   Individuals with thalassemia intermedia, such as Anne Niemick, could have inher-  ited two different defective alleles, one from each parent. One parent may be a "silent"  carrier, with one normal allele and one mildly affected allele. This parent produces  enough functional p-globin, so no clinical symptoms of thalassemia appear. (However,  they generally have a somewhat decreased amount of hemoglobin, resulting in micro-  cytic hypochromic red blood cells.) When this parent contributes the mildly defective  allele and the other heterozygous parent contributes a more severely defective allele,    LINE (Long INterspersed Elements)  make up about 5% of the human  genome. In some patients with  hemophilia (a disease in which blood does  not clot normally), a LINE sequence has  been inserted into exon 14 of the gene for  Factor VIII, a protein of the blood-clotting  system. The insertion of the LINE sequence  leads to the production of a nonfunctional  protein.   The mutations that cause the thalassemias affect the synthesis of either the a- or the p-chains of adult hemoglobin, causing an  anemia. They are classified by the chain affected (a- or p-) and by the amount of chain synthesized (0 for no synthesis and + for  synthesis of some functional chains). They are also classified as major, intermediate, or minor, according to the severity of the  clinical disorder, p-thalassemia major (also called homozygous p-thalassemia) is a clinically severe disorder requiring frequent  blood transfusions. It is caused by the inheritance of two alleles for a severe mutation. In p-thalassemia intermedia, the patient exhibits a  less severe clinical phenotype and is able to maintain hemoglobin levels above 6 g/dL. It is usually the result of two different mild muta-  tions or homozygosity for a mild mutation, ^-thalassemia minor (also known as ^-thalassemia trait) is a heterozygous disorder involving  a single mutation that is often clinically asymptomatic.   During embryonic and fetal life, the p-chain is replaced by the e and 7 chains. As a result, patients with severe mutations in the a-chain  tend to die in utero, whereas those with mutations in the p chains exhibit symptoms postnatally, as hemoglobin F is normally replaced  with adult hemoglobin A.   Recent studies have indicated that a  failure to properly dispose of cellu-  lar debris, a normal byproduct of  cell death, may lead to the induction of auto-  antibodies directed against chromatin in  patients with SLE. Normal cells have a finite  lifetime, and are programmed to die (apop-  tosis) through a distinct biochemical mecha-  nism. One of the steps in this mechanism is  the stepwise degradation of cellular DNA  (and other cellular components). If the nor-  mal intracellular components are exposed to  the immune system, auto-antibodies against  them may be generated. The enzyme in cells  that degrades DNA is deoxyribonuclease I  (DNase I), and individuals with SLE have  reduced serum activity levels of DNase I  compared with individuals who do not have  the disease. Through an understanding of  the molecular mechanism whereby auto-  antibodies are generated, it may be possible  to develop therapies to combat this disorder.   The reverse transcriptase that  copies the viral genome has a high  error rate because it does not have  proofreading capability. Because incorpora-  tion of mismatched bases leads to evolution  of the virus, new populations develop rapidly  in response to changes in the environment.  Mutations in the viral reverse transcriptase  gene cause the enzyme to become resistant  to drugs such as ZDV. Current vaccines  developed experimentally against the gp120  surface protein may not be effective because  this protein also mutates. As a consequence  of the rapid mutation rate of HIV, current  treatments for AIDS have limited effective-  ness, and a cure has proved elusive.   thalassemia intermedia occurs in the child. The child is thus heterozygous for two dif-  ferent defective alleles.   Ivy Sharer. Ivy Sharer was treated with a multidrug regimen for tuber-  culosis because the microbes that cause the disease frequently become  resistant to the individual drugs. Rifampin in combination with the drug  isoniazid (which affects metabolism of vitamin B 6 in the pathogenic bacteria) is  usually effective, but months of treatment are required.   Just as bacteria can become resistant to drugs, so can HIV. A great concern to physi-  cians treating patients with AIDS is the appearance of resistant strains of HIV-1 in  patients taking a single drug, such as ZDV (zidovudine), for 6 months or more. There-  fore, multidrug regimens are used that include other nucleoside reverse transcriptase  inhibitors, such as dideoxyinosine (didanosine, formerly called ddl) and dideoxycyti-  dine (zalcitabine, formerly called ddC). The multidrug therapy often includes nonnu-  cleoside inhibitors (e.g., efavirenz), which acts allosterically on reverse transcriptase,  and protease inhibitors (e.g., indinavir), which prevent the HIV polyprotein from being  cleaved into its mature products (see Biochemical Comments).   Amanda Tin. The toxin a-amanitin is capable of causing irreversible  hepatocellular and renal dysfunction through inhibition of mammalian  RNA polymerases. Fortunately, Amanda Tin's toxicity proved mild. She  developed only gastrointestinal symptoms and slight changes in her hepatic and  renal function, which returned to normal within a few weeks. Treatment was pri-  marily supportive, with fluid and electrolyte replacement for that lost through the  gastrointestinal tract. No effective antidote is available for the A. phalloides toxin.   Sis Lupus. SLE is a multisystem disease of unknown origin characterized  by inflammation related to the presence of autoantibodies in the blood.  These autoantibodies react with antigens normally found in the nucleus,  cytoplasm, and plasma membrane of the cell. Such "self antigen-antibody  (autoimmune) interactions initiate an inflammatory cascade that produces the broad  symptom profile of multiorgan dysfunction found in Sis Lupus.   Pharmacological therapy for SLE, directed at immunosuppression, includes high  doses of corticosteroids, which suppress the immune response. In refractory cases that  do not respond to corticosteroids, cytotoxic drugs inhibiting the synthesis of antibody-  producing cells by the bone marrow are also used to cause immunosuppression.   Production of the Virus That Causes AIDS. AIDS is caused by the human  immuno-deficiency virus (HIV). Two forms of the virus have been discovered, HIV-  1, which is prevalent in industrialized countries, and HIV-2, which is prevalent in  certain regions of Africa. Eight to ten years or more can elapse between the initial  infection and development of the full-blown syndrome.   Proteins in the viral coat bind to membrane receptors (named CD4) of helper T  lymphocytes, a class of cells involved in the immune response. Subsequently, con-  formational changes occur that allow the viral coat proteins to bind to a chemokine  coreceptor in the cell membrane. The lipid in the viral coat then fuses with the cell  membrane, and the viral core enters the cell, releasing its RNA and enzymes  (including the reverse transcriptase) by a process called "uncoating." Reverse tran-  scriptase uses the viral RNA as a template to produce a single-stranded DNA copy,  which then serves as a template for synthesis of a double- stranded DNA. An inte-  grase enzyme, also carried by the virus, enables this DNA to integrate into the host  cell genome as a provirus (Fig. 14.22).   Unspliced /X/X/X/X/X/X \ X/X/X/ Spliced /  RNA j \ | RNA /'   Fig. 14.22. Infection of a host cell by HIV. The HIV virus particle binds to the CD4 recep-  tor and a chemokine coreceptor in the host cell membrane. The virus enters the cell and  uncoats, releasing its RNA and proteins. The viral enzyme reverse transcriptase produces a  double- stranded DNA copy that is integrated into the host cell genome. HIV is now a  provirus. Transcripts of the viral DNA are spliced and translated to produce the proteins Tat,  Rev, and Nef. Tat stimulates transcription of the viral DNA, and Rev causes the viral RNA  transcripts to leave the nucleus unspliced. The unspliced RNA serves as the viral genome and  also codes for the proteins of the viral core and envelope. The envelope proteins (gp41 and  gpl20, which are derived from the env protein) enter the cell membrane. The viral core pro-  teins are synthesized as a polyprotein, which is cleaved by a protease as the viral particles  form and bud from the cell membrane. The particles carry membrane lipid as a coat that con-  tains gp41 and gpl20. Nef indirectly aids in the assembly of viral particles. Pol is the reverse  transcriptase produced from the viral RNA. 0 = stimulates.   Drugs currently used to treat AIDS  act on the viral reverse transcrip-  tase or the protease (see Fig.  14.22). The nonnucleoside drugs (e.g.,  efavirenz) bind to reverse transcriptase and  inhibit its action. The nucleoside analogs  (e.g., ZDV) add to the 3' end of the growing  DNA transcript produced by reverse tran-  scriptase and prevent further elongation.  The protease inhibitors (e.g., indinavir) bind  to the protease and prevent it from cleaving  the polyprotein.   In the initial stage of transcription of the provirus, the transcript is spliced, and  three proteins, Nef, Tat, and Rev, are produced. Tat stimulates transcription of the  viral genes. As Rev accumulates, it allows unspliced viral RNA to leave the nucleus  and to produce proteins of the viral envelope and viral core, including reverse tran-  scriptase. Two of the envelope glycoproteins (gp41 and gpl20, which are derived  from the env gene product) form a complex that embeds in the cell membrane. The  other proteins, which are translated as a polyprotein and cleaved by the viral pro-  tease, combine with the full-length viral RNA to form core viral particles, which  bud from the cell membrane. Thus, the virus obtains its lipid coat from the host cell  membrane, and the coat contains the viral proteins gp41 and gpl20. These surface  proteins of the virus bind to CD4 receptors on other human helper T lymphocytes,  and the infection spreads.   In an uninfected person, helper T lymphocytes usually number approximately  1,000/mL. Infection with HIV causes the number of these cells to decrease, which  results in a deficiency of the immune system. When the number of T lymphocytes  drops below 200/mL, the disease is in an advanced stage, and opportunistic infec-  tions, such as tuberculosis, occur. Although macrophages and dendritic cells lack  CD4 receptors, they can also become infected with HIV and can carry the virus to  the central nervous system.   The most effective means of combating HIV infection involves the use of drugs  that inhibit the viral reverse transcriptase or the viral protease. However, these drugs  only hold the infection at bay; they do not effect a cure.   Lewin B. Chapter 5: mRNA. In: Genes VII. Oxford: Oxford University Press, 2000:119-138.  Lewin B. Chapter 9: mRNA and Transcription. In: Genes VII. Oxford: Oxford University Press,  2000:232-272.   Lewin, B. Chapter 20: Initiation of transcription. In: Genes VII. Oxford: Oxford University Press,  2000:649-684.   Clumeck N. Choosing the best initial therapy for HIV-1 infection. N Engl J Med 1999;341:1925-1926.   [See also the accompanying articles.]  Frankel AD, Young JAT. HIV-1: Fifteen proteins and an RNA. Annu Rev Biochem 1998;67:1-25.  Weatherall DJ, Clegg JB, Higgs DR, Wood WG. The hemoglobinopathies. Vol. III. In: Scriver CR,   Beaudet AL, Sly WS, Valle B., eds. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed.   1. The short transcript AUCCGUACG would be derived from which of the following template DNA sequences? (Note all  sequences are written from 5' to 3')   2. Given that the LD 50 (the dose at which 50% of the recipients die) of amanitin is 0.1 mg per kg body weight, and that the aver-  age mushroom contains 7 mg amanitin, how many mushrooms must be consumed by Amanda Tin (50 kg body weight) to be  above the LD 50 ?   3. Which of the following eukaryotic DNA control sequences does not need to be in a fixed location, and is most responsible for  high rates of transcription of particular genes?   4. Which of the following is true of both eukaryotic and prokaryotic gene expression?   (A) After transcription, a 3 ' poly A tail and a 5 ' cap are added to mRNA.   (D) RNA polymerase binds at a promoter region upstream of the gene.   (E) Mature mRNA is always precisely co-linear to the gene from which it was transcribed.   5. In a segment of a transcribed gene, the nontemplate strand of DNA has the following sequence:  5'...AGCTCACTG...3'   What will be the corresponding sequence in the RNA produced from this segment of the gene?   Fig. 15.1. Binding of tRNA to a codon on  mRNA. The tRNA contains an amino acid at  its 3 '-end that corresponds to the codon on  mRNA with which the anticodon of the tRNA  can base-pair. Note that the codon-anticodon  pairing is complementary and antiparallel.   Proteins are produced by the process of translation, which occurs on ribosomes  and is directed by mRNA. The genetic message encoded in DNA is first tran-  scribed into mRNA, and the nucleotide sequence in the coding region of the  mRNA is then translated into the amino acid sequence of the protein.   Translation of the code. The portion of mRNA that specifies the amino acid  sequence of the protein is read in codons, which are sets of three nucleotides that  specify individual amino acids (Fig 15.1 ). The codons on mRNA are read sequen-  tially in the 5' to 3' direction, starting with the 5 '-AUG (or "start" codon) that  specifies methionine and sets the reading frame and ending with a 3' -termina-  tion (or "stop" ) codon (UAG, VGA, or UAA). The protein is synthesized from its  N -terminus to its C -terminus.   Each amino acid is carried to the ribosome by an aminoacyl-tRNA (i.e., a  tRNA with an amino acid covalently attached). Base -pairing between the anti-  codon of the tRNA and the codon on the mRNA ensures that each amino acid is  inserted into the growing polypeptide at the appropriate position.   Synthesis of the protein. Initiation involves formation of a complex containing  the initial methionyl-tRNA bound to the AUG "start" codon of the mRNA and to  the "P" site of the ribosome. It requires GTP and proteins known as eukaryotic  initiation factors (elFs).   Elongation of the polypeptide involves three steps: (a) binding of an aminoa-  cyl-tRNA to the "A" site on the ribosome where it base-pairs with the second  codon on the mRNA; (b) formation of a peptide bond between the first and sec-  ond amino acids; and (c) translocation, movement of the mRNA relative to the  ribosome, so that the third mRNA codon moves into the "A" site. These three  elongation steps are repeated until a termination codon aligns with the site on  the ribosome where the next aminoacyl-tRNA would normally bind. Release fac-  tors bind instead, causing the completed protein to be released from the ribosome.   After one ribosome binds and moves along the mRNA, translating the polypep-  tide, another ribosome can bind and begin translation. The complex of a single  mRNA with multiple ribosomes is known as a polysome.   Folding and modification and targeting of the protein. Folding of the  polypeptide into its three-dimensional configuration occurs as the polypeptide  is being translated. This process involves proteins called chaperones. Modifi-  cation of amino acid residues in a protein occurs during or after translation.  Proteins synthesized on cytosolic ribosomes are released into the cytosol or  transported into mitochondria, peroxisomes, and nucleus. Proteins synthesized  on ribosomes attached to the rough endoplasmic reticulum (RER) are destined  for lysosomes, cell membranes, or secretion from the cell. These proteins are  transferred to the Golgi complex, where they are modified and targeted to their  ultimate locations.   Anne Niemick, a 4-year old patient with (^-thalassemia intermedia (see  Chapter 14), showed no improvement in her symptoms at her second visit.  Her hemoglobin level was 7.0 g/dL (reference range for females = 12-16  g/dL).   Jay Sakz is a 9-month-old male infant of Ashkenazi Jewish parentage. His  growth and development were normal until age 5 months, when he began  to exhibit mild, generalized muscle weakness. By 7 months, he had poor  head control, slowed development of motor skills, and was increasingly inattentive  to his surroundings. His parents also noted unusual eye movements and staring  episodes. On careful examination of his retinae, his pediatrician observed a "cherry-  red" spot within a pale macula. The physician suspected Tay-Sachs disease and sent  whole blood samples to the molecular biology-genetics laboratory.   Neu Moania returned to his physician's office after 1 week of erythromy-  cin therapy (see Chapter 12). The sputum sample from his previous visit  had been cultured. The results confirmed that his respiratory infection was  caused by Streptococcus pneumoniae and that the organism was sensitive to peni-  cillin, macrolides (e.g., erythromycin, clarithromycin), tetracycline, and other  antibiotics.   Erna Nemdy, a 25 -year-old junior medical student ( "earn an M.D."),  brings her healthy 4-month-old daughter, Beverly, to the pediatrician for  her second diphtheria, pertussis, tetanus (DPT-2) immunization. Erna tells  the doctor that her great, great aunt had died of diphtheria during an epidemic many  years ago.   Transcription, the transfer of the genetic message from DNA to RNA, and transla-  tion, the transfer of the genetic message from the nucleotide language of nucleic  acids to the amino acid language of proteins, both depend on base-pairing. In the  late 1950s and early 1960s, molecular biologists attempting to decipher the process  of translation recognized two problems. The first involved decoding the relationship  between the "language" of the nucleic acids and the "language" of the proteins, and  the second involved determining the molecular mechanism by which translation  between these two languages occurs.   Twenty different amino acids are commonly incorporated into proteins, and,  therefore, the protein "alphabet" has 20 characters. The nucleic acid alphabet, how-  ever, has only four characters, corresponding to the four nucleotides of mRNA (A,  G, C, and U). If two nucleotides constituted the code for an amino acid, then only  4 2 or 16 amino acids could be specified. Therefore, the number of nucleotides that  code for an amino acid has to be three, providing 4 3 or 64 possible combinations or  "codons," more than required, but not overly excessive.   Scientists set out to determine the specific codons for each amino acid. In  1961, Marshall Nirenberg produced the first crack in the genetic code (the col-  lection of codons that specify all the amino acids found in proteins). He showed  that poly(U), a polynucleotide in which all the bases are uracil, produced  polyphenylalanine in a cell-free protein-synthesizing system. Thus, UUU must be    The results of tests performed in  the molecular biology laboratory  show that Jay Sakz has an insertion  in exon 11 of the (3-chain of the hex-  osaminidase A gene, the most common  mutation found in patients of Ashkenazi Jew-  ish background who have Tay-Sachs disease.   Fig. 15.2. Base-pairing of codons for alanine  with 5'-IGC-3'. A. Note that the variation is in  the third base. B. The first three of these  codons can pair with a tRNA that contains the  anticodon 5'-IGC-3\ Hypoxanthine (I) is an  unusual base found in tRNA that can form base  pairs with U, C, or A. It is formed by the deam-  ination of adenine.   Hypoxanthine is the base attached  \ \ ) to ribose in the nucleoside inosine.   The single-letter abbreviation for  hypoxanthine is "I," in reference to the  nucleoside inosine. (In other cases, the first  letter of the base is also the first letter of the  nucleoside and the single letter abbrevia-  tion. For example, A is the base adenine and  the nucleoside adenosine.)   the codon for phenylalanine. As a result of experiments using synthetic polynu-  cleotides in place of mRNA, other codons were identified.   The pioneering molecular biologists recognized that, because amino acids can-  not bind directly to the sets of three nucleotides that form their codons, "adapters"  are required. The "adapters" were found to be tRNA molecules. Each tRNA mole-  cule contains an anticodon and covalently binds a specific amino acid at its 3 '-end  (see Chapters 12 and 14). The anticodon of a tRNA molecule is a set of three  nucleotides that can interact with a codon on mRNA (Fig. 15.2). To interact, the  codon and anticodon must be complementary (i.e., they must be able to form base  pairs in an antiparallel orientation). Thus, the anticodon of a tRNA serves as the link  between an mRNA codon and the amino acid that the codon specifies.   Obviously, each codon present within mRNA must correspond to a specific  amino acid. Nirenberg found that trinucleotides of known base sequence could bind  to ribosomes and induce the binding of specific aminoacyl-tRNAs (i.e., tRNAs with  amino acids covalently attached). As a result of these and the earlier experiments,  the relationship between all 64 codons and the amino acids they specify (the entire  genetic code) was determined by the mid-1960s (Table 15.1).   Three of the 64 possible codons (UGA, UAG, and UAA) terminate protein syn-  thesis and are known as "stop" or nonsense codons. The remaining 61 codons spec-  ify amino acids. Two amino acids each have only one codon (AUG = methionine;  UGG = tryptophan). The remaining amino acids have multiple codons.   Because many amino acids are specified by more than one codon, the genetic code  is described as "degenerate," which means that an amino acid may have more than  one codon. However, each codon specifies only one amino acid, and the genetic  code is, thus, unambiguous.   Inspection of a codon table shows that in most instances of multiple codons for  a single amino acid, the variation occurs in the third base of the codon (see Table  15.1). Crick noted that the pairing between the 3' -base of the codon and the 5' -base  of the anticodon does not always follow the strict base-pairing rules that he and  Watson had previously discovered (i.e., A pairs with U, and G with C). This obser-  vation resulted in the "wobble" hypothesis.   At the third base of the codon (the 3' -position of the codon and the 5' -position  of the anticodon), the base pairs can "wobble", e.g., G can pair with U; and A, G,  or U can pair with the unusual base hypoxanthine (I) found in tRNA. Thus, three of   the four codons for alanine (GCU, GCC, and GCA) can pair with a single tRNA that  contains the anticodon 5'-IGC-3' (see Fig. 15.2). If each of the 61 codons for amino  acids required a distinct tRNA, cells would contain 61 tRNAs. However, because of  "wobble" between the codon and anticodon, fewer than 61 tRNAs are required to  translate the genetic code.   All organisms studied so far use the same genetic code, with some rare exceptions.  One exception occurs in human mitochondrial mRNA, where UGA codes for tryp-  tophan instead of serving as a stop codon, AUA codes for methionine instead of  isoleucine, and CUA codes for threonine instead of leucine.   mRNA does not contain punctuation to separate one codon from the next and the  codons do not overlap. Each nucleotide is read only once. Beginning with a start  codon (AUG) near the 5 '-end of the mRNA, the codons are read sequentially, end-  ing with a stop codon (UGA, UAG, or UAA) near the 3 '-end of the mRNA.   The start codon (AUG) sets the reading frame, the order in which the sequence of  bases in the mRNA is sorted into codons (Fig. 15.3). The order of the codons in the  mRNA determines the sequence in which amino acids are added to the growing  polypeptide chain. Thus, the order of the codons in the mRNA determines the lin-  ear sequence of amino acids in the protein.   Mutations that result from damage to the nucleotides of DNA molecules or from  unrepaired errors during replication (see Chapter 13) can be transcribed into  mRNA, and, therefore, can result in the translation of a protein with an abnormal  amino acid sequence. Various types of mutations can occur that have different  effects on the encoded protein (Table 15.2).   L T J L T J L 1~ J L T J L T J  Protein N terminal-- Met - His -Ser-Gly-Val C terminal   Fig. 15.3. Reading frame of mRNA. A. For any given mRNA sequence, there are three pos-  sible reading frames (1,2, and 3). B. An AUG near the 5 '-end of the mRNA (the start codon)  sets the reading frame for translation of a protein from the mRNA. The codons are read in  linear order, starting with this AUG. (The other potential reading frames are not used. They  would give proteins with different amino acid sequences.)   An addition of one or more bases  A loss of one or more bases   CGA -> CGG  Arg — > Arg  CGA -> CCA  Arg — » Pro  CGA -> UGA  Arg -> Stop   Sickle cell anemia is caused by a  missense mutation. In each of the  alleles for p-globin, Will Sichel's  DNA has a single base change (see Chapter  6). In the sickle cell gene, GTG replaces the  normal GAG. Thus, in the mRNA, the codon  GUG replaces GAG and a valine residue  replaces a glutamate residue in the protein.   Point mutations occur when only one base in DNA is altered, producing a change  in a single base of an mRNA codon. There are three basic types of point mutations:  silent mutations, missense mutations, and nonsense mutations. Point mutations are  said to be "silent" when they do not affect the amino acid sequence of the protein.  For example, a codon change from CGA to CGG does not affect the protein because  both of these codons specify arginine (see Table 15.1). In missense mutations, one  amino acid in the protein is replaced by a different amino acid. For example, a  change from CGA to CCA causes arginine to be replaced by proline. A "nonsense"  mutation causes the premature termination of a polypeptide chain. For example, a  codon change from CGA to UGA causes a codon for arginine to be replaced by a  stop codon and synthesis of the mutant protein terminates at this point.   One type of thalassemia is caused  by a nonsense mutation. Codon 17  of the p-globin chain is changed   Some types of thalassemia are  ■ B # caused by deletions in the globin   genes. Patients have been studied  who have large deletions in either the 5' or  the 3' coding region of the (3-globin gene,  removing almost one third of the DNA  sequence.   Is it likely that Anne Niemick has a large  deletion in the p-globin gene?   An insertion occurs when one or more nucleotides are added to DNA. If the inser-  tion does not generate a stop codon, a protein with more amino acids than normal  could be produced.   When one or more nucleotides are removed from DNA, the mutation is known  as a deletion. If the deletion does not affect the normal start and stop codons, a pro-  tein with fewer than the normal number of amino acids could be produced.   A frameshift mutation occurs when the number of inserted or deleted nucleotides  is not a multiple of three (Fig. 15.4). The reading frame shifts at the point where the  insertion or deletion begins. Beyond that point, the amino acid sequence of the pro-  tein translated from the mRNA differs from the normal protein.   A tRNA that contains an amino acid covalently attached to its 3' -end is called an  aminoacyl-tRNA and is said to be "charged." Aminoacyl-tRNAs are named both for  the amino acid and the tRNA that carries the amino acid. For example, the tRNA   Fig. 15.4. A frameshift mutation. The insertion of a single nucleotide (the A in the dotted box)  causes the reading frame to shift, so that the amino acid sequence of the protein translated  from the mRNA is different after the point of insertion. A similar effect can result from the  insertion or deletion of nucleotides if the number inserted or deleted is not a multiple of 3.   for alanine (tRNA Ala ) acquires alanine to become alanyl-tRNA ala . A particular  tRNA recognizes only the AUG start codon that initiates protein synthesis and not  other AUG codons that specify insertion of methionine within the polypeptide  chain. This initiator methionyl-tRNA Met is denoted by the subscript "i" in  methionyl-tRNAi Met .   Amino acids are attached to their tRNAs by highly specific enzymes known as  aminoacyl-tRNA synthetases. Twenty different synthetases exist, one for each  amino acid. Each synthetase recognizes a particular amino acid and all of the tRNAs  that carry that amino acid.   The formation of the ester bond that links the amino acid to the tRNA by an  aminoacyl-tRNA synthetase is an energy-requiring process that occurs in two steps.  The amino acid is activated in the first step when its carboxyl group reacts with  adenosine triphosphate (ATP) to form an enzyme/aminoacyl-AMP complex and  pyrophosphate (Fig. 15.5). The cleavage of a high-energy bond of ATP in this reac-  tion provides energy, and the subsequent cleavage of pyrophosphate by a pyrophos-  phatase helps to drive the reaction by removing one of the products. In the second  step, the activated amino acid is transferred to the 2'- or 3' -hydroxy 1 group (depend-  ing on the type of aminoacyl-tRNA synthetase catalyzing the reaction) of the 3 ' ter-  minal A residue of the tRNA, and AMP is released (recall that all tRNAs have a  CCA added to their 3' end posttranscriptionally). The energy in the aminoacyl-  tRNA ester bond is subsequently used in the formation of a peptide bond during the  process of protein synthesis.   Some aminoacyl-tRNA synthetases use the anticodon of the tRNA as a recog-  nition site as they attach the amino acid to the hydroxyl group at the 3 '-end of  the tRNA (Fig. 15.6). However, other synthetases do not use the anticodon but  recognize only bases located at other positions in the tRNA. Nevertheless, inser-  tion of the amino acid into a growing polypeptide chain depends solely on the  bases of the anticodon, through complementary base-pairing with the mRNA  codon.   Translation of a protein involves three steps: initiation, elongation, and termination.  It begins with the formation of the initiation complex. Subsequently, synthesis of  the polypeptide occurs by a series of elongation steps that are repeated as each    A nonsense mutation at codon 17  would cause premature termina-  tion of translation. A nonfunctional  peptide containing only 16 amino acids  would result, producing a p°-thalassemia if  the mutation occurred in both alleles. A  large deletion in the coding region of the  gene could also produce a truncated protein.  If Anne Niemick has a nonsense mutation or  a large deletion, it could only be in one  allele. The mutation in the other allele must  be milder, since she produces some normal  p-globin. Her hemoglobin is 7 g/dL, typical of  thalassemia intermedia (a (^-thalassemia).   Fig. 15.5. Formation of aminoacyl-tRNA. The  amino acid is first activated by reacting with  ATP. The amino acid is then transferred from  the aminoacyl- AMP to tRNA.   Fig. 15.6. Some aminoacyl-tRNA synthetase recognition sites on tRNA. Each aminoacyl-  tRNA synthetase is specific for one tRNA, which it "recognizes" by binding the sequences  of nucleotides called the recognition sites, shown in blue. In some cases, the anticodon is a  recognition site; in others, it is not. This is true for human tRNAs as well as those shown here.   Fig. 15.7. Overview of the process of translation. 1. Once a tRNA has donated its amino acid  to the growing polypeptide chain, it is released from the mRNA. 2. A new aminoacyl-tRNA  binds to the correct codon in the mRNA to donate its amino acid to the growing polypeptide  chain.   amino acid is added to the growing chain (Fig. 15.7). Termination occurs where the  mRNA contains an in-frame stop codon, and the completed polypeptide chain is  released.   Eukaryotic initiation factor 2 (elF2)  \ % ) and also elongation factor 1 (EF1)   are types of heterotrimeric G pro-  teins (see Chapter 11). They dramatically  change their conformation and actively form  complexes when they bind GTP but become  inactive and dissociate when they hydrolyze  this GTP to GDP. GTP can then displace the  bound GDP to reactivate the initiation factor  elF2 or the elongation factor EF1.   In eukaryotes, initiation of translation involves formation of a complex composed  of methionyl-tRNAi Met , mRNA, and a ribosome (Fig. 15.8). Methionyl-tRNA^  (also known as Met-tRNA^ 61 ) initially forms a complex with the protein eukaryotic  initiation factor 2 (eIF2), which binds GTR This complex then binds to the small  (40S) ribosomal subunit. The cap at the 5 '-end of the mRNA binds an initiation fac-  tor known as the cap binding protein (CBP). CBP contains a number of subunits,  including eIF4E. Several other elFs join, and the mRNA then binds to the elFs-Met-  tRNAi Met - 40S ribosome complex. In a reaction requiring hydrolysis of ATP (due  to the helicase activity of an elF subunit), this complex unwinds a hairpin loop in  the mRNA and scans the mRNA until it locates the AUG start codon (usually the    Fig. 15.8. Initiation of protein synthesis. P site = peptidyl site on the ribosome; A site = aminoacyl site on the ribosome (The A and P sites or  portions of them are indicated by dashed lines); elF = eukaryotic initiation factor.   Table 15.3. Differences between Eukaryotes and Prokaryotes in the Initia-  tion of Protein Synthesis   Cap at 5' end of mRNA binds elFs and  40S ribosomal subunit containing  tRNA met . mRNA is scanned for AUG  start codon   Shine-Dalgarno sequence  upstream of initiating AUG  binds to complementary  sequence in 16S rRNA   first AUG). GTP is hydrolyzed, the initiation factors are released, and the large ribo-  somal (60S) subunit binds. The ribosome is now complete. It contains one small and  one large subunit, and has two binding sites for tRNA, known as the P (peptidyl)  and A (aminoacyl) sites. During initiation, Met-tRNA^ 61 binds to the ribosome at  the P site.   The initiation process differs for prokaryotes and eukaryotes (Table 15.3). In bac-  teria, the initiating methionyl-tRNA is formylated, producing a formyl-methionyl-  tRNA f Met that participates in formation of the initiation complex (Fig. 15.9). Only  three initiation factors (IFs) are required to generate this complex in prokaryotes,  compared with the dozen or more required by eukaryotes. The ribosomes also differ  in size. Prokaryotes have 70S ribosomes, composed of 30S and 50S subunits, and  eukaryotes have 80S ribosomes, composed of 40S and 60S subunits. Unlike eukary-  otic mRNA, bacterial mRNA is not capped. Identification of the initiating AUG  triplet in prokaryotes occurs when a sequence in the mRNA (known as the  Shine-Dalgarno sequence) binds to a complementary sequence near the 3' -end of  the 16S rRNA of the small ribosomal subunit.   After the initiation complex is formed, addition of each amino acid to the growing  polypeptide chain involves binding of an aminoacyl-tRNA to the A site on the ribo-  some, formation of a peptide bond, and translocation of the peptidyl-tRNA to the P  site (Fig. 15.10). The peptidyl-tRNA contains the growing polypeptide chain.   When Met-tRNAi (or a peptidyl-tRNA) is bound to the P site, the mRNA codon in  the A site determines which aminoacyl-tRNA will bind to that site. An aminoacyl-  tRNA binds when its anticodon is antiparallel and complementary to the mRNA  codon. In eukaryotes, the incoming aminoacyl-tRNA first combines with elongation   Fig. 15.9. Bacterial tRNA containing formyl-methionine. The initial methionine is not  formylated in eukaryotic protein synthesis.   Insulin, an anabolic hormone, stim-  ulates general protein synthesis by  activating the initiation factor  elF4E. Normally, elF4E is bound to an  inhibitor protein, 4E binding protein (4E-BP).  When insulin binds to its cell surface recep-  tor, it initiates an intracellular sequence of  events resulting in phosphorylation of 4E-BR  Phosphorylated 4E-BP no longer binds to  elF4E, and elF4E is now free to participate in  the initiation of protein synthesis.   elF2 is a regulator of the initiation  step in protein synthesis. When it is  phosphorylated, it is inactive, and  protein synthesis cannot begin. Conditions  such as starvation, heat shock, and viral  infection result in phosphorylation of elF2 by  a specific kinase.   The regulation of globin synthesis by  heme in reticulocytes illustrates the role of  elF2 in regulation of translation. Reticulo-  cytes, which are the precursors of red blood  cells, synthesize the oxygen-carrying hemo-  globin molecules from the globin polypep-  tide chains and the Fe-binding pigment,  heme. In the absence of heme, the rate of  initiation of globin synthesis decreases.  Heme acts by inhibiting the phosphorylation  of the initiation factor elF2. Thus, elF2 is  active in the presence of heme and globin  synthesis is initiated.   Many antibiotics that are used to  combat bacterial infections in  humans take advantage of the dif-  ferences between the mechanisms for pro-  tein synthesis in prokaryotes and eukary-  otes. For example, streptomycin binds to the  30S ribosomal subunit of prokaryotes. It  interferes with initiation of protein synthesis  and causes misreading of mRNA.   Although the bacterium causing his  infection was sensitive to streptomycin, this  drug was not used to treat Neu Moania  because it can cause permanent hearing  loss. Its use is, therefore, confined mainly to  the treatment of tuberculosis or other infec-  tions that do not respond adequately to  other antibiotics.   The antibiotic tetracycline binds to  the 30S ribosomal subunit of  prokaryotes and inhibits binding of  aminoacyl-tRNA to the A site of the ribo-  some. The bacterium causing Neu Moania's  infection was found to be sensitive to tetra-  cycline.    Fig. 15.10. Elongation of a polypeptide chain. 1. Binding of valyl-tRNA Val to the A site. 2. Formation of a peptide bond. 3. Translocation. After  step 3, step 1 is repeated using the aminoacyl-tRNA for the new codon in the A site. Steps 2 and 3 follow. These three steps keep repeating until  termination occurs. (In prokaryotes, an exit site called the E site binds the t-RNA after it is displaced from the P site). EF = elongation factor.   factor EFla containing bound GTP before binding to the mRNA-ribosome com-  plex (EFla is the GTP-binding a subunit of a heterotrimeric G protein, which is  activated for association with other proteins when it contains GTP; see Chapter 11).  When the aminoacyl-tRNA-EFla-GTP complex binds to the A site, GTP is  hydrolyzed to GDP. This prompts dissociation of EFla-GDP from the aminoacyl-  tRNA ribosomal complex, thereby allowing protein synthesis to continue (Fig. 15.11).    Fig. 15.11. Recycling of EF1 in eukaryotes. Note that EF1 is a heterotrimeric G protein. Its a-subunit binds GTP and activates the process  whereby an aminoacyl-tRNA binds to the A site of the ribosome. GTP is hydrolyzed, and EFla binds to the EFlp7 subunits, releasing GDP.  GTP binds to the a subunit, the p7 subunits are released, and EFla GTP is ready for another round. In prokaryotes, EFla is EF-Tu and the pro-  tein corresponding to p7 is EF-Ts.   The free EFla-GDP reassociates with the EFIP7 subunits, and GDP is released.  Subsequently, GTP binds and the P7 subunits dissociate. Thus, EFla-GTP is ready  to bind another aminoacyl-tRNA molecule.   The process of elongation is very similar in prokaryotes, except that the corre-  sponding factor for EFla is named EF-Tu and the associating elongation factors are  called EF-Ts instead of EFIP7.   In the first round of elongation, the amino acid on the tRNA in the A site forms a  peptide bond with the methionine on the tRNA in the P site. In subsequent rounds  of elongation, the amino acid on the tRNA in the A site forms a peptide bond with  the peptide on the tRNA in the P site (see Fig. 15.10). Peptidyltransf erase, which is  not a protein but the rRNA of the large ribosomal subunit, catalyzes the formation  of the peptide bond. The tRNA in the A site now contains the growing polypeptide  chain, and the tRNA in the P site is uncharged (i.e., it no longer contains an amino  acid or peptide).   Translocation in eukaryotes involves another G protein, elongation factor EF2  (EF-G in prokaryotes) that complexes with GTP and binds to the ribosome, caus-  ing a conformational change that moves the mRNA and its base-paired tRNAs  with respect to the ribosome. The uncharged tRNA moves from the P site and is  released from the ribosome. The peptidyl-tRNA moves into the P site, and the  next codon of the mRNA occupies the A site. During translocation, GTP is  hydrolyzed to GDP, which is released from the ribosome along with the elongation  factor (see Fig. 15.10).   The three elongation steps are repeated until a termination (stop) codon moves into  the A site on the ribosome. Because no tRNAs with anticodons that can pair with  stop codons normally exist in cells, release factors bind to the ribosome instead,  causing peptidyltransferase to hydrolyze the bond between the peptide chain and  tRNA. The newly synthesized polypeptide is released from the ribosome, which  dissociates into its individual subunits, releasing the mRNA.   Currently, some parents are not having their children immunized. The decrease  in the incidence of infectious disease in the United States has led to compla-  cency. Other parents who lack health insurance cannot afford to have their chil-  dren vaccinated. Anyone who remembers the summertime fear of poliomyelitis in the  1940s and 1950s realizes that immunizations are a blessing that should be available to  all children.   Protein synthesis requires a considerable amount of energy. Formation of each  \\ ) aminoacyl-tRNA requires the equivalent of two high-energy phosphate bonds   because ATP is converted to AMP and pyrophosphate, which is cleaved to form  two inorganic phosphates. As each amino acid is added to the growing peptide chain,  two GTPs are hydrolyzed, one at the step involving EF1 and the second at the transloca-  tion step. Thus, four high-energy bonds are cleaved for each amino acid of the polypep-  tide. In addition, energy is required for initiation of synthesis of a polypeptide chain and  for synthesis from nucleotide triphosphate precursors of the mRNA, tRNA, and rRNA  involved in translation.    The macrolide antibiotics (e.g.,  erythromycin, clarithromycin) bind  to the 50S ribosomal subunit of  bacteria and inhibit translocation. Clar-  ithromycin was used to treat Neu Moania  because he had taken it previously without  difficulty. It has less serious side effects than  many other antibiotics and is used as an  alternative drug in patients, such as Mr.  Moania, who are allergic to penicillin. After 1  week of therapy, Mr. Moania recovered from  his infection.   Chloramphenicol is an antibiotic  that interferes with the peptidyl-  transferase activity of the 50S  ribosomal subunit of bacteria. It was not  used to treat Neu Moania because it is very  toxic to humans, partly because of its effect  on mitochondrial protein synthesis.   Diphtheria is a highly contagious  disease caused by a toxin secreted  by the bacterium Corynebacterium  diphtheriae. Although the toxin is a protein,  it is not produced by a bacterial gene, but by  a gene brought into the bacterial cell by an  infecting bacteriophage.   Diphtheria toxin is composed of two pro-  tein subunits. The B subunit binds to a cell  surface receptor, facilitating the entry of the  A subunit into the cell. In the cell, the A sub-  unit catalyzes a reaction in which the ADP-  ribose (ADPR) portion of NAD is transferred  to EF2 (ADP-ribosylation). In this reaction,  the ADPR is covalently attached to a post-  translationally modified histidine residue,  known as diphthamide. ADP-ribosylation of  EF2 inhibits protein synthesis, leading to cell  death. Children, including Erna Nemdy's  daughter, are usually immunized against this  often fatal disease at an early age.   Fig. 15.12. A polysome. The complex of  mRNA and multiple ribosomes, each of which  is producing a polypeptide chain, is called a  polysome.   As one ribosome moves along the mRNA, producing a polypeptide chain, a sec-  ond ribosome can bind to the vacant 5' -end of the mRNA. Many ribosomes can  simultaneously translate a single mRNA, forming a complex known as a  polysome (Fig. 15.12). A single ribosome covers approximately 80 nucleotides  of mRNA. Therefore, ribosomes are positioned on mRNA at intervals of approx-  imately 100 nucleotides. The growing polypeptide chains attached to the ribo-  somes become longer as each ribosome moves from the 5' -end toward the 3 '-end  of the mRNA.   Nascent polypeptide chains (i.e., polypeptides that are in the process of being synthe-  sized) are processed. As they are being produced, they travel through a tunnel in the  ribosome, which can hold roughly 30 amino acid residues. As polymerization of the  chain progresses, the amino acid residues at the N-terminal end begin to emerge from  this protected region within the ribosome and to fold and refold into the three-dimen-  sional conformation of the polypeptide. Proteins bind to the nascent polypeptide and  mediate the folding process. These mediators are called chaperones (see Chapter 7)  because they prevent improper interactions from occurring. Disulfide bond formation  between cysteine residues is catalyzed by disulfide isomerases and may also be  involved in producing the three-dimensional structure of the polypeptide.   After proteins emerge from the ribosome, they may undergo posttranslational mod-  ifications. The initial methionine is removed by specific proteases; methionine is  not the N-terminal amino acid of all proteins. Subsequently, other specific cleavages  also may occur that convert proteins to more active forms (e.g., the conversion of  proinsulin to insulin). In addition, amino acid residues within the peptide chain can  be enzymatically modified to alter the activity or stability of the proteins, direct it  to a subcellular compartment, or prepare it for secretion from the cell.   Amino acid residues are enzymatically modified by the addition of various types  of functional groups. (Box 15.1) For example, the N-terminal amino acid is some-  times acetylated, and methyl groups can be added to lysine residues. These changes  alter the charge on the protein. Proline and lysine residues can be modified by  hydroxylation. In collagen, hydroxylations lead to stabilization of the protein. Car-  boxylations are important, especially for the function of proteins involved in blood  coagulation. Formation of 7-carboxylglutamate allows these proteins to chelate  Ca 2+ , a step in clot formation. Fatty acids or other hydrophobic groups (e.g., prenyl  groups) anchor the protein in membranes. An ADP-ribose group can be transferred  from NAD + to certain proteins. The addition and removal of phosphate groups  (which bind covalently to serine, threonine, or tyrosine residues) serve to regulate  the activity of many proteins (e.g., the enzymes of glycogen degradation and regu-  lators of gene transcription.) Glycosylation, the addition of carbohydrate groups, is  a common modification that occurs mainly on proteins that are destined to be  secreted or incorporated into lysosomes or cellular membranes.   Many proteins are synthesized on polysomes in the cytosol. After they are  released from ribosomes, they remain in the cytosol, where they carry out their  functions. Other proteins synthesized on cytosolic ribosomes enter organelles,    Fig. 15.13. Synthesis of proteins on the RER. 1. Translation of the protein begins in the cytosol. 2. As the signal peptide emerges from the ribo-  some, a signal recognition particle (SRP) binds to it and to the ribosome and inhibits further synthesis of the protein. 3. The SRP binds to the  SRP receptor in the RER membrane, docking the ribosome on the RER. 4. The SRP is released and protein synthesis resumes. 5. As the signal  peptide moves through a pore into the RER, a signal peptidase removes the signal peptide. 6. Synthesis of the nascent protein continues, and the  completed protein is released into the lumen of the RER.   such as mitochondria or nuclei. These proteins contain amino acid sequences  called targeting sequences or signal sequences that facilitate their transport into  a certain organelle. Another group of proteins are synthesized on ribosomes  bound to the RER. These proteins are destined for secretion or for incorporation  into various subcellular organelles (e.g., lysosomes, endoplasmic reticulum  [ER], Golgi complex) or cellular membranes, including the plasma membrane.  Proteins that enter the RER as they are being synthesized have signal peptides  near their N- termini that do not have a common amino acid sequence. However,  they do contain a number of hydrophobic residues and are 15 to 30 amino acids  in length (Fig. 15.13). A signal recognition particle (SRP) binds to the ribosome  and to the signal peptide as the nascent polypeptide emerges from the tunnel in  the ribosome, and translation ceases. When the SRP subsequently binds to an  SRP receptor (docking protein) on the RER, translation resumes, and the  polypeptide begins to enter the lumen of the RER. The signal peptide is removed  by the signal peptidase, and the remainder of the newly synthesized protein  enters the lumen of the RER. These proteins are transferred in small vesicles to  the Golgi complex.   The Golgi complex serves to process the proteins it receives from the RER  and to sort them so that they are delivered to their appropriate destinations  (Fig. 15.14). Processing, which can be initiated in the endoplasmic reticulum,  involves glycosylation, the addition of carbohydrate groups, and modification of  existing carbohydrate chains. Sorting signals permit delivery of proteins to their  target locations. For example, glycosylation of enzymes destined to become  lysosomal enzymes results in the presence of a mannose 6-phosphate residue on  an oligosaccharide attached to the enzyme. This residue is recognized by the  mannose 6-phosphate receptor protein, which incorporates the enzyme into a  clathrin-coated vesicle. The vesicle travels to endosomes, and is eventually  incorporated into lysosomes. Other proteins containing a KDEL (lys-asp-glu-  leu) sequence at their carboxyl terminal are returned to the ER from the Golgi.  Proteins with hydrophobic regions can embed in various membranes. Some pro-  teins, whose sorting signals have not yet been determined, enter secretory vesi-  cles and travel to the cell membrane, where they are secreted by the process of  exocytosis.    l-cell disease (Mucolipidosis II) is a  disorder of protein targeting. Lyso-  somal proteins are not sorted prop-  erly from the Golgi to the lysosomes, and  lysosomal enzymes end up secreted from  the cell. This is because of a mutation in the  enzyme N-acetylglucosamine phosphotrans-  ferase, which is a required first step for  attaching the lysosomal targeting signal,  mannose-6-phosphate, to lysosomal pro-  teins. Thus, lysosomal proteins cannot be  targeted to the lysosomes, and these  organelles become clogged with materials  that cannot be digested, destroying overall  lysosomal function. This leads to a lysoso-  mal storage disease of severe consequence,  with death before the age of 8.    Fig. 15.14. Fate of proteins synthesized on the  RER. Proteins synthesized on ribosomes  attached to the ER travel in vesicles to the cis  face of the Golgi complex. After the mem-  branes fuse, the proteins enter the Golgi com-  plex. Structural features of the proteins deter-  mine their fate. Some remain in the Golgi  complex, and some return to the RER. Others  bud from the trans face of the Golgi complex  in vesicles. These vesicles can become lyso-  somes or secretory vesicles, depending on  their contents. Secretory proteins are released  from the cell when secretory vesicles fuse with  the cell membrane (exocytosis). Proteins with  hydrophobic regions embedded in the mem-  brane of secretory vesicles become cell mem-  brane proteins. See Chapter 10 for descriptions  of the endoplasmic reticulum, Golgi complex,  lysosomes, and the cell membrane, and also  for an explanation of the process of exocytosis.   Ann Niemick has a p + thalassemia classified clinically as p-tha-  lassemia intermedia. She produces an intermediate amount of functional p  globin chains (her hemoglobin is 7 g/dL; normal is 12-16). In (^-tha-  lassemia, little or none of the hemoglobin (3 chain is produced, p -thalassemia inter-  media is usually the result of two different mutations (one that mildly affects the  rate of synthesis of p -globin and one severely affecting its rate of synthesis), or, less  frequently, homozygosity for a mild mutation in the rate of synthesis, or a complex  combination of mutations. The mutations that cause the thalassemias have been  studied extensively and are summarized in Table 15.4. For each of these mutations,  you should now be able to explain whether it is most likely to result in a p + or (3°  thalassemia.   a Jay Sakz. The molecular biology genetics laboratory's report on Jay  ) Sakz's white blood cells indicated that he had a deficiency of hex-  osaminidase A caused by a defect in the gene encoding the a subunit of  this enzyme (variant B, Tay-Sachs disease). Hexosaminidases are lysosomal  enzymes necessary for the normal degradation of glycosphingolipids, such as the  gangliosides. Gangliosides are found in high concentrations in neural ganglia,  although they are produced in many areas of the nervous system. When the activity  of these degradative enzymes is absent or subnormal, partially degraded ganglio-  sides accumulate in lysosomes in various cells of the central nervous system, caus-  ing a wide array of neurologic disorders known collectively as gangliosidoses.   Data from Scriver CR, et al. The Metabolic and Molecular Basis of Inherited Diseases, Vol III. New York:  McGraw-Hill, 1995:3456-3457.   When the enzyme deficiency is severe, symptoms appear within the first 3-5  months of life. Eventually symptoms include upper and lower motor neuron  deficits, visual difficulties that can progress to blindness, seizures, and increasing  cognitive dysfunction. By the second year of life, the patient may regress into a  completely vegetative state, often succumbing to bronchopneumonia caused by  aspiration or an inability to cough.   Erna Nemdy. With the availability of diphtheria toxoid as part of the  almost universal DPT immunization practices in the United States, fatali-  ties due to infection by the Gram-positive bacillus C. diphtheriae are rare.  Most children, as is the case with Erna Nemdy 's daughter Beverly, are immunized.  In unimmunized individuals, however, symptoms are caused by a bacterial exotoxin  encoded by a phage that infects the bacterial cells. The toxin enters human cells,  inhibiting protein synthesis and, ultimately, causing cell death. Complications  related to cardiac and nervous system involvement are the major cause of morbid-  ity and mortality. Patients for whom a definitive diagnosis of diphtheria is estab-  lished are treated with equine diphtheria antitoxin.   Antibiotics That Inhibit Protein Synthesis. The processes of  translation on bacterial ribosomes and on the cytoplasmic ribosomes of  eukaryotic cells have many similarities, but there are a number of subtle  differences. Antibiotics act at steps at which these differences occur, and different  antibiotics target each of the major steps of protein synthesis (Table 15.5). There-  fore, these compounds can be used selectively to prevent bacterial protein synthesis  and inhibit bacterial proliferation, while having little or no effect on human cells.  Caution must be exercised in their use, however, because some of the antibiotics  affect human mitochondria, which have a protein-synthesizing system similar to  that of bacteria. Another problem with these drugs is that bacteria can become  resistant to their action. Mutations in genes encoding the proteins or RNA of bacte-  rial ribosomes can cause resistance. Resistance also results when bacteria take up  plasmids carrying genes for inactivation of the antibiotic. Because of the wide-  spread and often indiscriminate use of antibiotics, strains of bacteria are rapidly  developing that are resistant to all known antibiotics.   Streptomycin. Streptomycin inhibits initiation by binding to three pro-  teins and probably the 16S rRNA of the 30S ribosomal subunit of bacteria.  Abnormal initiation complexes, known as streptomycin monosomes, accu-  mulate. Streptomycin can also cause misreading of mRNA, resulting in premature  termination of translation or in the incorporation of incorrect amino acids into  polypeptide chains that already have been initiated. The use of this antibiotic is lim-  ited because it causes ototoxicity that can result in loss of hearing.   Streptomycin Binds to the 30S ribosomal subunit of prokaryotes, thereby preventing   formation of the initiation complex. It also causes misreading of mRNA   Tetracycline Binds to the 30S ribosomal subunit and inhibits binding of   Tetracycline. Tetracycline binds to the 30S ribosomal subunit of bac-  teria and prevents an aminoacyl-tRNA from binding to the A site on the  ribosome. This effect of the drug is reversible; thus, when the drug is  removed, bacteria resume protein synthesis and growth, resulting in a rekindling of  the infection. Furthermore, tetracycline is not absorbed well from the intestine, and  its concentration can become elevated in the contents of the gut, leading to changes  in the intestinal flora. Because it has been used to treat human infections and added  to animal feed to prevent animal infections, humans have had extensive exposure to  tetracycline. As a result, resistant strains of bacteria have developed.   Chloramphenicol. Chloramphenicol binds to the 50S ribosomal sub-  unit of bacteria and prevents binding of the amino acid portion of the  aminoacyl-tRNA, effectively inhibiting peptidyltransferase action. This  antibiotic is used only for certain extremely serious infections, such as meningitis  and typhoid fever. Chloramphenicol readily enters human mitochondria, where it  inhibits protein synthesis. Cells of the bone marrow often fail to develop in patients  treated with chloramphenicol, and use of this antibiotic has been linked to fatal  blood dyscrasias, including an aplastic anemia.   Erythromycin. Erythromycin and the other macrolide antibiotics bind to  the 50S ribosomal subunit of bacteria near the binding site for chlorampheni-  col. They prevent the translocation step, the movement of the peptidyl-tRNA  from the 4 A" to the "P" site on the ribosome. Because the side effects are less severe  and more readily reversible than those of many other antibiotics, the macrolides are  often used to treat infections in persons who are allergic to penicillin, an antibiotic that  inhibits bacterial cell wall synthesis. However, bacterial resistance to erythromycin is  increasing. Therefore, its close relative, clarithromycin, is often used.   Pestova TV, Hellen CUT Ribosome recruitment and scanning. TIBS 1999;24:85-87.  Lewin B. Genes VII. Protein synthesis. Translation: expressing genes as proteins. Oxford: Oxford Uni-  versity Press, 2000:139-166.  Tay-Sachs:   Gravel R, Clarke J, Kaback M, et al. The Gm2 gangliosidoses. In: Scriver C, Beaudet A, Sly W, Valle D  (eds). The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. Vol III. New York: McGraw-  Hill, 2001:3827-3876.   Weatherall D, Clegg J, Higgs D, Wood W. The hemoglobinopathies. In: Scriver C, Beaudet A, Sly W,  Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. Vol III. New York:  McGraw-Hill, 2001:4571-4636.   Hardman JG, Limbird LE, et al. Goodman and Gilman's The Pharmacological Basis of Therapeutics.  New York: McGraw-Hill, 1996:1103-1153.   In the read-out of the genetic code in prokaryotes, which one of the following processes acts before any of the others?   (C) Movement of the ribosome from one codon to the next   2. tRNA charged with cysteine can be chemically treated so that the amino acid changes its identity to alanine. If some of this  charged tRNA is added to a protein- synthesizing extract that contains ALL the normal components required for translation,  which of the following statements represents THE MOST LIKELY OUTCOME after adding an mRNA that has both cys and  ala codons in the normal reading frame?   (A) Cysteine would be added each time the alanine codon was translated.   (B) Alanine would be added each time the cysteine codon was translated.   3. The genetic code is said to be degenerate because of which of the following?   (A) Many codons have pairs of identical bases next to each other.   (B) Some triplets are made up of repeating purines or pyrimidines.   (C) Many of the amino acids have more than one triplet code.   (D) There is wobble in the bond between the first base of the anticodon and the third base of the codon.   4. Which of the following is responsible for retaining the enzyme protein disulfide isomerase in the ER?   (D) There are four possible bases at each of three codon positions.   (E) The more codons, the faster protein synthesis can be accomplished.   The prokaryotic bacterium  \\ ) Escherichia coli can use a wide   range of different nutrients to sus-  tain growth. It accomplishes this partially by  turning on and off expression of the genes  required for synthesis of enzymes in differ-  ent metabolic pathways. For example,  enzymes encoded by the lac operon allow  E. coli to use lactose for energy and the pres-  ence of lactose activates transcription of  these genes. Thus, the nutrient itself is  involved in regulating gene expression.   In contrast, the cells of the adult human  are exposed to a nearly constant and safe  environment composed of blood and inter-  stitial fluid. Hormones maintain this nearly  constant environment, in spite of changes in  nutrient demand and availability, partially by  regulating gene transcription. The immune  system protects cells against foreign organ-  isms, partially by controlling the expression  of genes required for the immune response.  However, some nutrient regulation of gene  expression also occurs. For example, iron  controls expression of the genes for its stor-  age and transport proteins at the level of  their mRNAs.   Gene expression, the generation of a protein or RNA product from a particular  gene, is controlled by complex mechanisms. Normally, only a fraction of the genes  in a cell are expressed at any time. Gene expression is regulated differently in  prokaryotes and eukaryotes.   Regulation of gene expression in prokaryotes. In prokaryotes, gene expres-  sion is regulated mainly by controlling the initiation of gene transcription. Sets of  genes encoding proteins with related functions are organized into operons, and  each operon is under the control of a single promoter (or regulatory region). Reg-  ulatory proteins called repressors bind to the promoter and inhibit the binding of  RNA polymerase (negative control), whereas activator proteins facilitate RNA  polymerase binding (positive control). Repressors are controlled by nutrients or  their metabolites, classified as inducers or corepressors. Regulation also may  occur through attenuation of transcription.   Eukaryotes: Regulation of gene expression at the level ofDNA. In eukary-  otes, activation of a gene requires changes in the state of chromatin (chromatin  remodeling) that are facilitated by acetylation ofhistones and methylation of  bases. These changes in DNA determine which genes are available for transcrip-  tion.   Regulation of eukaryotic gene transcription. Transcription of specific genes is  regulated by proteins (called specific transcription factors or transactivators)  that bind to gene regulatory sequences ( called promoter-proximal elements,  response elements, or enhancers) that activate or inhibit assembly of the basal  transcription complex and RNA polymerase at the TATA box. These specific tran-  scription factors, which may bind to DNA sequences some distance from the pro-  moter, interact with coactivators or corepressors that bind to components of the  basal transcription complex. These protein factors are said to work in "trans" ;  the DNA sequences to which they bind are said to work in "cis."   Other sites for regulation of eukaryotic gene expression. Regulation also  occurs during the processing of RNA, during RNA transport from the nucleus to  the cytoplasm, and at the level of translation in the cytoplasm. Regulation can  occur simultaneously at multiple levels for a specific gene, and many factors act  in concert to stimulate or inhibit expression of a gene.   Arlyn Foma, a 68 -year old man, complained of fatigue, loss of appetite,  and a low-grade fever. An open biopsy of a lymph node indicated the pres-  ence of non-Hodgkin's lymphoma, follicular type. Computed tomography  and other noninvasive procedures showed a diffuse process with bone marrow   involvement. He is receiving multidrug chemotherapy with AV/CM (doxorubicin  [adriamycin], vincristine, cyclophosphamide, and methotrexate). His disease is not  responding well to this regimen, and the follicular lymphoma appears to be evolv-  ing into a more aggressive process. Because recombinant interferon a-2b has been  reported to have synergistic or additive effects with these agents, it is added to the  protocol. Although resistance to methotrexate is considered, the drug is continued  as part of the combined therapeutic approach.   Mannie Weitzels is a 56-year-old male who complains of headaches,  weight loss related to a declining appetite for food, and a decreasing  tolerance for exercise. He notes discomfort and fullness in the left upper  quadrant of his abdomen. On physical examination, he is noted to be pale and  to have ecchymoses (bruises) on his arms and legs. His spleen is markedly  enlarged.   Initial laboratory studies show a hemoglobin of 10.4 g/dL (normal = 13.5-17.5  g/dL) and a leukocyte (white blood cell) count of 86,000 cells/mm 3 (normal =  4,500-11,000 cells/mm 3 ). Most of the leukocytes are granulocytes (white blood  cells arising from the myeloid lineage), some of which have an "immature" appear-  ance. The percentage of lymphocytes in the peripheral blood is decreased. A bone  marrow aspiration and biopsy show the presence of an abnormal chromosome (the  Philadelphia chromosome) in dividing marrow cells.    Each of the drugs used by Arlyn  Foma inhibits the proliferation of  cancer cells in a different way. Dox-  orubicin (adriamycin) is a large nonpolar  molecule synthesized by fungi that interca-  lates between DNA bases, inhibiting replica-  tion and transcription and forming DNA with  single- and double-stranded breaks. Vin-  cristine binds to tubulin and inhibits forma-  tion of the mitotic spindle, thereby prevent-  ing cell division. Cyclophosphamide is an  alkylating agent that damages DNA by cova-  lently attaching alkyl groups to DNA bases.  Methotrexate is an analogue of the vitamin  folate. It inhibits folate-requiring enzymes in  the pathways for synthesis of thymine and  purines, thereby depriving cells of precur-  sors for DNA synthesis.   Ann O'Rexia, who has anorexia nervosa, has continued on an almost  meat-free diet (see Chapters 1, 3, 9, and 11). She now appears emaciated  and pale. Her hemoglobin is 9.7 g/dL (normal = 12—16 g/dL), her hema-  tocrit (volume of packed red cells) is 31% (reference range for women = 36—46%),  and her mean corpuscular hemoglobin (the average amount of hemoglobin per red  cell) is 21 pg/cell (reference range = 26—34 pg/cell). These values indicate an ane-  mia that is microcytic (small red cells) and hypochromic (light in color, indicating  a reduced amount of hemoglobin per red cell). Her serum ferritin (the cellular stor-  age form of iron) was also subnormal. Her plasma level of transferrin (the iron  transport protein in plasma) was greater than normal, but its percent saturation with  iron was below normal. This laboratory profile is consistent with changes that occur  in an iron deficiency state.   Although most cells of an organism contain identical sets of genes, at any given  time only a small number of the total genes in each cell are expressed (that is, gen-  erate a protein or RNA product). The remaining genes are inactive. Organisms gain  a number of advantages by regulating the activity of their genes. For example, both  prokaryotic and eukaryotic cells adapt to changes in their environment by turning  the expression of genes on and off. Because the processes of RNA transcription and  protein synthesis consume a considerable amount of energy, cells conserve fuel by  making proteins only when they are needed.   In addition to regulating gene expression to adapt to environmental changes,  eukaryotic organisms alter expression of their genes during development. As a fer-  tilized egg becomes a multicellular organism, different kinds of proteins are syn-  thesized in varying quantities. In the human, as the child progresses through ado-  lescence and then into adulthood, physical and physiologic changes result from  variations in gene expression and, therefore, of protein synthesis. Even after an  organism has reached the adult stage, regulation of gene expression enables certain  cells to undergo differentiation to assume new functions.    E. coli is a facultative anaerobe,  which means that it can grow in  the presence or absence of oxy-  gen. The switch to oxygen-requiring path-  ways for fuel metabolism is under control of  arc, the aerobic respiration control gene.  When arc is activated, transcription is  increased by 1,000-fold or more for  enzymes in the pathways that ultimately  transfer electrons to oxygen (e.g., proteins  of the respiratory chain, the TCA cycle, and  fatty acid oxidation). In the absence of oxy-  gen (i.e., anaerobic, without air), these pro-  teins are not synthesized, an energy-saving  feature useful for bacteria growing in the  largely anaerobic colon. Most human cells,  in contrast, express constant (constitutive)  levels of respiratory enzymes and die with-  out oxygen.   Fig. 16.1. E. coli cell. In prokaryotes, DNA is  not separated from the rest of the cellular con-  tents by a nuclear envelope; therefore, simulta-  neous transcription and translation occur in  bacteria. Once a small piece of mRNA is syn-  thesized, ribosomes bind to the mRNA, and  translation begins.   Prokaryotes are single-celled organisms and, therefore, require less complex regu-  latory mechanisms than the multicellular eukaryotes (Fig. 16.1). The most exten-  sively studied prokaryote is the bacterium Escherichia coli, an organism that thrives  in the human colon, usually enjoying a symbiotic relationship with its host. Based  on the size of its genome (4 X 10 6 base pairs), E. coli should be capable of making  several thousand proteins. However, under normal growth conditions, they synthe-  size only about 600 to 800 different proteins. Obviously, many genes are inactive,  and only those genes are expressed that generate the proteins required for growth in  that particular environment.   All E. coli cells of the same strain are morphologically similar and contain an  identical circular chromosome (see Fig. 16.1). As in other prokaryotes, DNA is not  complexed with histones, no nuclear envelope separates the genes from the contents  of the cytoplasm, and gene transcripts do not contain introns. In fact, as mRNA is  being synthesized, ribosomes bind and begin to produce proteins, so that transcrip-  tion and translation occur simultaneously. The mRNAs in E. coli have very short  half-lives and are degraded within a few minutes so that an mRNA must be con-  stantly generated from transcription to maintain synthesis of its proteins. Thus, reg-  ulation of transcription, principally at the level of initiation, is sufficient to regulate  the level of proteins within the cell.   The genes encoding proteins are called structural genes. In the bacterial genome,  the structural genes for proteins involved in performing a related function (such as  the enzymes of a biosynthetic pathway) are often grouped sequentially into units  called operons (Fig. 16.2). The genes in an operon are coordinately expressed; that  is, they are either all "turned on" or all "turned off." When an operon is expressed,  all of its genes are transcribed. A single polycistronic mRNA is produced that codes  for all the proteins of the operon. This polycistronic mRNA contains multiple sets  of start and stop codons that allow a number of different proteins to be produced  from this single transcript at the translational level. Transcription of the genes in an  operon is regulated by its promoter, which is located in the operon at the 5 '-end,  upstream from the structural genes.   In bacteria, the principle means of regulating gene transcription is through repres-  sors, which are regulatory proteins that prevent the binding of RNA polymerase to  the promoter and, thus, act on initiation of transcription (Fig. 16.3). In general,   Fig. 16.2. An operon. The structural genes of an operon are transcribed as one long poly-  cistronic mRNA. During translation, different start (AUG) and stop (shown in blue) codons  lead to a number of distinct proteins being produced from this single mRNA.   regulatory mechanisms such as repressors that work through inhibition of gene tran-  scription are referred to as negative control, and mechanisms that work through  stimulation of gene transcription are called positive control.   The repressor is encoded by a regulatory gene (see Fig. 16.3). Although this  gene is considered part of the operon, it is not always located near the remainder  of the operon. Its product, the repressor protein, diffuses to the promoter and  binds to a region of the operon called the operator. The operator is located within  the promoter or near its 3' -end, just upstream from the transcription startpoint.  When a repressor is bound to the operator, the operon is not transcribed because  the repressor blocks the binding of RNA polymerase to the promoter. Two regu-  latory mechanisms work through controlling repressors: induction (an inducer  inactivates the repressor), and repression (a co-repressor is required to activate  the repressor).   Induction involves a small molecule, known as an inducer, which stimulates expres-  sion of the operon by binding to the repressor and changing its conformation so that  it can no longer bind to the operator (Fig. 16.4). The inducer is either a nutrient or  a metabolite of the nutrient. In the presence of the inducer, RNA polymerase can  therefore bind to the promoter and transcribe the operon. The key to this mechanism  is that in the absence of the inducer, the repressor is active, transcription is  repressed, and the genes of the operon are not expressed.   Consider, for example, induction of the lac operon of E. coli by lactose (Fig.  16.5). The enzymes for metabolizing glucose by glycolysis are produced constitu-  tively; that is, they are constantly being made. If the milk sugar lactose is available,  the cells adapt and begin to produce the three additional enzymes required for lac-  tose metabolism, which are encoded by the lac operon. A metabolite of lactose  (allolactose) serves as an inducer, binding to the repressor and inactivating it.  Because the inactive repressor no longer binds to the operator, RNA polymerase can  bind to the promoter and transcribe the structural genes of the lac operon, produc-  ing a polycistronic mRNA that encodes for the three additional proteins. However,  the presence of glucose can prevent activation of the lac operon (see "Stimulation  of RNA polymerase binding," below).   In a regulatory model called repression, the repressor is inactive until a small mol-  ecule called a corepressor (a nutrient or its metabolite) binds to the repressor, acti-  vating it (Fig. 16.6). The repressor-corepressor complex then binds to the operator,  preventing binding of RNA polymerase and gene transcription. Consider, for exam-  ple, the trp operon, which encodes the five enzymes required for the synthesis of the  amino acid tryptophan. When tryptophan is available, E. coli cells save energy by  no longer making these enzymes. Tryptophan is a corepressor that binds to the inac-  tive repressor, causing it to change conformation and bind to the operator, thereby  inhibiting transcription of the operon. Thus, in the repression model, the repressor  is inactive without a corepressor; in the induction model, the repressor is active  unless an inducer is present.   If one of the lac operon enzymes induced by lactose is lactose permease (which  \\ increases lactose entry into the cell), how does lactose initially get into the cell  to induce these enzymes? A small amount of the permease exists even in the  absence of lactose, and a few molecules of lactose enter the cell and are metabolized to  allolactose, which begins the process of inducing the operon. As the amount of the per-  mease increases, more lactose can be transported into the cell.   Fig. 16.3. Regulation of operons by repres-  sors. When the repressor protein is bound to  the operator, RNA polymerase cannot bind,  and transcription therefore does not occur.   Fig. 16.4. An inducible operon. In the absence  of an inducer, the repressor binds to the opera-  tor, preventing the binding of RNA poly-  merase. When the inducer is present, the  inducer binds to the repressor, inactivating it.  The inactive repressor no longer binds to the  operator. Therefore, RNA polymerase can bind  to the promoter region and transcribe the struc-  tural genes.   Fig. 16.5. The protein products of the lac operon. Lactose is a disaccharide that is hydrolyzed  to glucose and galactose by p-galactosidease (the Z gene). Both glucose and galactose can be  oxidized by the cell for energy. The permease (Y gene) enables the cell to take up lactose more  readily. The A gene produces a transacetylase that acetylates p-galactosides. The function of  this acetylation is not clear. The promoter binds RNA polymerase and the operator binds a  repressor protein. Lactose is converted to allolactose, an inducer that binds the repressor pro-  tein and prevents it from binding to the operator. Transcription of the lac operon also requires  activator proteins that are inactive when glucose levels are high.   In addition to regulating transcription by means of repressors that inhibit RNA  polymerase binding to promoters (negative control), bacteria regulate transcrip-  tion by means of activating proteins that bind to the promoter and stimulate the  binding of RNA polymerase (positive control). Transcription of the lac operon,  for example, can be induced by allolactose only if glucose is absent. The pres-  ence or absence of glucose is communicated to the promoter by a regulatory pro-  tein named the cyclic adenosine monophosphate (cAMP) receptor protein (CRP)   Fig. 16.6. A repressible operon. The repressor is inactive until a small molecule, the core-  pressor, binds to it. The repressor-corepressor complex binds to the operator and prevents  transcription.   Fig. 16.7. Catabolite repression of stimulatory proteins. The lac operon is used as an exam-  ple. A. The inducer allolactose (a metabolite of lactose) inactivates the repressor. However,  because of the absence of the required coactivator, cAMP-CRP, no transcription occurs  unless glucose is absent. B. In the absence of glucose, cAMP levels rise. cAMP forms a com-  plex with the cAMP receptor protein (CRP). The binding of the cAMP-CRP complex to a  regulatory region of the operon permits the binding of RNA polymerase to the promoter.  Now the operon is transcribed, and the proteins are produced.   (Fig. 16.7). This regulatory protein is also called a catabolite activator protein  (CAP). A decrease in glucose levels increases levels of the intracellular second  messenger cAMP by a mechanism that is not well understood. cAMP binds to  CRP, and the cAMP-CRP complex binds to a regulatory region of the operon,  stimulating binding of RNA polymerase to the promoter and transcription. When  glucose is present, cAMP levels decrease, CRP assumes an inactive conforma-  tion that does not bind to the operon, and transcription is inhibited. Thus, the  enzymes encoded by the lac operon are not produced if cells have an adequate  supply of glucose, even if lactose is present at very high levels.   Nutrient regulation of gene expres-  \\ ) sion may occur through the nutri-  *S ent itself, or through a metabolite  of the nutrient synthesized inside the cell.  Sometimes both the nutrient and its  metabolite are grouped into the term  "catabolite," as in "catabolite repression."   E. coli has only one RNA polymerase. Sigma factors bind to this RNA poly-  merase, stimulating its binding to certain sets of promoters, thus simultaneously  activating transcription of several operons. The standard sigma factor in E. coli is   a , a protein with a molecular weight of 70,000 daltons (see Chapter 14). Other  sigma factors also exist. For example, a 32 helps RNA polymerase recognize pro-  moters for the different operons that encode the "heat shock" proteins. Thus,  increased transcription of the genes for heat shock proteins, which prevent protein  denaturation at high temperatures, occurs in response to elevated temperatures.   Some operons are regulated by a process that interrupts (attenuates) transcription  after it has been initiated (Fig. 16.8). For example, high levels of tryptophan atten-  uate transcription of the E. coli trp operon, as well as repress its transcription. As  mRNA is being transcribed from the trp operon, ribosomes bind and rapidly begin  to translate the transcript. Near the 5 '-end of the transcript, there are a number of  codons for tryptophan. Initially, high levels of tryptophan in the cell result in high  levels of trp-tRNA trp and rapid translation of the transcript. However, rapid transla-  tion generates a hairpin loop in the mRNA that serves as a termination signal for  RNA polymerase, and transcription terminates. Conversely, when tryptophan levels  are low, levels of trp-tRNA trp are low, and ribosomes stall at codons for tryptophan.  A different hairpin loop forms in the mRNA that does not terminate transcription,  and the complete mRNA is transcribed.   Fig. 16.8. Attenuation of the trp operon. Sequences 2, 3, and 4 in the mRNA transcript can form base pairs (2 with 3 or 3 with 4) that generate  hairpin loops. When tryptophan levels are low, the ribosome stalls at the adjacent trp codons in sequence 1, the 2-3 loop forms, and transcrip-  tion continues. When tryptophan levels are high, translation is rapid and the ribosome blocks formation of the 2-3 loop. Under these conditions,  the 3^ loop forms and terminates transcription.   The tryptophan, histidine, leucine, phenylalanine, and threonine operons are reg-  ulated by attenuation. Repressors and activators also act on the promoters of some  of these operons, allowing the levels of these amino acids to be very carefully and  rapidly regulated.   Multicellular eukaryotes are much more complex than single-celled prokaryotes. As  the human embryo develops into a multicellular organism, different sets of genes  are turned on, and different groups of proteins are produced, resulting in differenti-  ation into morphologically distinct cell types able to perform different functions.  Even beyond the reproductive age, certain cells within the organism continue to dif-  ferentiate, such as those that produce antibodies in response to an infection, renew  the population of red blood cells, and replace digestive cells that have been  sloughed into the intestinal lumen. All of these physiologic changes are dictated by  complex alterations in gene expression.   Differences between eukaryotic and prokaryotic cells result in different mecha-  nisms for regulating gene expression. DNA in eukaryotes is organized into the  nucleosomes of chromatin, and genes must be in an active structure to be expressed  in a cell. Furthermore, operons are not present in eukaryotes, and the genes encod-  ing proteins that function together are usually located on different chromosomes.  Thus, each gene needs its own promoter. In addition, the processes of transcription  and translation are separated in eukaryotes by intracellular compartmentation  (nucleus and cytosol, or endoplasmic reticulum [ER]) and by time (eukaryotic  hnRNA must be processed and translocated out of the nucleus before it is trans-  lated). Thus, regulation of eukaryotic gene expression occurs at multiple levels:   • DNA and the chromosome, including chromosome remodeling and gene  rearrangement   • Transcription, primarily through transcription factors affecting binding of RNA  polymerase   Once a gene is activated through chromatin remodeling, the major mechanism of  regulating expression affects initiation of transcription at the promoter.   Once a haploid sperm and egg combine to form a diploid cell, the number of genes  in human cells remains approximately the same. As cells differentiate, different  genes are available for transcription. A typical nucleus contains chromatin that is  condensed (heterochromatin) and chromatin that is diffuse (euchromatin)(Fig. 16.9)  (see Chapter 12). The genes in heterochromatin are inactive, whereas those in  euchromatin produce mRNA. Long-term changes in the activity of genes occur dur-  ing development as chromatin goes from a diffuse to a condensed state or vice  versa.   The cellular genome is packaged together with histones into nucleosomes, and  initiation of transcription is prevented if the promoter region is part of a nucleo-  some. Thus, activation of a gene for transcription requires changes in the state of the  chromatin, called chromatin remodeling. The availability of genes for transcription   ^The globin chains of hemoglobin  provide an example of functionally  related proteins that are on differ-  ent chromosomes. The gene for the a-globin  chain is on chromosome 16, whereas the  gene for the p-globin chain is on chromo-  some 11. As a consequence of this spatial  separation, each gene must have its own  promoter. This situation is different from  that of bacteria, in which genes encoding  proteins that function together are often  sequentially arranged in operons controlled  by a single promoter.   Fig. 16.9. Inactivation of genes during devel-  opment of red blood cells. Diffuse chromatin  (euchromatin) is active in RNA synthesis.  Condensed chromatin (heterochromatin) is  inactive. As red blood cell precursors mature,  their chromatin becomes more condensed.  Eventually, the nucleus is extruded.   Fig. 16.10. Histone acetylation. Abbrevia-  tions: HAC, histone acetylase; HDAC, histone  deacetylase.   also can be affected in certain cells, or under certain circumstances, by gene  rearrangements, amplification, or deletion. For example, during lymphocyte matu-  ration, genes are rearranged to produce a variety of different antibodies.   The remodeling of chromatin generally refers to displacement of the nucleosome from  specific DNA sequences so that transcription of the genes in that sequence can be ini-  tiated. This occurs through two different mechanisms. The first mechanism is by an  adenosine triphosphate (ATP)-driven chromatin remodeling complex, which uses  energy from ATP hydrolysis to unwind certain sections of DNA from the nucleosome  core. The second mechanism is by covalent modification of the histone tails through  acetylation (Fig. 16.10). Histone acetyltransferases (HAT) transfer an acetyl group  from acetyl CoA to lysine residues in the tail (the amino terminal ends of histones  H2A, H2B, H3, and H4). This reaction removes a positive charge from the e-amino  group of the lysine, thereby reducing the electrostatic interactions between the histones  and the negatively charged DNA, making it easier for DNA to unwind from the his-  tones. The acetyl groups can be removed by histone deacetylases (HDAC). Each his-  tone has a number of lysine residues that may be acetylated and, through a complex  mixing of acetylated and nonacetylated sites, different segments of DNA can be freed  from the nucleosome. A number of transcription factors and co-activators also contain  histone acetylase activity, which facilitates the binding of these factors to the DNA and  simultaneous activation of the gene and initiation of its transcription.   Cytosine residues in DNA can be methylated to produce 5-methylcytosine. The methy-  lated cytosines are located in GC-rich sequences (called GC-islands), which are often  near or in the promoter region of a gene. In certain instances, genes that are methylated  are less readily transcribed than those that are not methylated. For example, globin  genes are more extensively methylated in nonerythroid cells (cells which are not a part  of the erythroid, or red blood cell, lineage) than in the cells in which these genes are  expressed (such as the erythroblast and reticulocyte). Methylation is a mechanism for  regulating gene expression during differentiation, particularly in fetal development.   Methylation has been implicated in genomic imprinting, a process occurring during the formation of the eggs or sperm that  blocks the expression of the gene in the fertilized egg. Males methylate a different set of genes than females. This sex-depen-  dant differential methylation has been most extensively studied in two human disorders, Prader-Willi syndrome and Angelman  syndrome. Both syndromes, which have very different symptoms, result from deletions of the same region of chromosome 15 (a  microdeletion of less than 5 megabases in size). If the deletion is inherited from the father, Prader-Willi syndrome is seen in the child; if  the deletion is inherited from the mother, Angelman's syndrome is observed. A disease occurs when a gene that is in the deleted region  of one chromosome is methylated on the other chromosome. The mother methylates different genes than the father, so different genes  are expressed depending on which parent transmitted the intact chromosome. For example, if genes 1, 2, and 3 are deleted in the pater-  nal chromosome in the Prader-Willi syndrome, and gene 2 is methylated in the maternal chromosome, only genes 1 and 3 will be  expressed. If in the Angelman syndrome, genes 1, 2 and 3 are deleted on the maternal chromosome and gene 1 is methylated on the pater-  nal chromosome, only genes 2 and 3 would be expressed.   Segments of DNA can move from one location to another in the genome, associating  with each other in various ways so that different proteins are produced (Fig. 16.11).  The most thoroughly studied example of gene rearrangement occurs in cells that  produce antibodies. Antibodies contain two light chains and two heavy chains, each  of which contains both a variable and a constant region (see Chapter 7, section V.B,  Fig. 7.19). Cells called B cells make antibodies. In the precursors of B cells, hun-  dreds of V H sequences, approximately 20 D H sequences, and approximately 6 J H  sequences are located in clusters within a long region of the chromosome. During  the production of the immature B cells, a series of recombinational events occur that  join one V H , one D H , and one J H sequence into a single exon. This now encodes the  variable region of the heavy chain of the antibody. Given the large number of imma-  ture B cells that are produced, virtually every recombinational possibility occurs,  such that all VDJ combinations are represented within this cell population. Later in  development, during differentiation of mature B cells, recombinational events join  a VDJ sequence to one of the nine heavy chain elements. When the immune system  encounters an antigen, the one immature B cell that can bind to that antigen  (because of its unique manner in forming the VDJ exon) is stimulated to proliferate  (clonal expansion) and to produce antibodies against the antigen.    Although rearrangements of short  DNA sequences are difficult to  detect, microscopists have  observed major rearrangements for many  years. Such major rearrangements, known  as translocations, can be observed in  metaphase chromosomes under the micro-  scope.   Mannie Weitzels has such a transloca-  tion, known as the Philadelphia chromo-  some because it was first observed in that  city. The Philadelphia chromosome is pro-  duced by a balanced exchange between  chromosomes 9 and 22.   Gene amplification is not the usual physiologic means of regulating gene expression  in normal cells, but it does occur in response to certain stimuli if the cell can obtain  a growth advantage by producing large amounts of a protein. In gene amplification,  certain regions of a chromosome undergo repeated cycles of DNA replication. The  newly synthesized DNA is excised and forms small, unstable chromosomes called  "double minutes." The double minutes integrate into other chromosomes through-  out the genome, thereby amplifying the gene in the process. Normally, gene ampli-  fication occurs through errors during DNA replication and cell division and, if the  environmental conditions are correct, cells containing amplified genes may have a  growth advantage over those without the amplification.   In fragile X syndrome, a GCC triplet is amplified on the 5'-side of a gene (FMR-1)  associated with the disease. This gene is located on the X chromosome. The  disease is named for the finding that in the absence of folic acid (which impairs  nucleotide production and hence, the replication of DNA) the X chromosome develops  single and double-stranded breaks in its DNA. These were termed fragile sites. It was  subsequently determined that the FMR-1 gene was located in one of these fragile sites.  A normal person has about 30 copies of the GCC triplet, but in affected individuals, thou-  sands of copies can be present. This syndrome, which is a common form of inherited  mental retardation, affects about 1 in 1,250 males and 1 in 2,000 females.   Arlyn Foma has been treated with a  combination of drugs that includes  methotrexate, a drug that inhibits  cell proliferation by inhibiting dihydrofolate  reductase. Dihydrofolate reductase reduces  dihydrofolate to tetrahydrofolate, a cofactor  required for synthesis of thymine and purine  nucleotides. Because Arlyn Foma has not  been responding well, the possibility that he  has become resistant to methotrexate was  considered. Sometimes, rapidly dividing  cancer cells treated with methotrexate  amplify the gene for dihydrofolate reduc-  tase, producing hundreds of copies in the  genome. These cells generate large  amounts of difhydrofolate reductase, and  normal doses of methotrexate are no longer  adequate. Gene amplification is one of the  mechanisms by which patients become  resistant to a drug.   Fig. 16.11. Rearrangement of DNA. The heavy chain gene from which lymphocytes produce immunoglobulins is generated by combining spe-  cific segments from among a large number of potential sequences in the DNA of precursor cells. The variable and constant regions of  immunoglobulins (antibodies) are described in Chapter 7.   The terminology used to describe  components of gene-specific regu-  lation varies somewhat, depending  on the system. For example, in the original  terminology, DNA regulatory sequences  called enhancers bound transactivators,  which bound coactivators. Similarly,  silencers bound corepressors. Hormones  bound to hormone receptors, which bound  to hormone response elements in DNA.  Although these terms are still used, they are  often replaced with more general terms such  as "DNA regulatory sequences" and "spe-  cific transcription factors," in recognition of  the fact that many transcription factors acti-  vate one gene while inhibiting another or  that a specific transcription factor may be  changed from a repressor to an activator by  phosphorylation.   Histone acetylase activity has been  associated with a number of tran-  scription factors and co-activators.  The proteins ACTR (activator of the thyroid  and retinoic acid receptor) and SRC-1  (steroid receptor co-activator) are involved  in activation of transcription by several lig-  and-bound nuclear receptors, and both con-  tain histone acetylase activity. TAF250, a  component of TFIID, also contains histone  acetylase activity, as does the co-activator  p300/CBP (CREB binding protein), which  interacts with the transcription factor CREB.   With a few exceptions, the deletion of genetic material is likewise not a normal  means of controlling transcription, although such deletions do result in disease.  Gene deletions can occur through errors in DNA replication and cell division and  are usually only noticed if a disease results. For example, various types of cancers  result from the loss of a good copy of a tumor suppressor gene, leaving the cell with  a mutated copy of the gene (see Chapter 18).   The transcription of active genes is regulated by controlling assembly of the basal  transcription complex containing RNA polymerase and its binding to the TATA box  of the promoter (see Chapter 14). The basal transcription complex contains the  TATA binding protein (TBP, a component of TFIID) and other proteins called gen-  eral (basal) transcription factors (such as TFIIA, etc.) that form a complex with  RNA polymerase II. Additional transcription factors that are ubiquitous to all pro-  moters bind upstream at various sites in the promoter region. They increase the fre-  quency of transcription and are required for a promoter to function at an adequate  level. Genes that are regulated solely by these consensus elements in the promoter  region are said to be constitutively expressed.   The control region of a gene also contains DNA regulatory sequences that are spe-  cific for that gene and may increase its transcription 1,000-fold or more (Fig. 16.12).  Gene-specific transcription factors (also called transactivators or activators) bind to  these regulatory sequences and interact with a mediator protein, such as a coactiva-  tor. By forming a loop in the DNA, coactivators interact with the basal transcription  complex and can activate its assembly at the initiation site on the promoter. These  DNA regulatory sequences might be some distance from the promoter and may be  either upstream or downstream of the initiation site.   The regulatory proteins that bind directly to DNA sequences are most often called  transcription factors or gene-specific transcription factors (if it is necessary to distin-  guish them from the general transcription factors of the basal transcription complex).  They also can be called activators (or transactivators), inducers, repressors, or  nuclear receptors. In addition to their DNA-binding domain, these proteins usually  have a domain that binds to mediator proteins (coactivators, corepressors, or TATA  binding protein associated factors-TAFs). Coactivators, corepressors, and other  mediator proteins do not bind directly to DNA but generally bind to components of  the basal transcription complex and mediate its assembly at the promoter. They can  be specific for a given gene transcription factor or general and bind many different  gene-specific transcription factors. Certain co-activators have histone acetylase  activity, and certain corepressors have histone deacetylase activity. When the appro-  priate interactions between the transactivators, coactivators, and the basal transcrip-  tion complex occur, the rate of transcription of the gene is increased (induction).   Some regulatory DNA binding proteins inhibit (repress) transcription and may be  called repressors. Repression can occur in a number of ways. A repressor bound to its  specific DNA sequence may inhibit binding of an activator to its regulatory sequence.  Alternately, the repressor may bind a corepressor that inhibits binding of a coactivator  to the basal transcription complex. The repressor may directly bind a component of the  basal transcription complex. Some steroid hormone receptors that are transcription fac-  tors bind either coactivators or corepressors, depending on whether the receptor con-  tains bound hormone. Furthermore, a particular transcription factor may induce tran-  scription when bound to the regulatory sequence of one gene and may repress  transcription when bound to the regulatory sequence of another gene.   Fig. 16.12. The gene regulatory control region consists of the promoter region and additional gene regulatory sequences, including enhancers  and hormone response elements (shown in blue). Gene regulatory proteins that bind directly to DNA (regulatory DNA binding proteins) are usu-  ally called specific transcription factors or transactivators; they may be either activators or repressors of the transcription of specific genes. The  specific transcription factors bind mediator proteins (co-activators or corepressors) that interact with the general transcription factors of the basal  transcription complex. The basal transcription complex contains RNA polymerase and associated general transcription factors (TFII factors) and  binds to the TATA box of the promoter, initiating gene transcription.   In the human, steroid hormones and other lipophilic hormones activate or inhibit tran-  scription of specific genes through binding to nuclear receptors that are gene-specific  transcription factors (Fig. 16. 13 A). The nuclear receptors bind to DNA regulatory  sequences called hormone response elements and induce or repress transcription of  target genes. The receptors contain a hormone (ligand) binding domain, a DNA bind-  ing domain, and a dimerization domain that permits two receptor molecules to bind to  each other, forming characteristic homodimers or heterodimers. A transactivation  domain binds the coactivator proteins that interact with the basal transcription com-  plex. The receptors also contain a nuclear localization signal domain that directs them  to the nucleus at various times after they are synthesized.   Various members of the steroid hormone/thyroid hormone receptor family work  in different ways. The glucocorticoid receptor, which binds the steroid hormone  Cortisol, resides principally in the cytosol bound to heat shock proteins. As Cortisol  binds, the receptor dissociates from the heat shock proteins, exposing the nuclear  localization signal (see Fig. 16.13B). The receptors form homodimers that are  translocated to the nucleus, where they bind to the hormone response elements (glu-  cocorticoid response elements-GRE) in the DNA control region of certain genes.  The transactivation domains of the receptor dimers bind mediator proteins, thereby  activating transcription of specific genes and inhibiting transcription of others.    In a condition known as testicular  feminization, patients produce  androgens (the male sex steroids),  but target cells fail to respond to these  steroid hormones because they lack the  appropriate intracellular transcription factor  receptors. Therefore, the transcription of the  genes responsible for masculinization is not  activated. A patient with this condition has  an XY (male) karyotype (set of chromo-  somes) but looks like a female. External  male genitalia do not develop, but testes are  present, usually in the inguinal region.   Fig. 16.13. Steroid hormone receptors. A. Domains of the steroid hormone receptor. The transactivation domain (TAD) binds coactivators; DNA-  binding domain (DBD) binds to hormone response element in DNA; ligand-binding domain (LBD) binds hormone; NLS is the nuclear local-  ization signal; the dimerization sites are the portions of the protein involved in forming a dimer. The inhibitor binding site binds heat shock pro-  teins and masks the nuclear localization signal. B. Transcriptional regulation by steroid hormone receptors. Additional abbreviations: HSP, heat  shock proteins; GRE, glucocorticoid response element; GIZ, glucocorticoid receptor.   Other members of the steroid hormone/thyroid hormone family of receptors are  also gene-specific transactivation factors but generally form heterodimers that con-  stitutively bind to a DNA regulatory sequence in the absence of their hormone lig-  and and repress gene transcription (Fig. 16.14). For example, the thyroid hormone  receptor forms a heterodimer with the retinoid X receptor (RXR) that binds to thy-  roid hormone response elements and to corepressors (including one with deacety-  lase activity), thereby inhibiting expression of certain genes. When thyroid hormone  binds, the receptor dimer changes conformation, and the transactivation domain  binds coactivators, thereby initiating transcription of the genes.   The RXR receptor, which binds the retinoid 9-cis retinoic acid, can form het-  erodimers with at least eight other nuclear receptors. Each heterodimer has a dif-  ferent DNA binding specificity. This allows the RXR to participate in the regulation  of a wide variety of genes, and to regulate gene expression differently, depending  on the availability of other active receptors.   Several unique structural motifs have been characterized for specific transcription  factors. Each of these proteins has a distinct recognition site (DNA binding domain)  that binds to the bases of a specific sequence of nucleotides in DNA. Four of the  best-characterized structural motifs are zinc fingers, b-zip proteins (including  leucine zippers), helix- turn-helix, and helix-loop-helix.   To regulate gene transcription, two estrogen receptors combine to form a  \\ j dimer that binds to a palindrome in the promoter region of certain genes (see  *r Figs. 16.12 and 16.13). A palindrome is a sequence of bases that is identical on  the antiparallel strand when read in the opposite direction. For example, the sequence  ATCGCGAT base-pairs to form the sequence TAGCGCTA, which when read in the oppo-  site direction is ATCGCGAG. Each estrogen receptor is approximately 73 amino acids  long and contains two zinc fingers. Each zinc is chelated to two cysteines in an a -helix  and two cysteines in a (3-sheet region. The position of the nucleotide recognition  sequence in an a-helix keeps the sequence in a relatively rigid conformation as it fits into  the major groove of DNA. The zinc finger that lies closest to the carboxyl terminal is  involved in dimerization with the second estrogen receptor, thus inverting the nucleotide  recognition sequence to match the other half of the palindrome. The dimer-palindrome  requirement enormously enhances the specificity of binding, and, consequently, only  certain genes are affected.   Zinc finger motifs (commonly found in the DNA binding domain of steroid hor-  mone receptors) contain a bound zinc chelated at four positions with either histidine  or cysteine in a sequence of approximately 20 amino acids (Fig. 16.15). The result  is a relatively small, tight, autonomously-folded domain. The zinc is required to  maintain the tertiary structure of this domain. Eukaryotic transcription factors gen-  erally have two to six zinc finger motifs that function independently. At least one of  the zinc fingers forms ana-helix containing a nucleotide recognition signal, a  sequence of amino acids that specifically fits into the major groove of DNA (Fig.  16.16A).   Leucine zippers also function as dimers to regulate gene transcription (see Fig.  16.16B). The leucine zipper motif is an a-helix of 30 to 40 amino acid residues that  contains a leucine every seven amino acids, positioned so that they align on the same  side of the helix. Two helices dimerize so that the leucines of one helix align with the   Fig. 16.14. Activity of the thyroid hormone  receptor-retinoid receptor dimer (TR-RXR) in  the presence and absence of thyroid hormone  (T 3 ). Abbrev: HAC, histone acetylase; HDAC,  histone deacetylase.   A wide variety of transcription factors contain the zinc finger motif, including  the steroid hormone receptors, such as the estrogen and the glucocorticoid  receptor. Other transcription factors that contain zinc finger motifs include Sp1  and polymerase III transcription factor TFIIIA (part of the basal transcription complex),  which has nine zinc finger motifs.   Leucine zipper transcription factors function as homodimers or heterodimers.  For example, AP1 is a heterodimer whose subunits are encoded by the genes  fos and jun.   Fig. 16.15. Zinc fingers of the estrogen receptor. In each of the two zinc fingers, one zinc ion is coordinated with four cysteine residues, shown  in blue. The region labeled a-helix with NRS forms an a-helix that contains a nucleotide recognition signal (NRS). This signal consists of a  sequence of amino acid residues that bind to a specific base sequence in the major groove of DNA. Regions enclosed in boxed arrows partici-  pate in rigid helices.    Fig. 16.16. Interaction of DNA binding proteins with DNA. (A) Zinc finger motifs consist of an a-helix and a p sheet in which 4 cysteine and/or  histidine residues coordinately bind a zinc ion. The nucleotide recognition signal (contained within the a-helix) of at least one zinc finger binds  to a specific sequence of bases in the major groove of DNA. (B) Leucine zipper motifs form from two distinct polypeptide chains. Each polypep-  tide contains a helical region in which leucine residues are exposed on one side. These leucines form hydrophobic interactions with each other,  causing dimerization. The remaining helices interact with DNA. (C) Helix-turn-helix motifs contain three (or sometimes four) helical regions,  one of which binds to DNA, whereas the others lie on top and stabilize the interaction. (D) Helix-loop-helix motifs contain helical regions that  bind to DNA like leucine zippers. However, their dimerization domains consist of two helices, each of which is connected to its DNA binding  helix by a loop.   The helix-turn-helix motif is found  Kj J in homeodomain proteins (pro-  *S teins that play critical roles in the  regulation of gene expression during devel-  opment).   motif are involved in cellular differ-  entiation (such as myogenin in skeletal  muscle, neurogenin in neurogenesis and  SCL/tal-1 in hematopoeisis, blood cell devel-  opment).   other helix through hydrophobic interactions to form a coiled coil. The portions of the  dimer adjacent to the zipper "grip" the DNA through basic amino acid residues (argi-  nine and lysine) that bind to the negatively charged phosphate groups. This DNA bind-  ing portion of the molecule also contains a nucleotide recognition signal.   In the helix-turn-helix motif, one helix fits into the major groove of DNA, mak-  ing most of the DNA binding contacts (see Fig. 16.16C). It is joined to a segment  containing two additional helices that lie across the DNA binding helix at right  angles. Thus, a very stable structure is obtained without dimerization.   Helix-loop-helix transcription factors are a fourth structural type of DNA bind-  ing protein (see Fig. 16.16D). They also function as dimers that fit around and grip  DNA in a manner geometrically similar to leucine zipper proteins. The dimerization  region consists of a portion of the DNA-gripping helix and a loop to another helix.  Like leucine zippers, helix-loop-helix factors can function as either hetero or  homodimers. These factors also contain regions of basic amino acids near the amino  terminus and are also called basic helix-loop-helix (bHLH) proteins.   The activity of gene-specific transcription factors is regulated in a number of different  ways. Because transcription factors need to interact with a variety of coactivators to  stimulate transcription, the availability of coactivators or other mediator proteins is crit-  ical for transcription factor function. If a cell upregulates or downregulates its synthe-  sis of coactivators, the rate of transcription can also be increased or decreased. Tran-  scription factor activity can be modulated by changes in the amount of transcription  factor synthesized (see section 5, "Multiple regulators of promoters"), by binding a  stimulatory or inhibitory ligand (such as steroid hormone binding to the steroid hor-  mone receptors), and by stimulation of nuclear entry (illustrated by the glucocorticoid  receptor). The ability of a transcription factor to influence the transcription of a gene is  also augmented or antagonized by the presence of other transcription factors. For  example, the thyroid hormone receptor is critically dependent on the concentration of  the retinoid receptor to provide a dimer partner. Another example is provided by the  phosphoenol pyruvate (PEP) carboxykinase gene, which is induced or repressed by a  variety of hormone-activated transcription factors (see section 5). Frequently, tran-  scription factor activity is regulated through phosphorylation.   Growth factors, cytokines, polypeptide hormones, and a number of other signal  molecules regulate gene transcription through phosphorylation of specific tran-  scription factors by receptor kinases.   For example, STAT proteins are transcription factors phosphorylated by JAK-  STAT receptors, and SMAD proteins are transcription factors phosphorylated by  serine-threonine kinase receptors such as the transforming growth factor-P(TGF-(3)  receptor (see Chapter 11).   Nonreceptor kinases, such as protein kinase A, also regulate transcription factors  through phosphorylation. Many hormones generate the second messenger cAMP,  which activates protein kinase A. Activated protein kinase A enters the nucleus and  phosphorylates the transcription factor CREB (cAMP response element binding  protein). CREB is constitutively bound to the DNA response element CRE (cAMP  response element) and is activated by phosphorylation. Other hormone signaling  pathways, such as the MAP kinase pathway, also phosphorylate CREB (as well as  many other transcription factors).   The same transcription factor inducer can activate transcription of many different  genes if the genes each contain a common response element. Furthermore, a single  inducer can activate sets of genes in an orderly, programmed manner (Fig. 16.17).  The inducer initially activates one set of genes. One of the protein products of this  set of genes can then act as a specific transcription factor for another set of genes.  If this process is repeated, the net result is that one inducer can set off a series of  events that result in the activation of many different sets of genes.   Fig. 16.17. Activation of sets of genes by a single inducer. Each gene in a set has a common  DNA regulatory element, so one regulatory protein can activate all the genes in the set. In the  example shown, the first regulatory protein stimulates the transcription of genes A and B,  which have a common DNA regulatory sequence in their control regions. The protein prod-  uct of gene B is itself a transcriptional activator, which in turn stimulates the transcription of  genes E, F, and G, which likewise contain common response elements.   Interferons, cytokines produced by  cells that have been infected with a  virus, bind to the JAK-STAT family  of cell surface receptors. When an interferon  binds, JAK (a receptor-associated tyrosine  kinase) phosphorylates STAT transcription  factors bound to the receptors (see Chap.  11). The phosphorylated STAT proteins are  released, dimerize, enter the nucleus, and  bind to specific gene regulatory sequences.  Different combinations of phosphorylated  STAT proteins bind to different sequences  and activate transcription of a different set of  genes. One of the genes activated by inter-  feron produces the oligonucleotide 2'-5'-  oligo(A), which is an activator of a ribonu-  clease. This RNase degrades mRNA, thus  inhibiting synthesis of the viral proteins  required for its replication.   In addition to antiviral effects, interferons  have antitumor effects. The mechanisms of  the antitumor effects are not well under-  stood, but are probably likewise related to  stimulation of specific gene expression by  STAT proteins. Interferon-a, produced by  recombinant DNA technology, has been  used to treat patients such as Arlyn Foma  who have certain types of nodular lym-  phomas and patients, such as Mannie  Weitzels, who have chronic myelogenous  leukemia.   ^An example of a transcriptional  cascade of gene activation is  observed during adipocyte (fat cell)  differentiation. Fibroblast-like cells can be  induced to form adipocytes by the addition  of dexamethasome (a steroid hormone) and  insulin to the cells. These factors induce the  transient expression of two similar transcrip-  tion factors named C/EPBp and C/EPB7. The  names stand for CCAAT enhancer binding  protein, and (3 and 7 are two forms of these  factors which recognize CCAAT sequences in  DNA. The C/EPB transcription factors then  induce the synthesis of yet another tran-  scription factor, named the peroxisome pro-  liferator-activated receptor 7 (PPAR7), which  forms heterodimers with RXR to regulate the  expression of two more transcription fac-  tors, C/EPB a and STAT5. The combination of  PPAR7, STAT5, and C/EPBa then leads to the  expression of adipocyte-specific genes.   The enzyme PEP carboxykinase  (PEPCK) is required for the liver to  produce glucose from amino acids  and lactate. Ann O'Rexia, who has an eating  disorder, needs to maintain a certain blood  glucose level to keep her brain functioning  normally. When her blood glucose levels  drop, Cortisol (a glucocorticoid) and  glucagon (a polypeptide hormone) are  released. In the liver, glucagon increases  intracellular cAMP levels, resulting in activa-  tion of protein kinase A and subsequent  phosphorylation of CREB. Phosphorylated  CREB binds to its response element in DNA,  as does the Cortisol receptor. Both transcrip-  tion factors enhance transcription of the  PEPCK gene (see Figure 16.17). Insulin,  which is released when blood glucose levels  rise after a meal, can inhibit expression of  this gene.   Fig. 16.18. A simplified view of the regula-  tory region of the PEPCK gene. Boxes repre-  sent various response elements in the 5 '-flank-  ing region of the gene. Not all elements are  labeled. Regulatory proteins bind to these  DNA elements and stimulate or inhibit the  transcription of the gene. This gene encodes  the enzyme phosphoenolpyruvate carboxyki-  nase (PEPCK), which catalyzes a reaction of  gluconeogenesis (the pathway for production  of glucose) in the liver. Synthesis of the  enzyme is stimulated by glucagon (by a  cAMP-mediated process), by glucocorticoids,  and by thyroid hormone. Synthesis of PEPCK  is inhibited by insulin. CRE = cAMP response  element; TRE = thyroid hormone response  element; GRE = glucocorticoid response ele-  ment; IRE = insulin response element.    An individual gene contains many different response elements and enhancers,  and genes that encode different protein products contain different combinations  of response elements and enhancers. Thus, each gene does not have a single,  unique protein that regulates its transcription. Rather, as different proteins are  stimulated to bind to their specific response elements and enhancers in a given  gene, they act cooperatively to regulate expression of that gene (Fig. 16.18).  Overall, a relatively small number of response elements and enhancers and a rel-  atively small number of regulatory proteins generate a wide variety of responses  from different genes.   After the gene is transcribed (i.e., posttranscription), regulation can occur during  processing of the RNA transcript (hnRNA) into the mature mRNA. The use of alter-  native splice sites or sites for addition of the poly(A) tail (polyadenylation sites) can  result in the production of different mRNAs from a single hnRNA and, conse-  quently, in the production of different proteins from a single gene.   Processing of the primary transcript involves the addition of a cap to the 5 '-end,  removal of introns, and polyadenylation (the addition of a poly (A) tail to the 3' -end)  to produce the mature mRNA (see Chapter 14). In certain instances, the use of alter-  native splicing and polyadenylation sites causes different proteins to be produced  from the same gene (Fig. 16.19). For example, genes that code for antibodies are  regulated by alterations in the splicing and polyadenylation sites, in addition to  undergoing gene rearrangement (Fig. 16.20). At an early stage of maturation, pre-B  lymphocytes produce IgM antibodies that are bound to the cell membrane. Later, a  shorter protein (IgD) is produced that no longer binds to the cell membrane, but  rather is secreted from the cell.   In some instances, RNA is "edited" after transcription. Although the sequence of the  gene and the primary transcript (hnRNA) are the same, bases are altered or  nucleotides are added or deleted after the transcript is synthesized so that the mature  mRNA differs in different tissues (Fig. 16.21).   E. Regulation at the Level of Translation and the  Stability of mRNA   In eukaryotes, regulation of gene transcription at the level of translation usually  involves the initiation of protein synthesis by elFs (eukaryotic initiation factors),  which are regulated through mechanisms involving phosphorylation (see Chap-  ter 15, section V.B.). For example, heme regulates translation of globin mRNA  in reticulocytes by controlling the phosphorylation of eIF2 (Fig. 16.22). In retic-  ulocytes (red blood cell precursors), globin is produced when heme levels in the  cell are high but not when they are low. Because reticulocytes lack nuclei, glo-  bin synthesis must be regulated at the level of translation rather than transcrip-  tion. Heme acts by preventing phosphorylation of eIF2 by a specific kinase  (heme kinase) that is inactive when heme is bound. Thus, when heme levels are  high, eIF2 is not phosphorylated and is active, resulting in globin synthesis. Sim-  ilarly, in other cells, conditions such as starvation, heat shock, or viral infections   Fig. 16.19. Alternative splicing of the calcitonin gene produces an mRNA for calcitonin in thyroid cells and an mRNA for CGRP in neurons. In  thyroid cells, the pre-mRNA from the calcitonin gene is processed to form an mRNA that codes for calcitonin. Cleavage occurs at poly(A) site  1, and splicing occurs along the blue dashed lines. In the brain, the pre-mRNA of this gene undergoes alternative splicing and polyadenylation  to produce calcitonin gene-related protein (CGRP). Cleavage occurs at poly(A) site 2, and splicing occurs along the black dashed lines. CGRP  is involved in the sensation of taste.   may result in activation of a specific kinase that phosphorylates eIF2 to an inac-  tive form. Another example is provided by insulin, which stimulates general pro-  tein synthesis by activating the phosphorylation of an inhibitor of eIF4E, called  4E-BP. When 4E-BP is phosphorylated, it dissociates, leaving eIF4E in the active  form.   A different mechanism for regulation of translation is illustrated by iron reg-  ulation of ferritin synthesis (Fig. 16.23). Ferritin, the protein involved in the stor-  age of iron within cells, is synthesized when iron levels increase. The mRNA for  ferritin has an iron response element (IRE), consisting of a hairpin loop near its  5' -end, which can bind a regulatory protein called the iron response element  binding protein (IRE-BP). When IRE-BP does not contain bound iron, it binds to  the IRE and prevents initiation of translation. When iron levels increase and IRE-  BP binds iron, it changes to a conformation that can no longer bind to the IRE  on the ferritin mRNA. Therefore, the mRNA is translated and ferritin is pro-  duced.   q q In addition to stimulating degrada-  [ /'Va ) tion of mRNA, interferon also  ™ causes elF2 to become phosphory-  lated and inactive. This is a second mecha-  nism by which interferon prevents synthesis  of viral proteins.   Stability of an mRNA also plays a role in regulating gene expression, because  mRNAs with long half-lives can generate a greater amount of protein than can those  with shorter half-lives. The mRNA of eukaryotes is relatively stable (with half-lives  measured in hours to days), although it can be degraded by nucleases in the nucleus  or cytoplasm before it is translated. To prevent degradation during transport from  the nucleus to the cytoplasm, mRNA is bound to proteins that help to prevent its  degradation. Sequences at the 3 '-end of the mRNA appear to be involved in deter-  mining its half-life and binding proteins that prevent degradation. One of these is  the poly (A) tail, which protects the mRNA from attack by nucleases. As mRNA  ages, its poly (A) tail becomes shorter.   An example of the role of mRNA degradation in control of translation is pro-  vided by the transferrin receptor mRNA (Fig. 16.24) The transferrin receptor is a   Fig. 16.20. Production of a membrane-bound antibody (IgM) and a smaller secreted antibody (IgD) from the same gene. Initially, the lympho-  cytes produce a long transcript that is cleaved and polyadenylated after the second stop codon. The intron that contains the first stop codon is  removed by splicing between the 5'- and 3 '-splice sites. Therefore, translation ends at the second stop codon, and the protein contains a  hydrophobic exon at its C-terminal end that becomes embedded in the cell membrane. After antigen stimulation, the cells produce a shorter tran-  script by using a different cleavage and polyadenylation site. This transcript lacks the 3 '-splice site for the intron, so the intron is not removed.  In this case, translation ends at the first stop codon. The IgD antibody does not contain the hydrophobic region at its C-terminus, so it is secreted  from the cell.   Fig. 16.21. RNA editing. In liver, the apoprotein B (ApoB) gene produces a protein that con-  tains 4,563 amino acids. In intestinal cells, the same gene produces a protein that contains  only 2,152 amino acids. Conversion of a C to a U (through deamination) in the RNA tran-  script generates a stop codon in the intestinal mRNA. Thus, the protein produced in the intes-  tine (B-48) is only 48% of the length of the protein produced in the liver (B-100).   Fig. 16.22. Heme prevents inactivation of  eIF2. When eIF2 is phosphorylated by heme  kinase, it is inactive, and protein synthesis can-  not be initiated. Heme inactivates heme kinase,  thereby preventing phosphorylation of eIF2  and activating translation of the globin mRNA.   Fig. 16.23. Translational regulation of ferritin synthesis. The mRNA for ferritin has an iron  response element (IRE). When the iron response element binding protein, IRE-BP does not  contain bound iron, it binds to IRE, preventing translation. When IRE-BP binds iron, it dis-  sociates, and the mRNA is translated.   protein located in cell membranes that permits cells to take up transferrin, the pro-  tein that transports iron in the blood. The rate of synthesis of the transferrin recep-  tor increases when intracellular iron levels are low, enabling cells to take up more  iron. Synthesis of the transferrin receptor, like that of the ferritin receptor, is regu-  lated by the binding of the iron response element binding protein (IRE-BP) to the  iron response elements (IRE). However, in the case of the transferrin receptor  mRNA, the IREs are hairpin loops located at the 3' -end of the mRNA, and not at  the 5 'end where translation is initiated. When the IRE-BP does not contain bound  iron, it has a high affinity for the IRE hairpin loops. Consequently, IRE-BP prevents  degradation of the mRNA when iron levels are low, thus permitting synthesis of  more transferrin receptor so that the cell can take up more iron. Conversely, when  iron levels are elevated, IRE-BP binds iron and has a low affinity for the IRE hair-  pin loops of the mRNA. Without bound IRE-BP at its 3' end, the mRNA is rapidly  degraded and the transferrin receptor is not synthesized. (Note: When IRE-BP is  bound to IRE hairpin loops at the 3' -end of the mRNA, the IRE-BP prevents degra-  dation of the mRNA)   Fig. 16.24. Regulation of degradation of the  mRNA for the transferrin receptor. Degrada-  tion of the mRNA is prevented by binding of  the iron response element binding protein  (IRE-BP) to iron response elements (IRE),  which are hairpin loops located at the 3 '-end of  the transferrin receptor mRNA. When iron lev-  els are high, IRE-BP binds iron and is not  bound to the mRNA. The mRNA is rapidly  degraded, preventing synthesis of the transfer-  rin receptor.   Arlyn Foma. Follicular lymphomas are the most common subset of  non-Hodgkin's lymphomas (25^10% of cases). Patients with a more  aggressive course, as seen in Arlyn Foma, die within 3 to 5 years after diag-  nosis if left untreated. In patients pretreated with multidrug chemotherapy (in this  case AV/CM), a response rate of 50% has been reported when interferon-a is added  to this regimen. In addition, a significantly longer event-free survival has been  reported when using this approach.   Ominipotent stem cells in the bone  marrow normally differentiate and  mature in a highly selective and  regulated manner, becoming red blood cells,  white blood cells, or platelets. Cytokines  stimulate differentiation of the stem cells  into the lymphoid and myeloid lineages. The  lymphoid lineage gives rise to B and T lym-  phocytes, which are white blood cells that  generate antibodies for the immune  response. The myeloid lineage gives rise to  three types of progenitor cells: erythroid,  granulocytic-monocytic, and megakary-  ocytic. The erythroid progenitor cells differ-  entiate into red blood cells (erythrocytes),  and the other myeloid progenitors give rise  to nonlymphoid white blood cells and  platelets. Various medical problems can  affect this process. In Mannie Weitzels, who  has chronic myelogenous leukemia (CML), a  single line of primitive myeloid cells pro-  duces leukemic cells that proliferate abnor-  mally, causing a large increase in the num-  ber of white blood cells in the circulation. In  Anne Niemick, who has a deficiency of red  blood cells caused by her (3 + thalassemia  (see Chapter 15), differentiation of precursor  cells into mature red blood cells is stimu-  lated to compensate for the anemia.   0 o Ann O'Rexia has a hypochromic  I oOA J anemia, which means that her red  ^ blood cells are pale because they  contain low levels of hemoglobin. Because  of her iron deficiency, she is not producing  adequate amounts of heme. Consequently,  elF2 is phosphorylated in her reticulocytes  and cannot activate inititation of globin  translation.   2 Mannie Weitzels. Mannie Weitzels has CML (chronic myeloge-  ) nous leukemia), a hematologic disorder in which the proliferating  leukemic cells are believed to originate from a single line of primitive  myeloid cells. Although classified as one of the myeloproliferative disorders,  CML is distinguished by the presence of a specific cytogenetic abnormality of  the dividing marrow cells known as the Philadelphia chromosome, found in more  than 90% of cases. In most instances, the cause of CML is unknown, but the dis-  ease occurs with an incidence of around 1.5 per 100,000 population in Western  societies.   ^ Ann O'Rexia. Ann O'Rexia's iron stores are depleted. Normally, about  ) 16 to 18% of total body iron is contained in ferritin, which contains a  spherical protein (apoferritin) that is capable of storing as many as 4,000  atoms of iron in its center. When an iron deficiency exists, serum and tissue ferritin  levels fall. Conversely, the levels of transferrin (the blood protein that transports  iron) and the levels of the transferrin receptor (the cell surface receptor for trans-  ferrin) increase.   Regulation of transcription by iron. A cell's ability to acquire and  store iron is a carefully controlled process. Iron obtained from the diet  is absorbed in the intestine and released into the circulation, where it is  bound by transferrin, the iron transport protein in plasma. When a cell requires  iron, the plasma iron-transferrin complex binds to the transferrin receptor in the  cell membrane and is internalized into the cell. Once the iron is freed from trans-  ferrin, it then binds to ferritin, which is the cellular storage protein for iron. Fer-  ritin has the capacity to store up to 4,000 molecules of iron per ferritin molecule.  Both transcriptional and translational controls work to maintain intracellular lev-  els of iron (see Figs. 16.23 and 16.24). When iron levels are low, the iron  response element binding protein (IRE-BP) binds to specific looped structures  on both the ferritin and transferrin receptor mRNAs. This binding event stabi-  lizes the transferrin receptor mRNA so that it can be translated and the number  of transferrin receptors in the cell membrane increased. Consequently, cells will  take up more iron, even when plasma transferrin/iron levels are low. The binding  of IRE-BP to the ferritin mRNA, however, blocks translation of the mRNA. With  low levels of intracellular iron, there is little iron to store and less need for intra-  cellular ferritin. Thus, the IRE-BP can stabilize one mRNA, and block transla-  tion from a different mRNA.   What happens when iron levels rise? Iron will bind to the IRE-BP, thereby  decreasing its affinity for mRNA. When the IRE-BP dissociates from the transfer-  rin receptor mRNA, the mRNA becomes destabilized and is degraded, leading to  less receptor being synthesized. Conversely, dissociation of the IRE-BP from the  ferritin mRNA allows that mRNA to be translated, thereby increasing intracellular  levels of ferritin and increasing the cells capacity for iron storage.   Why does an anemia result from iron deficiency? When an individual is defi-  cient in iron, the reticulocytes do not have sufficient iron to produce heme, the  required prosthetic group of hemoglobin. When heme levels are low, the eukary-  otic initiation factor eIF2 (see Fig. 16.22) is phosphorylated, and inactive. Thus,  globin mRNA cannot be translated because of the lack of heme. This results in  red blood cells with inadequate levels of hemoglobin for oxygen delivery, and an  anemia.   Lewin B. Genes VII. Oxford: Oxford University Press, 2000:273-318, 649-684.  Thalassemias:   Watson J, Gilman M, Witkowski J, Zoller M. Recombinant DNA. Scientific American Books. New York:   WH Freeman, 1992:540-544.  Weatherall DJ, Clegg JB, Higgs DR, Wood WG. The hemoglobinopathies. In Scriver CR, Beaudet AL,   Slv WS, Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. New York:   Goldman L, Bennet JC. Cecil Textbook of Medicine. 21st Ed. Philadelphia: WB Saunders,  2000:945-977.   1 . Which of the following explains why several different proteins can be synthesized from a typical prokaryotic mRNA?   (C) The gene contains several operator sequences from which to initiate translation.   (E) Many RNAs are organized in a series of consecutive translational cistrons.   2. In E. Coli, under high lactose, high glucose conditions, which of the following could lead to maximal transcription activation  of the lac operon?   (A) A mutation in the lac I gene (which encodes the repressor)   (B) A mutation in the CRP binding site leading to enhanced binding   3. A mutation in the I (repressor) gene of a "non-inducible" strain of E. coli resulted in an inability to synthesize any of the pro-  teins of the lac operon. Which of the following provides a rational explanation?   (C) A trans acting factor can no longer bind to the promoter.   4. Which of the following double- stranded DNA sequences shows perfect dyad symmetry (the same sequence of bases on both  strands)?   Which of the following describes a common theme in the structure of DNA binding proteins?   (A) The presence of a specific helix that lies across the major groove of DNA   (B) The ability to recognize RNA molecules with the same sequence   (C) The ability to form multiple hydrogen bonds between the protein peptide backbone and the DNA phosphodiester backbone   The rapid development of techniques in the field of molecular biology is revolu-  tionizing the practice of medicine. The potential uses of these techniques for the  diagnosis and treatment of disease are vast.   Clinical applications. Polymorphisms, inherited differences in DNA base  sequences, are abundant in the human population, and many alterations in DNA  sequences are associated with diseases. Tests for DNA sequence variations are  more sensitive than many other techniques (such as enzyme assays) and permit  recognition of diseases at earlier and therefore potentially more treatable stages.  These tests can also identify carriers of inherited diseases so they can receive  appropriate counseling. Because genetic variations are so distinctive, DNA  "fingerprinting" (analysis of DNA sequence differences) can be used to determine  family relationships or to help identify the perpetrators of a crime.   Techniques of molecular biology are used in the prevention and treatment of  disease. For example, recombinant DNA techniques provide human insulin for the  treatment of diabetes, Factor VIII for the treatment of hemophilia, and vaccines  for the prevention of hepatitis. Although treatment of disease by gene therapy is in  the experimental phase of development, the possibilities are limited only by the  human imagination and, of course, by ethical considerations.   Techniques. To recognize normal or pathologic genetic variations, DNA must  be isolated from the appropriate source, and adequate amounts must be available  for study. Techniques for isolating and amplifying genes and studying and manip-  ulating DNA sequences involve the use of restriction enzymes, cloning vectors,  polymerase chain reaction (PCR), gel electrophoresis, blotting onto nitrocellu-  lose paper, and the preparation of labeled probes that hybridize to the appropri-  ate target DNA sequences. Gene therapy involves isolating normal genes and  inserting them into diseased cells so that the normal genes are expressed, permit-  ting the diseased cells to return to a normal state. Students must have at least a  general understanding of recombinant DNA techniques to appreciate their current  use and the promise they hold for the future.   Erna Nemdy, a third-year medical student, has started working in the hos-  pital blood bank two nights per week (see Chapter 15 for an introduction  to Erna Nemdy and her daughter, Beverly). Because she will be handling  human blood products, she must have a series of hepatitis B vaccinations. She has  reservations about having these vaccinations and inquires about the efficacy and  safety of the vaccines currently in use.   Cystic fibrosis is a disease caused  by an inherited deficiency in the  CFTR (cystic fibrosis transmem-  brane conductance regulator) protein, which  is a chloride channel (see Chapter 10, Fig.  10.11). In the absence of chloride secretion,  dried mucus blocks the pancreatic duct,  resulting in decreased secretion of digestive  enzymes into the intestinal lumen. The  resulting malabsorption of fat and other  foodstuffs decreases growth and may lead  to varying degrees of small bowel obstruc-  tion. Liver and gallbladder secretions may  be similarly affected. Eventually, atrophy of  the secretory organs or ducts may occur.  Dried mucus also blocks the airways,  markedly diminishing air exchange and pre-  disposing the patient to stasis of secretions,  diminished immune defenses, and increased  secondary infections. Defects in the CFTR  chloride channel also affect sweat composi-  tion, increasing the sodium and chloride  contents of the sweat, thereby providing a  diagnostic tool.    Sissy Fibrosa is a 3 -year-old Caucasian girl who is diagnosed with cystic  fibrosis. Her growth rate has been in the lower 30th percentile over the last  year. Since birth, she has had occasional episodes of spontaneously  reversible and minor small bowel obstruction. These episodes are superimposed on  gastrointestinal symptoms that suggest a degree of dietary fat malabsorption, such  as bulky, glistening, foul-smelling stools two or three times per day. She has expe-  rienced recurrent flare-ups of bacterial bronchitis in the last 10 months, each time  caused by Pseudomonas aeruginosa. A quantitative sweat test was unequivocally  positive (Excessive sodium and chloride were found in her sweat on two occa-  sions.). Based on these findings, the pediatrician informed Sissy's parents that Sissy  probably has cystic fibrosis (CF). A sample of her blood was sent to a DNA testing  laboratory to confirm the diagnosis and to determine specifically which one of the  many potential genetic mutations known to cause CF was present in her cells.   Carrie Sichel, Will Sichel's 19-year-old sister, is considering marriage.  Her growth and development have been normal, and she is free of symp-  toms of sickle cell anemia. Because a younger sister, Amanda, was tested  and found to have sickle trait, and because of Will's repeated sickle crises, Carrie  wants to know whether she also has sickle trait (see Chapters 6 and 7 for Will  Sichel's history). A hemoglobin electrophoresis is performed that shows the com-  position of her hemoglobin to be 58% HbA, 39% HbS, 1% HbF, and 2% HbA 2 , a  pattern consistent with the presence of sickle cell trait. The hematologist who saw  her in the clinic on her first visit is studying the genetic mutations of sickle cell trait  and asks Carrie for permission to draw additional blood for more sophisticated  analysis of the genetic disturbance that causes her to produce HbS. Carrie informed  her fiance that she has sickle cell trait and that she wants to delay their marriage  until he is tested.   Victoria Tim (Vicky Tim) was a 21 -year-old woman who was the victim  of a rape and murder. She left her home and drove to the local convenience  store. When she had not returned home an hour later, her father drove to  the store, looking for Vicky. He found her car still parked in front of the store and  called the police. They searched the area around the store, and found Vicky Tim's  body in a wooded area behind the building. She had been sexually assaulted and  strangled. Medical technologists from the police laboratory collected a semen sam-  ple from vaginal fluid and took samples of dried blood from under the victim's fin-  gernails. Witnesses identified three men who spoke to Vicky Tim while she was at  the convenience store. DNA samples were obtained from these suspects to deter-  mine whether any of them was the perpetrator of the crime.   nlvy Sharer's cough is slightly improved on a multidrug regimen for pul-  1 monary tuberculosis, but she continues to have night sweats. She is toler-  — ating her current AIDS therapy well but complains of weakness and  fatigue. The man with whom she has shared "dirty" needles to inject drugs accom-  panies Ivy to the clinic and requests that he be tested for the presence of HIV   Techniques for joining DNA sequences into new combinations (recombinant DNA)  were originally developed as research tools to explore and manipulate genes but are  now also being used to identify defective genes associated with disease and to cor-  rect genetic defects. Even a cursory survey of the current literature demonstrates  that these techniques will soon replace many of the current clinical testing proce-  dures. At least a basic appreciation of recombinant DNA techniques is required to  understand the ways in which genetic variations among individuals are determined   and how these differences can be used to diagnose disease. The first steps in deter-  mining individual variations in genes involve isolating the genes (or fragments of  DNA) that contain variable sequences and obtaining adequate quantities for study.   A. Strategies for Obtaining Fragments of DNA and  Copies of Genes   Enzymes called restriction endonucleases enable molecular biologists to cleave seg-  ments of DNA from the genome of various types of cells or to fragment DNA  obtained from other sources. A restriction enzyme is an endonuclease that specifi-  cally recognizes a short sequence of DNA, usually 4 to 6 base pairs (bp) in length,  and cleaves a phosphodiester bond in both DNA strands within this sequence  (Fig. 17.1). A key feature of restriction enzymes is their specificity. A restriction  enzyme always cleaves at the same DNA sequence and only cleaves at that partic-  ular sequence. Most of the DNA sequences recognized by restriction enzymes are  palindromes, that is, both strands of DNA have the same base sequence when read  in a 5' to 3' direction. The cuts made by these enzymes are usually "sticky" (that is,  the products are single-stranded at the ends, with one strand overhanging the other).  However, sometimes they are blunt (the products are double- stranded at the ends,  with no overhangs). Hundreds of restriction enzymes with different specificities  have been isolated (Table 17.1).   Restriction fragments of DNA can be used to identify variations in base sequence  in a gene. However, they also can be used to synthesize a recombinant DNA (also  called chimeric DNA), which is composed of molecules of DNA from different   Fig. 17.1. Action of restriction enzymes. Note  that the DNA sequence shown is a palindrome;  each strand of the DNA, when read in a 5 ' to  3' direction, has the same sequence. Cleavage  of this sequence by EcoRl produces single-  stranded (or "sticky") ends or tails. Not shown  is an example of an enzyme that generates  blunt ends (see Table 17.1).   In sickle cell anemia, the point mutation that converts a glutamate residue to a  valine residue (GAG to GTG) occurs in a site that is cleaved by the restriction  enzyme Msfll (recognition sequence CCTNAGG, where N can be any base)  within the normal (3-globin gene. The sickle cell mutation causes the p-globin gene to  lose this Msfll restriction site. Therefore, because Will Sichel is homozygous for the sickle  cell gene, neither of the two alleles of his (3-globin gene will be cleaved at this site.   Arthrobacter luteus  Bacillus amyloliquefaciens H  Escherichia coli RY13  Haemophilus aegyptius  Haemophilus influenzae R d  Moraxella species  Microcoleus  Nocardia otitidis  Providencia stuartii 1 64  Serratia marcescens S b   * Restriction enzymes are named for the bacterium from which they were isolated (e.g., ECO is from  Escherichia coli.).   Which ONE of the following  sequences is most likely to be a  restriction enzyme recognition  sequence?   Restriction endonucleases were  \\ ) discovered in bacteria in the late   1960s and 1970s. These enzymes  were named for the fact that bacteria use  them to "restrict" the growth of viruses (bac-  teriophage) that infect the bacterial cells.  They cleave the phage DNA into smaller  pieces so the phage cannot reproduce in the  bacterial cells. However, they do not cleave  the bacterial DNA, because its bases are  methylated at the restriction sites by DNA  methylases. Restriction enzymes also  restrict uptake of DNA from the environ-  ment, and they restrict mating with nonho-  mologous species.   f^S^ The answer is C. C follows a palin-  dromic sequence of CTN AG, where  N can be any base. None of the  other sequences is this close to a palin-  drome. Although most restriction enzymes  recognize a "perfect" palindrome, where the  sequence of bases in each strand are the  same, others can have intervening bases  between the regions of identity, as in this  question. Note also the specificity of the  enzyme Msfll in Table 1.   Fig. 17.2. Production of recombinant DNA molecules with restriction enzymes and DNA  ligase. The dashes at the 5' and 3 '-ends indicate that this sequence is part of a longer DNA  molecule.   sources that have been recombined in vitro (outside the organism, e.g., in a test  tube). The sticky ends of two unrelated DNA fragments can be joined to each other  if they have sticky ends that are complementary. Complementary ends are obtained  by cleaving the unrelated DNAs with the same restriction enzyme (Fig. 17.2). After  the sticky ends of the fragments base-pair with each other, the fragments can be  covalently attached by the action of DNA ligase.   If mRNA transcribed from a gene is isolated, this mRNA can be used as a template  by the enzyme reverse transcriptase, which produces a DNA copy (cDNA) of the  RNA. In contrast to DNA fragments cleaved from the genome by restriction  enzymes, DNA produced by reverse transcriptase does not contain introns because  mRNA, which has no introns, is used as a template.   Automated machines can synthesize oligonucleotides (short molecules of single-  stranded DNA) up to 100 nucleotides in length. These machines can be pro-  grammed to produce oligonucleotides with a specified base sequence. Although  entire genes cannot yet be synthesized in one piece, oligonucleotides can be pre-  pared that will base-pair with segments of genes. These oligonucleotides can be  used in the process of identifying, isolating, and amplifying genes.   A probe is a single- stranded polynucleotide of DNA or RNA that is used to identify  a complementary sequence on a larger single-stranded DNA or RNA molecule  (Fig. 17.3). Formation of base pairs with a complementary strand is called anneal-  ing or hybridization. Probes can be composed of cDNA (produced from mRNA by  reverse transcriptase), fragments of genomic DNA (cleaved by restriction enzymes  from the genome), chemically synthesized oligonucleotides, or, occasionally, RNA.   To identify the target sequence, the probe must carry a label (see Fig. 17.3). If  the probe has a radioactive label such as 32 P, it can be detected by autoradiography.  An autoradiogram is produced by covering the material containing the probe with  a sheet of x-ray film. Electrons (p particles) emitted by disintegration of the   Fig. 17.3. Use of probes to identify DNA sequences. The probe can be either DNA or RNA.   radioactive atoms expose the film in the region directly over the probe. A number  of techniques can be used to introduce labels into these probes. Not all probes are  radioactive. Some are chemical adducts (compounds that bind covalently to DNA)  that can be identified, for example, by fluorescence.   Gel electrophoresis is a technique that uses an electrical field to separate molecules  on the basis of size. Because DNA contains negatively charged phosphate groups,  it will migrate in an electrical field toward the positive electrode (Fig. 17.4). Shorter  molecules migrate more rapidly through the pores of a gel than do longer mole-  cules, so separation is based on length. Gels composed of polyacrylamide, which  can separate DNA molecules that differ in length by only one nucleotide, are used  to determine the base sequence of DNA. Agarose gels are used to separate longer  DNA fragments that have larger size differences.   The bands of DNA in the gel can be visualized by various techniques. Staining with  dyes such as ethidium bromide allows direct visualization of DNA bands under ultra-  violet light. Specific sequences are generally detected by means of a labeled probe.   To detect specific sequences, DNA is usually transferred to a solid support, such as  a sheet of nitrocellulose paper. For example, if bacteria are growing on an agar  plate, cells from each colony will adhere to a nitrocellulose sheet pressed against the   Fig. 17.4. Gel electrophoresis of DNA. A. DNA samples are placed into depressions  ("wells") at one end of a gel, and an electrical field is applied. The DNA migrates toward the  positive electrode at a rate that depends on the size of the DNA molecules. Shorter molecules  migrate more rapidly than longer molecules. B. The gel is removed from the apparatus. The  bands are not visible until techniques are performed to visualize them (see Fig. 17.6).   Fig. 17.5. Identification of bacterial colonies  containing specific DNA sequences. The  autoradiogram can be used to identify bacterial  colonies on the original agar plate that contain  the desired DNA sequence. Note that an orien-  tation marker is placed on the nitrocellulose  and the agar plate so the results of the autora-  diogram can be properly aligned with the orig-  inal plate of bacteria.   agar, and an exact replica of the bacterial colonies can be transferred to the nitro-  cellulose paper (Fig. 17.5). A similar technique is used to transfer bands of DNA  from electrophoretic gels to nitrocellulose sheets. After bacterial colonies or bands  of DNA are transferred to nitrocellulose paper, the paper is treated with an alkaline  solution. Alkaline solutions denature DNA (that is, separate the two strands of each  double helix). The single- stranded DNA is then hybridized with a probe, and the  regions on the nitrocellulose blot containing DNA that base-pairs with the probe are  identified.   E. M. Southern developed the technique, which bears his name, for identifying  DNA sequences on gels. Southern blots are produced when DNA on a nitrocellu-  lose blot of an electrophoretic gel is hybridized with a DNA probe. Molecular biol-  ogists decided to continue with this geographic theme as they named two additional  techniques. Northern blots are produced when RNA on a nitrocellulose blot is  hybridized with a DNA probe. A slightly different but related technique, known as  a Western blot, involves separating proteins by gel electrophoresis and probing with  labeled antibodies for specific proteins (Fig. 17.6).   The most common procedure for determining the sequence of nucleotides in a DNA  strand was developed by Frederick Sanger and involves the use of dideoxynu-  cleotides. Dideoxynucleotides lack a 3' -hydroxy 1 group (in addition to lacking the  2' hydroxyl group normally absent from DNA deoxynucleotides). Thus, once they  are incorporated into the growing chain, the next nucleotide cannot add, and poly-  merization is terminated. In this procedure, only one of the four dideoxynucleotides  (ddATP, ddTTP, ddGTP, or ddCTP) is added to a tube containing all four normal  deoxynucleotides, DNA polymerase, a primer, and the template strand for the DNA  that is being sequenced (Fig. 17.7). As DNA polymerase catalyzes the sequential  addition of complementary bases to the 3' end, the dideoxynubleotide competes  with its corresponding normal nucleotide for insertion. Whenever the dideoxynu-  cleotide is incorporated, further polymerization of the strand cannot occur, and syn-  thesis is terminated. Some of the chains will terminate at each of the locations in the  template strand that is complementary to the dideoxnucleotide. Consider, for exam-  ple, a growing polynucleotide strand in which adenine (A) should add at positions  10, 15, and 17. Competition between ddATP and dATP for each position results in  some chains terminating at position 10, some at 15, and some at 17. Thus, DNA  strands of varying lengths are produced from a template. The shortest strands are  closest to the 5' -end of the growing DNA strand because the strand grows in a 5' to  3' direction.   Four separate reactions are performed, each with only one of the dideoxynu-  cleotides present (ddATP, ddTTP, ddGTP, ddCTP) plus a complete mixture of nor-  mal nucleotides (see Fig. 17.7B.). In each tube, some strands are terminated when-  ever the complementary base for that dideoxynucleotide is encountered. If these  strands are subjected to gel electrophoresis, the sequence 5 '—>3' of the DNA strand  complementary to the template can be determined by "reading" from the bottom to  the top of the gel, that is, by noting the lanes (A, G, C, or T) in which bands appear,  starting at the bottom of the gel and moving sequentially toward the top.   0 o Western blots are one of the tests  ( J for the AIDS virus. Viral proteins in  ™ the blood are detected by antibod-  ies. Tests performed on Ivy Sharer's friend  showed that he was HIV positive. Unlike Ivy,  however, he has not yet developed the  symptoms of AIDS.   Ivy Sharer is being treated with didanosine. This drug is a purine nucleoside  composed of the base hypoxanthine linked to dideoxyribose. In cells, didano-  sine is phosphorylated to form a nucleotide that adds to growing DNA strands.  Because dideoxynucleotides lack both 2'- and 3'-hydroxyl groups, DNA syn-  thesis is terminated. Reverse transcriptase has a higher affinity for the dideoxynu-  cleotides than does the cellular DNA polymerase, so the use of this drug will affect  reverse transcriptase to a greater extent than the cellular enzyme.   In the early studies on cystic fibrosis, DNA sequencing was used to determine  I the type of defect in patients. Buccal cells were obtained from washes of the  mucous membranes of the mouth, DNA isolated from these cells was amplified  by PCR, and DNA sequencing of the CF gene was performed. A sequencing gel for the  region in which the normal gene differs from the mutant gene is shown below.   What is the difference between the normal and the mutant CF gene sequence shown on  the gel, and what effect would this difference have on the protein produced from this  gene?   To study genes or other DNA sequences, adequate quantities of material must be  obtained. It is often difficult to isolate significant quantities of DNA from the orig-  inal source. For example, an individual cannot usually afford to part with enough  tissue to provide the amount of DNA required for clinical testing. Therefore, the  available quantity of DNA has to be amplified.   The first technique developed for amplifying the quantity of DNA is known as  cloning (Fig. 17.8). The DNA that you want amplified (the "foreign" DNA) is  attached to a vector (a carrier DNA), which is introduced into a host cell that makes  multiple copies of the DNA. The foreign DNA and the vector DNA are usually  cleaved with the same restriction enzyme, which produces complementary sticky  ends in both DNAs. The foreign DNA is then added to the vector. Base pairs form  between the complementary single-stranded regions, and DNA ligase joins the mol-  ecules to produce a chimera, or recombinant DNA. As the host cells divide, they  replicate their own DNA, and they also replicate the DNA of the vector, which  includes the foreign DNA.   If the host cells are bacteria, commonly used vectors are bacteriophage (viruses  that infect bacteria), plasmids (extrachromosomal pieces of circular DNA that are  taken up by bacteria), or cosmids (plasmids that contain DNA sequences from the  lambda phage). When eukaryotic cells are used as the host, the vectors are often  retroviruses, adenoviruses, free DNA, or DNA coated with a lipid layer (liposomes).  The foreign DNA sometimes integrates into the host cell genome or it exists as eip-  somes (extrachromosomal fragments of DNA) (See section III.D. of this chapter.)   Fig. 17.6. Southern, Northern, and Western  blots. For Southern blots, DNA molecules are  separated by electrophoresis, denatured, trans-  ferred to nitrocellulose paper (by "blotting"),  and hybridized with a DNA probe. For  Northern blots, RNA is electrophoresed and  treated similarly except that alkali is not used.  (First, alkali hydrolyzes RNA, and second,  RNA is already single stranded.) For Western  blots, proteins are electrophoresed, transferred  to nitrocellulose, and probed with a specific  antibody.   ^> A genomic "library" in molecular  \\ ) biologists' terms is a set of host  *S cells that collectively contain all of  the DNA sequences from the genome of  another organism. A cDNA library is a set of  host cells that collectively contain all the  DNA sequences produced by reverse tran-  scriptase from the mRNA obtained from  cells of a particular type. Thus, a cDNA  library contains all the genes expressed in  that cell type, at the stage of differentiation  when the mRNA was isolated.   v^—^v In individuals of northern Euro-  A •) pean descent, 70% of the cases of  cystic fibrosis (CF) are caused by a  deletion of three bases in the CF gene. In  the region of the gene shown on the gels,  the base sequence (read from the bottom to  the top of the gel) is the same for the nor-  mal and mutant gene for the first 6 posi-  tions, and the bases in positions 10 through  16 of the normal gene are the same as the  bases in positions 7 through 13 of the  mutant gene. Therefore, a 3-base deletion  in the mutant gene corresponds to bases 7  through 9 of the normal gene.   ' lie ' lie ' Gly '  Loss of 3 bp (indicated by the dashes) main-  tains the reading frame, so only the single  amino acid phenylalanine (F) is lost. Pheny-  lalanine would normally appear as residue  508 in the protein. Therefore, the deletion is  referred to as AF 508 - The rest of the amino  acid sequence of the normal and the mutant  proteins is identical.   DNA polymerase + ddATP and  other normal nucleotides  dGTP, dCTP, dTTP)   Fig. 17.7. The Sanger method. (A). A reaction mixtures contain one of the dideoxynucleotides,  such as ddATP, and some of the normal nucleotide, dATP, which compete for incorporation into  the growing polypeptide chain. When a T is encountered on the template strand (position 10),  some of the molecules will incorporate a ddATP, and the chain will be terminated. Those that  incorporate a normal dATP will continue growing until position 15 is reached, where they will  incorporate either a ddATP or the normal dATP. Only those that incorporate a dATP will con-  tinue growing to position 17. Thus, strands of different length from the 5' end are produced, cor-  responding to the position of a T in the template strand. (B). DNA sequencing by the dideoxynu-  cleotide method. Four tubes are used. Each one contains DNA polymerase, a DNA template  hybridized to a primer, plus dATP, dGTP, dCTP, and dTTP Either the primer or the nucleotides  must have a radioactive label, so bands can be visualized on the gel by autoradiography. Only  one of the four dideoxyribonucleotides (ddNTPs) is added to each tube. Termination of synthe-  sis occurs where the ddNTP is incorporated into the growing chain. The template is comple-  mentary to the sequence of the newly synthesized strand.   Host cells that contain recombinant DNA are called transformed cells if they  are bacteria, or transfected cells if they are eukaryotes. Markers in the vector  DNA are used to identify cells that have been transformed, and probes for the  foreign DNA can be used to determine that the host cells actually contain the for-  eign DNA. If the host cells containing the foreign DNA are incubated under con-  ditions in which they replicate rapidly, large quantities of the foreign DNA can  be isolated from the cells. With the appropriate vector and growth conditions that   Fig. 17.8. Simplified scheme for cloning of DNA in bacteria. A plasmid is a specific type of  vector, or carrier, which can contain inserts of foreign DNA of up to 2.0 kb in size. For clar-  ity, the sizes of the pieces of DNA are not drawn to scale (for example, the bacterial chro-  mosomal DNA should be much larger than the plasmid DNA).   permit expression of the foreign DNA, large quantities of the protein produced  from this DNA can be isolated.   PCR is an in vitro method that can be used for rapid production of very large  amounts of specific segments of DNA. It is particularly suited for amplifying  regions of DNA for clinical or forensic testing procedures because only a very small  sample of DNA is required as the starting material. Regions of DNA can be ampli-  fied by PCR from a single strand of hair or a single drop of blood or semen.   First, a sample of DNA containing the segment to be amplified must be iso-  lated. Large quantities of primers, the four deoxyribonucleoside triphosphates,  and a heat- stable DNA polymerase are added to a solution in which the DNA is  heated to separate the strands (Fig. 17.9). The primers are two synthetic oligonu-    Although only small amounts of  semen were obtained from Vicky  Tim's body, the quantity of DNA in  these specimens could be amplified by PCR.  This technique provided sufficient amounts  of DNA for comparison with DNA samples  from the three suspects.   Fig. 17.9. Polymerase chain reaction (PCR). Strand 1 and strand 2 are the original DNA strands. The short dark blue fragments are the primers.  After multiple heating and cooling cycles, the original strands remain, but most of the DNA consists of amplified copies of the segment (shown  in lighter blue) synthesized by the heat- stable DNA polymerase.   The DNA polymerase used for PCR  \\ } is isolated from Thermus aquati-  *r cus, a bacterium that grows in hot  springs. This polymerase can withstand the  heat required for separation of DNA strands.   cleotides; one oligonucleotide is complementary to a short sequence in one  strand of the DNA to be amplified, and the other is complementary to a sequence  in the other DNA strand. As the solution is cooled, the oligonucleotides form  base pairs with the DNA and serve as primers for the synthesis of DNA strands  by the heat-stable DNA polymerase. The process of heating, cooling, and new  DNA synthesis is repeated many times until a large number of copies of the DNA  are obtained. The process is automated, so that each round of replication takes  only a few minutes and in 20 heating and cooling cycles, the DNA is amplified  over a million-fold.   Polymorphisms are variations among individuals of a species in DNA sequences  of the genome. They serve as the basis for using recombinant DNA techniques  in the diagnosis of disease. The human genome probably contains millions of  different polymorphisms. Some polymorphisms involve point mutations, the  substitution of one base for another. Deletions and insertions are also responsi-  ble for variations in DNA sequences. Some polymorphisms occur within the cod-  ing region of genes. Others are found in noncoding regions closely linked to  genes involved in the cause of inherited disease, in which case they can be used  as a marker for the disease.   Occasionally, a point mutation occurs in a recognition site for one of the restric-  tion enzymes. The restriction enzyme therefore can cut at this restriction site in  DNA from most individuals, but not in DNA from individuals with this mutation.  Consequently, the restriction fragment that binds a probe for this region of the  genome will be larger for a person with the mutation than for most members of  the population. Mutations also can create restriction sites that are not commonly  present. In this case, the restriction fragment from this region of the genome will  be smaller for a person with the mutation than for most individuals. These vari-  ations in the length of restriction fragments are known as restriction fragment  length polymorphisms (RFLPs).   In some cases, the mutation that causes a disease affects a restriction site within  the coding region of a gene. However, in many cases, the mutation affects a restric-  tion site that is outside the coding region but tightly linked (i.e., physically close on  the DNA molecule) to the abnormal gene that causes the disease. This RFLP could  still serve as a biologic marker for the disease. Both types of RFLPs can be used for  genetic testing to determine whether an individual has the disease.   Other techniques have been developed to detect mutations, because many muta-  tions associated with genetic diseases do not occur within restriction enzyme  recognition sites or cause detectable restriction fragment length differences  when digested with restriction enzymes. For example, oligonucleotide probes  (containing 15-20 nucleotides) can be synthesized that are complementary to a  DNA sequence that includes a mutation. Different probes are produced for alle-  les that contain mutations and for those that have a normal DNA sequence. The  region of the genome that contains the abnormal gene is amplified by PCR, and  the samples of DNA are placed in narrow bands on nitrocellulose paper ("slot  blotting"). The paper is then treated with the radioactive probe for either the nor-  mal or the mutant sequence. Autoradiograms indicate whether the normal or  mutant probe has preferentially base-paired (hybridized) with the DNA, that is,  whether the alleles are normal or mutated. Carriers, of course, have two differ-  ent alleles, one that binds to the normal probe and one that binds to the mutant  probe.   So how does one determine the DNA sequence of a gene that contains a muta-  \\ ) tion to develop specific probes to that mutation? Initially the gene causing the  *S disease must be identified. This is done by a process known as positional  cloning, which involves linking polymorphic markers to the disease. Individuals who  express the disease should contain a specific variant of these polymorphic markers,  whereas individuals who do not express the disease would not contain these markers.  Once such polymorphic markers are identified, they are used as probes to screen a  human genomic library. This will identify pieces of human DNA containing the polymor-  phic marker. These pieces of DNA are then used as probes to expand the region of the  genome surrounding this marker. Potential genes within this region are identified (using  data available from the sequencing of the human genome), and the sequence of bases  within each gene compared with the sequence of bases in the genes of individuals with  the disease. The one gene that shows an altered sequence in disease-carrying individu-  als as compared with normal individuals is the tentative disease gene. Through the  sequencing of genes from many people afflicted with the disease, the types of mutations  that lead to this disease can be characterized and specific tests developed to look for  these specific mutations.   The mutation that causes sickle cell  anemia abolishes a restriction site  for the enzyme Msfll in the p-globin  gene. The consequence of this mutation is  that the restriction fragment produced by  Msfll that includes the 5'-end of the p-globin  gene is larger (1.3 kilobases [kb]) for individ-  uals with sickle cell anemia than for normal  individuals (1.1 kb). Analysis of restriction  fragments provides a direct test for the muta-  tion. In Will Sichel's case, both alleles for p-  globin lack the Msfll site and produce 1.3-kb  restriction fragments; thus, only one band is  seen in a Southern blot.   Carriers have both a normal and a mutant  allele. Therefore, their DNA will produce both  the larger and the smaller Msfll restriction  fragments. When Will Sichel's sister Carrie  Sichel was tested, she was found to have  both the small and the large restriction frag-  ments, and her status as a carrier of sickle cell  anemia, initially made on the basis of protein  electrophoresis, was confirmed.   Southern blot  of DNA cut with  Msfll and  hybridized  with P-globin  probe   Testing for cystic fibrosis by DNA  lB # sequencing is time-consuming and  expensive. Therefore, another  technique that uses allele-specific oligonu-  cleotide probes has been developed. Sissy  Fibrosa and her family were tested by this  method. Oligonucleotide probes, comple-  mentary to the region where the 3-base dele-  tion is located, have been synthesized. One  probe binds to the mutant (AF 508 ) gene, and  the other to the normal gene.   DNA was isolated from Sissy, her par-  ents, and two siblings and amplified by PCR.  Samples of the DNA were spotted on nitro-  cellulose paper, treated with the oligonu-  cleotide probes, and the following results  were obtained. (Dark spots indicate binding  of the probe.)   Which members of Sissy's family have CF,  which are normal, and which are carriers?   If an oligonucleotide complementary to a DNA sequence containing a mutation is used  as a primer for PCR, a DNA sample used as the template will be amplified only if it  contains the mutation. If the DNA is normal, the primer will not hybridize with it, and  the DNA will not be amplified. This concept is extremely useful for clinical testing. In  fact, a number of oligonucleotides, each specific for a different mutation and each con-  taining a different label, can be used as primers in a single PCR reaction. This proce-  dure results in rapid and relatively inexpensive testing for multiple mutations.   Human DNA contains many sequences that are repeated in tandem a variable  number of times at certain loci in the genome. These regions are called highly  variable regions because they contain a variable number of tandem repeats  (VNTR). Digestion with restriction enzymes that recognize sites that flank the  VNTR region produces fragments containing these loci, which differ in size  from one individual to another, depending on the number of repeats that are pres-  ent. Probes used to identify these restriction fragments bind to or near the  sequence that is repeated (Fig. 17.10).   The restriction fragment patterns produced from these loci can be used to iden-  tify individuals as accurately as the traditional fingerprint. In fact, this restriction  fragment technique has been called "DNA fingerprinting" and is gaining wide-  spread use in forensic analysis. Family relationships can be determined by this  method, and it can be used to help acquit or convict suspects in criminal cases.   Fig. 17.10. Restriction fragments produced from a gene with a variable number of tandem  repeats (VNTR). Each individual has two homologues of every somatic chromosome and  thus two genes each containing this region with a VNTR. Cleavage of each individual's  genomic DNA with a restriction enzyme produces two fragments containing this region. The  length of the fragments depends on the number of repeats they contain. Electrophoresis sep-  arates the fragments, and a labeled probe that binds to the fragments allows them to be visu-  alized. Each short blue block represents one repeat.   Obviously, the father and mother  are both carriers of the defective  allele, as is one of the two siblings  (Child 2). Sissy has the disease, and the  other sibling (Child 1) is normal.   Incubate with probe, wash  and perform autoradiography  to observe labeled DNA bands   Q o DNA samples were obtained from  f /~oA ] each of the three suspects in Vicky  ^ Tim's rape and murder case, and  these samples were compared with the vic-  tim's DNA by using DNA fingerprinting.  Because Vicky "Tim's sample size was small,  PCR was used to amplify the regions con-  taining the VNTRs. The results, using a probe  for one of the repeated sequences in human  DNA, are shown below to illustrate the  process. For more positive identification, a  number of different restriction enzymes and  probes were used. The DNA from suspect 2  produced the same restriction pattern as the  DNA from the semen obtained from the vic-  tim. If the other restriction enzymes and  probes corroborate this finding, suspect 2  can be identified by DNA fingerprinting as  the rapist/murderer.   The huge amount of information  fji ) now available from the sequencing  *S of the human genome, and the  results available from gene chip experi-  ments, has greatly expanded the field of  Bioinformatics. Bioinformatics can be  defined as the gathering, processing, data  storage, data analysis, information extrac-  tion, and visualization of biologic data.  Bioinformatics also provides the scientist  with the capability of organizing vast  amounts of data in a manageable form that  allows easy access and retrieval of data.  Powerful computers are required to perform  these analyses. As an example of an experi-  ment requiring these tools, suppose you  want to compare the effects of two different  immunosuppressant drugs on gene expres-  sion in lymphocytes. Lymphocytes would be  treated with either nothing (the control) or  with the drugs (experimental samples). RNA  would be isolated from the cells during drug  treatment, and the RNA converted to fluo-  rescent cDNA using the enzyme reverse  transcriptase and a fluorescent nucleotide  analog. The cDNA produced from your  three samples would be used as probes for  a gene chip containing DNA fragments from  more than 5,000 human genes. The samples  would be allowed to hybridize to the chips,  and you would then have 15,000 results to  interpret (the extent of hybridization of each  cDNA sample with each of the 5,000 genes  on the chip). Computers are used to analyze  the fluorescent spots on the chips and to  compare the levels of fluorescent intensity  from one chip to another. In this way, you  can group genes showing similar levels of  stimulation or inhibition in the presence of  the drugs and compare the two drugs with  respect to which genes have activated or  inhibited expression.   Individuals who are closely related genetically will have restriction fragment  patterns (DNA fingerprints) that are more similar than those who are more distantly  related. Only monozygotic twins will have identical patterns.   A recently introduced technique will permit the screening of many genes to deter-  mine which alleles of these genes are present in samples obtained from patients.  The surface of a small chip is dotted with thousands of pieces of single-stranded  DNA, each representing a different gene or segment of a gene. The chip is then  incubated with a sample of a patient's DNA, and the pattern of hybridization is  determined by computer analysis. The results of the hybridization analysis could be  used, for example, to determine which one of the many known mutations for a par-  ticular genetic disease is the specific defect underlying a patient's problem. An indi-  vidual's gene chip also may be used to determine which alleles of drug-metaboliz-  ing enzymes are present and, therefore, the likelihood of that individual having an  adverse reaction to a particular drug.   Another use for a DNA chip is to determine which genes are being expressed. If  the mRNA from a tissue specimen is used to produce a cDNA by reverse transcrip-  tase, the cDNA will hybridize with only those genes being expressed in that tissue.  In the case of a cancer patient, this technique could be used to determine the clas-  sification of the cancer much more rapidly and more accurately than the methods  traditionally used by pathologists. The treatment then could be more specifically  tailored to the individual patient. This technique also can be used to identify the  genes required for tissue specificity (e.g., the difference between a muscle cell and  a liver cell) and differentiation (the conversion of precursor cells into the different  cell types). Experiments using gene chips are helping us to understand differentia-  tion and may open the opportunity to artificially induce differentiation and tissue  regeneration in the treatment of disease.   Although development of this DNA or biochip technology is in its infancy, the  technique has astonishing potential for the future diagnosis and treatment of  disease.   III. USE OF RECOMBINANT DNA TECHNIQUES FOR THE  PREVENTION AND TREATMENT OF DISEASE   Before the advent of recombinant DNA technology, vaccines were made exclu-  sively from infectious agents that had been either killed or attenuated (altered so  that they can no longer multiply in an inoculated individual). Both types of vaccines  were potentially dangerous because they could be contaminated with the live, infec-  tious agent. In fact, in a small number of instances, disease has actually been caused  by vaccination. The human immune system responds to antigenic proteins on the  surface of an infectious agent. By recombinant DNA techniques, these antigenic  proteins can be produced, completely free of the infectious agent, and used in a vac-  cine. Thus, any risk of infection is eliminated. The first successful recombinant  DNA vaccine to be produced was for the hepatitis B virus.   When Erna Nemdy began working with patients, she received the hepatitis B vaccine. The hepatitis B virus (HBV) infects the liver,  causing severe damage. The virus contains a surface antigen (HBsAg) or coat protein for which the gene has been isolated. How-  ever, because the protein is glycosylated, it could not be produced in E. co\\. (Bacteria, because they lack subcellular organelles,  cannot produce glycosylated proteins.) Therefore, a yeast (eukaryotic) expression system was used that produced a glycosylated form of  the protein. The viral protein, separated from the small amount of contaminating yeast protein, is used as a vaccine for immunization  against HBV infection.   Recombinant DNA techniques are used to produce proteins that have therapeutic  properties. One of the first such proteins to be produced was human insulin.  Recombinant DNA corresponding to the A chain of human insulin was prepared  and inserted into plasmids that were used to transform E. coli cells. The bacteria  then synthesized the insulin chain, which was purified. A similar process was used  to obtain B chains. The A and B chains were then mixed and allowed to fold and  form disulfide bonds, producing active insulin molecules (Fig. 17.1 1). Insulin is not  glycosylated, so there was no problem with differences in glycosyltransferase activ-  ity between E. coli and human cell types.   Human growth hormone has also been produced in E. coli and is used to treat  children with growth hormone deficiencies. Before production of recombinant  growth hormone, growth hormone isolated from cadaver pituitary tissue was used,  which was in short supply.   Di Abietes is using a recombinant  human insulin called lispro (Huma-  log) (see Chapter 6, Fig. 6.13).  Lispro was genetically engineered so that  lysine is at position 28 and proline is at posi-  tion 29 of the B chain (the reverse of their  positions in normal human insulin). Di  injects a Humalog mixture that contains 75%  lispro protamine suspension (intermediate-  acting) and 25% lispro solution (rapid-act-  ing). The switch of position of the two amino  acids leads to a faster-acting insulin  homolog. The lispro is absorbed from the  site of injection much more quickly than  other forms of insulin, and it acts to lower  blood glucose levels much more rapidly  than the other insulin forms.   More complex proteins have been produced in mammalian cell culture using  recombinant DNA techniques. The gene for Factor VIII, a protein involved in blood  clotting, is defective in individuals with hemophilia. Before genetically engineered  Factor VIII became available, a number of hemophiliac patients died of AIDS or  hepatitis that they contracted from transfusions of contaminated blood or from  Factor VIII isolated from contaminated blood.   Tissue plasminogen activator (TPA) is a protease in blood that converts plas-  minogen to plasmin. Plasmin is a protease that cleaves fibrin (a major component  of blood clots), and, thus, TPA administration dissolves blood clots. Recombinant  TPA, produced in mammalian cell cultures, is frequently administered during or  immediately after a heart attack to dissolve the thrombi that occlude coronary arter-  ies and prevent oxygen from reaching the heart muscle.   Hematopoietic growth factors also have been produced in mammalian cell cul-  tures by recombinant DNA techniques. Erythropoietin can be used in certain types  of anemias to stimulate the production of red blood cells. Colony- stimulating fac-  tors (CSFs) and interleukins (ILs) can be used after bone marrow transplants and  after chemotherapy to stimulate white blood cell production and decrease the risk  of infection.   Recombinant p -interferon is the first drug known to decrease the frequency and  severity of episodes resulting from the effects of demyelination in patients with  multiple sclerosis.   A method for producing human proteins that is being tested involves transgenic  animals. These animals (usually goat or sheep) have been genetically engineered to  produce human proteins in the mammary gland and secrete them into milk. The  gene of interest is engineered to contain a promoter that is only active in the  mammary glands under lactating conditions. The vector containing the gene and  promoter is inserted into the nucleus of a freshly fertilized egg, which is then  implanted into a foster mother. The female animal progeny are tested for the pres-  ence of this transgene, and milk from the positive animals is collected. Large quan-  tities of the protein of interest can then be isolated from the relatively small number  of proteins present in milk.   One means of preventing disease is to avoid passing defective genes to offspring. If  individuals are tested for genetic diseases, particularly in families known to carry a   Fig. 17.11. Production of human insulin in E.  coli. Amp R = the gene for ampicillin resist-  ance. The presence of Amp R allows bacterial  cells that contain the vector to grow in the  presence of ampicillin. Cells lacking the Amp R  gene die in the presence of ampicillin. Because  E. coli cannot process preproinsulin, a syn-  thetic scheme was developed whereby each  individual chain of insulin was expressed, pro-  duced, and purified, and then the two chains  were linked together in a test tube.   Carrie Sichel's fiance decided to be  tested for the sickle cell gene. He  was found to have both the 1.3-kb  and the 1.1 -kb Msfll restriction fragments  that include a portion of the (3-globin gene.  Therefore, like Carrie, he also is a carrier for  the sickle cell gene.   Fig. 17.12. Use of retroviruses for gene ther-  apy. The retrovirus carries an RNA copy of the  therapeutic gene into the cell. The endosome  that contains the virus dissolves, and the RNA  and viral reverse transcriptase are released.  This enzyme copies the RNA, making a dou-  ble-stranded DNA that integrates into the host  cell genome. Transcription and translation of  this DNA (the therapeutic gene) produces the  therapeutic protein. (The virus does not multi-  ply because its genes were removed and  replaced by the RNA copy of the therapeutic  gene.)   defective gene, genetic counselors can inform them of their risks and options. With  this information, individuals can decide in advance whether to have children.   Screening tests, based on the recombinant DNA techniques outlined in this  chapter, have been developed for many inherited diseases. Although these tests  are currently rather expensive, particularly if entire families have to be screened,  the cost may be trivial compared with the burden of raising children with severe  disabilities. Obviously, ethical considerations must be taken into account, but  recombinant DNA technology has provided individuals with the opportunity to  make choices.   Screening can be performed on the prospective parents before conception. If they  decide to conceive, the fetus can be tested for the genetic defect. In some cases, if  the fetus has the defect, treatment can be instituted at an early stage, even in utero.  For certain diseases, early therapy leads to a more positive outcome.   The ultimate cure for genetic diseases is to introduce normal genes into individuals  who have defective genes. Currently, gene therapy is being attempted in animals,  cell cultures, and human subjects. It is not possible at present to replace a defective  gene with a normal gene at its usual location in the genome of the appropriate cells.  However, as long as the gene is expressed at the appropriate time and produces ade-  quate amounts of the protein to return the person to a normal state, the gene does  not have to integrate into the precise place in the genome. Sometimes the gene does  not even have to be in the cells that normally contain it.   Retroviruses were the first vectors used to introduce genes into human cells.  Normally, retroviruses enter target cells, their RNA genome is copied by reverse  transcriptase, and the double- stranded DNA copy is integrated into the host cell  genome (see Fig. 14.22). If the retroviral genes (e.g., gag, pol, and env) are first  removed and replaced with the therapeutic gene, the retroviral genes integrated into  the host cell genome will produce the therapeutic protein rather than the viral pro-  teins (Fig. 17.12). This process works only when the human host cells are undergo-  ing division, so it has limited applicability. Other problems with this technique are  that it can only be used with small genes ( = 8 kb), and it may disrupt other genes  because the insertion point is random, thereby possibly resulting in cancer.   q q A defect in the adenosine deaminase (ADA) gene causes severe combined  \©\u ) ' mmunoc ' e ^ c ' enc y syndrome (SCID). When ADA is defective, deoxyadenosine  Vj^x and dATP accumulate in rapidly dividing cells, such as lymphocytes, and prove  toxic to these cells. Cells of the immune system cannot proliferate at a normal rate, and  children with SCID usually die at an early age because they cannot combat infections. To  survive, they must be confined to a sterile, environmental "bubble." When an appropri-  ate donor is available, bone marrow transplantation can be performed with a reasonable  degree of success.   In 1990, a 4-year-old girl, for whom no donor was available, was treated with infu-  sions of her own lymphocytes that had been treated with a retrovirus containing a nor-  mal ADA gene. Although she had not responded to previous therapy, she improved sig-  nificantly after this attempt at gene therapy. This disease is still being treated with gene  therapy, in combination with replacement enzyme infusion.   In familial hypercholesterolemia, a condition associated with a high incidence  of heart attacks, the low-density lipoprotein (LDL) receptor is deficient.  Attempts to correct this defect with gene therapy involved removal of a seg-  ment of liver and preparation of hepatocytes that had been grown in tissue culture. After  these dividing cells were infected with a retrovirus containing the gene for the LDL recep-  tor, they were reinfused into the hepatic portal vein of the patient. The early efforts using  this approach met with only limited success.   Adenoviruses, which are natural human pathogens, can also be used as vectors.  As in retroviral gene therapy, the normal viral genes required for synthesis of viral  particles are replaced with the therapeutic genes. The advantages to using an aden-  ovirus are that the introduced gene can be quite large (~36 kb), and infection does  not require division of host cells. The disadvantage is that genes carried by the ade-  novirus do not stably integrate into the host genome, resulting in only transient  expression of the therapeutic proteins (but preventing disruption of host genes).  Thus, the treatment must be repeated periodically. Another problem with adenovi-  ral gene therapy is that the host can mount an immune response to the pathogenic  adenovirus, causing complications.   To avoid the problems associated with viral vectors, researchers are employing  treatment with DNA alone or with DNA coated with a layer of lipid (i.e., in lipo-  somes). Adding a ligand for a receptor located on the target cells could aid delivery  of the liposomes to the appropriate host cells. Many problems still plague the field  of gene therapy. In many instances, the therapeutic genes must be targeted to the  cells where they normally function — a difficult task at present. Deficiencies in dom-  inant genes are more difficult to treat than those in recessive genes, and the expres-  sion of the therapeutic genes often needs to be carefully regulated. Although the  field is moving forward, progress is slow.   Another approach to gene therapy involves the use of antisense oligonucleotides  rather than vectors. These oligonucleotides are designed to hybridize either with the  target gene to prevent transcription or with mRNA to prevent translation. Again  technical problems have plagued the development of therapy based on this theoret-  ically promising idea.   The introduction of normal genes into somatic cells with defective genes corrects  the defect only in the treated individuals, not in their offspring. To eliminate the  defect for future generations, the normal genes must be introduced into the germ  cell line (the cells that produce sperm in males or eggs in females). Experiments  with animals indicate that gene therapy in germ cells is feasible. Genes can be intro-  duced into fertilized eggs from which transgenic animals develop, and these trans-  genic animals can produce apparently normal offspring.   In fact, if the nucleus isolated from the cell of one animal is injected into the enu-  cleated egg from another animal of the same species and the egg is implanted in a  foster mother, the resulting offspring is a "clone" of the animal from which the  nucleus was derived (Fig. 17.13). Clones of sheep and pigs have been produced, and  similar techniques could be used to clone humans. Obviously, these experiments  raise many ethical questions that will be difficult to answer.   Adenoviral vectors have been used  in a aerosal spray to deliver normal  copies of the CFTR (cystic fibrosis  transmembrane conductance regulator)  gene to cells of the lung. Some cells took up  this gene, and the patients experienced  moderate improvement. However, stable  integration of the gene into the genome did  not occur, and cells affected by the disease  other than those in the lung (e.g., pancreatic  cells) did not benefit. Nevertheless, this  approach marked a significant forward step  in the development of gene therapy.   In an attempt to treat ornithine  transcarbamoylase deficiency (a  disorder of nitrogen metabolism)  using adenoviral vectors, a volunteer died of  a severe immune response to the vector.  This unfortunate result has led to a revalu-  ation of the safety of viral vectors for gene  therapy.   One approach to in vivo gene ther-  apy involves the direct injection of  DNA for certain HLA antigens into  malignant melanomas (skin cancers). The  HLA gene chosen for therapy should not be  the one expressed by the patient. Thus, if the  gene is incorporated into the cells and  expressed, the body should recognize the  tumor cells as foreign tissue and mount an  immune attack. Preliminary results using  this strategy were encouraging.   Erna Nemdy. In reading about development of the hepatitis B vaccine,  Erna Nemdy learned that the first vaccine available for HBV, marketed in  1982, was a purified and "inactivated" vaccine containing HBV virus that  had been chemically killed. The virus was derived from the blood of known HBV  carriers. Later, "attenuated" vaccines were used in which the virus remained live but  was altered so that it no longer multiplied in the inoculated host. Both the inacti-  vated and the attenuated vaccines are potentially dangerous because they can be  contaminated with live infectious HBV.   The modern "subunit" vaccines, first marketed in 1987, were made by recombi-  nant DNA techniques described earlier in this chapter. Because this vaccine consists  solely of the viral surface protein or antigen to which the immune system responds,  there is no risk for infection with HBV.   0 © The most common CF mutation is  | /^OA J a 3-bp deletion that causes the loss  ™ of phenylalanine at position 508  (delta 508). This mutation is present in more  than 70% of CFTR patients. The defective  protein is synthesized in the endoplasmic  reticulum, but is misfolded. It is therefore  not transported to the Golgi, but is degraded  by a proteolytic enzyme complex called the  proteosome. Other mutations responsible  for CF generate an incomplete mRNA  because of premature stop signals, frame  shifts, or abnormal splice sites or create a  CFTR channel in the membrane that does  not function properly.   ^ Sissy Fibrosa. Cystic fibrosis (CF) is a genetically determined autoso-  ) mal recessive disease that can be caused by a variety of mutations within  the CF gene located on chromosome 7. Sissy Fibrosa was found to have a  3-bp deletion at residue 508 of the CF gene (the mutation present in approximately  70% of Caucasian patients with CF in the United States). This mutation is generally  associated with a more severe clinical course than many other mutations causing the  disease. However, other genes and environmental factors may modify the clinical  course of the disease, so it is not currently possible to counsel patients accurately  about prognosis based on their genotype.   CF is a relatively common genetic disorder in the United States, with a carrier  rate of approximately 5% in Caucasians. The disease occurs in 1 per 1,600 to 2,000  Caucasian births in the country (1 per 17,000 in African Americans and 1 per  100,000 in Asians).   a Carrie Sichel. After learning the results of their tests for the sickle  ) cell gene, Carrie Sichel and her fiance consulted a genetic counselor.  The counselor informed them that, because they were both carriers of  the sickle cell gene, their chance of having a child with sickle cell anemia was  fairly high (approximately 1 in 4). She told them that prenatal testing was avail-  able with fetal DNA obtained from cells by amniocentesis or chorionic villus  sampling. If these tests indicated that the fetus had sickle cell disease, abortion  was a possibility. Carrie, because of her religious background, was not sure that  abortion was an option for her. But having witnessed her brother's sickle cell  crises for many years, she also was not sure that she wanted to risk having a child  with the disease. Her fiance also felt that, at 25 years of age, he was not ready to  deal with such difficult problems. They mutually agreed to cancel their marriage  plans.   Vicky Tim. DNA fingerprinting represents an important advance in  forensic medicine. Before development of this technique, identification of  criminals was far less scientific. The suspect in the rape and murder of  Vicky Tim was arrested and convicted mainly on the basis of the results of DNA  fingerprint analysis.   This technique has been challenged in some courts on the basis of technical prob-  lems in statistical interpretation of the data and sample collection. It is absolutely nec-  essary for all of the appropriate controls to be run, including samples from the victim's  DNA as well as the suspect's DNA. Another challenge to the fingerprinting procedure  has been raised because PCR is such a powerful technique that it can amplify minute  amounts of contaminating DNA from a source unrelated to the case.   Mapping of the Human Genome. The Human Genome Project  began in 1990, and by the summer of 2000, the entire human genome had  been mapped. This feat was accomplished in far less than the expected  time as a result of both cooperative and competitive interactions of laboratories in  the private as well as the public sector.   The human genome contains over 3 X 10 9 (3 billion) bp. A large percentage of  this genome (<95%) does not code for the amino acid sequences of proteins or for  functional RNA (such as rRNA or tRNA) but is composed of repetitive sequences,  introns, and other noncoding elements of unknown function. The human genome is  estimated to contain only about 30,000 to 50,000 genes.   As the announcement of the identification of a wayward gene appears in the  morning newspaper, the average citizen expects the cure for the genetic disease to  be described in the evening edition. Although knowledge of the chromosomal loca-  tion and the sequence of genes will result in the rapid development of tests to deter-  mine whether an individual carries a defective gene, the development of a treatment  for the genetic disease caused by the defective gene will not be that easy or that  rapid. As outlined in the section on gene therapy above, many technical problems  need to be solved before gene therapy becomes commonplace. In addition to solv-  ing the molecular puzzles involved in gene therapy, we also will have to deal with  many difficult questions.   Is it appropriate to replace defective genes in somatic cells to relieve human suf-  fering? Many people may agree with this goal. But there is a related question: is it  appropriate to replace defective genes in the germ cell line to relieve human suf-  fering? Fewer people may agree with this goal. Genetic manipulation of somatic  cells affects only one generation; these cells die with the individual. Germ cells,  however, live on, producing each successive generation.   The techniques developed to explore the human genome could be used for many  purposes. What are the limits for the application of the knowledge gained by advances  in molecular biology? Who should decide what the limits are, and who should serve as  the genetic police? If we permit experiments that involve genetic manipulation of the  human germ cell line, however nobly conceived, could we, in our efforts to "improve"  ourselves, genetically engineer the human race into extinction?   Through the analysis of 1,000 indi-  viduals of different ethnicities, one can  determine the frequency of a particular DNA  polymorphism within that distinct popula-  tion. By matching four or five polymor-  phisms from DNA at the crime scene with  DNA from a suspect, one can determine the  odds of that match happening by chance.  For example, let us assume that a suspect's  DNA was compared with DNA found at the  crime scene for four unique polymorphisms  within the suspect's ethnic group. The fre-  quency of polymorphism A in that popula-  tion is 1 in 20; of polymorphism B, 1 in 30; of  polymorphism C, 1 in 50; and of polymor-  phism D, 1 in 100. The odds of the suspect  DNA matching the DNA found at the crime  scene for all four polymorphisms would be  the product of each individual probability, or  (1/20) X (1/30) X (1/50) X (1/100). This comes  out to a 1 in 3 million chance that an individ-  ual would have the same polymorphisms in  their DNA as that found at the crime scene.  The question left to the courts is whether the  1 in 3 million match is sufficient to convict  the suspect of the crime. Given that there  may be 30 million individuals in the United  States within the same ethnic group as the  suspect, there would then be 30 people  within the country who would match the  DNA polymorphisms found at the scene of  the crime. Can the court be sure that the sus-  pect is the correct individual? Clearly, the  use of DNA fingerprinting is much clearer  when a match is not made, for that immedi-  ately indicates that the suspect was not at  the scene of the crime.   Housman D. DNA on trial — the molecular basis of DNA fingerprinting. N Engl J Med  1995;332:534-535.   Watson JD, Gilman M, Witkowski J, Zoller M. Recombinant DNA. 2nd Ed. New York: Scientific  American Books, WH Freeman, 1992.  • Gelehrter TD, Collins FS, Ginsburg D. Principles of Medical Genetics. 2nd Ed. Baltimore: Williams  &Wilkins, 1998.   Welsh MJ. Cystic Fibrosis. Chapter 76. In: Goldman L, Bennett JC, eds. Cecil Textbook of Medi-  cine. 21st Ed, Volume 1, pp 401-405. Philadelphia: WB Saunders, 2000.   1. Electrophoresis resolves double- stranded DNA fragments based on which of the following?   2. If a restriction enzyme recognizes a six-base sequence, how frequently, on average, will this enzyme cut a large piece of DNA?   3. Which of the following sets of reagents are required for dideoxy chain DNA synthesis in the Sanger technique for DNA  sequencing? .   4. Which of the following statements correctly describe a feature of DNA electrophoresis?   (C) DNA can be visualized using UV light and the dye ethidium bromide.   (D) Total human genomic DNA cut by a specific restriction endonuclease will generate three distinctly separable bands.   (E) DNA must be denatured before it can be run in the gel.   5. The best method to determine whether albumin is transcribed in the liver of a mouse model of hepatocarcinoma is which of  the following?   The term cancer applies to a group of diseases in which cells grow abnormally  and form a malignant tumor. Malignant cells can invade nearby tissues and  metastasize (i.e., travel to other sites in the body, where they establish secondary  areas of growth). This aberrant growth pattern results from mutations in genes  that regulate proliferation, differentiation, and survival of cells in a multicellu-  lar organism. Because of these genetic changes, cancer cells no longer respond to  the signals that govern growth of normal cells (Fig. 18.1.)   Oncogenes and Tumor Suppressor Genes. The genes involved in the develop-  ment of cancer are classified as oncogenes or tumor suppressor genes. Onco-  genes are mutated derivatives of normal genes (proto-oncogenes) whose function  is to promote proliferation or cell survival. These genes can code for growth fac-  tors, growth factor receptors, signal transduction proteins, intracellular kinases,  and transcription factors. The process of transformation into a malignant cell  may begin with a "gain of function" mutation in only one copy of a proto-onco-  gene. As the mutated cell proliferates, additional mutations can occur. Tumor  suppressor genes (normal growth suppressor genes) encode proteins that inhibit  proliferation, promote cell death, or repair DNA; both alleles need to be inacti-  vated for transformation (a loss of function). Growth suppressor genes have been  called the guardians of the cell.   Cell Cycle Suppression and Apoptosis. Normal cell growth depends on a bal-  anced regulation of cell cycle progression and apoptosis (programmed cell death)  by proto-oncogenes and growth suppressor genes. At checkpoints in the cell cycle  products of tumor suppressor genes slow growth in response to signals from the  cell's environment, including external growth inhibitory factors , or to allow time  for repair of damaged DNA, or in response to other adverse circumstances in  cells. Alternately, cells with damaged DNA are targeted for apoptosis so that they  will not proliferate. Many growth- stimulatory pathways involving proto-oncogenes,  and growth-inhibitory controls involving a variety of tumor suppressor genes, con-  verge to regulate the activity of some key protein kinases, the cyclin-dependent  kinases. These kinases act to control progression at specific points in the cell  growth cycle. Apoptosis is initiated by either death receptor activation or intracel-  lular signals leading to release of the mitochondrial protein, cytochrome c.   Mutations. Mutations in DNA that give rise to cancer may be inherited or  caused by chemical carcinogens, radiation, viruses, and by replication errors  that are not repaired. A cell population must accumulate multiple mutations for  transformation to malignant cells.    Fig. 18.1. Development of cancer. Accumula-  tion of mutations in a number of genes results  in transformation. Cancer cells change mor-  phologically, proliferate, invade other tissues,  and metastasize.   Patients with leukemia experience a  variety of hemorrhagic (bleeding)  manifestations caused by a  decreased number of platelets. Platelets are  small cells that initiate clot formation at the  site of endothelial injury. Because of the  uncontrolled proliferation of white cells within  the limited space of the marrow, the normal  platelet precursor cells (the megakaryocytes)  in the marrow are "squeezed" or crowded  and fail to develop into mature platelets. Con-  sequently, the number of mature platelets  (thrombocytes) in the circulation falls, and a  thrombocytopenia develops. Because there  are fewer platelets to contribute to clot forma-  tion, bleeding problems are common.   0 © Malignant neoplasms (new  ( /^oA ) growth, a tumor) of epithelial cell  ™ origin (including the intestinal lin-  ing, cells of the skin, and cells lining the air-  ways of the lungs) are called carcinomas. If  the cancer grows in a gland-like pattern, it is  an adenocarcinoma. Thus, Nick O'Tyne and  Colin Tuma have adenocarcinomas. Mel  Anoma had a carcinoma arising from  melanocytes, which is technically a  melanocarcinoma, but is usually referred to  as a melanoma.   Moles (also called nevi) are tumors  \\ ) of the skin. They are formed by   melanocytes that have been trans-  formed from highly dendritic single cells  interspersed among other skin cells to round  oval cells that grow in aggregates or  "nests." (Melanocytes produce the dark pig-  ment melanin that protects against sunlight  by absorbing UV light.) Additional mutations  may transform the mole into a malignant  melanoma.   Mannie Weitzels has chronic myelogenous leukemia, a disease in which a  single line of myeloid cells in the bone marrow proliferate abnormally,  causing a large increase in the number of nonlymphoid white blood cells  (see Chapter 16). His myeloid cells contain the abnormal Philadelphia chromo-  some, which increases their proliferation. He has recently complained of pain and  tenderness in various areas of his skeleton, possibly stemming from the expanding  mass of myeloid cells within his bone marrow. He also reports a variety of hemor-  rhagic signs, including bruises (ecchymoses), bleeding gums, and the appearance of  small red spots (petechiae caused by release of red cells into the skin).   Nick O'Tyne was diagnosed with a well-differentiated adenocarcinoma of  the lungs (see Chapter 13). He underwent anatomic staging procedures to  determine the location and severity of the tumor. As a result of these tests,  he was considered a candidate for surgical resection of the primary tumor aimed at  cure. He survived the surgery and was recovering uneventfully until 6 months later  when he complained of an increasingly severe right temporal headache. A CT scan  of his brain was performed. Results indicated that the cancer, which had originated  in his lungs, had metastasized to his brain.   Colin Tuma has had an intestinal adenocarcinoma resected, but there were  several metastatic nodules in his liver (see Chapters 12 and 13). He com-  pleted his second course of chemotherapy with 5-fluorouracil (5-FU) and  had no serious side effects. He assured his physician at his most recent checkup that,  this time, he intended to comply with any instructions his physicians gave him. He  ruefully commented that he wished he had returned for regular examinations after  his first round of surgery for benign intestinal polyps.   Mel Anoma returned to his physician after observing a brownish-black  irregular mole on his forearm (see Chapter 13). His physician thought the  mole looked suspiciously like a malignant melanoma, and performed an  excision biopsy (surgical removal for the purposes of biopsy).   Cancer is the term applied to a group of diseases in which cells no longer respond  to normal restraints on growth. Normal cells in the body respond to signals, such as  contact inhibition, that direct them to stop proliferating. Cancer cells do not require  growth stimulatory signals and they are resistant to growth inhibitory signals. They  are also resistant to apoptosis, the programmed death process whereby unwanted or    The study of cells in culture was a great impetus to the study of cancer, because the development of a tumor in animals could  take months. Once cells could be removed from an animal and propagated in a tissue culture dish, the onset of transformation  (the normal cell becoming a cancer cell) could be seen in days.   What are the criteria that distinguish transformed cells from normal cells in culture? The first is the requirement for serum in the cell  culture medium to stimulate growth. Transformed cells have a reduced requirement for serum, approximately 10% that required for nor-  mal cells to grow. The second is the ability to grow without attachment to a supporting matrix (anchorage dependence). Normal cells (such  as fibroblasts, smooth muscle cells) require adherence to a substratum (in this case, the bottom of the plastic dish) and will not grow if  suspended in a soft agar mixture (the consistency of loose jello). Transformed cells, however, have lost this anchorage dependence for  growth. An additional criterion used to demonstrate that cells are truly transformed is that they form tumors when injected into mice lack-  ing an immune system.   irreparably damaged cells self-destruct. They have an infinite proliferative capacity  and do not become senescent (i.e., they are immortalized). Furthermore, they can  grow independently of structural support, such as the extracellular matrix (loss of  anchorage dependence).   A single cell that divides abnormally eventually forms a mass called a tumor. A  tumor can be benign and harmless; the common wart is a benign tumor formed from  a slowly expanding mass of cells. In contrast, a malignant neoplasm (malignant  tumor) is a proliferation of rapidly growing cells that progressively infiltrate,  invade, and destroy surrounding tissue. Tumors develop angiogenic potential, which  is the capacity to form new blood vessels and capillaries. Thus, tumors can gener-  ate their own blood supply to bring in oxygen and nutrients. Cancer cells also can  metastasize, separating from the growing mass of the tumor and traveling through  the blood or lymph to unrelated organs, where they establish new growths of can-  cer cells.   The transformation of a normal cell to a cancer cell begins with damage to DNA  (base changes or strand breaks) caused by chemical carcinogens, UV light, viruses,  or replication errors (see Chapter 13). Mutations result from the damaged DNA if it  is not repaired properly or if it is not repaired before replication occurs. A mutation  that can lead to transformation also may be inherited. When a cell with one muta-  tion proliferates, this clonal expansion (proliferation of cells arising from a single  cell) results in a substantial population of cells containing this one mutation, from  which one cell may acquire a second mutation relevant to control of cell growth or  death. With each clonal expansion, the probability of another transforming mutation  increases. As mutations accumulate in genes controlling proliferation, subsequent  mutations occur even more rapidly until the cells acquire the multiple mutations (in  the range of 4-7) necessary for full transformation.   The transforming mutations occur in genes that regulate cellular proliferation  and differentiation (proto-oncogenes), suppress growth (tumor suppressor genes),  target irreparably damaged cells for apoptosis, or repair damaged DNA. The genes  regulating cellular growth are called proto-oncogenes, and their mutated forms are  called oncogenes. The term oncogene is derived from the Greek word "onkos"  meaning bulk or tumor. A transforming mutation in a proto-oncogene increases the  activity or amount of the gene product (a gain-of-function mutation). Tumor sup-  pressor genes (normal growth suppressor genes) and repair enzymes protect against  uncontrolled cell proliferation. A transforming mutation in these protective genes  results in a loss of activity or a decreased amount of the gene product. In summary,  cancer is caused by the accumulation of mutations in the genes involved in normal  cellular growth and differentiation. These mutations give rise to cancer cells capa-  ble of unregulated, autonomous, and infinite proliferation.   II. DAMAGE TO DNA LEADING TO MUTATIONS  A. Chemical and Physical Alterations in DNA   An alteration in the chemical structure of DNA, or of the sequence of bases in a  gene, is an absolute requirement for the development of cancer. The function of  DNA depends on the presence of various polar chemical groups in DNA bases,  capable of forming hydrogen bonds between DNA strands or other chemical reac-  tions. The oxygen and nitrogen atoms in DNA bases are targets for a variety of elec-  trophiles (electron- seeking chemical groups). A typical sequence of events leading  to a mutation is shown for dimethylnitrosamine in Figure 18.2. Chemical carcino-  gens (compounds that can cause transforming mutations) found in the environment  and ingested in foods are generally stable lipophilic compounds that, like dimethyl-  nitrosamine, must be activated by metabolism in the body to react with DNA (see  also benz[o]pyrene, Action of Mutagens, Chapter 13, section III.A.). Many   Drs. Michael Bishop and Harold  Varmus demonstrated that cancer  is not caused by unusual and novel  genes, but rather by mutation within exist-  ing cellular genes and that for every gene  that causes cancer (an oncogene) there was  a corresponding cellular gene, called the  proto-oncogene. Although this concept  seems straightforward today, it was a signif-  icant finding when it was first announced  and, in 1989, Drs. Bishop and Varmus were  awarded the Nobel Prize in Medicine.   The first experiments to show that  oncogenes were mutant forms of  proto-oncogenes in human tumors  involved cells cultured from a human blad-  der carcinoma. The DNA sequence of the ras  oncogene cloned from these cells differed  from the normal c-ras proto-oncogene. Sim-  ilar mutations were subsequently found in  the ras gene of lung and colon tumors. Colin  Tuma's malignant polyp had a mutation in  the ras proto-oncogene.   Fig. 18.2. Mutations in DNA caused by  nitrosamines. Nitrosamines are consumed in  many natural products and are produced in the  stomach from nitrites used as preservatives and  secondary amines found in foods such as fish.  They are believed to be responsible for the  high incidence of gastric cancer found in Japan  and Iceland, where salt-preserved fish was a  major dietary item. Nitrosamine metabolites  methylate guanine (the transferred methyl  group is shown in blue).   UV rays derived from the skin  induce an increased incidence of  all skin cancers, including squa-  mous cell carcinoma, basal cell carcinoma,  and malignant melanoma of the skin. The  wavelength of UV light most associated with  skin cancer is UVB (280-320 nm), which  forms pyrimidine dimers in DNA. This type  of DNA damage is repaired by nucleotide  excision repair pathways that require prod-  ucts of at least 20 genes. With excessive  exposure to the sun, the nucleotide excision  repair pathway is overwhelmed, and some  damage remains unrepaired.   Burkitt's lymphoma, a general  name for a number of types of B-  cell malignancies, results from a  translocation between chromosomes 8 and  14. The translocation of genetic material  moves the proto-oncogene transcription fac-  tor c-myc (normally found on chromosome  8) to chromosome 14. The translocated gene  is now under the control of the promoter  region for the immunoglobulin heavy chain  gene, which leads to inappropriate and over-  expression of c-myc. The result may be  uncontrolled cell proliferation and tumor  development. All subtypes of Burkitt's lym-  phoma contain this translocation. Epstein-  Barr virus infection of B cells is also associ-  ated with certain types of Burkitt's  lymphoma.   Mannie Weitzels' bone marrow  cells contain the Philadelphia chro-  mosome, typical of chronic myel-  ogenous leukemia (CML). The Philadelphia  chromosome results from a reciprocal  translocation between the long arms of chro-  mosome 9 and 22. As a consequence, a  fusion protein is produced containing the N-  terminal region of the Bcr protein from chro-  mosome 22 and the C-terminal region of the  Abl protein from chromosome 9. Abl is a  proto-oncogene, and the resulting fusion  protein (Bcr-Abl) has lost its regulatory  region and is constitutively active. When  active, Abl stimulates the Ras pathway of sig-  nal transduction, leading to cell proliferation.   The oncogene N-myc (a cell prolif-  eration transcription factor, related  to c-myc) is amplified in some neu-  roblastomas, and amplification of the erb-B2  oncogene (a growth factor receptor) is asso-  ciated with several breast carcinomas.   chemotherapeutic agents, which are designed to kill proliferating cells by interact-  ing with DNA, may also may act as carcinogens and cause new mutations and  tumors while eradicating the old. Structural alterations in DNA also occur through  radiation and through UV light, which causes the formation of pyrimidine dimers.  More than 90% of skin cancers occur in sunlight-exposed areas. Thus, each chem-  ical carcinogen or reactant creates a characteristic modification in a DNA base. The  DNA damage, if not repaired, introduces a mutation into the next generation when  the cell proliferates.   Proto-oncogenes are converted to oncogenes by mutations in the DNA that cause a  "gain- in-f unction," that is, the protein can now function better in the absence of the  normal activating events. Several mechanisms that lead to the conversion of proto-  oncogenes to oncogenes are known:   • Radiation and chemical carcinogens act (a) by causing a mutation in the regula-  tory region of a gene, increasing the rate of production of the proto-oncogene  protein, or (b) by producing a mutation in the coding portion of the oncogene  that results in the synthesis of a protein of slightly different amino acid compo-  sition capable of transforming the cell (Fig. 18. 3 A).   • The entire proto-oncogene or a portion of it may be transposed or translocated,  that is, moved from one position in the genome to another (Fig. 18.3B). In its  new location, the proto-oncogene may be controlled by a more active promoter  and, therefore, overexpressed (increased amounts of the protein product may be  produced). If only a portion of the proto-oncogene is translocated, it may be  expressed as a truncated protein with altered properties, or it may fuse with  another gene and produce a fusion protein containing portions of what normally  were two separate proteins. The truncated or fusion protein would be hyperac-  tive and cause inappropriate cell growth.   • The proto-oncogene may be amplified (Fig. 18.3C), so that multiple copies of  the gene are produced in a single cell. If more genes are active, more proto-  oncogene protein will be produced, increasing the growth rate of the cells.   • If an oncogenic virus infects a cell, its oncogene may integrate into the host cell  genome, permitting production of the abnormal oncogene protein. The cell may  be transformed and exhibit an abnormal pattern of growth. Rather than inserting  an oncogene, a virus may simply insert a strong promoter into the host cell  genome. This promoter may cause an increased or untimely expression of a nor-  mal proto-oncogene.   The important point to remember is that transformation results from abnormali-  ties in the normal growth regulatory program caused by gain-of-function mutations  in proto-oncogenes. However, loss-of-function mutations also must occur in the  tumor suppressor genes, repair enzymes, or activators of apoptosis for full transfor-  mation to a cancer cell.   Repair enzymes are the first line of defense preventing conversion of chemical  damage in DNA to a mutation (see Chapter 13, section III.B). DNA repair enzymes  are tumor suppressor genes in the sense that errors repaired before replication do  not become mutagenic. DNA damage is constantly occurring from exposure to sun-  light, background radiation, toxins, and replication error. If DNA repair enzymes  are absent, mutations accumulate much more rapidly, and once a mutation develops  in a growth regulatory gene, a cancer may arise. As an example, inherited mutations  in the tumor suppressor genes brcal and brca2 predispose women to the develop-  ment of breast cancer.   Proto-oncogene or  a portion of it is  fused with another  gene   Fig. 18.3. Transforming mutations in proto-oncogenes. (A) Effect of radiation or chemical carcinogens on proto-oncogenes or their promoters.  The mutations may be point mutations, deletions, or insertions. (B) Gene rearrangements as caused by transposition or translocation of a proto-  oncogene or proto-oncogene fragment. (C) Amplification of a proto-oncogene allows more protein to be produced. The proto-oncogene and the  resulting oncogene are shown in blue.   The protein products of these genes play roles in DNA repair, recombination,  and regulation of transcription. A second example, HNPCC (hereditary non-poly-  posis colorectal cancer), was previously introduced in Chapter 13. It results from  inherited mutations in enzymes involved in the DNA mismatch repair system.   Proto-oncogenes control normal cell growth and division. These genes encode  proteins that are growth factors, growth factor receptors, signal transduction pro-  teins, transcription factors, cell cycle regulators, and regulators of apoptosis  (Table 18.1). (The name representing the gene of an oncogene is referred to in  lowercase letters and italics [e.g., myc], but the name of the protein product is  capitalized and italics are not used [e.g., Myc]). The mutations in oncogenes  giving rise to transformation are usually gain-of-function mutations; either a  more active protein is produced or an increased amount of the normal protein is  synthesized.    Although mutations in both the  brcal and brca2 genes are linked to  breast cancer development in  women, there are some fundamental differ-  ences in how these genes interact with other  proteins at the molecular level. As a result,  there are some differences in the diseases  expressed by mutations within these genes.  For example, brcal mutations are also linked  to ovarian cancer and brca2 mutations are  not. Brca2 mutations have been linked to  pancreatic cancer, whereas brcal mutations  have not. Men with inherited brca2 muta-  tions develop breast cancer; but men who  carry brcal mutations do not.   Table 18.1 Classes of Oncogenes, Mechanism of Activation, and Associated Human Tumors   Squamous cell carcionoma of the lung  Breast, ovarian, lung, stomach cancer   Multiple cancers including lung, colon,  thyroid, pancreas, many leukemias  Ovarian carcinoma  Myeloid leukemia  Chronic myeloid leukemia  Acute lymphoblastic leukemia  Breast   0 © The gene for the human epidermal  ( /~0A ) growth factor receptor {HER2, c-  ^ erbB-2) is over-expressed in 10 to  20% of breast cancer cases. When this gene is  over-expressed, the prognosis for recovery is  poor, as the patients display shorter disease-  free intervals, increased risks for metastasis,  and a resistance to therapy. Clinical trials are  underway using a monoclonal antibody  directed against this receptor (herceptin) to  inactivate it. Preliminary results are encour-  aging in that use of this drug, either alone or  in combination with others, appears to control  the growth of some tumors overexpressing  the HER2 gene. However, not all tumors over-  expressing HER2 are responsive to herceptin.  Thus, it appears that a complete genotyping  of breast cancer cells may be necessary (using  the microarray techniques described in Chap-  ter 17) to specifically develop an effective ther-  apy for each patient with the disease.   All of the proteins in growth factor signal transduction cascades are proto-onco-  genes (Fig 18.4).   The genes for both growth factors and growth factor receptors are oncogenes.   Growth factors generally regulate growth by serving as ligands that bind to cel-  lular receptors located on the plasma membrane (cell-surface receptors) (see Chap-  ter 11). Binding of ligands to these receptors stimulates a signal transduction path-  way in the cell activating the transcription of certain genes. If too much of a growth  factor or a growth factor receptor is produced, the target cells may respond by pro-  liferating inappropriately. Growth factors receptors may also become oncogenic  through translocation or point mutations in domains affecting binding of the growth  factor, dimerization, kinase activity, or some other aspect of their signal transmis-  sion. In such cases, the receptor transmits a proliferative signal even though the  growth factor normally required to activate the receptor is absent. In other words,  the receptor is stuck in the "on" position.   Fig. 18.4. Proto-oncogene sites for transforming mutations in growth factor signaling path-  ways. I. The amount of growth factor. II. The receptor, which normally must bind the growth  factor to dimerize and activate a kinase domain. III. Signal transduction proteins. Some, such  as PI 3-kinase, form second messengers. IV. G proteins, and their regulators, which are also  signal transduction proteins. V. Nonreceptor protein kinase cascades, which lead to phos-  phorylation of transcription factors. VI. Nuclear transcription factors normally activated  through phosphorylation or binding of a ligand.   The genes encoding proteins involved in growth factor signal transduction cascades  may also be proto-oncogenes. Consider, for example, the monomeric G protein Ras.  Binding of growth factor leads to the activation of Ras (see Fig 11.11). When Ras  binds GTP, it is active, but Ras slowly inactivates itself by hydrolyzing its bound  GTP to GDP and Pi. This controls the length of time that Ras is active. Ras is con-  verted to an oncogenic form by point mutations that decrease the activity of the  GTPase domain of Ras, thereby increasing the length of time it remains in the active  form.   Ras, when active, activates the serine/threonine kinase Raf (a MAP kinase kinase  kinase), which activates MEK (a MAP kinase kinase), which activates MAP kinase  (Fig. 18.5). Activation of MAP kinase results in the phosphorylation of cytoplasmic  and nuclear proteins, followed by increased transcription of the transcription factor  proto-oncogenes myc and fos (see below). Note that mutations in the genes for any  of the proteins that regulate MAP kinase activity, as well as those proteins induced  by MAP kinase activation, can lead to uncontrolled cell proliferation.   Many transcription factors, such as Myc and Fos, are proto-oncoproteins (the prod-  ucts of proto-oncogenes). MAP kinase, in addition to inducing myc and fos, also  directly activates the AP-1 transcription factor through phosphorylation (see  Fig. 18.5). AP-1 is a heterodimer formed by the protein products of ihzfos and jun  families of proto-oncogenes. The targets of AP-1 activation are genes involved in   Fig. 18.5. Phosphorylation cascade leading to  activation of proto-oncogene transcription fac-  tors myc, fos, and jun.    The synthesis of the transcription  factor C-myc is tightly regulated in  normal cells, and it is expressed   only during the S phase of the cell cycle. In a  large number of tumor types, this regulated  expression is lost, and c-myc becomes inap-  propriately expressed or overexpressed  throughout the cell cycle, driving cells con-  tinuously to proliferation.   Fig. 18.6. Cyclin synthesis during different  phases of the cell cycle.   The proteins induced by E2F  rj ) include cyclin E, cyclin A, cdc25A   (an activating protein phos-  phatase), and proteins required to bind at ori-  gins of replication to initiate DNA synthesis.  The synthesis of cyclin E allows it to complex  with cdk2, forming another active cyclin  complex that retains activity into S phase  (see Fig. 18.6). Cyclin A activates Cdk2. Thus,  each phase of the cell cycle activates the next  through cyclin synthesis. The cyclins are  removed by regulated proteolysis.   cellular proliferation and progression through the cell cycle, as are the targets of the  myc transcription factor. The net result is increased production of the proteins that  carry out the processes required for proliferation.   The growth of human cells, involving DNA replication and cell division in the cell  cycle, is activated by growth factors, hormones, and other messengers. These acti-  vators work through cyclins and cyclin- dependent kinases (CDKs) that control pro-  gression from one phase of the cycle to another (Fig 18.6). For quiescent cells to  proliferate, they must leave G 0 and enter the Gl phase of the cell cycle (see Chap-  ter 13, Fig. 13.7). If the proper sequence of events occurs during Gl, the cells enter  the S phase and are committed to DNA replication and cell division. Similarly, dur-  ing G2 cells make a commitment to mitotic division. CDKs are made constantly  throughout the cell cycle but require binding of a specific cyclin to be active. Dif-  ferent cyclins made at different times in the cell cycle control each of the transitions  (Gl/S, S/G2, G2/M).   The activity of the cyclin-CDK complex is further regulated through phospho-  rylation and through inhibitory proteins called cyclin-dependent kinase inhibitors  (CKIs) (Fig. 18.7). CKIs slow cell cycle progression by binding and inhibiting the  CDK-cyclin complexes. CDKs are also controlled through activating phosphoryla-  tion by CAK (cyclin-activating kinases) and inhibitory hyperphosphorylation  kinases.   To illustrate the role of these proteins, consider some of the events occurring at  the Gl/S checkpoint (Fig. 18.8). Because the cell is committed to DNA replication  and division once it enters the S phase, multiple regulatory proteins are involved in  determining whether the cell is ready to pass this checkpoint. These regulatory pro-  teins include cdk4 and cdk6 (which are constitutively produced throughout the cell  cycle), cyclin D (whose synthesis is only induced after growth factor stimulation of  a quiescent cell), the retinoblastoma gene product (Rb), and a class of transcription  factors known collectively as E2F. In quiescent cells, Rb is complexed with E2F,  resulting in inhibition of these transcription factors. On growth factor stimulation,  the cyclin Ds are induced (there are three types of cyclin D; Dl, D2, and D3). They  bind to cdk4 and cdk6, converting them to active protein kinases. One of the targets  of cyclin/cdk phosphorylation is the Rb protein. Phosphorylation of Rb releases it  from E2F, and E2F is then free to activate the transcription of genes required for  entry into S. The Rb protein is a tumor suppressor gene (more below).   Progression through the cell cycle is opposed by the CKIs (see Fig. 18.8). The  CKIs regulating cyclin/cdk expression in the Gl phase of the cell cycle fall into two  categories: the Cip/Kip family and the INK4 family. The Cip/Kip family members  (p21, p27, and p57) have a broad specificity and inhibit all cyclin-CDK complexes.  The INK4 family, which consists of pi 5, pi 6, pi 8, and pi 9, are specific for the  cyclin D-cdk4/6 family of complexes (inhibitors of cyclin-dependent kinase-4).  The regulation of synthesis of different CKIs is complex, but some are induced by  DNA damage to the cell and halt cell cycle progression until the damage can be  repaired. For example, the CKI p21 (a protein of 21,000 Daltons) is a key member  of this group that responds to specific signals to block cell proliferation. If the dam-  age cannot be repaired, an apoptotic pathway is selected, and the cell dies.   0 o In addition to sunlight and a preexisting nevus, hereditary factors also play a  ( /^oA ) role in the development of malignant melanoma. Ten percent of melanomas  ^ tend to run in families. Some of the suspected melanoma-associated genes  include the tumor suppressor gene p16 (an inhibitor of cdk 4) and CDK4. Mel Anoma was  the single child of parents who had died of a car accident in their 50s, and thus, a famil-  ial tendency could not be assessed.   Fig. 18.8. Control of the Gl/S transition in the cell cycle. The genes encoding cyclins and CDKs are oncogenes, and the gene encoding the  Retinoblastoma protein (Rb) is a tumor suppressor gene. Abbreviations: CDK, cyclin- dependent kinase; CDKI, cyclin-dependent kinase inhibitor.   Like the oncogenes, the tumor suppressor genes encode molecules involved in the  regulation of cell proliferation. Table 18.2 provides several examples. The normal  function of tumor suppressor proteins is generally to inhibit proliferation in  response to certain signals such as DNA damage. The signal is removed when the  cell is fully equipped to proliferate; the effect of their elimination of tumor sup-  pressor genes is to remove the brakes on cell growth. They affect cell cycle regula-  tion, signal transduction, transcription, and cell adhesion. The products of tumor  suppressor genes frequently modulate pathways that are activated by the products  of proto-oncogenes.   Stomach cancer  Basal cell carcinoma  Colon cancer  Neurofibrosarcoma  Pancreatic and  colorectal cancer  Wilms tumor  Melanoma,  lung, pancreatic  cancer   Tumor suppressor genes contribute to the development of cancer when both  copies of the gene are inactivated. This is different from the case of proto-oncogene  mutations because only one allele of a proto-oncogene needs to be converted to an  oncogene to initiate transformation.   The two best understood cell cycle regulators that are also tumor suppressors are the  retinoblastoma (rb) and p53 genes.   Inheritance of a mutation in p53  leads to Li-Fraumeni syndrome,  which is characterized by multiple  types of tumors. Mutations in p53 are pres-  ent in more than 50% of human tumors.  These are secondary mutations within the  cell, and if p53 is mutated the overall rate of  cellular mutation will increase because there  is no p53 to check for DNA damage, to initi-  ate the repair of the damaged DNA, or to ini-  tiate apoptosis if the damage is not repaired.  Thus, damaged DNA is replicated, and the  frequency of additional mutations within the  same cell increases remarkably.   As previously discussed, the retinoblastoma gene product, Rb, functions in the tran-  sition from Gl to S phase and regulates the activation of members of the E2F fam-  ily of transcription factors (see Fig. 18.8). If an individual inherits a mutated copy  of the rb allele, there is a 100% chance of that individual developing retinoblastoma,  because of high probability that the second allele of rb will gain a mutation  (Fig. 18.9). This is considered familial retinoblastoma. Individuals who do not  inherit mutations in rb, but who develop retinoblastoma, are said to have sporadic  retinoblastoma, and acquire two specific mutations, one in each rb allele of the  retinoblast, during their lifetime.   The p53 protein is a transcription factor regulating the cell cycle and apoptosis, pro-  grammed cell death. Loss of both p53 alleles is found in over more than 50% of  human tumors. p53 acts as the "guardian of the genome" by halting replication in  cells that have suffered DNA damage and targeting unrepaired cells to apoptosis.    Offspring's cells all contain  one functional and  one nonfunctional  Rb gene   Fig. 18.9. Mutations in the retinoblastoma (Rb) gene. A. Sporadic retinoblastoma. B. Familial retinoblastoma.   Fig. 18.10. p53 and cell cycle arrest. Mechanisms that recognize DNA damage stop p53 degradation and modify the p53 protein (circle 1). p53  stimulates the transcription of p21 (circle 2) and GADD45 (circle 3). p21 blocks the cyclin/CDK phosphorylation of Rb, which continues to  inhibit the E2F family of transcription factors, thereby blocking cell progression through the cell cycle. GADD45 allows the DNA damage to be  repaired. If the damage is not repaired, apoptotic genes are activated (circle 4).   In response to DNA-damaging mutagens, ionizing radiation, or ultraviolet light,  the level of p53 rises (Fig. 18.10, circle 1). p53, acting as a transcription factor,  stimulates transcription of p21 (a member of the Cip/Kip family of CKIs), as shown  in Figure 18.10, circle 2. The p21 gene product inhibits the cyclin/CDK complexes,  which prevents the phosphorylation of Rb and release of E2F proteins. The cell is  thus prevented from entering S phase. P53 also stimulates the transcription of  GADD45 (Growth Arrest and DNA Damage), a DNA repair enzyme (Fig. 18.10,  circle 3). If the DNA is successfully repaired, p53 induces its own downregulation.  If the DNA repair was not successful, p53 activates two apoptosis genes, bax (dis-  cussed below) and IGF-BP3 (Fig. 18.10, circle 4). The IGF-BP3 protein product  binds the receptor for insulin-like growth factor, which presumably induces apop-  tosis by blocking the anti-apoptotic signaling by growth factors, and the cell enters  a growth factor deprivation mode.   Tumor suppressor genes may encode receptors, components of the signaling trans-  duction pathway, or transcription factors.   The Ras family of proteins is involved in signal transduction for many hormones  and growth factors (see above), and is, therefore, oncogenic. The activity of these  pathways is interrupted by GAPs (GTPase-activating proteins; see Chapter 9),  which vary among cell types. Neurofibromin, the product of the tumor suppressor  gene NF-1, is a nervous system- specific GAP that regulates the activity of Ras in  neuronal tissues (Fig. 18.11). The growth signal is transmitted so long as the Ras  protein binds GTP. Binding of NF-1 to Ras activates the GTPase domain of Ras,  which hydrolyzes GTP to GDP, thereby inactivating Ras. Without a functional neu-  rofibromin molecule, Ras is perpetually active.   A good example of tumor suppressors and oncogenes working together is provided  by the co-receptor genes patched and smoothened, which encode the receptor for  the hedgehog class of signaling peptides. These co-receptors normally function to  control growth during embryogenesis and illustrate the importance of maintaining   Fig.18.11. NF-1 (neurofibromin) binds to Ras  and activates its GTPase activity, thereby  decreasing cell proliferation. A mutation in  neurofibromin leads to prolonged GTP binding  and activation of Ras.   An inherited mutation in NF-1 can  lead to neurofibromatosis, a dis-  ease primarily of numerous  benign, but painful, tumors of the nervous  system. The movie Elephant Man was based  on an individual who was believed to have  this disease. Recent analysis of the patient's  remains, however, indicates that he may  have suffered from the rare Proteus syn-  drome, and not neurofibromatosis.   The strange names of some of the  \\ ) tumor suppressor genes arose  *r because they were first discovered  in Drosophila (fruit fly), and the names of  Drosophila mutations are often based on the  appearance of the fly expressing the mutation.  Once the human homolog is found, it is given  the same name as the Drosophila gene.   Fig. 18.12. A. Catenins and cadherins. E-cad-  herin molecules form intercellular, calcium-  dependent homodimers with cadherins from  another cell, resulting in cell-cell adhesion.  The cytoplasmic portion of E-cadherin is com-  plexed to a variety of catenins, which anchor  the cadherin to the actin cytoskeleton. B. p-  Catenin and APC in transcription. The APC  complex activates p-catenin for proteolytic  degradation. If APC is inactivated, p-catenin  levels increase. It acts as a transcription factor  that increases synthesis of myc and other genes  regulating cell cycle progression.   a balance between oncogenes and tumor suppressor genes. The Patched receptor  protein inhibits Smoothened, its co-receptor protein. Binding of a hedgehog ligand  to Patched releases the inhibition of Smoothened, which then transmits an activat-  ing signal to the nucleus, stimulating new gene transcription. Smoothened is a proto-  oncogene, and patched is a tumor suppressor gene. If patched loses its function  (definition of a tumor suppressor), then Smoothened can signal the cell to prolifer-  ate, even in the absence of a hedgehog signal. Conversely, if smoothened undergoes  a gain of function mutation (definition of an oncogene), it can signal in the absence  of the hedgehog signal, even in the presence of Patched. Inherited mutations in  either smoothened or patched will lead to an increased incidence of basal cell  carcinoma.   The cadherin family of glycoproteins mediates calcium- dependent cell-cell adhe-  sion. Cadherins frorm intercellular complexes binding cells together (Fig. 18.12A).  They are anchored intracellularly by catenins, which bind to actin filaments. Loss  of E-cadherin expression may contribute to the ability of cancer cells to detach and  migrate in metastasis. Individuals who inherit a mutation in E cadherin (this muta-  tion is designated as CDH1) are sharply predisposed to developing diffuse type  gastric cancer.   The catenin proteins have two functions; in addition to anchoring cadherins to  the cytoskeleton, they act as transcription factors (Fig. 18.12B). (3-Catenin also  binds to a complex containing the regulatory protein APC (adenomatous polyposis  coli), which activates it for degradation. When the appropriate signal activates APC,  p-catenin levels increase, and it travels to the nucleus, where it activates myc and  cyclin Dl transcription, leading to cell proliferation. APC is a tumor suppressor  gene. If it is inactivated, it cannot bind p-catenin and inhibit cell proliferation.  Mutations in APC or proteins interacting with it are found in the vast majority of  sporadic human colon cancer. Inherited mutations in APC lead to the most common  form of hereditary colon cancer, familial adenomatous polyposis.   In the body, superfluous or unwanted cells are destroyed by a pathway called apop-  tosis, or programmed cell death. Apoptosis is a regulated energy-dependent  sequence of events by which a cell self-destructs. In this suicidal process, the cell  shrinks, the chromatin condenses, and the nucleus fragments. The cell membrane  forms blebs (outpouches), and the cell breaks up into membrane-enclosed apoptotic  vesicles (apoptotic bodies) containing varying amounts of cytoplasm, organelles,  and DNA fragments. Phosphatidylserine, a lipid on the inner leaflet of the cell  membrane, is exposed on the external surface of these apoptotic vesicles. It is one  of the phagocytic markers recognized by macrophages and other nearby phagocytic  cells that engulf the apoptotic bodies.   Apoptosis is a normal part of multiple processes in complex organisms: embryo-  genesis, the maintenance of proper cell number in tissues, the removal of infected  or otherwise injured cells, the maintenance of the immune system, and aging. It can  be initiated by injury, radiation, free radicals or other toxins, withdrawal of growth   A form of apoptosis is a normal part of embryonic development. For example,  the development of the nervous system uses apoptosis to destroy neurons that  have not made the proper connections with target cells. Neurons are produced  in excess, and more than 50% of developing neurons are eliminated by programmed cell  death. Those neurons that have made the correct connections survive by secreting  growth factors that block apoptosis.   factors or hormones, binding of pro-apoptotic cytokines, or interactions with cyto-  toxic T cells in the immune system. Apoptosis can protect organisms from the neg-  ative effect of mutations by destroying cells with irreparably damaged DNA before  they proliferate. Just as an excess of a growth signal can produce an excess of  unwanted cells, the failure of apoptosis to remove excess or damaged cells can  contribute to the development of cancer.   Apoptosis can be divided into three general phases: an initiation phase, a signal  integration phase, and an execution phase. Apoptosis can be initiated by external  signals that work through death receptors, such as tumor necrosis factor (TNF), or  deprivation of growth hormones (Fig. 18.13). It can also be initiated by intracellu-  lar events that affect mitochondrial integrity (e.g., oxygen deprivation, radiation),  and irreparably damaged DNA. In the signal integration phase, these pro-apoptotic  signals are balanced against anti-apoptotic cell survival signals by several pathways,  including members of the Bcl-2 family of proteins. The execution phase is carried  out by proteolytic enzymes called caspases.   Caspases are cysteine proteases that cleave peptide bonds next to an aspartate  residue. They are present in the cell as procaspases, zymogen-type enzyme precur-  sors activated by proteolytic cleavage of the inhibitory portion of their polypeptide  chain. The different caspases are generally divided into two groups according to  their function: initiator caspases, which specifically cleave other procaspases, and  execution caspases, which cleave other cellular proteins involved in maintaining  cellular integrity (see Fig. 18.13). The initiator caspases are activated through two  major signaling pathways; the death receptor pathway and the mitochondrial  integrity pathway. They activate the execution caspases, which cleave protein  kinases involved in cell adhesion, lamins that form the inner lining of the nuclear  envelope, actin and other proteins required for cell structure, and DNA repair  enzymes. They also cleave an inhibitor protein of the endonuclease CAD (caspase-  activated DNase). With destruction of the nuclear envelope, additional endonucle-  ases (Ca 2+ - and Mg 2+ -dependent) also become activated.   The death receptors are a subset of TNF- 1 receptors, which includes Fas/CD95, TNF-  Receptor 1 (TNF-R1) and Death Receptor 3 (DR3). These receptors form a trimer that  binds TNF-1 or another death ligand on its external domain and adaptor proteins to its  intracellular domain (Fig. 18. 14). The activated TNF-receptor complex forms the scaf-  fold for binding two molecules of procaspase 8, which autocatalytically cleave each  other to form active caspase 8. Caspase 8 is an initiator caspase that activates execution  caspases 3, 6, and 7. Caspase 8 also cleaves a Bcl-2 protein, Bid, to a form that acti-  vates the mitochondrial integrity pathway to apoptosis.   Apoptosis is also induced by intracellular signals indicating that cell death should  occur. Examples of these signals include growth factor withdrawal, cell injury, the  release of certain steroids, and an inability to maintain low levels of intracellular  calcium. All of these treatments, or changes, lead to release of cytochrome c from  the mitochondria (Fig. 18.15). Cytochrome c is a necessary protein component of  the mitochondrial electron transport chain that is a loosely bound to the outside of  the inner mitochondrial membrane. Its release initiates apoptosis.   Fig. 18.14. The death receptor pathway to apoptosis. The ligand (usually a cell surface protein on another cell) binds to the death receptor, which  makes a scaffold for autocatalytic activation of caspase 8. Active caspase 8 directly cleaves apoptotic execution caspases. However, the pathway  also activates Bid, which acts on mitochondrial membrane integrity.   In the cytosol, cytochrome c binds Apaf (pro-apoptotic protease activating fac-  tor). The Apaf/cytochrome c complex binds caspase 9, an initiator caspase, to form  an active complex called the apoptosome. The apoptosome in turn activates execu-  tion caspases by zymogen cleavage.   Roughly 30 Bcl-2 family members are currently  known. These proteins play tissue-specific as well as  signal pathway-specific roles in regulating apoptosis.  The tissue-specificity is overlapping. For example,  Bcl-2 is expressed in hair follicles, kidney, small intes-  tines, neurons, and the lymphoid system, whereas  Bcl-x is expressed in the nervous system and  hematopoietic cells.   4. INTEGRATION OF PRO- AND ANTI-APOPTOTIC SIGNALS  BY THE BCL-2 FAMILY OF PROTEINS   The Bcl-2 family members are decision-makers that integrate prodeath and anti-  death signals to determine whether the cell should commit suicide. Both pro-apop-  totic and anti-apoptotic members of the Bcl-2 family exist (Table 18.3).   The antiapoptotic Bcl-2 -type proteins (including Bcl-2, Bcl-xL, Bcl-wL) have  at least two ways of antagonizing death signals. They insert into the outer mito-  chondrial membrane to antagonize channel-forming pro-apoptotic factors, therby  decreasing cytochrome c release. They may also bind cytoplasmic Apaf so that it  cannot form the apoptosome complex (Fig. 18.16).   These anti-apoptotic Bcl-2 proteins are opposed by pro-apoptotic family members  that fall into two categories: ion-channel forming members and the BH3-only mem-  bers. The pro-death ion channel forming members, such as Bax, are very similar to the  anti-apoptotic family members, except that they do not contain the binding domain for  Apaf. They have the other structural domains, however, and when they dimerize with   Q o When Bcl-2 is mutated, and oncogenic, it is usually overexpressed, for exam-  | /^OA J pie, in follicular lymphoma and CML (chronic myelogenous leukemia). Overex-  ™ pression of Bcl-2 disrupts the normal regulation of pro and anti-apoptotic fac-  tors and tips the balance to an anti-apoptotic stand. This leads to an inability to destroy  cells with damaged DNA, such that mutations can accumulate within the cell. Bcl-2 is  also a multi-drug resistance protein and if over-expressed will block the induction of  apoptosis by antitumor agents by rapidly removing them from the cell. Thus, strategies  are being developed to reduce Bcl-2 levels in tumors over-expressing it before initiating  drug or radiation treatment.   pro-apoptotic BH3-only members in the outer mitochondrial membrane, they form an  ion channel that promotes cytochrome c release rather than inhibiting it (see Fig.  18.16). The pro-death BH3-only proteins (e.g., Bim and Bid) contain only the struc-  tural domain that allows them to bind to other bcl-2 family members (the BH3  domain), and not the domains for binding to the membrane or forming ion channels.  Their binding activates the pro-death family members and inactivates the anti-apoptotic  members. When the cell receives a signal from a pro-death agonist, a BH3 protein like  Bid is activated (see Fig. 18.16). The BH3 protein activates Bax (an ion-channel form-  ing pro-apoptotic channel member), which stimulates release of cytochrome c. Nor-  mally Bcl-2 acts as a death antagonist by binding Apaf and keeping it in an inactive  state. However, at the same time that Bid is activating Bax, Bid also binds to Bcl-2,  thereby disrupting the Bcl-2/Apaf complex and freeing Apaf to bind to released  cytochrome c to form the apoptosome.   Apoptosis should be triggered by a number of stimuli, such as withdrawal of growth  factors, elevation of p53 in response to DNA damage, monitoring of DNA damage  by repair enzymes, or by release of TNF or other immune factors. However, muta-  tions in oncogenes can create apoptosis-resistant cells.   One of the ways this occurs is through activation of growth factor-dependent  signaling pathways that inhibit apoptosis, such as the PDGF/Akt/BAD pathway.  Nonphosphorylated BAD acts like Bid in promoting apoptosis (see Fig. 18.16).  Binding of the platelet-derived growth factor to its receptor activates PI-3 kinase,  which phosphorylates and activates the serine/threonine kinase Akt (protein kinase  B, see Chapter 11, section III.B.3). Activation of Akt results in the phosphorylation  of the pro-apoptotic BH3-only protein BAD, which inactivates it. The  PDGF/Akt/BAD pathway illustrates the requirement of normal cells for growth fac-  tor stimulation to prevent cell death. One of the features of neoplastic transforma-  tion is the loss of growth factor dependence for survival.   Fig. 18.15. The mitochondrial integrity path-  way releases cytochrome c, which binds to  Apaf and forms a multimeric complex called  the apoptosome. The apoptosome converts  procaspase 9 to active caspase, which it  releases into the cytosol.   The MAP kinase pathway is also involved in regulating apoptosis and sends  cell survival signals. MAP kinase kinase phosphorylates and activates another  protein kinase known as RSK. Like Akt, RSK phosphorylates BAD and inhibits  its activity. Thus, BAD acts as a site of convergence for the PI-3 kinase/Akt and MAP  kinase pathways in signaling cell survival. Gain-of-function mutations in the genes con-  trolling these pathways, such as ras, creates apoptosis- resistant cells.   Fig. 18.16. Roles of the Bcl-2 family members in regulating apoptosis. Bcl-2, which is anti-  apoptotic, binds Bid (or tBid) and blocks formation of channels that allow cytochrome c  release from the mitochondria. Death signals result in activation of a BH3-only protein such  as Bid, which can lead to mitochondrial pore formation, swelling, and release of cytochrome c.  Bid binds to and activates the membrane ion-channel protein Bax, activating cytochrome c  release, which binds to Apaf and leads to formation of the apoptosome.   Fig. 18.17. Possible steps in the development  of colon cancer. The changes do not always  occur in this order, but the most benign tumors  have the lowest frequency of mutations, and  the most malignant have the highest frequency.   Cancer takes a long time to develop in humans because multiple genetic alter-  ations are required to transform normal cells into malignant cells (see Fig. 18.1).  A single change in one oncogene or tumor suppressor gene in an individual cell is  not adequate for transformation. For example, if cells derived from biopsy speci-  mens of normal cells are not already "immortalized," that is, able to grow in cul-  ture indefinitely, addition of the ras oncogene to the cells is not sufficient for trans-  formation. However, additional mutations in a combination of oncogenes, for  example ras and myc, can result in transformation (Fig. 18.17). Epidemiologists  have estimated that four to seven mutations are required for normal cells to be  transformed.   Cells accumulate multiple mutations through clonal expansion. When DNA  damage occurs in a normally proliferative cell, a population of cells with that muta-  tion is produced. Expansion of the mutated population enormously increases the  probability of a second mutation in a cell containing the first mutation. After one or  more mutations in proto-oncogenes or tumor suppressor genes, a cell may prolifer-  ate more rapidly in the presence of growth stimuli and with further mutations grow  autonomously, that is, independent of normal growth controls. Enhanced growth  increases the probability of further mutations. Some families have a strong predis-  position to cancer. Individuals in these families have inherited a mutation or dele-  tion of one allele of a tumor suppressor gene, and as progeny of that cell prolifer-  ate, mutations can occur in the second allele, leading to a loss of control of cellular  proliferation. These familial cancers include familial retinoblastoma, familial ade-  nomatous polyps of the colon, and multiple endocrine neoplasia, one form of which  involves tumors of the thyroid, parathyroid, and adrenal medulla (MEN type II).   Studies of benign and malignant polyps of the colon show that these tumors have  a number of different genetic abnormalities. The incidence of these mutations  increases with the level of malignancy. In the early stages, normal cells of the intes-  tinal epithelium proliferate, develop mutations in the APC gene, and polyps  develop. This change is associated with a mutation in the ras proto-oncogene that  converts it to an active oncogene. Progression to the next stage is associated with a  deletion or alteration of a tumor suppressor gene on chromosome 5. Subsequently,  mutations occur in chromosome 18, inactivating a gene that may be involved in cell  adhesion, and in chromosome 17, inactivating the p53 tumor suppressor gene. The   Nick O'Tyne had been smoking for 40 years before he developed lung cancer. The fact that cancer takes so long to develop has  made it difficult to prove that the carcinogens in cigarette smoke cause lung cancer. Studies in England and Wales show that cig-  arette consumption by men began to increase in the early 1900s. Followed by a 20-year lag, the incidence in lung cancer in men  also began to rise. Women began smoking later, in the 1920s. Again the incidence of lung cancer began to increase after a 20-year lag.   Fig. 18.18. Estimated cancer deaths by site and sex. Data from The American Cancer Soci-  ety, Inc: Cancer Facts and Figures, 2003.   cells become malignant, and further mutations result in growth that is more aggres-  sive and metastatic. This sequence of mutations is not always followed precisely,  but an accumulation of mutations in these genes is found in a large percentage of  colon carcinomas.   More than 20% of the deaths in the United States each year are caused by cancer,  with tumors of the lung, large intestine, and the breast being the most common  (Fig. 18.18). Different cell types typically use different mechanisms through which  they lose the ability to control their own growth. An examination of the genes  involved in the development of cancer shows that a particular type of cancer can  arise in multiple ways. For example, Patched and Smoothened are the receptor and  co-receptor for the signaling peptide, sonic hedgehog. Either mutation of  smoothened, an oncogene, or inactivation of patched, a tumor suppressor gene, can  give rise to basal cell carcinoma. Similarly, transforming growth factor (3 and its sig-  nal transduction proteins SMAD4/DPC are part of the same growth-inhibiting path-  way, and either may be absent in colon cancer. Thus, treatments which are success-  ful for one patient with colon cancer may not be successful in a second patient with  colon cancer because of the differences in the molecular basis of each individual's  disease (this now also appears to be the case with breast cancer as well). Medical  practice in the future will require identifying the molecular lesions involved in a  particular disease and developing appropriate treatments accordingly. The use of  gene chip technology (see Chapter 17) to genotype tumor tissues will aid greatly in  allowing patient specific treatments to be developed.    A new treatment for CML based on  rational drug design was recently  introduced. The fusion protein Bcr-  Abl is found only in the transformed cells  expressing the Philadelphia chromosome  and not in normal cells. Once the structure of  Bcr-Abl was determined, the drug Gleevec  was designed to specifically bind to and  inhibit only the active site of the fusion pro-  tein and not the normal protein. Gleevec was  successful in blocking Bcr-Abl function,  thereby stopping cell proliferation, and in  some cells would induce apoptosis, so the  cells would die. Because normal cells do not  express the hybrid protein, they were not  affected by the drug. The problem with this  treatment is that some patients suffered  relapses, and when their Bcr-Abl proteins  were studied it was found in some patients  that the fusion protein had a single amino  acid substitution near the active site that pre-  vented Gleevec from binding to the protein.  Other patients had an amplification of the  Bcr-Abl gene product. Thus, Gleevec is a  promising first step in designing drugs  specifically targeted to tumor cells and is  leading the way for rational drug design in  the treatment of cancer.   Mannie Weitzels. The treatment of a symptomatic patient with  CML whose white blood cell count is in excess of 50,000 cells/mL is  usually initiated with busulfan. Alkylating agents such as cyclophos-   phamide have been used alone or in combination with busulfan. Purine and  pyrimidine antagonists and hydroxyurea (an inhibitor of the enzyme ribonu-  cleotide reductase, which converts ribonucleotides to deoxyribonucleotides for  DNA synthesis) are sometimes effective in CML as well. In addition, trials with  both 7- and p -interferon have shown promise in increasing survival in these  patients. Interestingly, the latter agents have been associated with the disappear-  ance of the Philadelphia chromosome in dividing marrow cells of some patients  treated in this way.   The TNM system standardizes the  \\ ) classification of tumors. The T  stands for the stage of tumor (the  higher the number, the worse the progno-  sis), the N stands for the number of lymph  nodes that are affected by the tumor (again,  the higher the number, the worse the prog-  nosis), and M stands for the presence of  metastasis (0 for none, 1 for the presence of  metastatic cells).   ^ Nick O' Tyne. Surgical resection of the primary lung cancer with an  ) attempt at cure was justified in Nick O'Tyne, who had a good prognosis  with a TjjN^Mq staging classification preoperatively. Without some evi-  dence of spread to the central nervous system at that time, a preoperative CT  scan of the brain would not have been justified. This conservative approach  would require scanning of all of the potential sites for metastatic disease from a  non-small cell cancer of the lung in all patients who present in this way. In an  era of runaway costs of health care delivery, such an approach could not be con-  sidered cost-effective.   Unfortunately, Mr. O'Tyne developed a metastatic lesion in the right tempo-  ral cortex of his brain. Because metastases were almost certainly present in other  organs, Mr. O'Tyne's brain tumor was not treated surgically. In spite of palliative  radiation therapy to the brain, Mr. O'Tyne succumbed to his disease just 9  months after its discovery, an unusually virulent course for this malignancy. On  postmortem examination, it was found that his body was riddled with metastatic  disease.   Mutations associated with malig-  nant melanomas include ras (gain-  of-function in growth signal trans-  duction oncogene), p53 (loss of function of  tumor suppressor gene), p16 (loss of func-  tion in Cdk inhibitor tumor suppressor  gene), Cdk4 (gain of function in a cell cycle  progression oncogene) and cadherin/p-  catenin regulation (loss of regulation that  requires attachment).   ^\ Colin Tuma. Colin Tuma requires yearly colonoscopies to check for  ) new polyps in his intestinal tract. Because the development of a metastatic  adenoma requires a number of years (because of the large numbers of  mutations that must occur), yearly checks will enable new polyps to be identified  and removed before malignant tumors develop.   Mel Anoma. The biopsy of Mel Anoma's excised mole showed that it  was not malignant. The most important clinical sign of a malignant  melanoma is a change in color in a pigmented lesion. Unlike benign  (nondysplastic) nevi, melanomas exhibit striking variations in pigmentation,  appearing in shades of black, brown, red, dark blue, and gray. Additional clinical  warning signs of a melanoma are: enlargement of a preexisting mole, itching or pain  in a preexisting mole, development of a new pigmented lesion during adult life, and  irregularity of the borders of a pigmented lesion. Mel Anoma was advised to con-  duct a monthly self-examination, to have a clinical skin examination once or twice  yearly, to avoid sunlight, and to use appropriate sunscreens.   Viruses and human cancer. Three RNA retroviruses are associated  with the development of cancer in humans: HTLV-1, HIV, and hepatitis C.  There are also DNA viruses associated with cancer.   HTLV-1- HTLV-1 causes adult T-cell leukemia. The HTLV-1 genome  encodes a protein Tax, which is a transcriptional coactivator. The cellular  proto-oncogenes c-sis and c-fos are activated by Tax, thereby altering the  normal controls on cellular proliferation and leading to malignancy. Thus, tax is a  viral oncogene without a counterpart in the host cell genome.   HIV. Infection with HIV, the virus causing acquired immunodeficiency  disease (AIDS), leads to the development of neoplastic disease through  several mechanisms. HIV infection leads to immunosuppression and, con-  sequently, loss of immune-mediated tumor surveillance. HIV-infected individuals  are predisposed to non-Hodgkins lymphoma, which results from an overproduction  of T cell lymphocytes. The HIV genome encodes a protein, Tat, a transcription fac-  tor that activates transcription of the interleukin-6 and interleukin-10 genes in  infected T cells. IL-6 and IL-10 are growth factors that promote proliferation of T  cells and, thus, their increased production may contribute to the development of  non-Hodgkins lymphoma. Tat can also be released from infected cells and act as an  angiogenic (blood vessel forming) growth factor. This property is thought to con-  tribute to the development of Kaposi's sarcoma.   DNA viruses. Some DNA viruses also cause human cancer, but by dif-  ferent mechanisms. Three DNA tumor virus families, SV40, papillo-  mavirus, and adenovirus, encode proteins that inactivate pRb and p53. By  interfering with the Gl/S checkpoint, these oncoproteins increase the probability  that mutations in oncogenes and tumor suppressor genes will be incorporated into  the genome of infected cells, thereby increasing the probability of transformation.  The Epstein-Barr virus encodes a Bcl-2 protein that restricts apoptosis of the  infected cell.   Culotta E, Koshland D Jr. Molecule of the year: p53 sweeps through cancer research. Science  1993;262:1958-1959.   Tamm I, Schriever F, and Dorhen B. Apoptosis: implications of basic research for clinical oncology. The   Lancet Oncology 2001;2:33-42.  Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal tumor development.   N Engl J Med 1988;319:525.  Bertram JS. The molecular biology of cancer. Molecular Aspects Med 2000;21:167-223.  Alberts B, Johnson A, Lewis J, Raff M, et al. Cancer. Chapter 23. In Molecular Biology of the Cell, 4th   Ed. New York: Garland Scientific, 2002:1313-1362.  Weinberg RA. How cancer arises. Sci Am 1996:275:62-70.   1. The ras oncogene in Colin Tuma's malignant polyp differs from the c-ras proto-oncogene only in the region that encodes the  N-terminus of the protein. This portion of the normal and mutant sequences is shown below:   This mutation is similar to the mutation found in the ras oncogene in various tumors. What type of mutation converts the ras  proto-oncogene to an oncogene?   (A) An insertion that disrupts the reading frame of the protein   (B) A deletion that disrupts the reading frame of the protein   (C) A mis sense mutation that changes one amino acid within the protein   (D) A silent mutation that has no change in amino acid sequence of the protein   (E) An early termination that creates a stop codon in the reading frame of the protein   336 SECTION THREE / GENE EXPRESSION AND THE SYNTHESIS OF PROTEINS   2. The mechanism through which Ras becomes an oncogenic protein is which of the following?   3. Which of the following statements best describes a characteristic of oncogenes?   (B) Retroviral oncogenes were originally obtained from a cellular host chromosome.   (C) Proto-oncogenes are genes, found in retroviruses, that have the potential to transform normal cells when inappropriately  expressed.   (D) The oncogenes that lead to human disease are different from those that lead to tumors in animals.   4. When p53 increases in response to DNA damage, which of the following events occur?   5. A tumor suppressor gene is best described by which of the following?   (C) When expressed, the gene suppresses viral genes from being expressed.   All physiologic processes in living cells require energy transformation.  Cells convert the chemical bond energy in foods into other forms,  such as an electrochemical gradient across the plasma membrane, or  the movement of muscle fibers in an arm, or assembly of complex  molecules such as DNA (Fig. 1). These energy transformations can be  divided into three principal phases: (1) oxidation of fuels (fat, carbo-  hydrate, and protein), (2) conversion of energy from fuel oxidation into the high-  energy phosphate bonds of ATP, and (3) utilization of ATP phosphate bond energy  to drive energy-requiring processes.   The first two phases of energy transformation are part of cellular respiration, the  overall process of using 0 2 and energy derived from oxidizing fuels to generate  ATP. We need to breathe principally because our cells require 0 2 to generate ade-  quate amounts of ATP from the oxidation of fuels to C0 2 Cellular respiration uses  over 90% of the 0 2 we inhale.   In phase 1 of respiration, energy is conserved from fuel oxidation by enzymes  that transfer electrons from the fuels to the electron-accepting coenzymes NAD +  and FAD, which are reduced to NADH and FAD(2H), respectively (Fig. 2). The  pathways for the oxidation of most fuels (glucose, fatty acids, ketone bodies, and  many amino acids) converge in the generation of the activated 2-carbon acetyl  group in acetyl Co A. The complete oxidation of the acetyl group to C0 2 occurs in  the tricarboxylic acid (TCA) cycle, which collects the energy mostly as NADH and  FAD(2H).   In phase 2 of cellular respiration, the energy derived from fuel oxidation is con-  verted to the high-energy phosphate bonds of ATP by the process of oxidative phos-  phorylation (see Fig. 2). Electrons are transferred from NADH and FAD(2H) to 0 2  by the electron transport chain, a series of electron transfer proteins that are located  in the inner mitochondrial membrane. Oxidation of NADH and FAD(2H) by 0 2  generates an electrochemical potential across the inner mitochondrial membrane in  the form of a transmembrane proton gradient (Ap). This electrochemical potential  drives the synthesis of ATP form ADP and Pi by a transmembrane enzyme called  ATP synthase (or FoFiATPase).   In phase 3 of cellular respiration, the high-energy phosphate bonds of ATP are  used for processes such as muscle contraction (mechanical work), maintaining low  intracellular Na + concentrations (transport work), synthesis of larger molecules  such as DNA in anabolic pathways (biosynthetic work), or detoxification (bio-  chemical work). As a consequence of these processes, ATP is either directly or indi-  rectly hydrolyzed to ADP and inorganic phosphate (Pi), or to AMP and pyrophos-  phate (PPi).   Cellular respiration occurs in mitochondria (Fig. 3). The mitochondrial matrix,  which is the compartment enclosed by the inner mitochondrial membrance, con-  tains almost all of the enzymes for the TCA cycle and oxidation of fatty acids,    Fig. 1. Energy transformations in fuel metab-  olism. When ATP energy is transformed into  cellular responses, such as muscle contraction,  ATP is cleaved to ADP and Pi (inorganic phos-  phate). In cellular respiration, 0 2 is used for  regenerating ATP from oxidation of fuels to   Definitions of prefixes and suffixes  used in describing clinical condi-  tions:  Without  Blood  Excessive,   ketone bodies, and most amino acids. The inner mitochondrial membrane contains  the protein complexes of the electron transport chain and ATP synthase, the enzyme  complex that generates ATP from ADP and Pi. Some of the subunits of these com-  plexes are encoded by mitochondrial DNA, which resides in the matrix. ATP is gen-  erated in the matrix, but most of the energy-using processes in the cell occur out-  side of the mitochondrion. As a consequence, newly generated ATP must be  continuously transported to the cytosol by protein transporters in the impermeable  inner mitochondrial membrane and by diffusion through pores in the more perme-  able outer mitochondrial membrane.   The rates of fuel oxidation and ATP utilization are tightly coordinated through  feedback regulation of the electron transport chain and the pathways of fuel oxida-  tion. Thus, if less energy is required for work, more fuel is stored as glycogen or fat  in adipose tissue. The basal metabolic rate (BMR), caloric balance, and AG (the  change in Gibbs free energy, which is the amount of energy available to do useful  work) are quantitative ways of describing energy requirements and the energy that  can be derived from fuel oxidation. The various types of enzyme regulation  described in Chapter 9 are all used to regulate the rate of oxidation of different fuels  to meet energy requirements.   Fatty acids are a major fuel in the body. After eating, we store excess fatty acids  and carbohydrates that are not oxidized as fat (triacylglycerols) in adipose tissue.  Between meals, these fatty acids are released and circulate in blood bound to albu-  min. In muscle, liver, and other tissues, fatty acids are oxidized to acetyl CoA in the  pathway of p -oxidation. NADH and FAD(2H) generated from (3 -oxidation are reox-  idized by 0 2 in the electron transport chain, thereby generating ATP (see Fig. 2).  Small amounts of certain fatty acids are oxidized through other pathways that con-  vert them to either oxidizable fuels or urinary excretion products (e.g., peroxisomal  p -oxidation).   Not all acetyl CoA generated from (3-oxidation enters the TCA cycle. In the liver,  acetyl CoA generated from (3 -oxidation of fatty acids can also be converted to the   ketone bodies acetoacetate and p-hydroxybutyrate. Ketone bodies are taken up by  muscle and other tissues, which convert them back to acetyl Co A for oxidation in  the TCA cycle. They become a major fuel for the brain during prolonged fasting.   Amino acids derived from dietary or body proteins are also potential fuels that  can be oxidized to acetyl CoA, or converted to glucose and then oxidized (see  Fig. 2). These oxidation pathways, like those of fatty acids, generate NADH or  FAD(2H). Ammonia, which can be formed during amino acid oxidation, is toxic. It  is therefore converted to urea in the liver and excreted in the urine. There are more  than 20 different amino acids, each with a somewhat different pathway for oxida-  tion of the carbon skeleton and conversion of its nitrogen to urea. Because of the  complexity of amino acid metabolism, use of amino acids as fuels is considered  separately in Section Seven, Nitrogen Metabolism.   Glucose is a universal fuel used to generate ATP in every cell type in the body  (Fig. 4). In glycolysis, 1 mole of glucose is converted to 2 moles of pyruvate and 2  moles of NADH by cytosolic enzymes. Small amounts of ATP are generated when  high-energy pathway intermediates transfer phosphate to ADP in a process termed  substrate level phosphorylation. In aerobic glycolysis, the NADH produced from  glycolysis is reoxidized by 0 2 via the electron transport chain, and pyruvate enters  the TCA cycle. In anaerobic glycolysis, the NADH is reoxidized by conversion of  pyruvate to lactate, which enters the blood. Although anaerobic glycolysis has a low  ATP yield, it is important for tissues with a low oxygen supply and few mitochon-  dria (e.g., the kidney medulla), or tissues experiencing diminished blood flow  (ischemia).   All cells continuously use ATP and require a constant supply of fuels to provide  energy for the generation of ATP. Chapters 1 through 3 of this text outline the basic  patterns of fuel utilization in the human and provide information about dietary com-  ponents.   The pathologic consequences of metabolic problems in fuel oxidation can be  grouped into 2 categories: (1) lack of a required product, or (2) excess of a sub-  strate or pathway intermediate. The product of fuel oxidation is ATP, and an inad-  equate rate of ATP production occurs under a wide variety of medical conditions.  Extreme conditions that interfere with ATP generation from oxidative phosphory-  lation, such as complete oxygen deprivation (anoxia), or cyanide poisoning, are  fatal. A myocardial infarction is caused by a lack of adequate blood flow to regions  of the heart (ischemia), thereby depriving cardiomyocytes of oxygen and fuel.  Hyperthyroidism is associated with excessive heat generation from fuel oxidation,  and in hypothyroidism, ATP generation can decrease to a fatal level. Conditions  such as malnutrition, anorexia nervosa, or excessive alcohol consumption may  decrease availability of thiamine, Fe 2+ , and other vitamins and minerals required  by the enzymes of fuel oxidation. Mutations in mitochondrial DNA or nuclear  DNA result in deficient ATP generation from oxidative metabolism.   In contrast, problems arising from an excess of substrate or fuel are seen in dia-  betes mellitus, which may result in a potentially fatal ketoacidosis. Lactic acidosis  occurs with problems of oxidative metabolism.   Fig. 3. Oxidative metabolism in mitochondria.  The inner mitochondrial membrane forms  infoldings, called cristae, which enclose the  mitochondrial matrix. Most of the enzymes for  the TCA cycle, the p-oxidation of fatty acids,  and for mitochondrial DNA synthesis are  found in the matrix. ATP synthase and the pro-  tein complexes of the electron transport chain  are embedded in the inner mitochondrial mem-  brane. The outer mitochondrial membrane is  permeable to small ions, but the inner mito-  chondrial membrane is impermeable.   Fig. 4. Glycolysis. In glycolysis, glucose is  converted to pyruvate. If the pyruvate is  reduced to lactate, the pathway does not  require 0 2 and is called anaerobic glycolysis. If  this pyruvate is converted instead to acetyl  CoA and oxidized in the TCA cycle, glycoly-  sis requires 0 2 and is aerobic.   The ATP-ADP cycle. In cells, the chemical bond energy of fuels is trans-  formed into the physiologic responses necessary for life. The central role of the  high-energy phosphate bonds of ATP in these processes is summarized in the  ATP-ADP cycle (Fig. 19.1). To generate ATP through cellular respiration, fuels  are degraded by oxidative reactions that transfer most of their chemical bond  energy to NAD + and FAD to generate the reduced form of these coenzymes,  NADH and FAD(2H). When NADH and FAD(2H) are oxidized by 0 2 in the elec-  tron transport chain, the energy is used to regenerate ATP in the process of oxida  tive phosphorylation. Energy available from cleavage of the high-energy phos-  phate bonds of ATP can be used directly for mechanical work (e.g., muscle  contraction) or for transport work (e.g., a Na + gradient generated by Na + , K + -  ATPase). It can also be used for biochemical work (energy -re quiring chemical  reactions), such as anabolic pathways (biosynthesis of large molecules like pro-  teins) or detoxification reactions. Phosphoryl transfer reactions, protein confor-  mational changes, and the formation of activated intermediates containing high  energy bonds (e.g., UDP -sugars) facilitate these energy transformations. Energy  released from foods that is not used for work against the environment is trans-  formed into heat.   (homeo, same; stasis, state). Regardless of whether the level of cellular fuel uti-  lization is high (with increased ATP consumption), or low (with decreased ATP  consumption), the available ATP within the cell is maintained at a constant level  by appropriate increases or decreases in the rate of fuel oxidation. Problems in  ATP homeostasis and energy balance occur in obesity, hyperthyroidism, and  myocardial infarction.   Energy from Fuel Oxidation. Fuel oxidation is exergonic; it releases energy.  The maximum quantity of energy released that is available for useful work (e.g.,  ATP synthesis) is called AG 0 ', the change in Gibbs free energy at pH 7.0 under  standard conditions. Fuel oxidation has a negative AG 0 ', that is, the products  have a lower chemical bond energy than the reactants and their formation is  energetically favored. ATP synthesis from ADP and inorganic phosphate is ender-  gonic; it requires energy and has a positive AG 0 '. To proceed in our cells, all  pathways must have a negative AG 0 '. How is this accomplished for anabolic path  ways such as glycogen synthesis? These metabolic pathways incorporate reac-  tions that expend high-energy bonds to compensate for the energy-requiring steps  Because the AG 0 ' s for a sequence of reactions are additive, the overall pathway  becomes energetically favorable.   Fuels are oxidized principally by donating electrons to NAD + and FAD,  which then donate electrons to 0 2 in the electron transport chain. The caloric  value of a fuel is related to its AG 0 ' for transfer of electrons to 0 2 , and its  reduction potential, E°' (a measure of its willingness to donate, or accept,   In the thermodynamic perspective  \% ) of energy expenditure, where  \Wr\/ / energy intake to the body exceeds  energy expended, the difference is effec-  tively stored as fat.   The portion of food that is metabolized is  regulated to match the total energy expendi-  ture of the body. A certain amount of the  energy is obligatory (the amount of energy  expended to do the work of the cells, the  BMR). Some energy is also expended for  adaptive thermogenesis, heat generated in  response to cold or diet. An additional  amount of energy is used for physical exer-  cise (work against the environment). To vol-  untarily store less energy as fat, we can vary  our caloric intake through dietary changes or  our energy expenditure through changes in  our physical exercise.   Cora Nari suffered a heart attack 8  months ago and had a significant  loss of functional heart muscle.  The pain she is experiencing is called angina  pectoris, which is a crushing or constricting  pain located in the center of the chest, often  radiating to the neck or arms (see Ann Jeina,  Chapters 6 and 7). The most common cause  of angina pectoris is partial blockage of coro-  nary arteries from atherosclerosis. The heart  muscle cells beyond the block receive an  inadequate blood flow and oxygen, and die  when ATP production falls too low.   electrons). Because fatty acids are more reduced than carbohydrates, they have  a higher caloric value. The high affinity of oxygen for electrons (a high positive  reduction potential) drives fuel oxidation forward, with release of energy that  can be used for ATP synthesis in oxidative phosphorylation. However, smaller  amounts of ATP can be generated without the use of 0 2 in anaerobic  glycolysis.   Fuel oxidation can also generate NADPH, which usually donates electrons to  biosynthetic pathways and detoxification reactions. For example, in some reac-  tions catalyzed by oxygenases, NADPH is the electron donor and 0 2 the electron  acceptor.   • Otto Shape is a 26-year old medical student who has completed his first  HD ) year of medical school. He is 70 inches tall and began medical school   " weighing 154 lb, within his ideal weight range (see Chapter 1). By the time  he finished his last examination in his first year, he weighed 187 lb. He had calcu-  lated his BMR at approximately 1,680 kcal, and his energy expenditure for physical  exercise equal to 30% of his BMR. He planned on returning to his premedical school  weight in 6 weeks over the summer by eating 576 kcal less each day and playing 7  hours of tennis every day. However, he did a summer internship instead of playing  tennis. When Otto started his second year of medical school, he weighed 210 lb.   X.S. Teefore (excess T 4 ) is a 26-year-old man who noted heat intolerance  with heavy sweating, heart palpitations, and tremulousness. Over the past  4 months, he has lost weight in spite of a good appetite. He is sleeping  poorly and describes himself as feeling "jittery inside."   On physical examination, his heart rate is rapid (116 beats/min) and he appears  restless and fidgety. His skin feels warm, and he is perspiring profusely. A fine hand  tremor is observed as he extends his arms in front of his chest. His thyroid gland  appears to be diffusely enlarged and, on palpation, is approximately 3 times normal  size. Thyroid function tests confirm that Mr. Teefore 's thyroid gland is secreting  excessive amounts of the thyroid hormones T 4 (tetraiodothyronine) and T 3 (tri-  iodothyronine), the major thyroid hormones present in the blood.   • Cora Nari is a 64-year-old woman who had a myocardial infarction 8  flP ) months ago. Although she managed to lose 6 lb since that time, she   " remains overweight and has not reduced the fat content of her diet ade-  quately. The graded aerobic exercise program she started 5 weeks after her infarc-  tion is now followed irregularly, falling far short of the cardiac conditioning inten-  sity prescribed by her cardiologist. She is readmitted to the hospital cardiac care  unit (CCU) after experiencing a severe "viselike pressure" in the mid-chest area  while cleaning ice from the windshield of her car. The electrocardiogram (ECG)  shows evidence of a new posterior wall myocardial infarction. Signs and symptoms  of left ventricular failure are present.   The basic principle of the ATP-ADP cycle is that fuel oxidation generates ATP, and  hydrolysis of ATP to ADP provides the energy to perform most of the work required  in the cell. ATP has therefore been called the energy currency of our cells. Like the   one dollar bill, it has a defined value, is required to obtain goods and services, and  disappears before we know it. To keep up with the demand, we must constantly  replenish our ATP supply through the use of 0 2 for fuel oxidation.   The amount of energy from ATP cleavage available to do useful work is related  to the difference in energy levels between the products and substrates of the reac-  tion and is called the change in Gibbs free energy, AG (A, difference; G, Gibbs free  energy). In cells, the A G for energy production from fuel oxidation must be greater  than the AG of energy-requiring processes, such as protein synthesis and muscle  contraction, for life to continue.   The amount of energy released or required by bond cleavage or formation is deter-  mined by the chemical properties of the substrates and products. The bonds between  the phosphate groups in ATP are called phosphoanhydride bonds (Fig. 19.2). When  these bonds are hydrolyzed, energy is released because the products of the reaction  (ADP and phosphate) are more stable, with lower bond energies, than the reactants  (ATP and H 2 0). The instability of the phosphoanhydride bonds arises from their  negatively charged phosphate groups, which repel each other and strain the bonds  between them. It takes energy to make the phosphate groups stay together. In con-  trast, there are fewer negative charges in ADP to repel each other. The phosphate  group as a free anion is more stable than it is in ATP because of an increase in res-  onance structures (i.e., the electrons of the oxygen double bond are shared by all the  oxygen atoms). As a consequence, ATP hydrolysis is energetically favorable and  proceeds with release of energy as heat.   In the cell, ATP is not directly hydrolyzed. Energy released as heat from ATP  hydrolysis cannot be transferred efficiently into energy-requiring processes, such as  biosynthetic reactions or maintaining an ion gradient. Instead, cellular enzymes  directly transfer the phosphate group to a metabolic intermediate or protein that is  part of the energy-requiring process (a phosphoryl transfer reaction).   How much energy can be obtained from ATP hydrolysis to do the work required  in the cell? The maximum amount of useful energy that can be obtained from a    The heart is a specialist in the  transformation of ATP chemical  bond energy into mechanical work.  Each single heartbeat uses approximately  2% of the ATP in the heart. If the heart were  not able to regenerate ATP, all its ATP would  be hydrolyzed in less than 1 minute. Because  the amount of ATP required by the heart is  so high, it must rely on the pathway of  oxidative phosphorylation for generation of  this ATP. In Cora Nari's heart, hypoxia is  affecting her ability to generate ATP.   Fig. 19.2. Hydrolysis of ATP to ADP and inorganic phosphate (Pi). Cleavage of the phosphoanhydride bonds between either the p and 7 phos-  phates or the a and p phosphates releases the same amount of energy, approximately — 7.3 kcal/mole. However, hydrolysis of the phosphate-  adenosine bond (a phosphoester bond) releases less energy (—3.4 kcal/mole), and consequently, this bond is not considered a high-energy phos-  phate bond. During ATP hydrolysis, the change in disorder during the reaction is small and so AG values at physiologic temperature (37°C) are  similar to those at standard temperature (25°C). AG is affected by pH, which alters the ionization state of the phosphate groups of ATP and by  the intracellular concentration of Mg 2+ ions, which bind to the p and 7 phosphate groups of ATP.   The reaction catalyzed by phos-  phoglucomutase (PGM) is  reversible and functions in the syn-  thesis of glycogen from glucose as well as  the degradation of glycogen back to glucose.  If the AG 0 ' for conversion of glucose 6-P to  glucose 1-P is +1.65 kcal/mole, what is the  AG 0 ' of the reverse reaction?    reaction is called AG, the change in Gibbs free energy. The value of AG for a  reaction can be influenced by the initial concentration of substrates and products,  by temperature, pH, and pressure. The AG 0 for a reaction refers to the energy  change for a reaction starting at 1 M substrate and product concentrations and  proceeding to equilibrium (equilibrium, by definition, occurs when there is no  change in substrate and product concentrations with time). AG 0 ' is the value for  AG 0 under standard conditions (pH = 7.0, [H 2 0] = 55 M, and 25°C), as well as  standard concentrations (Table 19.1).   AG 0 ' is equivalent to the chemical bond energy of the products minus that of the  reactants, corrected for energy that has gone into entropy (an increase in amount of  molecular disorder). This correction for change in entropy is very small for most  reactions occurring in cells, and, thus, the AG 0 ' for hydrolysis of various chemical  bonds reflects the amount of energy available from that bond.   The value of —7.3 kcal/mole (-30.5 kJ/mole) that is generally used for the  AG 0 ' of ATP hydrolysis is, thus, the amount of energy available from hydrolysis  of ATP under standard conditions that can be spent on energy-requiring  processes; it defines the "monetary value" of our "ATP currency." Although the  difference between cellular conditions (pH 7.3, 37°C) and standard conditions is  very small, the difference between cellular concentrations of ATP, ADP, and Pi  and the standard 1-M concentrations is huge and greatly affects the availability  of energy in the cell.   The value of AG 0 'tells you whether the reaction requires or releases energy, the  amount of energy involved, and the ratio of products to substrates at equilibrium.  The negative value for the AG 0 ' of ATP hydrolysis indicates that, if you begin with  equimolar (1 M) concentrations of substrates and products, the reaction proceeds in   AG 0 Standard free energy change, AG at 1 M concentrations of substrates and products   K' eq Equilibrium constant at 25 Q C, pH 7.0, incorporating [H 2 0] = 55.5 M and [H + ] = 10~ 7 M   ~P Biochemical symbol for a high-energy phosphate bond, i.e., a bond which is  hydrolyzed with the release of more than about 7 kcal/mole  of heat    First law of thermodynamics, the conservation of energy: In any physical or chemical   surroundings, remains constant.  Second law of thermodynamics: The universe tends toward disorder. In all natural    Units of AG and AH = cal/mole or J/mole: 1 cal = 4.1 8 J  T, Absolute temperature: K, Kelvin = 273 + Q C (25 Q C = 298 Q K)  R, Universal gas constant: 1 .99 cal/mole-K or 8.31 J/mole-K  F, Faraday constant: F = 23 kca l/mole-volt or 96,500 J/V-mole  Units of E 0 ', volts   To generalize the expression for AG, consider a reaction in which   The small letters denote a moles of A will combine with b moles of B to produce c moles of  C and d moles of D.   the forward direction with the release of energy. From initial concentrations of 1 M,  the ATP concentration will decrease, and ADP and Pi will increase until equilibrium  is reached.   For a reaction in which a substrate S is converted to a product P, the ratio of  the product concentration to the substrate concentration at equilibrium is given  by:   (see Table 19.2. for a more general form of this equation; R is equal to the gas con-  stant (1.98 calories/mole-degree Kelvin), and T is equal to the temperature in  degrees Kelvin).   Thus, the difference in chemical bond energies of the substrate and product  (AG 0 ') determines the concentration of each at equilibrium.   Reactions such as ATP hydrolysis are exergonic (release energy) or exothermic  (release heat). They have a negative AG 0 ' and release energy while proceeding in  the forward direction to equilibrium. Endergonic, or endothermic, reactions have a  positive AG 0 ' for the forward direction (the direction shown), and the backward  direction is favored. For example, in the pathway of glycogen synthesis, phospho-  glucomutase converts glucose-6-P to glucose- 1 -P. Glucose- 1-P has a higher phos-  phate bond energy than glucose-6-P because the phosphate is on the aldehyde car-  bon (Fig 19.3). The AG 0 ' for the forward direction (glucose- 1-P — > glucose-6-P) is,  therefore, positive. Beginning at equimolar concentrations of both compounds,  there is a net conversion of glucose- 1-P back to glucose-6-P and, at equilibrium, the  concentration of glucose-6-P is higher than glucose- 1-P. The exact ratio is  determined by AG 0 ' for the reaction.   It is often said that a reaction with a negative AG' proceeds spontaneously in the  forward direction, meaning that products accumulate at the expense of reactants.  However, AG' is not an indicator of the velocity of the reaction, or the rate at which  equilibrium can be reached. In the cell, the velocity of the reaction depends on the  efficiency and amount of enzyme available to catalyze the reaction (see Chapter 9),  and, therefore, "spontaneously" in this context can be misleading.   ^"^\ The AG 0 ' for the reverse reaction is  •] -1.65 kcal. The change in free  energy is the same for the forward  and reverse directions, but has an opposite  sign. Because negative AG 0 ' values indicate  favorable reactions, this reaction under stan-  dard conditions favors the conversion of glu-  cose 1-P to glucose 6-R   Fig. 19.3. The phosphoglucomutase reaction.  The forward direction is involved in converting  glucose to glycogen, and the reverse direction  in converting glycogen to glucose 6-P.   The AG 0 ' for the conversion of glu-  §~1 # cose 6-P to glucose 1-P is +1.65  kcal/mole. What is the ratio of [glu-  cose-1-P] to [glucose-6-P] at equilibrium?   To do work in the cell, a mechanism must be available for converting the chemical  bond energy of ATP into another form, such as an Na + gradient across a membrane.  These energy transformations usually involve intermediate steps in which ATP is  bound to a protein, and cleavage of the bound ATP results in a conformational  change of the protein.   Fig. 19.4. A simplified diagram of myosin ATPase. Muscle fiber is made of thick filaments composed of bundles of the protein myosin, and thin  filaments composed of the protein actin (which is activated by Ca 2+ binding). At many positions along the actin filament, a terminal domain of  a myosin molecule, referred to as the "head," binds to a specific site on the actin. The myosin head has an ATP binding site and is an ATPase; it  can hydrolyze ATP to ADP and Pi. (1) As ATP binds to myosin, the conformation of myosin changes, and it dissociates from the actin. (2) Myosin  hydrolyzes the ATP, again changing conformation. (3) When Pi dissociates, the myosin head reassociates with activated actin at a new position.  (4) As ADP dissociates, the myosin again changes conformation, or tightens. This change of conformation at multiple association points between  actin and myosin slides the actin filament forward (5).   ®ln equation 1 of Table 19.2, AG 0 ' =  RT In K eq . For this reaction, K eq =  [glucose-1-phosphate]/[glucose-6-  phosphate]. The constant R is 1.99 X 10~ 3  kcal/mole- °K, and T is (273 + 25) °K, so RT  equals 0.593 kcal/mole. Substituting in equa-  tion 1 then gives 1.65 = -0.593 In [glucose-1-  P]/[glucose-6-P]. Thus, ln[glucose 1-P]/[glu-  cose 6-P] = -2.78, and [glucose-1-P]/  [glucose-6-phosphate] = e" 2 78 , or 0.062. So  the ratio of [glucose-1-P] to [glucose-6-P] at  equilibrium is 0.062.   Q o Otto Shape has not followed his  f /^oA } proposed diet and exercise regi-  ™ men and has been gaining weight.  He has a positive caloric balance, because  his daily energy expenditure is less than his  daily energy intake (see Chapter 2).  Although the energy expenditure for physi-  cal exercise is only approximately 30% of the  BMR (basal metabolic rate) in a sedentary  individual, it can be 100% or more of the  BMR in a person who exercises strenuously  for several hours or more. The large increase  in ATP utilization for muscle contraction dur-  ing exercise accounts for its contribution to  the daily energy expenditure.   In mechanical work, the high-energy phosphate bond of ATP is converted into  movement by changing the conformation of a protein (Fig. 19.4.). For example, in  contracting muscle fibers, the hydrolysis of ATP while it is bound to myosin ATPase  changes the conformation of myosin so that it is in a "cocked" position ready to  associate with the sliding actin filament. Thus, exercising muscle fibers have almost  a hundred-fold higher rate of ATP utilization and caloric requirements than resting  muscle fibers. Motor proteins, such as kinesins that transport chemicals along  fibers, provide another example of mechanical work in a cell.   In transport work, called active transport, the high-energy phosphate bond of ATP is  used to transport compounds against a concentration gradient (see Chapter 10,   The equations for calculating AG are based on the first law of thermodynamics  (see Table 19.1). The change in chemical bond energy that occurs during a reac-  tion is AH, the change in enthalpy of the reaction. At constant temperature and  pressure, AH is equivalent to the chemical bond energy of the products minus that of the  reactants. AG, the maximum amount of useful work available from a reaction, is equal  to AH minus TAS. TAS is a correction for the amount of energy that has gone into an  increase in the entropy (disorder in arrangement of molecules) of the system.   where AH = the change in enthalpy, T is the temperature of the system in Kelvin, and AS  is the change in entropy, or increased disorder of the system. AS is often negligible in  reactions such as ATP hydrolysis in which the number of substrates (H 2 0, ATP) and prod-  ucts (ADP, Pi) are equal and no gas is formed. Under these conditions, the values for AG  at physiologic temperature (37°C) are similar to those at standard temperature (25°C)   Fig. 10. 12). In P-ATPases (plasma membrane ATPases) and V-ATPases (vesicular  ATPases), the chemical bond energy of ATP is used to reversibly phosphorylate the  transport protein and change its conformation. For example, as Na + , K + -ATPase binds  and cleaves ATP, it becomes phosphorylated and changes its conformation to release 3  Na + ions to the outside of the cell, thereby building up a higher extracellular than intra-  cellular concentration of Na + . Na + re-enters the cell on cotransport proteins that drive  the uptake of amino acids and many other compounds into the cell. Thus, Na + must  be continuously transported back out. The expenditure of ATP for Na + transport occurs  even while we sleep and is estimated to account for 10 to 30% of our BMR.   A large number of other active transporters also convert ATP chemical bond energy  into an ion gradient (membrane potential). Vesicular ATPases pump protons into lyso-  somes. Ca 2+ ATPases in the plasma membrane move Ca 2+ out of the cell against a con-  centration gradient. Similar Ca 2+ ATPases pump Ca 2+ into the lumen of the endoplas-  mic reticulum and the sarcoplasmic reticulum (in muscle). Thus, a considerable  amount of energy is expended in maintaining a low cytoplasmic Ca 2+ level.    X.S. Teefore has increased blood  levels of thyroid hormones, which  accelerate basal metabolic  processes that use ATP in our organs (e.g.,  Na + ,K + -ATPase), thereby increasing the  BMR. An increased BMR was used for a pre-  sumptive diagnosis of hyperthyroidism  before development of the tests to measure  T3 and T4. Because X.S. Teefore did not fully  compensate for his increased ATP require-  ments with an increased caloric intake, he  was in negative caloric balance and lost  weight.   The high-energy phosphate bonds of ATP are also used for biochemical work. Bio-  chemical work occurs in anabolic pathways, which are pathways that synthesize  large molecules (e.g., DNA, glycogen, triacylglycerols, and proteins) from smaller  compounds. Biochemical work also occurs when toxic compounds are converted to  nontoxic compounds that can be excreted (e.g., the liver converts NH 4 + ions to urea  in the urea cycle). In general, formation of chemical bonds between two organic  molecules (e.g., C-C bonds in fatty acid synthesis or C-N bonds in protein synthe-  sis) requires energy and is therefore biochemical work. How do our cells get these  necessary energy-requiring reactions to occur?   To answer this question, the next sections consider how energy is used to syn-  thesize glycogen from glucose (Fig 19.5). Glycogen is a storage polysaccharide  consisting of glucosyl units linked together through glycosidic bonds. If an anabolic  pathway, such as glycogen synthesis, were to have an overall positive AG 0 ', the cell  would be full of glucose and intermediates of the pathway, but very little glycogen  would be formed. To avoid this, cells do biochemical work and spend enough of  their ATP currency to give anabolic pathways an overall negative AG 0 '.   Approximately 70% of our resting daily energy requirement arises from work  carried out by our largest organs: the heart, brain, kidneys, and liver. Using  their rate of oxygen consumption and a P/O ratio of 2.5, it can be estimated that  each of these organs is using and producing several times its own weight in ATP each  day. The heart, which rhythmically contracts, is using this ATP for mechanical work. In  contrast, skeletal muscles in a resting individual use far less ATP per gram of tissue. The  kidney has an ATP consumption per gram of tissue similar to that of the heart and is  using this ATP largely for transport work to recover usable nutrients and maintain pH and  electrolyte balance. The brain, likewise, uses most of its ATP for transport work, main-  taining the ion gradients necessary for conduction of the nerve impulse. The liver, in con-  trast, has a high rate of ATP consumption and utilization to carry out metabolic work  (biosynthesis and detoxification).  Estimated Daily Use of ATP  (g ATP/g tissue)   Fig. 19.5. Energetics of glycogen synthesis.  Compounds containing high-energy bonds are  shown in blue. (1) Glucose is transported into  the cell. (2) Glucose phosphorylation uses the  high-energy phosphate bond (~P) of ATP in a  phosphoryl transfer step. (4) Conversion of  glucose 6-phosphate to glucose 1 -phosphate  by phosphoglucomutase. (5) UDP-Glucose  pyrophosphorylase cleaves a ~P bond in UTP,  releasing pyrophosphate and forming UDP-  glucose, an activated intermediate. (6) The  pyrophosphate is hydrolyzed, releasing addi-  tional energy. (7) The phosphoester bond of  UDP-glucose is cleaved during the addition of  a glucosyl unit to the end of a glycogen poly-  saccharide chain. The UDP-glucose acts as the  leaving group in this reaction. Glucose-6-  phosphate also can be metabolized via glycol-  ysis (3) when energy is required.   Given a AG 0 ' of + 1 .65 kcal/mole for  ■ B* the conversion of glucose-6-P to   glucose-1-P, and a AG 0 ' of -4.0  kcal/mole for the conversion of glucose +  ATP to glucose-6-P + ADP, what is the value  of AG 0 ' for the conversion of glucose to glu-  cose-1-P?   Reactions in which chemical bonds are formed between two organic molecules are  usually catalyzed by enzymes that transfer energy from cleavage of ATP in a phos-  phoryl transfer reaction or by enzymes that cleave a high-energy bond in an acti-  vated intermediate of the pathway. Because the AG 0 ' values in a reaction sequence  are additive, the pathway acquires an overall negative AG 0 ', and the reactions in the  pathway will occur to move toward an equilibrium state where the concentration of  final products is greater than that of the initial reactants.   One of the characteristics of Gibbs free energy is that AG 0 values for consecutive  steps or reactions in a sequence can be added together to obtain a single value for  the overall process. Thus, the high-energy phosphate bonds of ATP can be used to  drive a reaction forward that would otherwise be highly unfavorable energetically.  Consider, for example, synthesis of glucose 6-P from glucose, the first step in gly-  colysis and glycogen synthesis (see Fig. 19.5, circle 2). If the reaction were to pro-  ceed by addition of inorganic phosphate to glucose, glucose-6-P synthesis would  have a positive AG 0 ' value of 3.3 kcal/mole (Table 19.3). However, when this reac-  tion is coupled to cleavage of the high-energy ATP bond through a phosphoryl  transfer reaction, the AG 0 for glucose-6-P synthesis acquires a net negative value of  minus 4.0 kcal/mole, which can be calculated from the sum of the two reactions.  Glucose 6-P cannot be transported back out of the cell, and therefore the net  negative AG 0 for glucose 6-P synthesis helps the cell to trap glucose for its own  metabolic needs.   The net value for synthesis of glucose 6-P from glucose and ATP would be the  same whether or not the two reactions were catalyzed by the same enzyme, were  catalyzed by two separate enzymes, or were not catalyzed by an enzyme at all,  because it is dictated by the amount of energy in the chemical bonds being broken  and formed.   To synthesize glycogen from glucose, energy is provided by the cleavage of 3 high-  energy phosphate bonds in ATP, UTP, and pyrophosphate (PPi)(see Fig. 19.5, Steps  2, 5, and 6). Energy transfer is facilitated by phosphoryl group transfer and by for-  mation of an activated intermediate (UDP-glucose). Step 4, the conversion of glu-  cose 6-phosphate to glucose 1-P, has a positive AG 0 '. This step is pulled and pushed  in the desired direction by the accumulation of substrate and removal of product in  reactions that have a negative AG 0 ' from cleavage of high-energy bonds. In Step 5,  the UTP high-energy phosphate bond is cleaved to form the activated sugar, UDP-  glucose (Fig 19.6). This reaction is further facilitated by cleavage of the high-energy  bond in the pyrophosphate (Step 6) that is released in Step 5 (approximately —7.7  kcal). In Step 7, cleavage of the bond between UDP and glucose in the activated  intermediate provides the energy for attaching the glucose moiety to the end of the  glycogen molecule (approximately —3.3 kcal). In general, the amount of ATP phos-  phate bond energy used in an anabolic pathway, or detoxification pathway, must  provide the pathway with an overall negative AG 0 ', so that the concentration of  products is favored over that of reactants.   Fig. 19.6. UDP-glucose contains a high-energy pyrophosphate bond, shown in blue.   AG 0 ' reflects the energy difference between reactants and products at specific con-  centrations (each at 1 M) and standard conditions (pH 7.0, 25°C). However, these  are not the conditions prevailing in cells, where variations from "standard condi-  tions" are relevant to determining actual free energy changes and hence the direc-  tion in which reactions are likely to occur. One aspect of free energy changes con-  tributing to the forward direction of anabolic pathways is the dependence of AG, the  free energy change of a reaction, on the initial substrate and product concentrations.  Reactions in the cell with a positive AG 0 ' can proceed in the forward direction if the  concentration of substrate is raised to high enough levels, or if the concentration of  product is decreased to very low levels. Product concentrations can be very low if,  for example, the product is rapidly used in a subsequent energetically favorable  reaction, or if the product diffuses or is transported away.   AG 0 ' for the overall reaction is the  | /\?) sum of the individual reactions,   Thus, the cleavage of ATP has made the  synthesis of glucose-1-P from glucose ener-  getically favorable.   The driving force toward equilibrium starting at any concentration of substrate and  product is expressed by AG, and not by AG 0 ', which is the free energy change to  reach equilibrium starting with 1 M concentrations of substrate and product. For a  reaction in which the substrate S is converted to the product P:   The expression for AG has two terms: AG 0 ', the energy change to reach equilib-  rium starting at equal and 1 M concentrations of substrates and products, and the  second term, the energy change to reach equal concentrations of substrate and  product starting from any initial concentration. (When [P] = [S] and [P]/[S] = 1,  the In of [P]/[S] is 0, and AG = AG 0 '). The second term will be negative for all con-  centrations of substrate greater than product, and the greater the substrate concen-  tration, the more negative this term will be. Thus, if the substrate concentration is  suddenly raised high enough or the product concentration decreased low enough,  AG (the sum of the first and second terms) will also be negative, and conversion of  substrate to product becomes thermodynamically favorable.   The effect of substrate and product concentration on AG and the direction of a reac-  tion in the cell can be illustrated with conversion of glucose-6-P to glucose- 1-P, the   The AG of ATP hydrolysis in cells  \\ ) can be very different than AG 0 '.   The synthesis of ATP rather than its  hydrolysis becomes the energetically favor-  able direction if the [ATP] drops just a little or  the [ADP] or [Pi] increase just a little over  equilibrium values. Thus, as ATP is  hydrolyzed in energy-requiring reactions,  the change in the sign of AG promotes ATP  synthesis.   reaction catalyzed by phosphoglucomutase in the pathway of glycogen synthesis  (see Fig. 19.3). The reaction has a small positive AG 0 ' for glucose 1-P synthesis  (+1.65.kcal/mole) and at equilibrium, the ratio of [glucose l-P]/[glucose 6-P] is  approximately 6 to 94 (which you calculated in Question 2). However, if another  reaction uses glucose 1-P such that this ratio suddenly becomes 3 to 94, there is now  a driving force for converting more glucose 6-P to glucose 1-P and restoring the  equilibrium ratio. Substitution in equation 2 gives AG, the driving force to equilib-  rium, as + 1.65 + RT In [G1P]/[G6P] = 1.65 + (-2.06) = -0.41, which is a neg-  ative value. Thus, a decrease in the ratio of product to substrate has converted the  synthesis of glucose 1-P from a thermodynamically unfavorable to a thermody-  namically favorable reaction that will proceed in the forward direction until equi-  librium is reached.   Many biochemical pathways form activated intermediates containing high-energy  bonds to facilitate biochemical work. The term "high-energy bond" is a biologic  term defined by the AG 0 ' for ATP hydrolysis; any bond that can be hydrolyzed with  the release of approximately as much, or more, energy than ATP is called a high-  energy bond. The high-energy bond in activated intermediates, such as UDP-  glucose in glycogen synthesis, facilitate energy transfer.   Cells use GTP and CTP, as well as UTP and ATP, to form activated intermediates.  Different anabolic pathways generally use different nucleotides as their direct  source of high phosphate bond energy: UTP is used for combining sugars, CTP in  lipid synthesis, and GTP in protein synthesis.   The high-energy phosphate bonds of UTP, GTP, and CTP are energetically  equivalent to ATP and are synthesized from ATP by nucleoside diphosphokinases  and nucleoside monophosphokinases. For example, UTP is formed from UDP by a  nucleoside diphosphokinase in the reaction:   ADP is converted back to ATP by the process of oxidative phosphorylation, using  energy supplied by fuel oxidation.   Energy-requiring reactions often generate the nucleoside diphosphate ADP.  Adenylate kinase, an important enzyme in cellular energy balance, is a nucleoside  monophosphate kinase that transfers a phosphate from one ADP to another ADP to  form ATP and AMP:   Fig. 19.7. Some compounds with high-energy  bonds. 1,3-bisphosphoglycerate and phospho-  enolpyruvate are intermediates of glycolysis.  Creatine phosphate is a high-energy phosphate  reservoir and shuttle in brain, muscle, and  spermatozoa. Acetyl CoA is a precursor of the  TCA cycle. The high-energy bonds are shown  in blue.   This enzyme, thus, can regenerate ATP under conditions in which ATP utiliza-  tion is required.   In addition to the nucleoside triphosphates, other compounds containing high-  energy bonds are formed to facilitate energy transfer in anabolic and catabolic path-  ways (e.g., 1,3- bisphosphoglycerate in glycolysis and acetyl CoA in the TCA  cycle) (Fig. 19.7). Creatine phosphate contains a high-energy phosphate bond that  allows it to serve as an energy reservoir for ATP synthesis and transport in muscle  cells, neurons, and spermatozoa. All of these high-energy bonds are "unstable," and  their hydrolysis yields substantial free energy because the products are much more  stable, as a result of electron resonance within their structures.   According to the first law of thermodynamics, energy cannot be destroyed. Thus,  energy from oxidation of a fuel (its caloric content) must be equal to the amount of  heat released, the work performed against the environment, and the increase in  order of molecules in our bodies. Some of the energy from fuel oxidation is con-  verted into heat as the fuel is oxidized and some heat is generated as ATP is used to  do work. If we become less efficient in converting energy from fuel oxidation into  ATP, or if we use an additional amount of ATP for muscular contraction, we will  oxidize an additional amount of fuel to maintain ATP homeostasis (constant cellu-  lar ATP levels). With the oxidation of additional fuel, we release additional heat.  Thus, heat production is a natural consequence of "burning fuel."   Thermogenesis refers to energy expended for the purpose of generating heat in  addition to that expended for ATP production. To maintain our body at 37°C, despite  changes in environmental temperature, it is necessary to regulate fuel oxidation and  its efficiency (as well as heat dissipation). In shivering thermogenesis, we respond  to sudden cold with asynchronous muscle contractions (shivers) that increase ATP  utilization and, therefore, fuel oxidation and the release of energy as heat. In non-  shivering thermogenesis (adaptive thermogenesis), the efficiency of converting  energy from fuel oxidation into ATP is decreased. More fuel needs to be oxidized  to maintain constant ATP levels and, thus, more heat is generated.   Fuel oxidation provides energy for bodily processes principally through generation  of the reduced coenzymes, NADH and FAD(2H). They are used principally to gen-  erate ATP in oxidative phosphorylation. However, fuel oxidation also generates  NADPH, which is most often used directly in energy-requiring processes. Carbo-  hydrates also may be used to generate ATP through a nonoxidative pathway, called  anaerobic glycolysis.   Fuel oxidation is our major source of ATP and our major means of transferring  energy from the chemical bonds of the fuels to cellular energy-requiring processes.  The amount of energy available from a fuel is equivalent to the amount of heat that  is generated when a fuel is burned. To conserve this energy for the generation of  ATP, the process of cellular respiration transforms the energy from the chemical  bonds of fuels into the reduction state of electron- accepting coenzymes, NAD + and  FAD (circle 1, Fig. 19.8). As these compounds transfer electrons to 0 2 in the elec-  tron transport chain, most of this energy is transformed into an electrochemical gra-  dient across the inner mitochondrial membrane (circle 2, Fig. 19.8). Much of the  energy in the electrochemical gradient is used to regenerate ATP from ADP in  oxidative phosphorylation (phosphorylation that requires 0 2 ).   Oxidation-reduction reactions always involve a pair of chemicals: an electron  donor, which is oxidized in the reactions, and an electron acceptor, which is reduced  in the reaction. In fuel metabolism, the fuel donates electrons, and is oxidized, and  NAD + and FAD accept electrons, and are reduced.   When is NAD + , rather than FAD, used in a particular oxidation-reduction reaction?  It depends on the chemical properties of the electron donor and the enzyme catalyzing  the reaction. In oxidation reactions, NAD + accepts two electrons as a hydride ion to  form NADH, and a proton (H + ) is released into the medium (Fig 19.9). It is generally  used for metabolic reactions involving oxidation of alcohols and aldehydes. In contrast,    X.S. Teefore has increased thyroid  hormone levels that increase his  rate of ATP utilization and fuel oxi-  dation. An excess of thyroid hormones also  may affect the efficiency of ATP production,  resulting in fewer ATP produced for a given  0 2 consumption. The increased rate of ATP  utilization and diminished efficiency stimu-  lates oxidative metabolism, resulting in a  much greater rate of heat production. The  hyperthyroid patient, therefore, complains  of constantly feeling hot (heat intolerance)  and sweaty. (Perspiration allows dissipation  of excess heat through evaporation from the  skin surface.)   <^f\ Oxidation is the loss of electrons,  and reduction is the gain of elec-  trons. Remember LEO GER:  Loss of Electrons = Oxidation;  Gain of Electrons = Reduction.  Compounds are oxidized in the body in  essentially three ways: (1) the transfer of  electrons from the compound as a hydrogen  atom or a hydride ion, (2) the direct addition  of oxygen from 0 2 , and (3) the direct dona-  tion of electrons (e.g., Fe 2+ — > Fe 3+ ) (see  Chapter 5). Fuel oxidation involves the trans-  fer of electrons as a hydrogen atom or a  hydride ion and, thus, reduced compounds  have more hydrogen relative to oxygen than  the oxidized compounds. Consequently,  aldehydes are more reduced than acids, and  alcohols are more reduced than aldehydes.    Fig. 19.8. Overview of energy transformations in oxidative phosphorylation. The electro-  chemical potential gradient across the mitochondrial membrane is represented by ApH, the  proton gradient, and A^P, the membrane potential. The role of the electrochemical potential  in oxidative phosphorylation is discussed in more depth in Chapter 21.   FAD accepts two electrons as hydrogen atoms, which are donated singly from separate  atoms (e.g., formation of a double bond or a disulfide) (Fig. 19.10).   As the reduced coenzymes donate these electrons to 0 2 through the electron  transport chain, they are reoxidized. The energy derived from reoxidation of NADH  and FAD(2H) is available for the generation of ATP by oxidative phosphorylation.  In our analogy of ATP as currency, the reduced coenzymes are our "paychecks" for  oxidizing fuels. Because our cells spend ATP so fast, we must immediately convert  our paychecks into ATP cash.    Fig. 19.9. Reduction of NAD + and NADP + . These structurally related coenzymes are  reduced by accepting two electrons as H:~, the hydride ion.   Fig. 19.10. Reduction of FAD. FAD accepts two electrons as two hydrogen atoms and is  reduced. The reduced coenzyme is denoted in this text as FAD(2H) because it often accepts  a total of two electrons one at a time, never going to the fully reduced form, FADH 2 . FMN  (flavin mononucleotide) consists of riboflavin with one phosphate group attached.   Each oxidation/reduction reaction makes or takes a fixed amount of energy,  (AG 0 '), which is directly proportional to the AE°' (the difference in reduction  potentials of the oxidation-reduction pair). The reduction potential of a com-  pound, E°', is a measure in volts of the energy change when that compound  accepts electrons (becomes reduced); minus E°' is the energy change when the  compound donates electrons (becomes oxidized). E°' can be considered an  expression of the willingness of the compound to accept electrons. Some exam-  ples of reduction potentials are shown in Table 19.4. Oxygen, which is the best  electron acceptor, has the largest positive reduction potential (i.e., is the most  willing to accept electrons and be reduced). As a consequence, the transfer of  electrons from all compounds to 0 2 is energetically favorable and occurs with  energy release.   The more negative the reduction potential of a compound, the greater is the  energy available for ATP generation when that compound passes its electrons to  oxygen. The AG 0 ' for transfer of electrons from NADH to 0 2 is greater than the  transfer from FAD(2H) to 0 2 (see the reduction potential values for NADH and  FAD(2H) in Table 19.4). Thus, the energy available for ATP synthesis from NADH  is approximately —53 kcal, and approximately —41 kcal from the FAD -containing  flavoproteins in the electron transport chain.   Cytochrome a-Fe 3+  CoQ + 2H + + 2 e"^CoQH 2  Fumarate + 2H + + 2 e" — > succinate  Oxalacetate + 2H + + 2e"^ malate  Acetaldehyde + 2H + + 2e"^ ethanol  Pyruvate + 2H + + 2e"^ lactate  Riboflavin + 2H + + 2e"^ riboflavin-H 2  NAD + + 2H + + 2 e" -> NADH + H +  Acetate + 2H + + 2e"^ acetaldehyde   To calculate the free energy change  of an oxidation-reduction reaction,  the reduction potential of the elec-  tron donor (NADH) is added to that of the  acceptor (0 2 ). The AE 0 ' for the net reaction is  calculated from the sum of the half reac-  tions. For NADH donation of electrons, it is =  + 0.320 volts, opposite of that shown in Table  4 (remember, Table 4 shows the E 0 ' for  accepting electrons), and for 0 2 acceptance,  it is +0.816. The number of electrons being  transferred is 2 (so, n = 2). The direct rela-  tionship between the energy changes in oxi-  dation-reduction reactions and AG 0 ' is  expressed by the equation   AG 0 ' = -n F AE 0 '  where n is the number of electrons trans-  ferred and F is Faraday's constant (23  kcal/mole - volt). Thus, a value of approxi-  mately -53 kcal/mole is obtained for the  energy available for ATP synthesis by trans-  ferring two electrons from NADH to oxygen.   Otto Shape decided to lose weight  lB # by decreasing his intake of fat and  alcohol (ethanol), and increasing  his content of carbohydrates. Compare the  structure of ethanol with that of glucose and  fatty acids (below). On the basis of their oxi-  dation state, which compound provides the  most energy (calories) per gram?   HOH 2 C - (HC - OH) 4 - C - H  Glucose   Fig. 19.11. Anaerobic glycolysis. Phosphate  is transferred from high-energy intermediates  of the pathway to ADR Because NADH from  the pathway is reoxidized by reduction of  pyruvate to lactate, no oxygen is required.   Fig. 19.12. Oxidases and oxygenases. The  fate of 0 2 is shown in blue. S represents an  organic substrate.   The caloric value of a food is directly related to its oxidation state, which is a meas-  ure of AG 0 ' for transfer of electrons from that fuel to 0 2 . The electrons donated by  the fuel are from its C-H and C-C bonds. Fatty acids such as palmitate  (CH 3 (CH 2 ) 14 COOH) have a caloric value of roughly 9 kcal/g. Glucose is already  partially oxidized and has a caloric value of only about 4 kcal/g. The carbons, on an  average, contain fewer C-H bonds from which to donate electrons.   The caloric value of a food is applicable in humans only if our cells have  enzymes that can oxidize that fuel by transferring electrons from the fuel to  NAD + , NADP + , or FAD. When we burn wood in a fireplace, electrons are trans-  ferred from cellulose and other carbohydrates to 0 2 , releasing energy as heat.  However, wood has no caloric content for humans; we cannot digest it and con-  vert cellulose to a form that can be oxidized by our enzymes. Cholesterol,  although a lipid, also has no caloric value for us because we cannot oxidize the  carbons in its complex ring structure in reactions that generate NADH,  FAD(2H), or NADPH.   NADP + is similar to NAD + and has the same reduction potential. However, NADP +  has an extra phosphate group on the ribose, which affects its enzyme binding (see  Fig. 19.9). Consequently, most enzymes use either NAD + or NADP + , but seldom  both. In certain reactions, fuels are oxidized by transfer of electrons to NADP + to  form NADPH. For example, glucose 6-P dehydrogenase, in the pentose phosphate  pathway, transfers electrons from glucose 6-P to NADP + instead of NAD + .  NADPH usually donates electrons to biosynthetic reactions such as fatty acid syn-  thesis, and to detoxification reactions that use oxygen directly. Consequently, the  energy in its reduction potential is usually used in energy-requiring reactions with-  out first being converted to ATP currency.   Not all ATP is generated by fuel oxidation. In anaerobic glycolysis, glucose is  degraded in reactions that form high-energy phosphorylated intermediates of the  pathway (Fig. 19. 11). These activated high-energy intermediates provide the energy  for the generation of ATP from ADP without involving electron transfer to 0 2 .  Therefore, this pathway is called anaerobic glycolysis, and ATP is generated from  substrate level phosphorylation rather than oxidative phosphorylation (see Chapter  22). Anaerobic glycolysis is a critical source of ATP for cells that have a decreased  0 2 supply, either because they are physiologically designed that way (e.g., cells in  the kidney medulla), or because their supply of 0 2 has been pathologically  decreased (e.g., coronary artery disease).   Approximately 90 to 95% of the oxygen we consume is used by the terminal oxi-  dase in the electron transport chain for ATP generation via oxidative phosphoryla-  tion. The remainder of the 0 2 is used directly by oxygenases and other oxidases,  enzymes that oxidize a compound in the body by transferring electrons directly to  0 2 (Fig. 19.12). The large positive reduction potential of 0 2 makes all of these reac-  tions extremely favorable thermodynamically, but the electronic structure of 0 2  slows the speed of electron transfer. These enzymes, therefore, contain a metal ion  that facilitates reduction of 0 2 .   Oxidases transfer electrons from the substrate to 0 2 , which is reduced to water  (H 2 0) or to hydrogen peroxide (H 2 0 2 ). The terminal protein complex in the elec-  tron transport chain, called cytochrome oxidase, is an oxidase because it accepts  electrons donated to the chain by NADH and FAD(2H) and uses these to reduce 0 2  to water. Most of the other oxidases in the cell form hydrogen peroxide (H 2 0 2 ),  instead of H 2 0, and are called peroxidases. Peroxidases are generally confined to  peroxisomes to protect DNA and other cellular components from toxic free radicals  (compounds containing single electrons in an outer orbital) generated by hydrogen  peroxide.   Oxygenases, in contrast to oxidases, incorporate one or both of the atoms of oxy-  gen into the organic substrate (see Fig 19.12). Monooxygenases, enzymes that  incorporate one atom of oxygen into the substrate and the other into H 2 0, are often  named hydroxylases (e.g., phenylalanine hydroxylase, which adds a hydroxyl group  to phenylalanine to form tyrosine) or mixed function oxidases. Monooxygenases  require an electron donor- substrate, such as NADPH, a coenzyme such as FAD,  which can transfer single electrons, and a metal or similar compound that can form  a reactive oxygen complex (Fig. 19. 13). They are usually found in the endoplasmic  reticulum, and occasionally in mitochondria. Dioxygenases, enzymes that incorpo-  rate both atoms of oxygen into the substrate, are used in the pathways for convert-  ing arachidonate into prostaglandins, thromboxanes, and leukotrienes.   Our total energy expenditure is equivalent to our oxygen consumption (Fig.  19.14). The resting metabolic rate (energy expenditure of a person at rest, at  25 °C, after an overnight fast) accounts for approximately 60 to 70% of our total  energy expenditure and 0 2 consumption, and physical exercise accounts for the   Fig. 19.14. Estimated contribution of processes to energy utilization in standard state.  Copied, with permission, from Rolfe DFS, Brown GC. Cellular energy utilization and molec-  ular origin of standard metabolic rate in mammals. Physiol Rev 1997;77:731-758.   In palmitate and other fatty acids,  most carbons are more reduced  than those in glucose or ethanol  (more of the carbons have electrons in car-  bon-hydrogen bonds). Therefore, fatty acids  have the greatest caloric content/gram, 9  kcal. In glucose, the carbons have already  formed bonds with oxygen, and fewer elec-  trons in C-H bonds are available to generate  energy. Thus, the complete oxidation of glu-  cose gives roughly 4 kcal/g. In ethanol, one  carbon is a methyl group with C-H bonds,  and one has an OH group. Therefore the oxi-  dation state is intermediate between glucose  and fatty acids, and ethanol thus has 7  kcal/g.   Fig. 19.13. Cytochrome P450 mono-oxyge-  nases. Electrons are donated by NADPH to 0 2  and the substrate. The flavin coenzymes FAD  and FMN in one subunit transfer single elec-  trons to cytochrome P450, which is an Fe-  heme containing protein that absorbs light at a  wavelength of 450 nm. The enzyme is embed-  ded in a membrane, usually the endoplasmic  reticulum.   remainder. Of the resting metabolic rate, approximately 90 to 95% of 0 2 con-  sumption is used by the mitochondrial electron transport chain, and only 5 to  10% is required for nonmitochondrial oxidases and oxygenases and is not related  to ATP synthesis. Approximately 20 to 30% of the energy from this mitochondr-  ial 0 2 consumption is lost by proton leak back across the mitochondrial mem-  brane, which dissipates the electrochemical gradient without ATP synthesis. The  remainder of our 0 2 consumption is used for ATPases that maintain ion gradients  and for biosynthetic pathways.   ATP homeostasis refers to the ability of our cells to maintain constant levels of  ATP despite fluctuations in the rate of utilization. Thus, increased utilization of ATP  for exercise or biosynthetic reactions increases the rate of fuel oxidation. The major  mechanism employed is feedback regulation; all of the pathways of fuel oxidation  leading to generation of ATP are feedback-regulated by ATP levels, or by com-  pounds related to the concentration of ATP. In general, the less ATP used, the less  fuel will be oxidized to generate ATP.   According to the first law of thermodynamics, the energy (cal) in our consumed  fuel can never be lost. Consumed fuel is either oxidized to meet the energy demands  of the basal metabolic rate + exercise, or it is stored as fat. Thus, an intake of calo-  ries in excess of those expended results in weight gain. The simple statement, "If  you eat too much and don't exercise, you will get fat," is really a summary of the  bioenergetics of the ATP-ADP cycle.   ^ Otto Shape. Otto Shape visited his physician, who noted the increased  ) weight. He recommended several diet modifications to Otto that would  decrease the caloric content of his diet and pointed out the importance of  exercise for weight reduction. He reminded Otto that the American Heart Associ-  ation and the American Cancer Society recommended 45 to 60 minutes of moder-  ate-to-vigorous exercise 5 to 7 days per week. He also reminded Otto that he would  want to be a role model for his patients. Otto decided to begin an exercise regimen  that includes an hour of running each day.   The thyroid gland secretes the thy-  \\ ) roid hormones tetraiodothyronine  V^^x (T 4 ) and triiodothyronine (T 3 ) (see  Fig. 11.8 for the structure of T 3 ). T 3 is the  most active form of the hormone. T 4 is syn-  thesized and secreted in approximately 10  times greater amounts than T 3 Hepatocytes  (liver cells) and other cells contain a deiodi-  nase that removes one of the iodines from  T 4 , converting it to T 3 . T 3 exerts its effects on  tissues by regulating the transcription of  specific genes involved in energy metabo-  lism (see Chapter 16, section W.C.2., Fig.  16.14).   ^\ X.S. Teefore. Mr. Teefore exhibited the classical signs and symptoms  J of hyperthyroidism (increased secretion of the thyroid hormones, T 3 and  T 4 ) including a goiter (enlarged thyroid gland). Thyroid function tests  confirmed this diagnosis.   Thyroid hormones (principally T 3 ) modulate cellular energy production and  utilization through their ability to increase the gene transcription of many pro-  teins involved in intermediary metabolism, including enzymes in the TCA cycle  and oxidative phosphorylation. They increase the rate of ATP utilization by Na + ,  K + -ATPase, and other enzymes. They also affect the efficiency of energy trans-  formations, so that either more fuel must be oxidized to maintain a given level of  ATP, or more ATP must be expended to achieve the desired physiological  response. The loss of weight experienced by X.S. Teefore, in spite of a very good  appetite, reflects his increased caloric requirements and a less efficient utiliza-  tion of fuels. The result is an enhanced oxidation of adipose tissue stores as well  as a catabolic effect on muscle and other protein-containing tissues. Through  mechanisms that are not well understood, increased levels of thyroid hormone in  the blood also increase the activity or "tone" of the sympathetic (adrenergic)  nervous system. An activated sympathetic nervous system leads to a more rapid  and forceful heartbeat (tachycardia and palpitations), increased nervousness  (anxiety and insomnia), tremulousness (a sense of shakiness or jitteriness), and  other symptoms.   Cora Nari. Cora Nari was in left ventricular heart failure (LVF) when  she presented to the hospital with her second heart attack in 8 months. The  diagnosis of LVF was based, in part, on her rapid heart rate (104  beats/min) and respiratory rate. On examining her lungs, her physician heard res-  piratory rales, caused by inspired air bubbling in fluid that had filled her lung air  spaces secondary to LVF. This condition is referred to as congestive heart failure.   Cora Nari's rapid heart rate (tachycardia) resulted from a reduced capacity of  her ischemic, failing left ventricular muscle to eject a normal amount of blood into  the arteries leading away from the heart with each contraction. The resultant drop  in intraarterial pressure signaled a reflex response in the central nervous system  that, in turn, caused an increase in heart rate in an attempt to bring the total amount  of blood leaving the left ventricle each minute (the cardiac output) back toward a  more appropriate level to maintain systemic blood pressure.   Treatment of Cora's congestive heart failure will include efforts to reduce the  workload of the heart with diuretics and other "load reducers," attempts to improve  the force of left ventricular contraction with digitalis and other "inotropes," and the  administration of oxygen by nasal cannula to reduce the injury caused by lack of  blood flow (ischemia) to the viable heart tissue in the vicinity of the infarction.   Active Transport and Cell Death. Most of us cannot remember  when we first learned that we would die if we stopped breathing. But  exactly how cells die from a lack of oxygen is an intriguing question.  Pathologists generally describe two histologically distinct types of cell death:  necrosis and apoptosis (programmed cell death). Cell death from a lack of 0 2 , such  as occurs during a myocardial infarction, can be very rapid, and is considered necro-  sis. The lack of ATP for the active transport of Na + and Ca 2+ triggers some of the  death cascades leading to necrosis (Fig. 19.15).   The influx of Na + and loss of the Na + gradient across the plasma membrane is  an early event accompanying ATP depletion during interruption of the 0 2 supply.  One consequence of the increased intracellular Na + concentration is that other  transport processes driven by the Na + gradient are impaired. For example, the  Na + / H + exchanger, which normally pumps out H + generated from metabolism in  exchange for extracellular Na + , can no longer function, and intracellular pH may  drop. The increased intracellular H + may impair ATP generation from anaerobic  glycolysis. As a consequence of increased intracellular ion concentrations, water  enters the cells and hydropic swelling occurs. Swelling is accompanied by the  release of creatine kinase MB subunits, troponin I, and troponin C into the blood.  These enzymes are measured in the blood as indicators of a myocardial infarction  (see Chapters 6 and 7). Swelling is an early event and is considered a reversible  stage of cell injury.   Normally, intracellular Ca 2+ concentration is carefully regulated to fluctuate at  low levels (intracellular Ca 2+ concentration is less than 10" 7 M, compared with  approximately 10" 3 M in extracellular fluid). Fluctuations of Ca 2+ concentration at  these low levels regulate myofibrillar contraction, energy metabolism, and other  cellular processes. However, when Ca 2+ concentration is increased above this nor-  mal range, it triggers cell death (necrosis). High Ca 2+ concentrations activate a  phospholipase that increases membrane permeability, resulting in further loss of ion  gradients across the cell membrane. They also trigger opening of the mitochondrial  permeability transition pore, which results in loss of mitochondrial function and fur-  ther impairs oxidative phosphorylation.   Intracellular Ca 2+ levels may increase as a result of cell swelling, the lack of  ATP for ATP-dependent Ca 2+ pumps, or the loss of the Na + gradient. Normally,   Congestive heart failure occurs  when the weakened pumping  action of the ischemic left ventricu-  lar heart muscle causes back pressure to  increase in the vessels which bring oxy-  genated blood from the lungs to the left side  of the heart. The pressure inside these pul-  monary vessels eventually reaches a critical  level above which water from the blood  moves down a "pressure gradient" from the  capillary lumen into alveolar air spaces of  the lung (transudation). The patient experi-  ences shortness of breath as the fluid in the  air spaces interferes with oxygen exchange  from the inspired air into arterial blood,  causing hypoxia. The hypoxia then stimu-  lates the respiratory center in the central  nervous system, leading to a more rapid  respiratory rate in an effort to increase the  oxygen content of the blood. As the patient  inhales deeply, the physician hears gurgling  sounds (known as inspiratory rales) with a  stethoscope placed over the posterior lung  bases. These sounds represent the bubbling  of inspired air as it enters the fluid-filled pul-  monary alveolar air spaces.   Fig. 19.15. Hypoxia, Ca 2+ , Na + , and cell  death. Without an adequate 0 2 supply,  decreased ATP synthesis from oxidative phos-  phorylation results in an increase of cytoplas-  mic Na + and Ca 2+ ions. Increased ions levels  can trigger death cascades that involve  increased permeability of the plasma mem-  brane, loss of ion gradients, decreased cytoso-  lic pH, mitochondrial Ca 2+ overload, and a  change in mitochondrial permeability called  the mitochondrial permeability transition. The  solid lines show the first sequence of events;  the dashed lines show how these events feed-  back to accelerate the mitochondrial deteriora-  tion, making recovery of oxidative phosphory-  lation impossible.   Ca -ATPases located in the plasma membrane pump Ca - out of the cell. Ca -  ATPases in the endoplasmic reticulum, and in the sarcoplasmic reticulum of heart  and other muscles, sequester Ca 2+ within the membranes, where it is bound by a low-  affinity binding protein. Ca 2+ is released from the sarcoplasmic reticulum in response  to a nerve impulse, which signals contraction, and the increase of Ca 2+ stimulates  both muscle contraction and the oxidation of fuels. Within the heart, another Ca 2+  transporter protein, the Na + /Ca 2+ exchange transporter, coordinates the efflux of  Ca 2+ in exchange for Na + , so that Ca 2+ is extruded with each contraction.   Nelson DL, Lehninger AL, Cox MM. Lehninger Principles of biochemistry. Chapter 14. In: Principles of   Bioenergetics. New York: Worth, 2000:490-526.  Hanson RW. The role of ATP in metabolism. Biochem Educ 1989; 17:86-92.   Rolfe DES, Brown GC. Cellular energy utilization and molecular origin of standard metabolic rate in  mammals. Physiol Rev 1997;77:731-758.   1 . The highest-energy phosphate bond in ATP is located between which of the following groups?   2. Which of the following bioenergetic terms or phrases is correctly defined?   (A) The first law of thermodynamics states that the universe tends towards a state of increased order.   (B) The second law of thermodynamics states that the total energy of a system remains constant.   (C) The change in enthalpy of a reaction is a measure of the total amount of heat that can be released from changes in the  chemical bonds.   (D) AG°' of a reaction is the standard free energy change measured at 37°C and a pH of 7.4.   (E) A high-energy bond is a bond that releases more than 3 kcal/mole of heat when it is hydrolyzed.   3. Which statement best describes the direction a chemical reaction will follow?   (A) A reaction with a positive free energy will proceed in the forward direction if the substrate concentration is raised high  enough.   (B) Under standard conditions, a reaction will proceed in the forward direction if the free energy AG°' is positive.   (C) The direction of a reaction is independent of the initial substrate and product concentrations because the direction is  determined by the change in free energy.   (D) The concentration of all of the substrates must be higher than all of the products to proceed in the forward direction.   (E) The enzyme for the reaction must be working at better than 50% of its maximum efficiency for the reaction to proceed  in the forward direction.   4. A patient, Mr. Perkins, has just suffered a heart attack. As a consequence, his heart would display which of the following  changes?   5. Which of the following statements correctly describes reduction of one of the electron carriers, NAD + or FAD?   (A) NAD + accepts two electrons as hydrogen atoms to form NADH 2   (B) NAD + accepts two electrons that are each donated from a separate atom of the substrate.   (C) NAD + accepts two electrons as a hydride ion to form NADH.   The TCA cycle is frequently called  the Krebs cycle because Sir Hans  Krebs first formulated its reactions  into a cycle. It is also called the "citric acid  cycle" because citrate was one of the first  compounds known to participate. The most  common name for this pathway, the tricar-  boxylic acid or TCA cycle, denotes the  involvement of the tricarboxylates citrate  and isocitrate.   The major pathways of fuel oxida-  tion generate acetyl CoA, which is  the substrate for the TCA cycle. In  the first step of the TCA cycle, the acetyl por-  tion of acetyl CoA combines with the 4-  carbon intermediate oxaloacetate to form  citrate (6 carbons), which is rearranged to  form isocitrate. In the next two oxidative  decarboxylation reactions, electrons are  transferred to NAD + to form NADH, and 2  molecules of electron-depleted C0 2 are  released. Subsequently, a high- energy  phosphate bond in GTP is generated from  substrate level phosphorylation. In the  remaining portion of the TCA cycle, succi-  nate is oxidized to oxaloacetate with the  generation of one FAD(2H) and one NADH.  The net reaction of the TCA cycle, which is  the sum of the equations for individual  steps, shows that the two carbons of the  acetyl group have been oxidized to two mol-  ecules of C0 2 , with conservation of energy  as three molecules of NADH, one of  FAD(2H), and one of GTP.   The TCA cycle (tricarboxylic acid cycle) accounts for over two thirds of the  ATP generated from fuel oxidation. The pathways for oxidation of fatty acids,  glucose, amino acids, acetate, and ketone bodies all generate acetyl CoA,  which is the substrate for the TCA cycle. As the activated 2-carbon acetyl  group is oxidized to two molecules of C0 2 , energy is conserved as NADH,  FAD(2H), and GTP (Fig. 20.1 ). NADH and FAD(2H) subsequently donate  electrons to 0 2 via the electron transport chain, with the generation of ATP  from oxidative phosphorylation. Thus, the TCA cycle is central to energy gen-  eration from cellular respiration.   Within the TCA cycle, the oxidative decarboxylation of a-ketoglutarate is cat-  alyzed by the multisubunit a-ketoglutarate dehydrogenase complex, which con-  tains the coenzymes thiamine -pyrophosphate, lipoate, and FAD. A similar com-  plex, the pyruvate dehydrogenase complex (PDC), catalyzes the oxidation of  pyruvate to acetyl CoA, thereby providing a link between the pathways of glycoly-  sis and the TCA cycle (see Fig. 20.1 )   The two-carbon acetyl group is the ultimate source of the electrons that are  transferred to NAD + and FAD and also the carbon in the two C0 2 molecules that  are produced. Oxaloacetate is used and regenerated in each turn of the cycle ( see  Fig. 20.1). However, when cells use intermediates of the TCA cycle for   Acetyl CoA + 3NAD + + FAD ► 2C0 2 + CoASH + 3 NADH + 3H +   biosynthetic reactions, the carbons of oxaloacetate must be replaced by  anaplerotic (filling up) reactions, such as the pyruvate carboxylase reaction.   The TCA cycle occurs in the mitochondrion, where its flux is tightly coordi-  nated with the rate of the electron transport chain and oxidative phosphorylation  through feedback regulation that reflects the demand for ATP. The rate of the  TCA cycle is increased when ATP utilization in the cell is increased through the  response of several enzymes to ADP levels, the NADHI NAD + ratio, the rate of  FAD(2H) oxidation or the Ca 2+ concentration. For example, isocitrate dehydro-  genase is allosterically activated by ADP.   There are two general consequences to impaired functioning of the TCA cycle:  (1) an inability to generate ATP from fuel oxidation, and (2) an accumulation of  TCA cycle precursors. For example, inhibition of pyruvate oxidation in the TCA  cycle results in its reduction to lactate, which can cause a lactic acidosis. The  most common situation leading to an impaired function of the TCA cycle is a rela-  tive lack of oxygen to accept electrons in the electron transport chain.   Otto Shape, a 26-year-old medical student, has faithfully followed his diet  and aerobic exercise program of daily tennis and jogging (see Chapter 19).  He has lost a total of 33 lb and is just 23 lb from his college weight of 154  lb. His exercise capacity has markedly improved; he can run for a longer time at a  faster pace before noting shortness of breath or palpitations of his heart. Even his  test scores in his medical school classes have improved.   Ann O' Rexia suffers from anorexia nervosa (see Chapters 1,3, and 9). In  addition to a low body weight, decreased muscle mass, glycogen, and fat  stores, she has iron-deficiency anemia (see Chapter 16). She has started to  gain weight, and is trying a daily exercise program. However, she constantly feels weak  and tired. When she walks, she feels pain in her calf muscles. On this visit to her nutri-  tionist, they discuss the vitamin content of her diet, and its role in energy metabolism.   Al Martini has been hospitalized for congestive heart failure (see Chapter  8) and for head injuries sustained while driving under the influence of alco-  hol (Chapters 9 and 10). He completed an alcohol detoxification program,  enrolled in a local Alcoholics Anonymous (AA) group, and began seeing a psy-  chologist. During this time, his alcohol-related neurologic and cardiac manifesta-  tions of thiamine deficiency partially cleared. However, in spite of the support he  was receiving, he began drinking excessive amounts of alcohol again while eating  poorly. Three weeks later, he was readmitted with symptoms of "high output" heart  failure.   In the TCA cycle, the 2-carbon acetyl group of acetyl CoA is oxidized to 2 C0 2 mol-  ecules (see Fig. 20.1). The function of the cycle is to conserve the energy from this oxi-  dation, which it accomplishes principally by transferring electrons from intermediates  of the cycle to NAD + and FAD. The eight electrons donated by the acetyl group even-  tually end up in three molecules of NADH and one of FAD(2H) (Fig. 20.2). As a con-  sequence, ATP can be generated from oxidative phosphorylation when NADH and  FAD(2H) donate these electrons to 0 2 via the electron transport chain.   Fig. 20.2. The acetyl group of acetyl CoA.  Acetyl CoA donates eight electrons to the  TCA cycle, which are shown in blue, and two  carbons. The high-energy bond is shown by  a ~. The acetyl group is the ultimate source of  the carbons in the two molecules of C0 2 that  are produced, and the source of electrons in the  one molecule of FAD(2H) and 3 molecules  NADH, which have each accepted two elec-  trons. However, the same carbon atoms and  electrons that enter from one molecule of  acetyl CoA do not leave as C0 2 , NADH, or  FAD(2H) within the same turn of the cycle.   Synthases, such as citrate synthase,  \ \ ) catalyze condensation of two  *S organic molecules to form a car-  bon-carbon bond. Dehydrogenases, such as  isocitrate dehydrogenase, are enzymes that  remove electron-containing hydrogen or  hydride atoms from a substrate and transfer  them to electron-accepting coenzymes, such  as NAD + or FAD. Aconitase is an isomerase,  an enzyme that catalyzes an internal  rearrangement of atoms or electrons. In  aconitase, a hydroxyl group is being trans-  ferred from one carbon to another. An iron  cofactor in the enzyme facilitates this transfer.   Initially, the acetyl group is incorporated into citrate, an intermediate of the TCA  cycle (Fig. 20.3). As citrate progresses through the cycle to oxaloacetate, it is oxi-  dized by four dehydrogenases (isocitrate dehydrogenase, a-ketoglutarate dehydro-  genase, succinate dehydrogenase, and malate dehydrogenase), which transfer elec-  trons to NAD + or FAD. The isomerase aconitase rearranges electrons in citrate,  thereby forming isocitrate, to facilitate an electron transfer to NAD + .   Although no 0 2 is introduced into the TCA cycle, the two molecules of C0 2 pro-  duced have more oxygen than the acetyl group. These oxygen atoms are ultimately  derived from the carbonyl group of acetyl CoA, two molecules of water added by  fumarase and citrate synthase, and the P0 4 2 " added to GDP.   The overall yield of energy-containing compounds from the TCA cycle is 3  NADH, 1 FAD(2H), and 1 GTP. The high-energy phosphate bond of GTP is gener-  ated from substrate level phosphorylation catalyzed by succinate thiokinase (suc-  cinyl CoA synthetase). As the NADH and FAD(2H) are reoxidized in the electron  transport chain, approximately 2.5 ATP are generated for each NADH, and 1.5 ATP   Fig. 20.3. Reactions of the TCA cycle. The oxidation-reduction enzymes and coenzymes are shown in blue. Entry of the two carbons of acetyl  CoA into the TCA cycle are indicated with blue dashed boxes. The carbons released as C0 2 are shown with black dashed boxes.   for the FAD(2H). Consequently, the net energy yield from the TCA cycle and oxida-  tive phosphorylation is about 10 high-energy phosphate bonds for each acetyl group  oxidized.   The TCA cycle begins with condensation of the activated acetyl group and oxaloac-  etate to form the 6-carbon intermediate citrate, a reaction catalyzed by the enzyme  citrate synthase (see Fig. 20.3). Because oxaloacetate is regenerated with each turn  of the cycle, it is not really considered a substrate of the cycle, or a source of elec-  trons or carbon.   In the next step of the TCA cycle, the hydroxyl (alcohol) group of citrate is  moved to an adjacent carbon so that it can be oxidized to form a keto group. The  isomerization of citrate to isocitrate is catalyzed by the enzyme aconitase, which is  named for an intermediate of the reaction. The enzyme isocitrate dehydrogenase  catalyzes the oxidation of the alcohol group and the subsequent cleavage of the  carboxyl group to release C0 2 (an oxidative decarboxylation).   The next step of the TCA cycle is the oxidative decarboxylation of a-ketoglutarate  to succinyl CoA, catalyzed by the a-ketoglutarate dehydrogenase complex (see Fig.  20.3). The dehydrogenase complex contains the coenzymes thiamine pyrophos-  phate, lipoic acid, and FAD.   In this reaction, one of the carboxyl groups of a-ketoglutarate is released as C0 2 ,  and the adjacent keto group is oxidized to the level of an acid, which then combines  with CoASH to form succinyl CoA (see Fig. 20.3). Energy from the reaction is con-  served principally in the reduction state of NADH, with a smaller amount present  in the high-energy thioester bond of succinyl CoA.   Energy from the succinyl CoA thioester bond is used to generate GTP from GDP and  Pi in the reaction catalyzed by succinate thiokinase (see Fig. 20.3). This reaction is an  example of substrate level phosphorylation. By definition, substrate level phosphoryla-  tion is the formation of a high-energy phosphate bond where none previously existed  without the use of molecular 0 2 (in other words, NOT oxidative phosphorylation). The  high-energy phosphate bond of GTP is energetically equivalent to that of ATP, and can  be used directly for energy-requiring reactions like protein synthesis.   Up to this stage of the TCA cycle, two carbons have been stripped of their available  electrons and released as C0 2 . Two pairs of these electrons have been transferred to  2 NAD + , and one GTP has been generated. However, two additional pairs of elec-  trons arising from acetyl CoA still remain in the TCA cycle as part of succinate. The  remaining steps of the TCA cycle transfer these two pairs of electrons to FAD and  NAD + and add H 2 0, thereby regenerating oxaloacetate.   The sequence of reactions converting succinate to oxaloacetate begins with the  oxidation of succinate to fumarate (see Fig. 20.3). Single electrons are transferred  from the two adjacent -CH 2 - methylene groups of succinate to an FAD bound to  succinate dehydrogenase, thereby forming the double bond of fumarate. From the  reduced enzyme-bound FAD, the electrons are passed into the electron transport  chain. An OFT group and a proton from water add to the double bond of fumarate,  converting it to malate. In the last reaction of the TCA cycle, the alcohol group of  malate is oxidized to a keto group through the donation of electrons to NAD + .   Otto Shape's exercise program  increases his rate of ATP utilization  and his rate of fuel oxidation in the  TCA cycle. The TCA cycle generates NADH  and FAD(2H), and the electron transport  chain transfers electrons from NADH and  FAD(2H) to 0 2 , thereby creating the electro-  chemical potential that drives ATP synthesis  from ADP As ATP is used in the cell, the rate  of the electron transport chain increases.  The TCA cycle and other fuel oxidative path-  ways respond by increasing their rates of  NADH and FAD(2H) production.   Succinate thiokinase is also known  as succinyl CoA synthetase. Both  names refer to the reverse direc-  tion of the reaction, i.e., the conversion of  succinate to the thioester succinyl CoA, uti-  lizing energy from GTP. Synthases, such as  citrate synthase, differ from synthetases in  that synthetases cleave a high- energy phos-  phate bond in ATP, UTP, CTP, or GTP, and  synthases do not.   SFrom Figure 20.3, which enzymes  | in the TCA cycle release C0 2 ? How  many moles of oxaloacetate are  consumed in the TCA cycle for each mole of  C0 2 produced?   The succinate to oxaloacetate  sequence of reactions — oxidation  through formation of a double  bond, addition of water to the double bond,  and oxidation of the resultant alcohol to a  ketone is found in many oxidative pathways  in the cell, such as the pathways for the oxi-  dation of fatty acids, and oxidation of the  branched chain amino acids.   Ann O'Rexia has been malnour-  ished for some time, and has  developed subclinical deficiencies  of many vitamins, including riboflavin. The  coenzymes FAD (flavin adenine dinu-  cleotide) and FMN (flavin mononucleotide)  are synthesized from the vitamin riboflavin.  Riboflavin is actively transported into cells,  where the enzyme flavokinase adds a phos-  phate to form FMN. FAD synthetase then  adds AMP to form FAD. FAD is the major  coenzyme in tissues and is generally found  tightly bound to proteins, with about 10%  being covalently bound. Its turnover in the  body is very slow, and people can live for  long periods on low intakes without display-  ing any signs of a riboflavin deficiency.   /^v Isocitrate dehydrogenase releases  •) the first C0 2 , and a-ketoglutarate  \^^y dehydrogenase releases the sec-  ond C0 2 . There is no net consumption of  oxaloacetate in the TCA cycle — the first step  use an oxaloacetate, and the last step pro-  duces one. The utilization and regeneration  of oxaloacetate is the "cycle" part of the TCA  cycle.   One of Otto Shape's tennis part-  ners told him that he had heard  about a health food designed for  athletes that contained succinate. The adver-  tisement made the claim that succinate  would provide an excellent source of energy  during exercise because it could be metabo-  lized directly without oxygen. Do you see  anything wrong with this statement?   With regeneration of oxaloacetate, the TCA cycle is complete; the chemical bond  energy, carbon, and electrons donated by the acetyl group have been converted to  C0 2 , NADH, FAD(2H), GTP, and heat.   The enzymes of the TCA cycle rely heavily on coenzymes for their catalytic func-  tion. Isocitrate dehydrogenase and malate dehydrogenase use NAD + as a coenzyme,  and succinate dehydrogenase uses FAD. Citrate synthase catalyzes a reaction that  uses a CoA derivative, acetyl CoA. The a-ketoglutarate dehydrogenase complex  uses thiamine pyrophosphate, lipoate and FAD as bound coenzymes, and NAD + and  CoASH as substrates. Each of these coenzymes has unique structural features that  enable it to fulfill its role in the TCA cycle.   Both FAD and NAD + are electron-accepting coenzymes. Why is FAD used in some  reactions and NAD + in others? Their unique structural features enable FAD and  NAD + to act as electron acceptors in different types of reactions, and play different  physiological roles in the cell. FAD is able to accept single electrons (H«), and  forms a half-reduced single electron intermediate (Fig. 20.4). It thus participates in  reactions in which single electrons are transferred independently from two different  atoms, which occurs in double bond formation (e.g., succinate to fumarate) and  disulfide bond formation (e.g., lipoate to lipoate disulfide in the a-ketoglutarate   Fig. 20.4. One-electron steps in the reduction of FAD. When FAD and FMN accept single electrons, they are converted to the half-reduced semi-  quinone, a semistable free radical form. They can also accept two electrons to form the fully reduced form, FADH 2 . However, in most dehydro-  genases, FADH 2 is never formed. Instead, the first electron is shared with a group on the protein as the next electron is transferred. Therefore, in  this text, overall acceptance of two electrons by FAD has been denoted by the more general abbreviation, FAD(2H).   Fig. 20.5. Oxidation and decarboxylation of isocitrate. The alcohol group (C — OH) is oxi-  dized to a ketone, with the C — H electrons donated to NAD + as the hydride ion. Subsequent  electron shifts in the pyridine ring remove the positive charge. The H of the OH group dis-  sociates into water as a proton, H + . NAD + , the electron acceptor, is reduced.   The claim that succinate oxidation  could produce energy without oxy-  gen is wrong. It was probably  based on the fact that succinate is oxidized  to fumarate by the donation of electrons to  FAD. However, ATP can only be generated  from this process when these electrons are  donated to oxygen in the electron transport  chain. The energy generated by the electron  transport chain is used for ATP synthesis in  the process of oxidative phosphorylation.  After the covalently bound FAD(2H) is oxi-  dized back to FAD by the electron transport  chain, succinate dehydrogenase can oxidize  another succinate molecule.   dehydrogenase reaction). In contrast, NAD + accepts a pair of electrons as the  hydride ion (H~), which is attracted to the carbon opposite the positively-charged  pyridine ring (Fig. 20.5). This occurs, for example, in the oxidation of alcohols to  ketones by malate dehydrogenase and isocitrate dehydrogenase. The nicotinamide  ring accepts a hydride ion from the C-H bond, and the alcoholic hydrogen is  released into the medium as a positively charged proton, H + .   The free radical, single-electron forms of FAD are very reactive, and FADH can  lose its electron through exposure to water or the initiation of chain reactions. As a  consequence, FAD must remain very tightly, sometimes covalently, attached to  its enzyme while it accepts and transfers electrons to another group bound on the  enzyme (Fig 20.6). Because FAD interacts with many functional groups on amino  acid side chains in the active site, the E 0 ' for enzyme-bound FAD varies greatly and  can be greater or much less than that of NAD + . In contrast, NAD + and NADH are  more like substrate and product than coenzymes.   NADH plays a regulatory role in balancing energy metabolism that FAD(2H)  cannot because FAD(2H) remains attached to its enzyme. Free NAD + binds to a  dehydrogenase and is reduced to NADH, which is then released into the medium  where it can bind and inhibit a different dehydrogenase. Consequently, oxidative  enzymes are controlled by the NADH/NAD + ratio, and do not generate NADH  faster than it can be reoxidized in the electron transport chain. The regulation of the  TCA cycle and other pathways of fuel oxidation by the NADH/NAD + ratio is part  of the mechanism for coordinating the rate of fuel oxidation to the rate of ATP  utilization.   CoASH, the acylation coenzyme, participates in reactions through the formation of  a thioester bond between the sulfur (S) of CoASH and an acyl group (e.g., acetyl   FAD has been referred to as a married coenzyme, and NAD + is its promiscuous  cousin. FAD faithfully accepts only electrons from a substrate that is bound to  the same enzyme (or enzyme complex), and donates these without leaving that  enzyme. It does this repeatedly while still attached to its enzyme. NAD + , conversely, may  accept electrons when bound to any dehydrogenase, and leaves the enzyme immedi-  ately afterward. It donates these electrons while bound to a different dehydrogenase,  such as NADH dehydrogenase in the electron transport chain. It really gets around!   Fig. 20.6. Succinate dehydrogenase contains  covalently bound FAD. As a consequence, suc-  cinate dehydrogenase and similar flavopro-  teins reside in the inner mitochondrial mem-  brane where they can directly transfer  electrons into the electron transport chain. The  electrons are transferred from the covalently  bound FAD to an Fe-S complex on the  enzyme, and then to coenzyme Q in the elec-  tron transport chain (see Chapter 21). Thus,  FAD does not have to dissociate from the  enzyme to transfer its electrons. All the other  enzymes of the TCA cycle are found in the  mitochondrial matrix.   CoASH is synthesized from the  vitamin pantothenate in a  sequence of reactions which phos-  phorylate pantothenate, add the sulfhydryl  portion of CoA from cysteine, and then add  AMP and an additional phosphate group  from ATP (see Fig. 8.12). Pantothenate is  widely distributed in foods (pantos means  everywhere), so it is unlikely that Ann  O'Rexia has developed a pantothenate defi-  ciency. Although CoA is required in approxi-  mately 100 different reactions in mammalian  cells, no Recommended Daily Allowance  (RDA) has been established for pantothen-  ate, in part because indicators have not yet  been found which specifically and sensi-  tively reflect a deficiency of this vitamin in  the human. The reported symptoms of pan-  tothenate deficiency (fatigue, nausea, and  loss of appetite) are characteristic of vitamin  deficiencies in general.   Fig. 20.7. Utilization of the high-energy thioester bond of acyl Co As. Energy transforma-  tions are shown in blue. A. The energy released by hydrolysis of the thioester bond of acetyl  CoA in the citrate synthase reaction contributes a large negative AG 0 ' to the forward direc-  tion of the TCA cycle. B. The energy of the succinyl CoA thioester bond is used for the syn-  thesis of the high-energy phosphate bond of GTP.   Fig. 20.8. Oxidative decarboxylation of a-  ketoglutarate. The a-ketoglutarate dehydroge-  nase complex oxidizes a-ketoglutarate to suc-  cinyl CoA. The carboxyl group is released as  C0 2 . The keto group on the a-carbon is oxi-  dized, and then forms the acyl CoA thioester,  succinyl CoA. The a, p, 7, and 8 on succinyl  CoA refer to the sequence of atoms in a-ketog-  lutarate.   CoA, succinyl CoA) (Fig. 20.7). The complete structure of CoASH and its vitamin  precursor, pantothenate, is shown in Figure 8.12. A thioester bond differs from a  typical oxygen ester bond because S, unlike O, does not share its electrons and par-  ticipate in resonance formations. One of the consequences of this feature of sulfur  chemistry is that the carbonyl carbon, the a-carbon and the p -carbon of the acyl  group in a CoA thioester can be activated for participation in different types of reac-  tions (e.g., in the citrate synthase reaction, the a-carbon methyl group is activated  for condensation with oxaloacetate, see Figs. 20.3 and 20. 7A). Another conse-  quence is that the thioester bond is a high-energy bond that has a large negative  AG 0 ' of hydrolysis (approximately- 13 kcal/mole).   The energy from cleavage of the high-energy thioester bonds of succinyl CoA  and acetyl CoA is used in two different ways in the TCA cycle. When the succinyl  CoA thioester bond is cleaved by succinate thiokinase, the energy is used directly  for activating an enzyme-bound phosphate that is transferred to GDP (see Fig.  20.7B). In contrast, when the thioester bond of acetyl CoA is cleaved in the citrate  synthase reaction, the energy is released, giving the reaction a large negative AG 0 '  of -7.7 kcal/mole. The large negative AG 0 ' for citrate formation helps to keep the  TCA cycle going in the forward direction.   The a-ketoglutarate dehydrogenase complex is one of a three-member family of  similar a-keto acid dehydrogenase complexes. The other members of this family are  the pyruvate dehydrogenase complex, and the branched chain amino acid a-keto  acid dehydrogenase complex. Each of these complexes is specific for a different a-  keto acid structure. In the sequence of reactions catalyzed by the complexes, the a-  ketoacid is decarboxylated (i.e., releases the carboxyl group as C0 2 ) (Fig. 20.8). The  keto group is oxidized to the level of a carboxylic acid, and then combined with  CoASH to form an acyl CoA thioester (e.g., succinyl CoA).   All of the a-ketoacid dehydrogenase complexes are huge enzyme complexes  composed of multiple subunits of three different enzymes, E 1? E 2 , and E 3  (Fig. 20.9). Ej is an a-ketoacid decarboxylase which contains thiamine  pyrophosphate (TPP); it cleaves off the carboxyl group of the a-keto acid. E 2 is  a transacylase containing lipoate; it transfers the acyl portion of the a-keto acid  from thiamine to CoASH. E 3 is dihydrolipoyl dehydrogenase, which contains   Fig. 20.9. Mechanism of a-keto acid dehydrogenase complexes (including a-ketoglutarate  dehydrogenase, pyruvate dehydrogenase and the branched-chain a-keto acid dehydrogenase  complex). R represents the portion of the a-ketoacid that begins with the p carbon. In a-  ketoglutarate, R is CH 2 -CH 2 -COOH. In pyruvate, R is CH 3 . The individual steps in the oxida-  tive decarboxylation of a-keto acids are catalyzed by three different subunits: E 1? a-ketoacid  decarboxylase (a-ketoglutarate decarboxylase); E 2 , transacylase (trans-succinylase), and E 3 ,  dihydrolipoyl dehydrogenase. Circle 1: Thiamine pyrophosphate (TPP) on E x decarboxylates  the a-ketoacid and forms a covalent intermediate with the remaining portion. Circle 2: The  acyl portion of the a-keto acid is transferred by TPP on E x to lipoate on E 2 , which is a  transacylase. Circle 3: E 2 transfers the acyl group from lipoate to CoASH. This process has  reduced the lipoyl disulfide bond to sulfhydryl groups (dihydrolipoyl). Circle 4: E 3 , dihy-  drolipoyl dehydrogenase (DH) transfers the electrons from reduced lipoate to its tightly  bound FAD molecule, thereby oxidizing lipoate back to its original disulfide form. Circle 5:  The electrons are then transferred from FAD(2H) to NAD + to form NADH.   FAD; it transfers electrons from reduced lipoate to NAD + . The collection of 3  enzyme activities into one huge complex enables the product of one enzyme to  be transferred to the next enzyme without loss of energy. Complex formation  also increases the rate of catalysis because the substrates for E 2 and E 3 remain   -0.32. Thus, transfer of electrons from  FAD(2H) to NAD + is energetically unfavor-  able. How do the a-keto acid dehydrogenase  complexes allow this electron transfer to  occur?   Thiamine pyrophosphate is synthesized from the vitamin thiamine by the addition  of pyrophosphate (see Fig. 8.11). The pyrophosphate group binds magnesium,  which binds to amino acid side chains on the enzyme. This binding is relatively  weak for a coenzyme, so thiamine turns over rapidly in the body, and a deficiency  can develop rapidly in individuals on a thiamine-free or low thiamine diet.   The general function of thiamine pyrophosphate is the cleavage of a carbon-  carbon bond next to a keto group. In the a-ketoglutarate, pyruvate, and branched  chain a-keto acid dehydrogenase complexes, the functional carbon on the thiazole  ring forms a covalent bond with the a-keto carbon, thereby cleaving the bond  between the a-keto carbon and the adjacent carboxylic acid group (see Fig. 8.11 for  the mechanism of this reaction). Thiamine pyrophosphate is also a coenzyme for  transketolase in the pentose phosphate pathway, where it similarly cleaves the car-  bon-carbon bond next to a keto group. In thiamine deficiency, a-ketoglutarate,  pyruvate, and other a-keto acids accumulate in the blood.   Lipoate is a coenzyme found only in a-keto acid dehydrogenase complexes. It is  synthesized in the human from carbohydrate and amino acids, and does not require   In Al Martini's heart failure, which  is caused by a dietary deficiency of  the vitamin thiamine, pyruvate  dehydrogenase, a-ketoglutarate dehydroge-  nase, and the branched chain a-keto acid  dehydrogenase complexes are less func-  tional than normal. Because heart muscle,  skeletal muscle, and nervous tissue have a  high rate of ATP production from the NADH  produced by the oxidation of pyruvate to  acetyl CoA and of acetyl CoA to C0 2 in the  TCA cycle, these tissues present with the  most obvious signs of thiamine deficiency.   In Western societies, gross thiamine defi-  ciency is most often associated with alco-  holism. The mechanism for active absorp-  tion of thiamine is strongly and directly  inhibited by alcohol. Subclinical deficiency  of thiamine from malnutrition or anorexia  may be common in the general population  and is usually associated with multiple vita-  min deficiencies.   The E 0 ' values were calculated in a test tube under standard conditions. When  FAD is bound to an enzyme, as it is in the a-keto acid dehydrogenase com-  plexes, amino acid side chains can alter its E 0 ' value. Thus, the transfer of elec-  trons from the bound FAD(2H) to NAD + in dihydrolipoyl dehydrogenase is actually ener-  getically favorable.    q q Arsenic poisoning is caused by the  f /~oA ) presence of a large number of dif-  ™ ferent arsenious compounds that  are effective metabolic inhibitors. Acute  accidental or intentional arsenic poisoning  requires high doses and involves arsenate  (As0 4 2 ~) and arsenite (AsO 2 ). Arsenite,  which is 10 times more toxic than arsenate,  binds to neighboring sulfhydryl groups,  such as those in dihydrolipoate and in  nearby cysteine pairs (vicinal) found in a-  keto acid dehydrogenase complexes and in  succinic dehydrogenase. Arsenate weakly  inhibits enzymatic reactions involving phos-  phate, including the enzyme glyceraldehyde  3-P dehydrogenase in glycolysis (see Chap-  ter 22). Thus both aerobic and anaerobic ATP  production can be inhibited. The low doses  of arsenic compounds found in water sup-  plies are a major public health concern, but  are associated with increased risk of cancer  rather than direct toxicity.   CH 2 CH CH 2 CH 2 N-lysine-  \ / H transacylase   Fig. 20.10. Function of lipoate. Lipoate is  attached to the e-amino group on the lysine  side chain of the tranacylase enzyme (E 2 ). The  oxidized lipoate disulfide form is reduced as it  accepts the acyl group from thiamine  pyrophosphate (TPP) attached to The  example shown is for the a-ketoglutarate  dehydrogenase complex.   a vitamin precursor. Lipoate is attached to the transacylase enzyme through its  carboxyl group, which is covalently bound to the terminal -NH 2 of a lysine in the  protein (Fig. 20.10). At its functional end, lipoate contains a disulfide group that  accepts electrons when it binds the acyl fragment of a-ketoglutarate. It can thus act  like a long flexible -CH 2 - arm of the enzyme that reaches over to the decarboxylase  to pick up the acyl fragment from thiamine and transfer it to the active site contain-  ing bound CoASH. It then swings over to dihydrolipoyl dehydrogenase to transfer  electrons from the lipoyl sulfhydryl groups to FAD.   FAD on dihydrolipoyl dehydrogenase accepts electrons from the lipoyl sulfhydryl  groups and transfers them to bound NAD + . FAD thus accepts and transfers elec-  trons without leaving its binding site on the enzyme. The direction of the reaction  is favored by interactions of FAD with groups on the enzyme, which change its  reduction potential and by the overall release of energy from cleavage and oxida-  tion of a-ketoglutarate.   Like all metabolic pathways, the TCA cycle operates with an overall net negative  AG 0 ' (Fig 20.11). The conversion of substrates to products is, therefore, energeti-  cally favorable. However, some of the reactions, such as the malate dehydrogenase  reaction, have a positive value.   Fig. 20.11. Approximate AG 0 ' values for the reactions in the TCA cycle, given for the for-  ward direction. The reactions with large negative AG 0 ' values are shown in blue. The stan-  dard free energy (AG 0 ') refers to the free energy change for conversion of 1 mole of substrate  to 1 mole of product under standard conditions (see Chapter 19).   The reactions of the TCA cycle are extremely efficient in converting energy in the  chemical bonds of the acetyl group to other forms. The total amount of energy avail-  able from the acetyl group is about 228 kcal/mole (the amount of energy that could  be released from complete combustion of 1 mole of acetyl groups to C0 2 in a bomb  calorimeter). The products of the TCA cycle (NADH, FAD(2H), and GTP) contain  about 207 kcal (Table 20.1). Thus, the TCA cycle reactions are able to conserve  about 90% of the energy available from the oxidation of acetyl Co A.   Three reactions in the TCA cycle have large negative values for AG 0 ' that strongly  favor the forward direction: the reactions catalyzed by citrate synthase, isocitrate  dehydrogenase, and a-ketoglutarate dehydrogenase (see Fig. 20.1 1). Within the TCA  cycle, these reactions are physiologically irreversible for two reasons: the products  do not rise to high enough concentrations under physiological conditions to over-  come the large negative AG 0 ' values, and the enzymes involved catalyze the reverse  reaction very slowly. These reactions make the major contribution to the overall neg-  ative AG 0 ' for the TCA cycle, and keep it going in the forward direction.   In contrast to these irreversible reactions, the reactions catalyzed by aconitase  and malate dehydrogenase have a positive AG 0 ' for the forward direction, and are  thermodynamically and kinetically reversible. Because aconitase is rapid in both  directions, equilibrium values for the concentration ratio of products to substrates is  maintained, and the concentration of citrate is about 20 times that of isocitrate. The  accumulation of citrate instead of isocitrate facilitates transport of excess citrate to  the cytosol, where it can provide a source of acetyl CoA for pathways like fatty acid  and cholesterol synthesis. It also allows citrate to serve as an inhibitor of citrate syn-  thase when flux through isocitrate dehydrogenase is decreased. Likewise, the equi-  librium constant of the malate dehydrogenase reaction favors the accumulation of  malate over oxaloacetate, resulting in a low oxaloacetate concentration that is influ-  enced by the NADH/NAD + ratio. Thus, there is a net flux of oxaloacetate towards  malate in the liver during fasting (due to fatty acid oxidation, which raises the  NADH/NAD + ratio), and malate can then be transported out of the mitochondria to  provide a substrate for gluconeogenesis.   The oxidation of acetyl CoA in the TCA cycle and the conservation of this energy  as NADH and FAD(2H) is essential for generation of ATP in almost all tissues in  the body. In spite of changes in the supply of fuels, type of fuels in the blood, or rate  of ATP utilization, cells maintain ATP homeostasis (a constant level of ATP). The  rate of the TCA cycle, like that of all fuel oxidation pathways, is principally regu-  lated to correspond to the rate of the electron transport chain, which is regulated by  the ATP/ADP ratio and the rate of ATP utilization (see Chapter 21). The major sites  of regulation are shown in Fig 20.12.   Two major messengers feed information on the rate of ATP utilization back to  the TCA cycle: (a) the phosphorylation state of ATP, as reflected in ATP and ADP  levels, and (b) the reduction state of NAD + , as reflected in the ratio of  NADH/NAD + . Within the cell, even within the mitochondrion, the total adenine  nucleotide pool (AMP, ADP, plus ATP) and the total NAD pool (NAD + plus NADH)  are relatively constant. Thus, an increased rate of ATP utilization results in a small  decrease of ATP concentration and an increase of ADP. Likewise, increased NADH  oxidation to NAD + by the electron transport chain increases the rate of pathways  producing NADH. Under normal physiological conditions, the TCA cycle and other   Chapter 19 explains the values given for energy yield  from NADH and FAD(2H).   ^The net standard free energy  change for the TCA cycle, AG 0 ', can  be calculated from the sum of the  AG 0 ' values for the individual reactions. The  AG 0 ', -13 kcal, is the amount of energy lost  as heat. It can be considered the amount of  energy spent to ensure that oxidation of the  acetyl group to C0 2 goes to completion. This  value is surprisingly small. However, oxida-  tion of NADH and FAD(2H) in the electron  transport chain helps to make acetyl oxida-  tion more energetically favorable and pull  the TCA cycle forward.   Otto Shape had difficulty losing  weight because human fuel utiliza-  tion is too efficient. His adipose tis-  sue fatty acids are being converted to acetyl  CoA, which is being oxidized in the TCA  cycle, thereby generating NADH and  FAD(2H). The energy in these compounds is  used for ATP synthesis from oxidative phos-  phorylation. If his fuel utilization were less  efficient and his ATP yield were lower, he  would have to oxidize much greater  amounts of fat to get the ATP he needs for  exercise.   As Otto Shape exercises, his  myosin ATPase hydrolyzes ATP to  provide the energy for movement  of myofibrils. The decrease of ATP and  increase of ADP stimulates the electron  transport chain to oxidize more NADH and  FAD(2H). The TCA cycle is stimulated to pro-  vide more NADH and FAD(2H) to the elec-  tron transport chain. The activation of the  TCA cycle occurs through a decrease of the  NADH/NAD + ratio, an increase of ADP con-  centration, and an increase of Ca 2+ .  Although regulation of the transcription of  genes for TCA cycle enzymes is too slow to  respond to changes of ATP demands during  exercise, the number and size of mitochon-  dria increase during training. Thus, Otto  Shape is increasing his capacity for fuel oxi-  dation as he trains.    Fig. 20.12. Major regulatory interactions in the TCA cycle. The rate of ATP hydrolysis controls the rate of ATP synthesis, which controls the  rate of NADH oxidation in the electron transport chain (ETC). All NADH and FAD(2H) produced by the cycle donate electrons to this chain  (shown on the right). Thus, oxidation of acetyl CoA in the TCA cycle can go only as fast as electrons from NADH enter the electron transport  chain, which is controlled by the ATP and ADP content of the cells. The ADP and NADH concentrations feed information on the rate of oxida-  tive phosphorylation back to the TCA cycle. Isocitrate dehydrogenase (DH), a-ketoglutarate dehydrogenase (DH), and malate dehydrogenase  (DH) are inhibited by increased NADH concentration. The NADH/NAD + ratio changes the concentration of oxaloacetate. Citrate is a product  inhibitor of citrate synthase. ADP is an allosteric activator of isocitrate dehydrogenase. During muscular contraction, increased Ca 2+ concentra-  tions activate isocitrate DH and a-ketoglutarate dehydrogenase (as well as pyruvate dehydrogenase).   oxidative pathways respond so rapidly to increased ATP demand that the ATP con-  centration does not significantly change.   The principles of pathway regulation are summarized in Table 20.2. In pathways  subject to feedback regulation, the first step of the pathway must be regulated so that    1 . Regulation matches function. The type of regulation use depends on the function of the pathway. Tissue-specific isozymes may allow the fea-  tures of regulatory enzymes to match somewhat different functions of the pathway in different tissues.   2. Regulation of metabolic pathways occurs at rate-limiting steps, the slowest steps, in the pathway. These are reactions in which a small  change of rate will affect the flux through the whole pathway.   3. Regulation usually occurs at the first committed step of a pathway or at metabolic branchpoints. In human cells, most pathways are intercon-  nected with other pathways and have regulatory enzymes for every branchpoint.   4. Regulatory enzymes often catalyze physiologically irreversible reactions. These are also the steps that differ in biosynthetic and degradative  pathways.   5. Many pathways have "feedback" regulation, that is, the endproduct of the pathway controls the rate of its own synthesis. Feedback regula-  tion may involve inhibition of an early step in the pathway (feedback inhibition) or regulation of gene transcription.   6. Human cells use compartmentation to control access of substrate and activators or inhibitors to different enzymes.   7. Hormonal regulation integrates responses in pathways requiring more than one tissue. Hormones generally regulate fuel metabolism by:   b. Changing the amount of enzyme present by changing its rate of synthesis (often induction or repression of mRNA synthesis) or degradation.   precursors flow into alternate pathways if product is not needed. Citrate synthase,  which is the first enzyme of the TCA cycle, is a simple enzyme that has no allosteric  regulators. Its rate is controlled principally by the concentration of oxaloacetate, its sub-  strate, and the concentration of citrate, a product inhibitor, competitive with oxaloac-  etate. (see Fig. 20.12). The malate-oxaloacetate equilibrium favors malate, so the  oxaloacetate concentration is very low inside the mitochondrion, and is below the  K mapv (see Chapter 9, section I.A.4) of citrate synthase. When the NADH/NAD + ratio  decreases, the ratio of oxaloacetate to malate increases. When isocitrate dehydroge-  nase is activated, the concentration of citrate decreases, thus relieving the product  inhibition of citrate synthase. Thus, both increased oxaloacetate and decreased citrate  levels regulate the response of citrate synthase to conditions established by the elec-  tron transport chain and oxidative phosphorylation. In the liver, the NADH/NAD +  ratio helps determine whether acetyl CoA enters the TCA cycle or goes into the alter-  nate pathway for ketone body synthesis.   Another generalization that can be made about regulation of metabolic pathways is  that it occurs at the enzyme that catalyzes the rate-limiting (slowest) step in a  pathway (see Table 20.2). Isocitrate dehydrogenase is considered one of the rate-  limiting steps of the TCA cycle, and is allosterically activated by ADP and inhib-  ited by NADH (Fig. 20.13). In the absence of ADP, the enzyme exhibits positive  cooperativity; as isocitrate binds to one subunit, other subunits are converted to an  active conformation (see Chapter 9, section III.A on allosteric enzymes). In the  presence of ADP, all of the subunits are in their active conformation, and isocitrate  binds more readily. Consequently, the K mapp (the S 0 5 ) shifts to a much lower value.  Thus, at the concentration of isocitrate found in the mitochondrial matrix, a small  change in the concentration of ADP can produce a large change in the rate of the  isocitrate dehydrogenase reaction. Small changes in the concentration of the prod-  uct, NADH, and of the cosubstrate, NAD + , also affect the rate of the enzyme more  than they would a nonallosteric enzyme.   The a-ketoglutarate dehydrogenase complex, although not an allosteric enzyme, is  product-inhibited by NADH and succinyl CoA, and may also be inhibited by GTP (see  Fig. 20.12). Thus, both a-ketoglutarate dehydrogenase and isocitrate dehydrogenase  respond directly to changes in the relative levels of ADP and hence the rate at which  NADH is oxidized by electron transport. Both of these enzymes are also activated by  Ca 2+ . In contracting heart muscle, and possibly other muscle tissues, the release of  Ca 2+ from the sarcoplasmic reticulum during muscle contraction may provide an addi-  tional activation of these enzymes when ATP is being rapidly hydrolyzed.   Regulation of the TCA cycle serves two functions: it ensures that NADH is gener-  ated fast enough to maintain ATP homeostasis and it regulates the concentration of  TCA cycle intermediates. For example, in the liver, a decreased rate of isocitrate  dehydrogenase increases citrate concentration, which stimulates citrate efflux to the  cytosol. A number of regulatory interactions occur in the TCA cycle, in addition to  those mentioned above, that control the levels of TCA intermediates and their flux  into pathways that adjoin the TCA cycle.   Fig. 20.13. Allosteric regulation of isocitrate  dehydrogenase (ICDH). Isocitrate dehydroge-  nase has eight subunits, and two active sites.  Isocitrate, NAD + , and NADH bind in the  active site; ADP and Ca 2+ are activators and  bind to separate allosteric sites. A. A graph of  velocity versus isocitrate concentration shows  positive cooperativity (sigmoid curve) in the  absence of ADR The allosteric activator ADP  changes the curve into one closer to a rectan-  gular hyperbola, and decreases the K m (S 0 5 )  for isocitrate. B. The allosteric activation by  ADP is not an all-or-nothing response. The  extent of activation by ADP depends on its  concentration. C. Increases in the concentra-  tion of product, NADH, decrease the velocity  of the enzyme through effects on the allosteric  activation.   Compounds enter the TCA cycle as acetyl CoA or as an intermediate that can be  converted to malate or oxaloacetate. Compounds that enter as acetyl CoA are   Acetate (acetic acid) is present in  \\ ) the diet, and can be produced from   the oxidation of ethanol. Roman  soldiers carried vinegar, a dilute solution of  acetic acid. The acidity of the vinegar made  it a relatively safe source of drinking water  because many kinds of pathogenic bacteria  do not grow well in acid solutions. The  acetate, which is activated to acetyl CoA,  provided an excellent fuel for muscular  exercise.   oxidized to C0 2 . Compounds that enter as TCA cycle intermediates replenish inter-  mediates that have been used in biosynthetic pathways, such as gluconeogenesis or  heme synthesis, but cannot be fully oxidized to C0 2 .   Acetyl CoA serves as a common point of convergence for the major pathways of  fuel oxidation. It is generated directly from the p -oxidation of fatty acids and degra-  dation of the ketone bodies p -hydroxy butyrate and acetoacetate (Fig. 20.14). It is  also formed from acetate, which can arise from the diet or from ethanol oxidation.  Glucose and other carbohydrates enter glycolysis, a pathway common to all cells,  and are oxidized to pyruvate. The amino acids alanine and serine are also converted  to pyruvate. Pyruvate is oxidized to acetyl CoA by the pyruvate dehydrogenase  complex. A number of amino acids, such as leucine and isoleucine are also oxidized  to acetyl CoA. Thus, the final oxidation of acetyl CoA to C0 2 in the TCA cycle is  the last step in all the major pathways of fuel oxidation.   The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA, thus  linking glycolysis and the TCA cycle. In the brain, which is dependent on the oxi-  dation of glucose to C0 2 to fulfill its ATP needs, regulation of the PDC is a life and  death matter.   PDC belongs to the a-ketoacid dehydrogenase complex family and, thus, shares  structural and catalytic features with the a-ketoglutarate dehydrogenase complex and  the branched chain a-ketoacid dehydrogenase complex (Fig. 20.15). It contains the  same three basic types of catalytic subunits: (1) pyruvate decarboxylase subunits that  bind thiamine-pyrophosphate (Ej); (2) transacetylase subunits that bind lipoate (E 2 ),  and (3) dihyrolipoyl dehydrogenase subunits that bind FAD (E 3 ) (see Fig. 20.9).  Although the E r and E 2 enzymes in PDC are relatively specific for pyruvate, the same  dihydrolipoyl dehydrogenase participates in all of the a-ketoacid dehydrogenase   Fig. 20.15. Pyruvate dehydrogenase complex  (PDC) catalyzes oxidation of the a-ketoacid  pyruvate to acetyl CoA.   Fig. 20.14. Origin of the acetyl group from various fuels. Acetyl CoA is derived from the  oxidation of fuels. The portions of fatty acids, ketone bodies, glucose, pyruvate, the amino  acid alanine, and ethanol that are converted to the acetyl group of acetyl CoA are shown in  blue.   complexes. In addition to these three types of subunits, the PDC complex contains one  additional catalytic subunit, protein X, which is a transacetylase. Each functional  component of the PDC complex is present in multiple copies (e.g., bovine heart PDC  has 30 subunits of E 1? 60 subunits of E 2 , and 6 subunits each of E 3 and X). The E 1  enzyme is itself a tetramer of two different types of subunits, a and p.   PDC activity is controlled principally through phosphorylation by pyruvate dehy-  drogenase kinase, which inhibits the enzyme, and dephosphorylation by pyruvate  dehydrogenase phosphatase, which activates it (Fig. 20.16). Pyruvate dehydroge-  nase kinase and pyruvate dehydrogenase phosphatase are regulatory subunits within  the PDC complex and act only on the complex. PDC kinase transfers a phosphate  from ATP to specific serine hydroxyl (ser-OH) groups on pyruvate decarboxylase  (Ej). PDC phosphatase removes these phosphate groups by hydrolysis. Phosphory-  lation of just one serine on the PDC E 1 a subunit can decrease its activity by over  99%. PDC kinase is present in complexes as tissue- specific isozymes that vary in  their regulatory properties.   PDC kinase is, itself, inhibited by ADP and pyruvate. Thus, when rapid ATP uti-  lization results in an increase of ADP, or when activation of glycolysis increases  pyruvate levels, PDC kinase is inhibited, and PDC remains in an active, nonphos-  phorylated form. PDC phosphatase requires Ca 2+ for full activity. In the heart,  increased intramitochondrial Ca 2+ during rapid contraction activates the phos-  phatase, thereby increasing the amount of active, nonphosphorylated PDC.   PDC is also regulated through inhibition by its products, acetyl CoA and NADH.  This inhibition is stronger than regular product inhibition because their binding to   Deficiencies of the pyruvate dehy-  drogenase complex (PDC) are  among the most common inher-  ited diseases leading to lacticacidemia and,  like pyruvate carboxylase deficiency, are  grouped into the category of Leigh's disease.  In its severe form, PDC deficiency presents  with overwhelming lactic acidosis at birth,  with death in the neonatal period. In a sec-  ond form of presentation, the lactic acade-  mia is moderate, but there is profound psy-  chomotor retardation with increasing age. In  many cases, concomitant damage to the  brain stem and basal ganglia lead to death in  infancy. The neurological symptoms arise  because the brain has a very limited ability  to use fatty acids as a fuel, and is, therefore,  dependent on glucose metabolism for its  energy supply.   The most common PDC genetic defects  are in the gene for the a subunit of E v The E q  a-gene is X-linked. Because of its impor-  tance in central nervous system metabolism,  pyruvate dehydrogenase deficiency is a  problem in both males and females, even if  the female is a carrier. For this reason, it is  classified as an X-linked dominant disorder.    Fig. 20.16. Regulation of pyruvate dehydrogenase complex (PDC). PDC kinase, a subunit of  the enzyme, phosphorylates PDC at a specific serine residue, thereby converting PDC to an  inactive form. The kinase is inhibited by ADP and pyruvate. PDC phosphatase, another sub-  unit of the enzyme, removes the phosphate, thereby activating PDC. The phosphatase is acti-  vated by Ca 2+ . When the substrates, pyruvate and CoASH, are bound to PDC, the kinase  activity is inhibited and PDC is active. When the products acetyl CoA and NADH bind to  PDC, the kinase activity is stimulated, and the enzyme is phosphorylated to the inactive form.  E x and the kinase exist as tissue-specific isozymes with overlapping tissue specificity, and  somewhat different regulatory properties.   PDC stimulates its phosphorylation to the inactive form. The substrates of the  enzyme, CoASH and NAD + , antagonize this product inhibition. Thus, when an  ample supply of acetyl CoA for the TCA cycle is already available from fatty acid  oxidation, acetyl CoA and NADH build up and dramatically decrease their own fur-  ther synthesis by PDC.   PDC can also be rapidly activated through a mechanism involving insulin, which  plays a prominent role in adipocytes. In many tissues, insulin may, slowly over time,  increase the amount of pyruvate dehydrogenase complex present.   The rate of other fuel oxidation pathways that feed into the TCA cycle is also  increased when ATP utilization increases. Insulin, other hormones and diet control  the availability of fuels for these oxidative pathways.   Pyruvate, citrate, a-ketoglutarate  KJ ) and malate, ADP, ATP, and phos-  v^^x phate (as well as many other com-  pounds) have specific transporters in the  inner mitochondrial membrane that trans-  port compounds between the mitochondrial  matrix and cytosol in exchange for a com-  pound of similar charge. In contrast, CoASH,  acetyl CoA, other CoA derivatives, NAD + and  NADH, and oxaloacetate, are not trans-  ported at a metabolically significant rate. To  obtain cytosolic acetyl CoA, many cells  transport citrate to the cytosol, where it is  cleaved to acetyl CoA and oxaloacetate by  citrate lyase.   The intermediates of the TCA cycle serve as precursors for a variety of different path-  ways present in different cell types (Fig. 20.17). This is particularly important in the  central metabolic role of the liver. The TCA cycle in the liver is often called an "open  cycle" because there is such a high efflux of intermediates. After a high carbohydrate  meal, citrate efflux and cleavage to acetyl CoA provides acetyl units for cytosolic fatty  acid synthesis. During fasting, gluconeogenic precursors are converted to malate,  which leaves the mitochondria for cytosolic gluconeogenesis. The liver also uses TCA  cycle intermediates to synthesize carbon skeletons of amino acids. Succinyl CoA may  be removed from the TCA cycle to form heme in cells of the liver and bone marrow.  In the brain, a-ketoglutarate is converted to glutamate and then to 7-aminobutyric acid  (GABA), a neurotransmitter. In skeletal muscle, a-ketoglutarate is converted to gluta-  mine, which is transported through the blood to other tissues.   Removal of any of the intermediates from the TCA cycle removes the 4 carbons that  are used to regenerate oxaloacetate during each turn of the cycle. With depletion of  oxaloacetate, it is impossible to continue oxidizing acetyl CoA. To enable the TCA   Fig. 20.17. Efflux of intermediates from the TCA cycle. In the liver, TCA cycle intermedi-  ates are continuously withdrawn into the pathways of fatty acid synthesis, amino acid syn-  thesis, gluconeogenesis, and heme synthesis. In brain, a-ketoglutarate is converted to gluta-  mate and GABA, both neurotransmitters.   cycle to keep running, cells have to supply enough four-carbon intermediates from  degradation of carbohydrate or certain amino acids to compensate for the rate of  removal. Pathways or reactions that replenish the intermediates of the TCA cycle  are referred to as anaplerotic ("filling up").   Pyruvate carboxylase is one of the major anaplerotic enzymes in the cell. It cat-  alyzes the addition of C0 2 to pyruvate to form oxaloacetate (Fig. 20.18). Like most  carboxylases, pyruvate carboxylase contains biotin, which forms a covalent inter-  mediate with C0 2 in a reaction requiring ATP and Mg 2+ (see Fig. 8.12, Chap. 8).  The activated C0 2 is then transferred to pyruvate to form the carboxyl group of  oxaloacetate.   Pyruvate carboxylase is found in many tissues, such as liver, brain, adipocytes,  and fibroblasts, where its function is anaplerotic. Its concentration is high in liver  and kidney cortex, where there is a continuous removal of oxaloacetate and malate  from the TCA cycle to enter the gluconeogenic pathway.   Pyruvate carboxylase is activated by acetyl CoA and inhibited by high concen-  trations of many acyl CoA derivatives. As the concentration of oxaloacetate is  depleted through the efflux of TCA cycle intermediates, the rate of the citrate syn-  thase reaction decreases and acetyl CoA concentration rises. The acetyl CoA then  activates pyruvate carboxylase to synthesize more oxaloacetate.   Fig. 20.18. Pyruvate carboxylase reaction.  Pyruvate carboxylase adds a carboxyl group  from bicarbonate (which is in equilibrium with  C0 2 ) to pyruvate to form oxaloacetate. Biotin  is used to activate and transfer the C0 2 . The  energy to form the covalent biotin-C0 2 com-  plex is provided by the high-energy phosphate  bond of ATP, which is cleaved in the reaction.  The enzyme is activated by acetyl CoA.   The pathways for oxidation of many amino acids convert their carbon skeletons  into 5- and 4-carbon intermediates of the TCA cycle that can regenerate oxaloac-  etate (Fig 20.19). Alanine and serine carbons can enter through pyruvate car-  boxylase (see Fig. 20. 19, circle 1). In all tissues with mitochondria (except for,  surprisingly, the liver), oxidation of the two branched chain amino acids  isoleucine and valine to succinyl CoA forms a major anaplerotic route (see  Fig.20.19, circle 3). In the liver, other compounds forming propionyl CoA (e.g.,  methionine, thymine and odd-chain length or branched fatty acids) also enter the  TCA cycle as succinyl CoA. In most tissues, glutamine is taken up from the  blood, converted to glutamate, and then oxidized to a-ketoglutarate, forming  another major anaplerotic route (see Fig.20.19, circle 2). However, the TCA  cycle cannot be resupplied with intermediates by even chain length fatty acid  oxidation, or ketone body oxidation, which forms only acetyl CoA. In the TCA  cycle, two carbons are lost from citrate before succinyl CoA is formed, and,  therefore, there is no net conversion of acetyl carbon to oxaloacetate.   Biotin is a vitamin. A deficiency of  \\ ) biotin is very rare in humans  *r because it is required in such small  amounts and is synthesized by intestinal  bacteria. However, an interesting case of  biotin deficiency arose in a man eating a diet  composed principally of peanuts and raw  egg whites. Egg whites contain a biotin bind-  ing protein, avidin. Since he did not dena-  ture avidin by cooking the egg whites, it  depleted his diet of biotin.   Pyruvate carboxylase deficiency is one of the genetic diseases grouped  together under the clinical manifestations of Leigh's disease (subacute necro-  tizing encephalopathy). In the mild form, the patient presents early in life with  delayed development and a mild-to-moderate lactic acidemia. Patients who survive are  severely mentally retarded, and there is a loss of cerebral neurons. In the brain, pyruvate  carboxylase is present in the astrocytes, which use TCA cycle intermediates to synthe-  size glutamine. This pathway is essential for neuronal survival. The major cause of the  lactic acidemia is that cells dependent on pyruvate carboxylase for an anaplerotic supply  of oxaloacetate cannot oxidize pyruvate in the TCA cycle (because of low oxaloacetate  levels), and the liver cannot convert pyruvate to glucose (because the pyruvate carboxy-  lase reaction is required for this pathway to occur), so the excess pyruvate is converted  to lactate.    Fig. 20.19. Major anaplerotic pathways of the TCA cycle. 1 and 3 (blue arrows) are the two  major anabolic pathways. (1) Pyruvate carboxylase (2) Glutamate is reversibly converted to  a-ketoglutarate by transaminases (TA) and glutamate dehydrogenase (GDH) in many tissues.  (3) The carbon skeletons of valine and isoleucine, a 3-carbon unit from odd chain fatty acid  oxidation, and a number of other compounds enter the TCA cycle at the level of succinyl  CoA. Other amino acids are also degraded to fumarate (4) and oxaloacetate (5), principally  in the liver.   In skeletal muscle and other tis-  \\ ) sues, ATP is generated by anaero-   bic glycolysis when the rate of aer-  obic respiration is inadequate to meet the  rate of ATP utilization. Under these circum-  stances, the rate of pyruvate production  exceeds the cell's capacity to oxidize NADH  in the electron transport chain, and hence, to  oxidize pyruvate in the TCA cycle. The  excess pyruvate is reduced to lactate.  Because lactate is an acid, its accumulation  affects the muscle and causes pain and  swelling.   ' system, and skeletal muscle occur in response to regular graded exercise.  The pumping efficiency of the heart increases, allowing a greater cardiac output  with fewer beats per minute and at a lower rate of oxygen utilization. The lungs  extract a greater percentage of oxygen from the inspired air, allowing fewer respi-  rations per unit of activity. The vasodilatory capacity of the arterial beds in skeletal  muscle increases, promoting greater delivery of oxygen and fuels to exercising mus-  cle. Concurrently, the venous drainage capacity in muscle is enhanced, ensuring that  lactic acid will not accumulate in contracting tissues. These adaptive changes in  physiological responses are accompanied by increases in the number, size, and  activity of skeletal muscle mitochondria, along with the content of TCA cycle  enzymes and components of the electron transport chain. These changes markedly  enhance the oxidative capacity of exercising muscle.   /^~^\ Ann O'Rexia. Ann O'Rexia is experiencing fatigue for a number of  reasons. She has iron deficiency anemia, which affects both iron-  containing hemoglobin in her red blood cells, iron in aconitase and   succinic dehydrogenase, as well as iron in the heme proteins of the electron   transport chain. She may also be experiencing the consequences of multiple  vitamin deficiencies, including thiamine, riboflavin, and niacin (the vitamin  precursor of NAD + ). It is less likely, but possible, that she also has subclinical  deficiencies of pantothenate (the precursor of CoA) or biotin. Because of this,  Ann's muscle must use glycolysis as their primary source of energy, which  results in sore muscles.   Riboflavin deficiency generally occurs in conjunction with other water-  soluble vitamin deficiencies. The classic deficiency symptoms are cheilosis  (inflammation of the corners of the mouth), glossitis (magenta tongue), and seb-  orrheic ("greasy") dermatitis. It is also characterized by sore throat, edema of the  pharyngeal and oral mucus membranes, and normochromic, normocytic anemia  associated with pure red cell cytoplasia of the bone marrow. However, it is not  known whether the glossitis and dermatitis are actually due to multiple vitamin  deficiencies.   ^ Al Martini. Al Martini presents a second time with an alcohol-related  J high output form of heart failure sometimes referred to as "wet" beriberi,  or as the "beriberi heart" (see Chapter 9). The term "wet" refers to the fluid  retention which may eventually occur when left ventricular contractility is so com-  promised that cardiac output, although initially relatively "high," cannot meet the  "demands" of the peripheral vascular beds, which have dilated in response to the  thiamine deficiency.   The cardiomyopathy is directly related to a reduction in the normal biochemical  function of the vitamin thiamine in heart muscle. Inhibition of the a-keto acid dehy-  drogenase complexes causes accumulation of a-keto acids in heart muscle (and in  blood), resulting in a chemically-induced cardiomyopathy. Impairment of two other  functions of thiamine may also contribute to the cardiomyopathy. Thiamine  pyrophosphate serves as the coenzyme for transketolase in the pentose phosphate  pathway, and pentose phosphates accumulate in thiamine deficiency. In addition,  thiamine triphosphate (a different coenzyme form) may function in Na + conduc-  tance channels.   Immediate treatment with large doses (50-100 mg) of intravenous thiamine may  produce a measurable decrease in cardiac output and increase in peripheral vascu-  lar resistance as early as 30 minutes after the initial injection. Dietary supplemen-  tation of thiamine is not as effective because ethanol consumption interferes with  thiamine absorption. Because ethanol also affects the absorption of most water-  soluble vitamins, or their conversion to the coenzyme form, Al Martini was also  given a bolus containing a multivitamin supplement.   Compartmentation of Mitochondrial Enzymes. The mito-  chondrion forms a structural, functional, and regulatory compartment  within the cell. The inner mitochondrial membrane is impermeable to  anions and cations, and compounds can cross the membrane only on specific trans-  port proteins. The enzymes of the TCA cycle, therefore, have more direct access to  products of the previous reaction in the pathway than they would if these products  were able to diffuse throughout the cell. Complex formation between enzymes also  restricts access to pathway intermediates. Malate dehydrogenase and citrate syn-  thase may form a loosely associated complex. The multienzyme pyruvate dehydro-  genase and a-ketoglutarate dehydrogenase complexes are examples of substrate  channeling by tightly bound enzymes; only the transacylase enzyme has access to  the thiamine-bound intermediate of the reaction, and only lipoamide dehydrogenase  has access to reduced lipoic acid.   Riboflavin has a wide distribution  \\ J in foods, and small amounts are  *S present as coenzymes in most  plant and animal tissues. Eggs, lean meats,  milk, broccoli, and enriched breads and cere-  als are especially good sources. A portion of  our niacin requirement can be met by syn-  thesis from tryptophan. Meat (especially red  meat), liver, legumes, milk, eggs, alfalfa,  cereal grains, yeast, and fish are good  sources of niacin and tryptophan.   Beri-beri, now known to be caused  by thiamine deficiency, was attrib-  uted to lack of a nitrogenous com-  ponent in food by Takaki, a Japanese sur-  geon, in 1884. In 1890, Eijkman, a Dutch  physician working in Java, noted that the  polyneuritis associated with beri-beri could  be prevented by rice bran that had been  removed during polishing. Thiamine is pres-  ent in the bran portion of grains, and abun-  dant in pork and legumes. In contrast to  most vitamins, milk and milk products,  seafood, fruits, and vegetables are NOT  good sources of thiamine.   "Now polished rice isn't nice",  Said the Dutchman Eijkman.  Whole grains are a far better  Source of thiamine.  For beri-beri is very, very  Hard on your nerves, you see.  Polyneuritis and an enlarged heart  May both accompany  A very bad diet, a very sad diet  A diet thiamine-free.  And many who dine, only on wine  Or consume brandy, whiskey or gin  May never recover, if you don't discover  They can't absorb thiamine.  Wernicke-Korsakoff describe the signs  And the confusion in the minds  Of patients with this deficiency.  So good doctors remember, try to recall  Before you charge your fee,  To give an injection, im or iv  Of this vitamin B.   Fig. 20.20. Model for the import of nuclear-  encoded proteins into the mitochondrial  matrix. The matrix preprotein with its posi-  tively charged N-terminal presequence is  shown in blue. Abbreviations: OM, outer mito-  chondrial membrane; IMS, intramembrane  space; IM, inner mitochondrial membrane;  TOM, translocases of the outer mitochondrial  membrane; TIM, translocases of the inner  mitochondrial membrane; mthsp70, mitochon-  drial heat shock protein 70.   Compartmentation plays an important role in regulation. The close association  between the rate of the electron transport chain and the rate of the TCA cycle is  maintained by their mutual access to the same pool of NADH and NAD + in the  mitochondrial matrix. NAD + , NADH, Co ASH, and acyl Co A derivatives have no  transport proteins and cannot cross the mitochondrial membrane. Thus, all of the  dehydrogenases compete for the same NAD + molecules, and are inhibited when  NADH rises. Likewise, accumulation of acyl CoA derivatives (e.g., acetyl Co A)  within the mitochondrial matrix affects other CoA-utilizing reactions, either by  competing at the active site or limiting Co ASH availability.   Import of Nuclear Encoded Proteins. All mitochondrial matrix  proteins, such as the TCA cycle enzymes, are encoded by the nuclear  genome. They are imported into the mitochondrial matrix as unfolded pro-  teins that are pushed and pulled through channels in the outer and inner mitochon-  drial membranes (Fig. 20.20). Proteins destined for the mitochondrial matrix have  a targeting N-terminal presequence of about 20 amino acids that includes several  positively charged amino acid residues. They are synthesized on free ribosomes in  the cytosol and maintain an unfolded conformation by binding to hsp70 chaper-  onins. This basic presequence binds to a receptor in a TOM complex (translocators  of the outer membrane) (see Fig. 20.20, step 1). The TOM complexes consist of  channel proteins, assembly proteins and receptor proteins with different specifici-  ties (e.g., TOM20 binds the matrix protein presequence). Negatively charged acidic  residues on the receptors and in the channel pore assist in translocation of the matrix  protein through the channel, presequence first.   The matrix preprotein is translocated across the inner membrane through a TIM  complex (translocases of the inner membrane) (see Fig. 20.20, step 2). Insertion of  the preprotein into the TIM channel is driven by the potential difference across the  membrane, Ai|i. Mitochondrial hsp70 (mthsp70), which is bound to the matrix side  of the TIM complex, binds the incoming preprotein and may "ratchet" it through the  membrane. ATP is required for binding of mthsp70 to the TIM complex and again  for the subsequent dissociation of the mthsp70 and the matrix preprotein. In the  matrix, the preprotein may require another heat shock protein, hsp60, for proper  folding. The final step in the import process is cleavage of the signal sequence by a  matrix processing protease (see Fig. 20.20, step 3).   Proteins of the inner mitochondrial membrane are imported through a similar  process, using TOM and TIM complexes containing different protein components.   Robinson, BH. Lactic acidemia: Disorders of pyruvate carboxylase and pyruvate dehydrogenase. In:  Scriver CR, Beudet AL, Sly WS, Valle D, eds: The Metabolic and Molecular Bases of Inherited  Disease. Vol. I. 8th Ed. New York: McGraw-Hill, 2001:4451-4480.   1 . Which of the following coenzymes is unique to a-ketoacid dehydrogenase complexes?   2. A patient diagnosed with thiamine deficiency exhibited fatigue and muscle cramps. The muscle cramps have been related to  an accumulation of metabolic acids. Which of the following metabolic acids is most likely to accumulate in a thiamine  deficiency?   3. Succinate dehydrogenase differs from all other enzymes in the TCA cycle in that it is the only enzyme that displays which of  the following characteristics?   4. During exercise, stimulation of the tricarboxylic acid cycle results principally from which of the following?   (C) A rapid decrease in the concentration of four carbon intermediates   (E) Stimulation of the flux through a number of enzymes by a decreased NADH/NAD + ratio   5. Coenzyme A is synthesized from which of the following vitamins?   Q o Most cells are dependent on oxida-  ( / vA ) t ' ve phosphorylation for ATP  ™ homeostasis. The ability to gener-  ate ATP depends on 0 2 and an intact inner  mitochondrial membrane. During oxygen  deprivation from ischemia (a low blood  flow), an inability to generate energy from  the electron transport chain results in an  increased permeability of this membrane  and mitochondrial swelling. Mitochondrial  swelling is a key element in the pathogene-  sis of irreversible cell injury leading to cell  lysis and death (necrosis).   Energy from fuel oxidation is converted to the high-energy phosphate bonds of  adenosine triphosphate (ATP) by the process of oxidative phosphorylation. Most  of the energy from oxidation of fuels in the TCA cycle and other pathways is con-  served in the form of the reduced electron-accepting coenzymes, NADH and  FAD(2H). The electron transport chain oxidizes NADH and FAD(2H), and  donates the electrons to 0 2 , which is reduced to H 2 0 (Fig. 21.1 ). Energy from  reduction of 0 2 is used for phosphorylation of adenosine diphosphate (ADP) to  ATP by ATP synthase (F 0 FjATPase). The net yield of oxidative phosphorylation  is approximately 2.5 moles of ATP per mole of NADH oxidized, or 1.5 moles of  ATP per mole of FAD(2H) oxidized.   Chemiosmotic model of ATP synthesis. The chemiosmotic model explains  how energy from transport of electrons to 0 2 is transformed into the high-  energy phosphate bond of ATP (see Fig. 21.1). Basically, the electron transport  chain contains three large protein complexes (I, HI, and IV) that span the  inner mitochondrial membrane. As electrons pass through these complexes in a  series of oxidation-reduction reactions, protons are transferred from the mito-  chondrial matrix to the cytosolic side of the inner mitochondrial membrane.  The pumping of protons generates an electrochemical gradient (Ap) across the  membrane composed of the membrane potential and the proton gradient. ATP  synthase contains a proton pore through the inner mitochondrial membrane  and a catalytic headpiece that protrudes into the matrix. As protons are driven   Fig. 21.1. Oxidative phosphorylation. Blue arrows show the path of electron transport from NADH to 0 2 . As electrons pass through the chain,  protons are pumped from the mitochondrial matrix to the intermembrane space, thereby establishing an electochemical potential gradient, Ap,  across the inner mitochondrial membrane. The positive and negative charges on the membrane denote the membrane potential (Ai|/). Ap drives  protons into the matrix through a pore in ATP synthase, which uses the energy to form ATP from ADP and Pi.   into the matrix through the pore, they change the conformation of the head-  piece, which releases ATP from one site and catalyzes formation of ATP from  ADP and Pi at another site.   Deficiencies of electron transport. In cells, complete transfer of electrons  from NADH and FAD(2H) through the chain to 0 2 is necessary for ATP gener-  ation. Impaired transfer through any complex can have pathologic conse-  quences. Fatigue can result from iron-defeciency anemia, which decreases Fe  for Fe-S centers and cytochromes. Cytochrome c 1 oxidase, which contains the  0 2 binding site, is inhibited by cyanide. Mitochondrial DN A (mtDNA), which  is maternally inherited, encodes some of the subunits of the electron transport  chain complexes and ATP synthase. Oxphos diseases are caused by mutations  in nuclear DN A or mt DNA that decrease mitochondrial capacity for oxidative  phosphorylation .   Regulation of oxidative phosphorylation. The rate of the electron transport  chain is coupled to the rate of ATP synthesis by the transmembrane  electochemical gradient. As ATP is used for energy-requiring processes and  ADP levels increase, proton influx through the ATP synthase pore generates  more ATP, and the electron transport chain responds to restore Ap. In  uncoupling, protons return to the matrix by a mechanism that bypasses the  ATP synthase pore, and the energy is released as heat. Proton leakage,  chemical uncouplers, and regulated uncoupling proteins increase our  metabolic rate and heat generation.   Mitochondria and cell death. Although oxidative phosphorylation is a  mitochondrial process, most ATP utilization occurs outside of the mitochon-  drion. ATP synthesized from oxidative phosphorylation is actively transported  from the matrix to the intermembrane space by adenine nucleotide translocase  (ANT). Porins form voltage-dependent anion channels (VDAC) through the  outer mitochondrial membrane for the diffusion of H 2 0, ATP metabolites, and  other ions. Under certain types of stress, ANT, VDAC, and other proteins form  a nonspecific open channel known as the mitochondrial permeability  transition pore. This pore is associated with events that lead rapidly to  necrotic cell death.   • Cora Nari was recovering uneventfully from her heart attack 1 month  flP ) earlier (see Chapter 19), when she won the Georgia State lottery. When   " she heard her number announced over television, she experienced crush-  ing chest pain, grew short of breath, and passed out. She regained consciousness  as she was being rushed to the hospital emergency room.   On initial examination, her blood pressure was extremely high and her heart  rhythm irregular. Her blood levels of CK-MB and Tnl (troponin I) were elevated.  An electrocardiogram showed unequivocal evidence of severe lack of oxygen  (ischemia) in the muscles of the anterior and lateral walls of her heart. Life sup-  port measures including nasal oxygen were initiated. An intravenous drip of nitro-  prusside, a vasodilating agent, was started in an effort to reduce her hypertension.  After her blood pressure was well controlled, a decision was made to administer  intravenous tissue plasminogen activator (TPA) in an attempt to break up any  intracoronary artery blood clots in vessels supplying the ischemic myocardium  (thrombolytic therapy).    Cora Nari is experiencing a second  myocardial infarction. Ischemia (a  low blood flow) has caused  hypoxia (low levels of oxygen) in her heart  muscle, resulting in inadequate generation  of ATP for the maintenance of low intracellu-  lar Na + and Ca 2+ levels (see Chapter 19). As  a consequence, cells have become swollen  and the cytosolic proteins creatine kinase  (MB isoform) and troponin (heart isoform)  have leaked into the blood. (See Ann Jeina,  Chapters 6 and 7).   An I thyroid uptake and scan performed on X.S. Teefore confirmed that  his hyperthyroidism was the result of Graves disease (see Chapter 19).  Graves disease, also known as diffuse toxic goiter, is an autoimmune genetic  disorder caused by the generation of human thyroid- stimulating immunoglobulins.  These immunoglobulins stimulate enlargement of the thyroid gland (goiter) and  excess secretion of the thyroid hormones, T 3 and T 4 As a consequence, Mr. Teefore 's  heat intolerance and sweating were growing worse with time.   Ivy Sharer, an intravenous drug abuser, appeared to be responding well to  her multidrug regimens to treat pulmonary tuberculosis and AIDS (see  Chapters 11, 12, 15, and 16). In the past 6 weeks, however, she has devel-  oped increasing weakness in her extremities to the point that she has difficulty car-  rying light objects or walking. Physical examination indicates a diffuse proximal  and distal muscle weakness associated with muscle atrophy. The muscles are nei-  ther painful on motion nor tender to compression. The blood level of the muscle  enzymes, creatine phosphokinase (CK) and aldolase, are normal. An electromyo-  gram (EMG) revealed a generalized reduction in the muscle action potentials, sug-  gestive of a primary myopathic process. Proton spectroscopy of her brain and upper  spinal cord showed no anatomic or biochemical abnormalities. The diffuse and pro-  gressive skeletal muscle weakness was out of proportion to that expected from her  AIDS or her tuberculosis. This information led her physicians to consider the pos-  sibility that her skeletal muscle dysfunction might be drug induced.   Arlyn Foma, who has a follicular  type non-Hodgkin's lymphoma,  was being treated with the anthra-  cycline drug doxorubicin (see Chapter 16).  During the course of his treatment, he devel-  oped biventricular heart failure. Although  doxorubicin is a highly effective anticancer  agent against a wide variety of human  tumors, its clinical use is limited by a spe-  cific, cumulative, dose-dependent cardiotox-  icity. Impairment of mitochondrial function  may play a major role in this toxicity. Dox-  orubicin binds to cardiolipin, a lipid compo-  nent of the inner membrane of mitochon-  dria, where it might directly affect  components of oxidative phosphorylation.  Doxorubicin inhibits succinate oxidation,  inactivates cytochrome oxidase, interacts  with CoQ, affects ion pumps, and inhibits  ATP synthase, resulting in decreased ATP  levels and mildly swollen mitochondria. It  decreases the ability of the mitochondria to  sequester Ca 2+ and increases free radicals  (highly reactive single-electron forms) lead-  ing to damage of the mitochondrial mem-  brane (see Chapter 24). It also might affect  heart function indirectly through other  mechanisms.   Generation of ATP from oxidative phosphorylation requires an electron donor  (NADH or FAD(2H)), an electron acceptor (0 2 ), an intact inner mitochondrial  membrane that is impermeable to protons, all the components of the electron trans-  port chain, and ATP synthase. It is regulated by the rate of ATP utilization.   Our understanding of oxidative phosphorylation is based on the chemiosmotic  hypothesis, which proposes that the energy for ATP synthesis is provided by an  electrochemical gradient across the inner mitochondrial membrane. This electro-  chemical gradient is generated by the components of the electron transport chain,  which pump protons across the inner mitochondrial membrane as they sequentially  accept and donate electrons (see Fig. 21.1). The final acceptor is 0 2 , which is  reduced to H 2 0.   In the electron transport chain, electrons donated by NADH or FAD(2H) are passed  sequentially through a series of electron carriers embedded in the inner mitochon-  drial membrane. Each of the components of the electron transfer chain is oxidized  as it accepts an electron, and then reduced as it passes the electrons to the next mem-  ber of the chain. From NADH, electrons are transferred sequentially through  NADH dehydrogenase (complex I), CoQ (coenzyme Q), the cytochrome b-c x com-  plex (complex III), cytochrome c, and finally cytochrome c oxidase (complex IV).  NADH dehydrogenase, the cytochrome b-c l complex and cytochrome c oxidase are  each multisubunit protein complexes that span the inner mitochondrial membrane.  CoQ is a lipid soluble quinone that is not protein-bound and is free to diffuse in the  lipid membrane. It transports electrons from complex I to complex III and is an  intrinsic part of the proton pumps for each of these complexes. Cytochome c is a  small protein in the inner membrane space that transfers electrons from the b-c 1   complex to cytochrome oxidase. The terminal complex, cytochrome c oxidase,  contains the binding site for 0 2 . As 0 2 accepts electrons from the chain, it is reduced  to H 2 0.   At each of the three large membrane- spanning complexes in the chain, electron  transfer is accompanied by proton pumping across the membrane. There is an  energy drop of approximately 16 kcal in reduction potential as electrons pass  through each of these complexes, which provides the energy required to move pro-  tons against a concentration gradient. The membrane is impermeable to protons, so  they cannot diffuse through the lipid bilayer back into the matrix. Thus, in actively  respiring mitochondria, the intermembrane space and cytosol may be approxi-  mately 0.75 pH units lower than the matrix.   The transmembrane movement of protons generates an electrochemical gra-  dient with two components: the membrane potential (the external face of the  membrane is charged positive relative to the matrix side) and the proton gradient  (the inter membrane space has a higher proton concentration and is therefore  more acidic than the matrix) (Fig. 21.2). The electrochemical gradient is some-  times called the proton motive force because it is the energy pushing the protons  to re-enter the matrix to equilibrate on both sides of the membrane. The protons  are attracted to the more negatively charged matrix side of the membrane, where  the pH is more alkaline.   Fig. 21.2. Proton motive force (electrochemi-  cal gradient) across the inner mitochondrial  membrane. The proton motive force consists of  a membrane potential, Ai|/, and a proton gradi-  ent, denoted by ApH for the difference in pH  across the membrane. The electrochemical  potential is called the proton motive force  because it represents the potential energy driv-  ing protons to return to the more negatively  charged alkaline matrix.   ATP synthase (FoFiATPase), the enzyme that generates ATP, is a multisubunit  enzyme containing an inner membrane portion (F 0 ) and a stalk and headpiece (F t )  that project into the matrix (Fig. 21.3). The 12 c subunits in the membrane form a  rotor that is attached to a central asymmetric shaft composed of the s and y sub-  units. The headpiece is composed of three a (3 subunit pairs. Each (3 subunit contains  a catalytic site for ATP synthesis. The headpiece is held stationary by a 8 subunit  attached to a long b subunit connected to subunit a in the membrane.   Fig. 21.4. Binding change mechanism for  ATP synthesis. The three ap subunit pairs of  the ATP synthase headpiece have binding sites  that can exist in three different conformations,  depending on the position of the 7 stalk sub-  unit. Step 1 : When ADP + Pi bind to an open  site and the proton influx rotates the 7 spindle  (represented by the arrow), the conformation  of the subunits change and ATP is released  from one site. (ATP dissociation is, thus, the  energy -requiring step). Bound ADP and Pi  combine to form ATP at another site. Step 2:  As the ADP + Pi bind to the new open site,  and the 7 shaft rotates, the conformations of  the sites change again, and ATP is released.  ADP and Pi combine to form another ATP.   The influx of protons through the proton channel turns the rotor. The proton  channel is formed by the c subunits on one side and the a subunit on the other side.  Although continuous, it has two offset portions, one portion directly open to the  intermembrane space and one portion directly open to the matrix. In the current  model, each c subunit contains a glutamyl carboxyl group that extends into the pro-  ton channel. As this carboxyl group accepts a proton from the intermembrane space,  the c subunit rotates into the hydrophobic lipid membrane. The rotation exposes a  different proton-containing c subunit to the portion of the channel directly open to  the matrix side. Because the matrix has a lower proton concentration, the glutamyl  carboxylic acid group releases a proton into the matrix portion of the channel. Rota-  tion is completed by an attraction between the negatively charged glutamyl residue  and a positively charged arginyl group on the a subunit.   According to the binding change mechanism, as the asymmetric shaft rotates to a  new position, it forms different binding associations with the a(3 subunits (Fig. 21.4).  The new position of the shaft alters the conformation of one p subunit so that it releases  a molecule of ATP and another subunit spontaneously catalyzes synthesis of ATP from  inorganic phosphate, one proton, and ADP. Thus, energy from the electrochemical gra-  dient is used to change the conformation of the ATP synthase subunits so that the newly  synthesized ATP is released. Twelve c subunits are hypothesized, and it takes 12 pro-  tons to complete one turn of the rotor and synthesize three ATP.   Electron transport to 0 2 occurs via a series of oxidation-reduction steps in which  each successive component of the chain is reduced as it accepts electrons and  oxidized as it passes electrons to the next component of the chain. The  oxidation-reduction components of the chain include flavin mononucleotide  (FMN), Fe-S centers, CoQ, and Fe in the cytochromes b, c 1? c, a, and a 3 . Cu is also  a component of cytochromes a and a 3 (Fig. 21.5). With the exception of CoQ, all of  these electron acceptors are tightly bound to the protein subunits of the carriers.   The reduction potential of each complex of the chain is at a lower energy level  than the previous complex, so that energy is released as electrons pass through each  complex. This energy is used to move protons against their concentration gradient,  so that they become concentrated on the cytosolic side of the inner membrane.   NADH dehydrogenase is an enormous 42- subunit complex that contains a binding  site for NADH, several FMN and iron-sulfur (Fe-S) center binding proteins, and  binding sites for CoQ (see Fig 21.5). An FMN accepts two electrons from NADH  and is able to pass single electrons to the Fe-S centers (Fig. 21.6). Fe-S centers,  which are able to delocalize single electrons into large orbitals, transfer electrons to  and from CoQ. Fe-S centers are also present in other enzyme systems, such as other  proteins, which transfer electrons to CoQ, in the cytochrome b-c r complex, and in  aconitase in the TCA cycle.   FMN, like FAD, is synthesized from  the vitamin riboflavin. It contains  the electron-accepting flavin ring  structure, but not the adenosine monophos-  phate (AMP) portion of FAD (see Fig. 19.10).  Severe riboflavin deficiency decreases the  ability of mitochondria to generate ATP from  oxidative phosphorylation due to the lack of  FMN in the electron carriers.   In addition to NADH dehydrogenase, succinic dehydrogenase and other flavopro-  teins in the inner mitochondrial membrane also pass electrons to CoQ (see  Fig. 21.5). Succinate dehydrogenase is part of the TCA cycle. ETF-CoQ oxidore-  ductase accepts electrons from ETF (electron transferring fiavoprotein), which  acquires them from fatty acid oxidation and other pathways. Both of these flavo-  proteins have Fe-S centers. a-Glycerophosphate dehydrogenase is a fiavoprotein  that is part of a shuttle for reoxidizing cytosolic NADH.   Fig. 21.5. Components of the electron transfer chain. NADH dehydrogenase (complex I) spans the membrane and has a proton pumping mech-  anism involving CoQ. The electrons go from CoQ to cytochrome b-cl complex (complex III), and electron transfer does NOT involve complex  II. Succinate dehydrogenase (complex II), glycerol 3-phosphate dehydrogenase, and ETF:Q oxidoreductase (shown in blue) all transfer electrons  to CoQ, but do not span the membrane and do not have a proton pumping mechanism. As CoQ accepts protons from the matrix side, it is con-  verted to QH 2 . Electrons are transferred from complex III to complex IV (cytochrome c oxidase) by cytochrome c, a small cytochrome in the  intermembrane space that has reversible binding sites on the b-c x complex and cytochrome c oxidase.   The free energy drop between NADH and CoQ of approximately —13 to —14  kcal is able to support movement of four protons. However, the FAD in succinate  dehydrogenase (as well as ETF-CoQ oxidoreductase and a-glycerophosphate dehy-  drogenase) is at roughly the same energy level as CoQ, and there is no energy  released as they transfer electrons to CoQ. These proteins do not span the membrane  and consequently do not have a proton pumping mechanism.   CoQ is the only component of the electron transport chain that is not protein bound.  The large hydrophobic side chain of 10 isoprenoid units (50 carbons) confers lipid  solubility, and CoQ is able to diffuse through the lipids of the inner mitochondrial  membrane (Fig. 21.7). When the oxidized quinone form accepts a single electron,  it forms a free radical (a compound with a single electron in an orbital). The trans-  fer of single electrons makes it the major site for generation of toxic oxygen free  radicals in the body (see Chapter 24).   The long side chain of CoQ has 10 of the 5-carbon isoprenoid units, and is  sometimes called CoQ 10 . It is also called ubiquinone (the quinone found every-  where) because quinones with similar structures are found in all plants and  animals. CoQ can be synthesized in the human from precursors derived from carbohy-  drates and fat. The long isoprenoid side chain is formed in the pathway that produces  the isoprenoid precursors of cholesterol. CoQ 10 is sometimes prescribed for patients  recovering from a myocardial infarction, in an effort to increase their exercise capacity.   Fig. 21.6. Fe 4 S 4 center. In Fe-S centers, the Fe  is chelated to free sulfur (S) atoms, and to cys-  teine sulfhydryl groups on proteins. Other Fe-  S centers contain Fe 2 S 2 . The protein subunits  are sometimes called non-heme iron proteins.  When these proteins are treated with acid, the  free sulfur produces hydrogen sulfide (H 2 S) —  the familiar smell of rotten eggs.   Fig. 21.7. Coenzyme Q contains a quinone with a long lipophilic side chain comprising 10 isoprenoid units (thus, it is sometimes called CoQ 10 .)  CoQ can accept one electron (e~) to become the half-reduced form, or 2 e~ to become fully reduced.   The semiquinone can accept a second electron and two protons from the matrix  side of the membrane to form the fully reduced quinone. The mobility of CoQ in  the membrane, its ability to accept one or two electrons, and its ability to accept and  donate protons enable it to participate in the proton pumps for both complexes I and  III as it shuttles electrons between them (see Section I.C.).   0 o Although iron deficiency anemia is  ( / \u ) cnaracter ' ze d by decreased levels  ™ of hemoglobin and other iron-  containing proteins in the blood, the iron-  containing cytochromes and Fe-S centers of  the electron transport chain in tissues such  as skeletal muscle are affected as rapidly.  Fatigue in iron deficiency anemia, in patients  such as Ann O'Rexia (see Chapter 16),  results, in part, from the lack of electron  transport for ATP production.   The remainder of the components in the electron transport chain are cytochromes  (see Fig. 21.5). Each cytochrome is a protein that contains a bound heme (i.e., an Fe  atom bound to a porphyrin nucleus similar in structure to the heme in hemoglobin)  (Fig. 21.8).   Because of differences in the protein component of the cytochromes and small  differences in the heme structure, each heme has a different reduction potential. The  cytochromes of the b-c x complex have a higher energy level than those of  cytochrome oxidase (a and a 3 ). Thus, energy is released by electron transfer  between complexes III and IV. The iron atoms in the cytochromes are in the Fe 3+  state. As they accept an electron, they are reduced to Fe 2+ . As they are reoxidized  to Fe 3+ , the electrons pass to the next component of the electron transport chain.   The last cytochrome complex is cytochrome oxidase, which passes electrons  from cytochrome c to 0 2 (see Fig. 21.5). It contains cytochromes a and a 3 and  the oxygen binding site. A whole oxygen molecule, 0 2 , must accept four  electrons to be reduced to 2 H 2 0. Bound copper (Cu + ) ions in the cytochrome  oxidase complex facilitate the collection of the four electrons and the reduction  of 0 2 .   Cytochrome oxidase has a much lower K m for 0 2 than myoglobin (the heme-  containing intracellular oxygen carrier) or hemoglobin (the heme-containing oxygen  transporter in the blood). Thus, 0 2 is "pulled" from the erythrocyte to myoglobin,  and from myoglobin to cytochrome oxidase, where it is reduced to H 2 0.   ^ cytochromes, never changes its  oxidation state (it is Fe 2+ in hemoglobin). If  the iron in hemoglobin were to become oxi-  dized (Fe 3+ ), the oxygen-binding capacity of  the molecule would be lost. Normally, the  protein structures binding the heme either  protect the iron from oxidation (such as the  globin proteins), or allow oxidation to occur  (such as happens in the cytochromes). How-  ever, in hemoglobin M, a rare hemoglobin  variant found in the human population, a  tyrosine is substituted for the histidine at  position F8 in the normal hemoglobin A. This  tyrosine stabilizes the Fe 3+ form of heme,  and these subunits cannot bind oxygen. This  is a lethal condition if homozygous.   Fig. 21.8. Heme A. Heme A is found in cytochromes a and a 3 . Cytochromes are proteins con-  taining a heme chelated with an iron atom. Hemes are derivatives of protoporphyrin IX. Each  cytochrome has a heme with different modifications of the side chains (indicated with dashed  lines), resulting in a slightly different reduction potential and, consequently, a different posi-  tion in the sequence of electron transfer.   One of the tenets of the chemiosmotic theory is that energy from the oxidation-  reduction reactions of the electron transport chain is used to transport protons from the  matrix to the intermembrane space. This proton pumping is generally facilitated by the  vectorial arrangement of the membrane spanning complexes. Their structure allows  them to pick up electrons and protons on one side of the membrane and release pro-  tons on the other side of the membrane as they transfer an electron to the next compo-  nent of the chain. The direct physical link between proton movement and electron  transfer can be illustrated by an examination of the Q cycle for the b-q complex  (Fig. 21.9). The Q cycle involves a double cycle of CoQ reduction and oxidation. CoQ  accepts two protons at the matrix side together with two electrons; it then releases pro-  tons into the intermembrane space while donating one electron back to another com-  ponent of the cytochrome b-q complex and one to cytochrome c.   The mechanism for pumping protons at the NADH dehydrogenase complex is  not well understood, but it involves a Q cycle in which the Fe-S centers and FMN  might participate. However, transmembrane proton movement at cytochrome c  oxidase probably involves direct transport of the proton through a series of bound  water molecules or amino acid side chains in the protein, a mechanism that has been  described as a "proton wire."   The significance of the direct link between the electron transfer and proton  movement is that one cannot occur without the other. Thus, when protons are not  being used for ATP synthesis, the proton gradient and the membrane potential build  up. This "proton back-pressure" controls the rate of proton pumping, which controls  electron transport and 0 2 consumption.   The overall free energy release from oxidation of NADH by 0 2 is approximately  — 53 kcal, and from FAD(2H), it is approximately —41 kcal. This AG 0 ' is so negative   Fig. 21.9. The proton motive Q cycle for the b-Cj complex. (1) From 2 QH 2 , electrons go  down two different paths: one path is through an FeS center protein (ISP) toward cytochrome  c (shown with blue arrows). Another path is "backward" to one of the b cytochromes, shown  with dashed arrows. (2) Electrons are transferred from ISP through cytochrome c x .  Cytochrome c, which is in the intermembrane space, binds to the b-q complex to accept an  electron. (3) Returning electrons go through another b cytochrome and are directed toward  the matrix. (4) At the matrix side, electrons and 2H + are accepted by Q. Q, Coenzyme Q; Q«~,  CoQ semiquinone; QH 2 , CoQ hydroquinone.   that the chain is never reversible; we never synthesize oxygen from H 2 0. It is so  negative that it drives NADH and FAD(2H) formation from the pathways of fuel  oxidation, such as the TCA cycle and glycolysis, to completion.   Overall, each NADH donates two electrons, equivalent to the reduction of Vi of  an 0 2 molecule. A generally (but not universally) accepted estimate of the stoi-  chiometry of ATP synthesis is that four protons are pumped at complex I, four pro-  tons at complex III, and two at complex IV. With four protons translocated for each  ATP synthesized, an estimated 2.5 ATPs are formed for each NADH oxidized and  1.5 ATPs for each of the other FAD(2H)-containing flavoproteins that donate elec-  trons to CoQ. (This calculation neglects proton requirements for the transport of  phosphate and substrates from the cytosol, as well as the basal proton leak.) Thus,  only approximately 30% of the energy available from NADH and FAD(2H) oxidation  by 0 2 is used for ATP synthesis. Some of the remaining energy in the electrochem-  ical potential is used for the transport of anions and Ca 2+ into the mitochondrion.  The remainder of the energy is released as heat. Consequently, the electron trans-  port chain is also our major source of heat.   In the cell, electron flow in the electron transport chain must be sequential from  NADH or a flavoprotein all the way to 0 2 to generate ATP (see Fig. 21.5). In  the absence of 0 2 , there is no ATP generated from oxidative phosphorylation  because electrons back up in the chain. Even complex I cannot pump protons to  generate the electrochemical gradient, because every molecule of CoQ already has  electrons that it cannot pass down the chain without an 0 2 to accept them at the end.  The action of the respiratory chain inhibitor cyanide, which binds to cytochrome  oxidase, is similar to that of anoxia; it prevents proton pumping by all three com-  plexes. Complete inhibition of the b-Cj complex prevents pumping at cytochrome   ^ Cyanide binds to the Fe 3+ in the heme of the cytochrome aa 3 component of cytochrome c oxidase and prevents electron trans-  port to 0 2 . Mitochondrial respiration and energy production cease, and cell death rapidly occurs. The central nervous system is  the primary target for cyanide toxicity. Acute inhalation of high concentrations of cyanide (e.g., smoke inhalation during a fire)  provokes a brief central nervous system stimulation rapidly followed by convulsion, coma, and death. Acute exposure to lower amounts  can cause lightheadedness, breathlessness, dizziness, numbness, and headaches.   Cyanide is present in the air as hydrogen cyanide (HCN), in soil and water as cyanide salts (e.g., NaCN), and in foods as cyanoglycosides.  Most of the cyanide in the air usually comes from automobile exhaust. Examples of populations with potentially high exposures include active  and passive smokers, people who are exposed to house or other building fires, residents who live near cyanide- or thiocyanate-containing haz-  ardous waste sites, and workers involved in a number of manufacturing processes (e.g., photography or pesticide application.)   Cyanoglycosides such as amygdalin are present in edible plants such as almonds, pits from stone fruits (e.g., apricots, peaches, plums,  cherries), sorghum, cassava, soybeans, spinach, lima beans, sweet potatoes, maize, millet, sugar cane, and bamboo shoots.   HCN is released from cyanoglycosides by p-glucosidases present in the plant or in intestinal bacteria. Small amounts are inactivated  in the liver principally by rhodanase, which converts it to thiocyanate.   In the United States, toxic amounts have been ingested as ground apricot pits, either due to health food promotion or as a treatment  for cancer. The drug Laetrile (amygdalin) was used as a cancer therapeutic agent, although it was banned in the United States because it  was ineffective and potentially toxic. Commercial fruit juices made from unpitted fruit could provide toxic amounts of cyanide, particularly  in infants or children. In countries in which cassava is a dietary staple, improper processing results in retention of its high cyanide con-  tent at potentially toxic levels.   Cora Nari has a lack of oxygen in  the anterior and lateral walls of her  heart caused by severe ischemia  (lack of blood flow) resulting from clots  formed at the site of ruptured atherosclerotic  plaques. The limited availability of 0 2 to act  as an electron acceptor will decrease proton  pumping and generation of an electrochem-  ical potential gradient across the inner mito-  chondrial membrane. As a consequence, the  rate of ATP generation in her heart will  decrease, thereby triggering events that lead  to irreversible cell injury.   Intravenous nitroprusside rapidly  lowers elevated blood pressure  through its direct vasodilating  action. Fortunately, it was only required in  Cora Nari's case for several hours. During  prolonged infusions of 24 to 48 hours or  more, nitroprusside is converted to cyanide,  an inhibitor of the cytochrome c oxidase  complex. Because small amounts of cyanide  are detoxified in the liver by conversion to  thiocyanate, which is excreted in the urine,  the conversion of nitroprusside to cyanide  can be monitored by following blood thio-  cyanate levels.   oxidase because there is no donor of electrons; it prevents pumping at complex I  because there is no electron acceptor. Although complete inhibition of any one com-  plex inhibits proton pumping at all of the complexes, partial inhibition of proton  pumping can occur when only a fraction of the molecules of a complex contain  bound inhibitor. The partial inhibition results in a partial decrease of the maximal  rate of ATP synthesis.   A decreased activity of the electron  €Bj) transport chain can result from  inhibitors as well as from muta-  tions in mtDNA and nuclear DNA. Why does  an impairment of the electron transport  chain result in lactic acidosis?   Clinical diseases involving components of oxidative phosphorylation (referred to as  OXPHOS diseases) are among the most commonly encountered degenerative dis-  eases. The clinical pathology may be caused by gene mutations in either mitochon-  drial DNA (mtDNA) or nuclear DNA (nDNA) that encode proteins required for  normal oxidative phosphorylation.   The mtDNA is a small 16,569 nucleotide pair, double-stranded, circular DNA. It  encodes 13 subunits of the complexes involved in oxidative phosphorylation: 7 of  the 42 subunits of complex I (NADH dehydrogenase complex), 1 of the 1 1 subunits  of complex III (cytochrome b-c 1 complex), 3 of 13 of the subunits of complex IV  (cytochrome oxidase), and two subunits of the F 0 portion ATP-synthase complex.  In addition, mtDNA encodes the necessary components for translation of its  mRNA: a large and small rRNA and 22 tRNAs. Mutations in mtDNA have been  identified as deletions, duplications, or point mutations (Table 21.1).   The genetics of mutations in mtDNA are defined by maternal inheritance, replica-  tive segregation, threshold expression, a high mtDNA mutation rate, and the accumu-  lation of somatic mutations with age. The maternal inheritance pattern reflects the  exclusive transmission of mtDNA from the mother to her children. The egg contains  approximately 300,000 molecules of mtDNA packaged into mitochondria. These are    Oxidative phosphorylation  (OXPHOS) is responsible for pro-  ducing most of the ATP that our  cells require. The genes responsible for the  polypeptides that comprise the OXPHOS  complexes within the mitochondria are  located within either the nuclear DNA (nDNA)  or the mitochondrial DNA (mtDNA). A broad  spectrum of human disorders (the OXPHOS  diseases) may result from genetic mutations  or nongenetic alterations (spontaneous muta-  tions) in either the nDNA or the mtDNA.  Increasingly, such changes appear to be  responsible for at least some aspects of com-  mon disorders, such as Parkinson's disease,  dilated and hypertrophic cardiomyopathies,  diabetes mellitus, Alzheimer disease, depres-  sive disorders, and a host of less well-known  clinical entities.   Onset before 20 years of age, characterized by  opthalmoplegia, atypical retinitis pigmentosa,  mitochondrial myopathy, and one of the following:  cardiac conduction defect, cerebellar syndrome,  or elevated CSF proteins.   Progressive myoclonic epilepsy, a mitochondrial  myopathy with ragged-red fibers, and a slowly  progressive dementia. Onset of symptoms: late  childhood to adult   Progressive neurodengenerative disease characterized  by stoke-like episodes first occurring between 5 and  15 years of age and a mitochondrial myopathy   Mean age of onset, 1 .5-5 years; clinical manifestations  included optic atrophy, ophthalmoplegia, nystagmus,  respiratory abnormalities, ataxia, hypotonia, spasticity,  and developmental delay or regression.   Deletion of contiguous segments of tRNA  and OXPHOS polypeptides, or duplication mutations  consisting of tandemly arranged normal mtDNA and  an mtDNA with a deletion mutation.   90% of European and Asian cases result from  mutation in NADH dehydrogenase.   The effect of inhibition of electron  transport is an impaired oxidation  of pyruvate, fatty acids, and other  fuels. In many cases, the inhibition of mito-  chondrial electron transport results in higher  than normal levels of lactate and pyruvate in  the blood and an increased lactate/pyruvate  ratio. NADH oxidation requires the com-  pleted transfer of electrons from NADH to  0 2 , and a defect anywhere along the chain  will result in the accumulation of NADH and  decrease of NAD + . The increase in  NADH/NAD + inhibits pyruvate dehydroge-  nase and causes the accumulation of pyru-  vate. It also increases the conversion of  pyruvate to lactate, and elevated levels of  lactate appear in the blood. A large number  of genetic defects of the proteins in respira-  tory chain complexes have, therefore, been  classified together as "congenital lactic  acidosis."   A patient experienced sponta-  neous muscle jerking (myoclonus)  in her mid-teens, and her condition  progressed over 10 years to include debili-  tating myoclonus, neurosensory hearing  loss, dementia, hypoventilation, and mild  cardiomyopathy. Energy metabolism was  affected in the central nervous system,  heart, and skeletal muscle, resulting in lactic  acidosis. A history indicated that the  patient's mother, her grandmother, and two  maternal aunts had symptoms involving  either nervous or muscular tissue (clearly a  case of maternal inheritance). However, no  other relative had identical symptoms. The  symptoms and history of the patient are  those of myoclonic epileptic ragged red fiber  disease (MERRF). The affected tissues (cen-  tral nervous system and muscle) are two of  the tissues with the highest ATP require-  ments. Most cases of MERRF are caused by  a point mutation in mitochondrial tRNA lys  (mtRNA lys ). The mitochondria, obtained by  muscle biopsy, are enlarged and show  abnormal patterns of cristae. The muscle tis-  sue also shows ragged red fibers.   retained during fertilization, whereas those of the sperm do not enter the egg or are  lost. Usually, some mitochondria are present with the mutant mtDNA and some with  normal (wild-type) DNA. As cells divide during mitosis and meiosis mitochondria  replicate by fission, but various amounts of mitochondria with mutant and wild-type  DNA are distributed to each daughter cell {replicative segregation). Thus, any cell can  have a mixture of mitochondria, each with mutant or wild-type mtDNAs (hetero-  plasmy). The mitotic and meiotic segregation of the heteroplasmic mtDNA mutation  results in variable oxidative phosphorylation deficiencies between patients with the  same mutation, and even among a patient's own tissues.   The disease pathology usually becomes worse with age, because a small amount  of normal mitochondria might confer normal function and exercise capacity while  the patient is young. As the patient ages, somatic (spontaneous) mutations in  mtDNA accumulate from the generation of free radicals within the mitochondria  (see Chapter 24). These mutations frequently become permanent, partly because  mtDNA does not have access to the same repair mechanisms available for nuclear  DNA {high mutation rate). Even in normal individuals, somatic mutations result in  a decline of oxidative phosphorylation capacity with age {accumulation of somatic  mutations with age). At some stage, the ATP-generating capacity of a tissue falls  below the tissue-specific threshold for normal function {threshhold expression). In  general, symptoms of these defects would appear in one or more of the tissues with  the highest ATP demands: nervous tissue, heart, skeletal muscle, and kidney.   Genetic mutations also have been reported for mitochondrial proteins encoded by  nuclear DNA. Most of the estimated 1,000 proteins required for oxidative phospho-  rylation are encoded by nuclear DNA, whereas mtDNA encodes only 13 subunits of  the oxidative phosphorylation complexes (including ATP synthase). Nuclear DNA  encodes the additional 70 or more subunits of the oxidative phosphorylation com-  plexes, as well as adenine nucleotide translocase (ANT) and other anion transloca-  tors. Coordinate regulation of expression of nuclear and mtDNA, import of proteins  into the mitochondria, assembly of the complexes, and regulation of mitochondrial  fission are nuclear encoded.   Nuclear DNA mutations differ from mtDNA mutations in several important  respects. These mutations do not show a pattern of maternal inheritance but are usu-  ally autosomal recessive. The mutations are uniformly distributed to daughter cells  and therefore are expressed in all tissues containing the allele for a particular tissue-  specific isoform. However, phenotypic expression still will be most apparent in tis-  sues with high ATP requirements.   The electrochemical gradient couples the rate of the electron transport chain to the  rate of ATP synthesis. Because electron flow requires proton pumping, electron  flow cannot occur faster than protons are used for ATP synthesis (coupled oxidative  phosphorylation) or returned to the matrix by a mechanism that short circuits the  ATP synthase pore (uncoupling).   ^The nuclear respiratory factors (NRF-1 and NRF-2) are nuclear transcription fac-  tors that bind to and activate promotor regions of the nuclear genes encoding  subunits of the respiratory chain complexes, including cytochrome c. They also  activate the transcription of the nuclear gene for the mitochondrial transcription factor  (mTF)-A. The protein product of this gene translocates into the mitochondrial matrix,  where it stimulates transcription and replication of the mitochondrial genome.   As ATP chemical bond energy is used by energy-requiring reactions, ADP and Pi  concentrations increase. The more ADP present to bind to the ATP synthase, the  greater will be proton flow through the ATP synthase pore, from the intermembrane  space to the matrix. Thus, as ADP levels rise, proton influx increases, and the elec-  trochemical gradient decreases (Fig 21.10). The proton pumps of the electron trans-  port chain respond with increased proton pumping and electron flow to maintain the  electrochemical gradient. The result is increased 0 2 consumption. The increased  oxidation of NADH in the electron transport chain and the increased concentration  of ADP stimulate the pathways of fuel oxidation, such as the TCA cycle, to supply  more NADH and FAD(2H) to the electron transport chain. For example, during  exercise, we use more ATP for muscle contraction, consume more oxygen, oxidize  more fuel (which means burn more calories), and generate more heat from the elec-  tron transport chain. If we rest, and the rate of ATP utilization decreases, proton  influx decreases, the electrochemical gradient increases, and proton "back-  pressure" decreases the rate of the electron transport chain. NADH and FAD(2H)  cannot be oxidized as rapidly in the electron transport chain, and consequently, their  build-up inhibits the enzymes that generate them.   The system is poised to maintain very high levels of ATP at all times. In most tis-  sues, the rate of ATP utilization is nearly constant over time. However, in skeletal  muscles, the rates of ATP hydrolysis change dramatically as the muscle goes from  rest to rapid contraction. Even under these circumstances, ATP concentration  decreases by only approximately 20% because it is so rapidly regenerated. In the  heart, Ca 2+ activation of TCA cycle enzymes provides an extra push to NADH gen-  eration, so that neither ATP nor NADH levels fall as ATP demand is increased. The  electron transport chain has a very high capacity and can respond very rapidly to  any increase in ATP utilization.   When protons leak back into the matrix without going through the ATP synthase  pore, they dissipate the electrochemical gradient across the membrane without gen-  erating ATP. This phenomenon is called "uncoupling" oxidative phosphorylation. It  occurs with chemical compounds, known as uncouplers, and it occurs physiologi-  cally with uncoupling proteins that form proton conductance channels through the  membrane. Uncoupling of oxidative phosphorylation results in increased oxygen  consumption and heat production as electron flow and proton pumping attempt to  maintain the electrochemical gradient.   ©Shivering results from muscular  contraction, which increases the  rate of ATP hydrolysis. As a conse-  quence of proton entry for ATP synthesis,  the electron transport chain is stimulated.  Oxygen consumption increases, as does the  amount of energy lost as heat by the elec-  tron transport chain.   Fig. 21.10. The concentration of ADP (or the  phosphate potential -[ATP]/[ADP][PJ) con-  trols the rate of oxygen consumption. (1) ADP  is phosphorylated to ATP by ATP synthase. (2)  The release of the ATP requires proton flow  through ATP synthase into the matrix. (3) The  use of protons from the intermembrane space  for ATP synthesis decreases the proton gradi-  ent. (4) As a result, the electron transport chain  pumps more protons, and oxygen is reduced to  H 2 0. (5) As NADH donates electrons to the  electron transport chain, NAD + is regenerated  and returns to the TCA cycle or other NADH-  producing pathways.   Chemical uncouplers, also known as proton ionophores, are lipid-soluble com-  pounds that rapidly transport protons from the cytosolic to the matrix side of the  inner mitochondrial membrane (Fig. 21.11). Because the proton concentration is  higher in the intermembrane space than in the matrix, uncouplers pick up protons  from the intermembrane space. Their lipid solubility enables them to diffuse  through the inner mitochondrial membrane while carrying protons and release these    A skeletal muscle biopsy performed on Ivy Sharer indicated proliferation of subsarcolemmal mitochondria with degeneration of  muscle fibers (ragged-red fibers) in approximately 55% of the total fibers observed. An analysis of mitochondrial (mtDNA) indi-  cated no genetic mutations but did show a moderate quantitative depletion of mtDNA.   Ivy Sharer's AIDS was being treated with zidovudine (AZT), which also can act as an inhibitor of the mitochondrial DNA polymerase  (polymerase 7). A review of the drugs' potential adverse effects showed that, rarely, it may cause varying degrees of mtDNA depeletion  in different tissues, including skeletal muscle. The depletion may cause a severe mitochondrial myopathy, including "ragged-red fiber"  accumulation within the skeletal muscle cells associated with ultrastructural abnormalities in their mitochondria.   High [H + ] causes outside Low [H + ] inside causes protons   Fig. 21.11. Dinitrophenol (DNP) is lipid soluble and can therefore diffuse across the mem-  brane. It has a dissociable proton with a pK a near 7.2. Thus, in the intermembrane space  where [H + ] is high (pH low), DNP picks up a proton, which it carries across the membrane.  At the lower proton concentration of the matrix, the H + dissociates. As a consequence, cells  cannot maintain their electrochemical gradient or synthesize ATP. DNP was once recom-  mended in the United States as a weight loss drug, based on the principle that decreased  [ATP] and increased electron transport stimulate fuel oxidation. However, several deaths  resulted from its use.   protons on the matrix side. The rapid influx of protons dissipates the electrochemi-  cal potential gradient; therefore, the mitochondria are unable to synthesize ATP.  Eventually, mitochondrial integrity and function are lost.   Q o Salicylate, which is a degradation  ( /~oA 1 product of aspirin in the human, is  ™ lipid soluble and has a dissociable  proton. In high concentrations, as in salicy-  late poisoning, salicylate is able to partially  uncouple mitochondria. The decline of ATP  concentration in the cell and consequent  increase of AMP in the cytosol stimulates  glycolysis. The overstimulation of the gly-  colytic pathway (see Chapter 22) results in  increased levels of lactic acid in the blood  and a metabolic acidosis. Fortunately,  Dennis Veere did not develop this conse-  quence of aspirin poisoning (see Chapter 4).   Uncoupling proteins (UCPs) form channels through the inner mitochondrial mem-  brane that are able to conduct protons from the intermembrane space to the matrix,  thereby short-circuiting ATP synthase.   UCP1 (thermogenin) is associated with heat production in brown adipose tissue.  The major function of brown adipose tissue is nonshivering thermogenesis, whereas  the major function of white adipose tissue is the storage of triacylglycerols in white  lipid droplets. The brown color arises from the large number of mitochondria that  participate. Human infants, who have little voluntary control over their environment  and may kick their blankets off at night, have brown fat deposits along the neck, the  breastplate, between the scapulae, and around the kidneys to protect them from  cold. However, there is very little brown fat in most adults.   In response to cold, sympathetic nerve endings release norepinephrine, which  activates a lipase in brown adipose tissue that releases fatty acids from triacylglyc-  erols (Fig 21.12). Fatty acids serve as a fuel for the tissue (i.e., are oxidized to gen-  erate the electrochemical potential gradient and ATP) and participate directly in the  proton conductance channel by activating UCP1 along with reduced CoQ. When  UCP1 is activated by purine nucleotides, fatty acids, and CoQ, it transports protons  from the cytosolic side of the inner mitochondrial membrane back into the mito-  chondrial matrix without ATP generation. Thus, it partially uncouples oxidative  phosphorylation and generates additional heat.   The uncoupling proteins exist as a family of proteins: UCP1 (thermogenin) is  expressed in brown adipose tissue; UCP2 is found in most cells, UCP3 is found  principally in skeletal muscle; UCP4 and UCP5 are found in the brain. These are  highly regulated proteins that, when activated, increase the amount of energy from  fuel oxidation that is being released as heat. Because they affect metabolic effi-  ciency, differences in the level of UCPs (particularly skeletal muscle UCP3) may  contribute to the tendency toward obesity in some individuals or populations. UCPs  also may decrease the amount of reduced CoQ available to form oxygen free radicals,  thereby decreasing mitochondrial and cell injury.   Fig. 21.12. Brown fat is a tissue specialized for nonshivering thermogenesis. Cold or exces-  sive food intake stimulates the release of norepinephrine from the sympathetic nerve endings.  As a result, a lipase is activated that releases fatty acids for oxidation. The proton conduc-  tance protein, thermogenin, is activated, and protons are brought into the matrix. This stim-  ulates the electron transport chain, which increases its rate of NADH and FAD(2H) oxidation  and produces more heat.   A low level of proton leak across the inner mitochondrial membrane occurs in our  mitochondria all of the time, and our mitochondria thus are normally partially  uncoupled. It has been estimated that more than 20% of our resting metabolic rate  is the energy expended to maintain the electrochemical gradient dissipated by our  basal proton leak (also referred to as global proton leak). Some of the proton leak  results from permeability of the membrane associated with proteins embedded in  the lipid bilayer. An unknown amount may result from uncoupling proteins.   Most of the newly synthesized ATP that is released into the mitochondrial matrix  must be transported out of the mitochondria, where it is used for energy-requiring  processes such as active ion transport, muscle contraction, or biosynthetic reactions.  Likewise, ADP, phosphate, pyruvate, and other metabolites must be transported into  the matrix. This requires transport of compounds through both the inner and outer  mitochondrial membranes.   The inner mitochondrial membrane forms a tight permeability barrier to all polar  molecules, including ATP, ADP, P i? anions such as pyruvate, and cations such as  Ca 2+ , H + , and K + . Yet the process of oxidative phosphorylation depends on rapid  and continuous transport of many of these molecules across the inner mitochondr-  ial membrane (Fig. 21.13). Ions and other polar molecules are transported across the  inner mitochondrial membrane by specific protein translocases that nearly balance  charge during the transport process. Most of the exchange transport is a form of  active transport that generally uses energy from the electrochemical potential gra-  dient, either the membrane potential or the proton gradient.   The inner and outer membranes  \\ ) differ substantially in their lipid   content. The inner mitochondrial  membrane is 22% cardiolipin and contains  almost no cholesterol. The outer membrane  resembles the cell membrane; it is less than  3% cardiolipin and approximately 45% cho-  lesterol.   Fig. 21.13. Transport of compounds across the inner and outer mitochondrial membranes. The electrochemical potential gradient drives the  transport of ions across the inner mitochondrial membrane on specific translocases. Each translocase is composed of specific membrane-  spanning helices that bind only specific compounds (ANT; adenine nucleotide translocase). In contrast, the outer membrane contains relatively  large unspecific pores called VDAC (voltage-dependent anion channels) through which a wide range of ions diffuse. These bind cytosolic pro-  teins such as hexokinase (HK), which enables HK to have access to newly exported ATP.   ANT is an antiport, an exchange  K I ) protein that translocates one ion in  exchange for a molecule of similar  charge. In contrast, the phosphate transporter  and the pyruvate transporter are symports,  which are translocases that co-transport two  molecules of opposite charge. The Ca 2+ chan-  nel is called a uniporter because no other ions  are involved. All of the metabolites entering  or leaving the TCA cycle are transported  across the inner mitochondrial membrane by  specific transport proteins. This includes the  dicarboxylate transporter (phosphate-malate  exchange), the tricarboxylate transporter (cit-  rate— malate exchange), the aspartate-gluta-  mate transporter, and the malate-a-ketoglu-  tarate transporter.   Investigators reported finding anti-  €B # bodies against cardiac ATP-ADP  translocase in an individual who  died of a viral cardiomyopathy. How could  these antibodies result in death?   ATP-ADP translocase (also called ANT for adenine nucleotide translocase) trans-  ports ATP formed in the mitochondrial matrix to the intermembrane space in a specific  1 : 1 exchange for ADP produced from energy-requiring reactions outside of the mito-  chondria (see Fig. 21.13). Because ATP contains four negative charges and ADP only  three, the exchange is promoted by the electrochemical potential gradient, because the  net effect is the transport of one negative charge from the matrix to the cytosol. Simi-  lar antiports exist for most metabolic anions. In contrast, inorganic phosphate and pyru-  vate are transported into the mitochondrial matrix on specific transporters called sym-  ports together with a proton. A specific transport protein for Ca 2+ uptake, called the  Ca 2+ uniporter, is driven by the electrochemical potential gradient, which is negatively  charged on the matrix side of the membrane relative to the cytosolic side.   Whereas the inner mitochondrial membrane is highly impermeable, the outer mito-  chondrial membrane is permeable to compounds with a molecular weight up to  approximately 6,000 daltons because it contains large nonspecific pores called volt-  age-dependent anion channels (VDAC) that are formed by mitochondrial porins  (see Fig. 21.13). Unlike most transport proteins, which are membrane- spanning  helices with specific binding sites, VDACs are composed of porin homodimers that  form a p -barrel with a relatively large nonspecific water- filled pore through the cen-  ter. These channels are "open" at low transmembrane potential, with a preference  for anions such as phosphate, chloride, pyruvate, citrate, and adenine nucleotides.   As ATP is hydrolyzed during muscular contraction, ADP is formed. This ADP must  • J exchange into the mitochondria on ATP-ADP translocase to be converted back to  ATP Inhibition of ATP-ADP translocase results in rapid depletion of the cytosol ATP  levels and loss of cardiac contractility.   VDACs thus facilitate translocation of these anions between the intermembrane  space and the cytosol. A number of cytosolic kinases, such as the hexokinase that  initiates glycolysis, bind to the cytosolic side of the channel, where they have ready  access to newly synthesized ATP.   The mitochondrial permeability transition involves the opening of a large nonspecific  pore (called MPTP, the mitochondrial permeability transition pore) through the inner  mitochondrial membrane and outer membranes at sites where they form a junction  (Fig. 21.14). The basic components of the mitochondrial permeability transition pore  are adenine nucleotide translocase (ANT), the voltage-dependant anion channel  (VDAC), and cyclophilin D (which is a cis-trans isomerase for the proline peptide  bond). Normally ANT is a closed pore that functions specifically in a 1:1 exchange of  matrix ATP for ADP in the intermembrane space. However, increased mitochondrial  matrix Ca 2+ , excess phosphate, or ROS (reactive oxygen species that form oxygen or  oxygen-nitrogen radicals) can activate opening of the pore. Conversely, ATP on the  cytosolic side of the pore (and possibly a pH below 7.0) and a membrane potential  across the inner membrane protect against pore opening. Opening of the MPTP can be  triggered by ischemia (hypoxia), which results in a temporary lack of 0 2 for maintain-  ing the proton gradient and ATP synthesis. When the proton gradient is not being gen-  erated by the electron transport chain, ATP synthase runs backward and hydrolyzes  ATP in an attempt to restore the gradient, thus rapidly depleting cellular levels of ATP.  As ATP is hydrolyzed to ADP, the ADP is converted to adenine, and the nucleotide pool  is no longer able to protect against pore opening. This can lead to a downward spiral  of cellular events. A lack of ATP for maintaining the low intracellular Ca 2+ can con-  tribute to pore opening. When the MPTP pore opens, protons will flood in, and main-  taining a proton gradient becomes impossible. Anions and cations enter the matrix,  mitochondrial swelling ensues, and the mitochondria become irreversibly damaged.  The result is cell lysis and death (necrosis).   v Cora Nari. Thrombolysis stimulated by intravenous recombinant tissue  ) plasminogen activator (TPA) restored 0 2 to Cora Nari's heart muscle and  successfully decreased the extent of ischemic damage. The rationale for  the use of TPA within 4 to 6 hours after the onset of a myocardial infarction relates   In addition to a normal role in move-  \ \ ) ment of molecules across the mito-   chondrial outer membrane, VDACs  appear to have roles in processes leading to  cell death. These roles are influenced by pro-  teins that bind to the VDACs. By forming a  component of the mitochondrial permeability  transition pore, VDACs may contribute to  events leading to cell necrosis. Alternatively,  binding of pro-apoptotic or anti-apoptotic  members of the Bcl-2 family may change the  permeability of the outer membrane so as to  either favor or block events leading to pro-  grammed cell death (apoptosis).   Fig. 21.14. The mitochondrial permeability  transition pore (MPTP). In the MPTP, ANT is  thought to complex with VDAC. The confor-  mation of ANT is regulated by cyclophilin D  (CD), and Ca 2+ . VDACs bind a number of pro-  teins, including Bcl2 and Bax, which regulate  apoptosis. The change to an open pore is acti-  vated by Ca 2+ , depletion of adenine  nucleotides, and oxygen radicals (ROS) that  alter SH groups. It is inhibited by the electro-  chemical potential gradient (Ap), by cytosolic  ATP, and by a low cytosolic pH.   0 © As infusion of TPA lysed the clot blocking blood flow to Cora Nari's heart, oxygenated blood was reintroduced into the ischemic  I /~0A J heart. Although oxygen may rapidly restore the capacity to generate ATP, it often increases cell death, a phenomenon called  ™ ischemia-reperfusion injury.   During ischemia, a number of factors may protect heart cells against ireversible injury and cell death until oxygen is reintroduced. The  stimulation of anaerobic glycolysis in the cytosol generates ATP without oxygen as glucose is converted to lactic acid. Lactic acid  decreases cytosolic pH. Both cytosolic ATP and a lowering of the pH protect against opening of the MPTP. In addition, Ca 2+ uptake by mito-  chondria requires a membrane potential, and it is matrix Ca 2+ that activates opening of the MPTP. Thus, depending on the severity of the  ischemic insult, the MPTP may not open, or may open and reseal, until oxygen is reintroduced. Then, depending on the sequence of  events, reestablishment of the proton gradient, mitochondrial uptake of Ca 2+ , or an increase of pH above 7.0 may activate the MPTP before  the cell has recovered. In addition, the reintroduction of 0 2 generates oxygen free radicals, particularly through free radical forms of CoQ  in the electron transport chain. These also may open the MPTP. The role of free radicals in ischemia-reperfusion injury is covered in more  detail in Chapter 24.   to the function of the normal intrinsic fibrinolytic system (see Chapter 45). This sys-  tem is designed to dissolve unwanted intravascular clots through the action of the  enzyme plasmin, a protease that digests the fibrin matrix within the clot. TPA stim-  ulates the conversion of plasminogen to its active form, plasmin. The result is a lysis  of the thrombus and improved blood flow through the previously obstructed vessel,  allowing fuels and oxygen to reach the heart cells. The human TPA protein used in  Mrs. Nari is produced by recombinant DNA technology (see Chapter 17). This  treatment rapidly restored oxygen supply to the heart.    In addition to increased transcrip-  tion of genes encoding TCA cycle  enzymes and certain other  enzymes of fuel oxidation, thyroid hor-  mones increase the level of UCP2 and UCP3.  In hyperthyroidism, the efficiency with  which energy is derived from the oxidation  of these fuels is significantly less than nor-  mal. As a consequence of the increased rate  of the electron transport chain, hyperthy-  roidism results in increased heat production.  Patients with hyperthyroidism, such as X.S.  Teefore, complain of constantly feeling hot  and sweaty.   ^ X.S- Teefore. Mr. Teefore could be treated with antithyroid drugs, by  ) subtotal resection of the thyroid gland, or with radioactive iodine. Suc-  cessful treatment normalizes thyroid hormone secretion, and all of the  signs, symptoms, and metabolic alterations of hyperthyroidism quickly subside.   ^\ Ivy Sharer. In the case of Ivy Sharer, a diffuse myopathic process was  ) superimposed on her AIDS and her pulmonary tuberculosis, either of  which could have caused progressive weakness. In addition, she could  have been suffering from a congenital mtDNA myopathy, symptomatic only as she  ages. A systematic diagnostic process, however, finally led her physician to con-  clude that her myopathy was caused by a disorder of oxidative phosphorylation  induced by her treatment with zidovudine (AZT). Fortunately, when AZT was dis-  continued, Ivy's myopathic symptoms gradually subsided. A repeat skeletal muscle  biopsy performed 4 months later showed that her skeletal muscle cell mtDNA had  been restored to normal and that she had experienced a reversible drug-induced dis-  order of oxidative phosphorylation.   ing factor (Apaf-1), which activates  the initiator caspase-9. Caspase-9, in turn,  activates effector caspases-3, -6, and -7  through proteolytic cleavage, which then  degrade cytoplasmic proteins. AIF, which  has a nuclear targeting sequence, is trans-  ported into the nucleus, where it initiates  chromatin condensation and degradation.  Caspase-2 and caspase-9 also activate CAD,  which migrates to the nucleus and  hydrolyzes bonds in nuclear DNA.   ©Mitochondria and Apoptosis The loss of mitochondrial integrity  is a major route initiating apoptosis (see Chapter 18, section V). The inter-  membrane space contains procaspases —2, — 3, and —9, which are prote-  olytic enzymes that are in the zymogen form (i.e., must be proteolytically cleaved  to be active). It also contains apoptosis initiating factor (AIF) and caspase-activated  DNAase (CAD). Cytochrome c, which is loosely bound to the outer mitochondrial  membrane, may also enter the intermembrane space when the electrochemical  potential gradient is lost. The release of cytochrome c and the other proteins into the  cytosol initiates apoptosis.   But how are cytochrome c and the other proteins released? The VDAC pore is not  large enough to allow the passage of proteins. A number of theories have been pro-  posed, each supported and contradicted by experimental evidence. One is that Bax (a  member of the Bcl-2 family of proteins that forms an ion channel in the outer mito-  chondrial membrane) allows the entry of ions into the intermembrane space, causing  swelling of this space and rupture of the outer mitochondrial membrane. Another the-  ory is that Bax and VDAC (which is known to bind Bax and other Bcl-2 family mem-  bers) combine to form an extremely large pore, much larger than formed by either  alone. Finally, it is possible that the MPTP or ANT participate in rupture of the outer  membrane, but close in a way that still provides the energy for apoptosis.   Boyer PD. A perspective of the binding change mechanism for ATP synthesis. FASEB J  1989;3:2164-2178.   Brown GC. Control of respiration and ATP synthesis in mammalian mitochondria and cells. Biochem J  1992;284:1-11.   Lactic acidosis and mitochondrial myopathy in a young woman. Nutr Rev 1988;46:157-163.  Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfu-   sion injury. Biochem Soc Symp 1999;66:181-203.  Lowell BB, Spiegelman BM. Towards a molecular understanding of adaptive thermogenesis. Nature   Olson RD, Mushlin PS. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J  1990;4:3076-3086.   Shoffner JM. Oxidative phosphorylation diseases. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The  Metabolic and Molecular Bases of Inherited Disease, vol II. 8th Ed. New York: McGraw-Hill,  2001:2367-2424.    1 . Consider the following experiment. Carefully isolated liver mitochondria are incubated in the presence of a limiting amount  of malate. Three minutes after adding the substrate, cyanide is added, and the reaction is allowed to proceed for another 7 min-  utes. At this point, which of the following components of the electron transfer chain will be in an oxidized state?   2. Consider the following experiment. Carefully isolated liver mitochondria are placed in a weakly buffered solution. Malate is  added as an energy source, and an increase in oxygen consumption confirms that the electron transfer chain is functioning  properly within these organelles. Valinomycin and potassium are then added to the mitochondrial suspension. Valinomycin is  a drug that allows potassium ions to freely cross the inner mitochondrial membrane. What is the effect of valinomycin on the  proton motive force that had been generated by the oxidation of malate?   (A) The proton motive force will be reduced to a value of zero   (B) There will be no change in the proton motive force   (D) The proton motive force will be decreased, but to a value greater than zero   (E) The proton motive force will be decreased to a value less than zero   3. Dinitrophenol acts as an uncoupler of oxidative phosphorylation by which of the following mechanisms?   4. A 25-year-old female presents with chronic fatigue. A series of blood tests are ordered, and the results suggest that her red  blood cell count is low because of iron deficiency anemia. Such a deficiency would lead to fatigue because of which of the  following?   (A) Her decrease in Fe-S centers is impairing the transfer of electrons in the electron transport chain.   (B) She is not producing as much H 2 0 in the electron transport chain, leading to dehydration, which has resulted in  fatigue.   (C) Iron forms a chelate with NADH and FAD(2H) that is necessary for them to donate their electrons to the electron trans-  port chain.   (D) Iron acts as a cofactor for a-ketoglutarate DH in the TCA cycle, a reaction required for the flow of electrons through  the electron transport chain.   (E) Iron accompanies the protons that are pumped from the mitochondrial matrix to the cytosolic side of the inner mito-  chondrial membrane. Without iron, the proton gradient cannot be maintained to produce adequate ATP.   Which of the following would be expected for a patient with an OXPHOS disease?   (D) A high activity of complex II of the electron transport chain   (E) A defect in the integrity of the inner mitochondrial membrane   Glucose is the universal fuel for human cells. Every cell type in the human is able  to generate adenosine triphosphate (ATP) from glycolysis, the pathway in which  glucose is oxidized and cleaved to form pyruvate. The importance of glycolysis in  our fuel economy is related to the availability of glucose in the blood, as well as  the ability of glycolysis to generate ATP in both the presence and absence of0 2 .  Glucose is the major sugar in our diet and the sugar that circulates in the blood  to ensure that all cells have a continuous fuel supply. The brain uses glucose  almost exclusively as a fuel.   Glycolysis begins with the phosphorylation of glucose to glucose 6-phosphate  (glucose-6-P) by hexokinase (HK). In subsequent steps of the pathway, one glu-  cose-6-P molecule is oxidized to two pyruvate molecules with generation of two  molecules ofNADH (Fig. 22.1 ). A net generation of two molecules of ATP occurs  through direct transfer of high-energy phosphate from intermediates of the path-  way to ADP (substrate level phosphorylation).   Glycolysis occurs in the cytosol and generates cytosolic NADH. Because  NADH cannot cross the inner mitochondrial membrane, its reducing equivalents  are transferred to the electron transport chain by either the malate -aspartate  shuttle or the glycerol 3 -phosphate shuttle (see Fig. 22.1). Pyruvate is then oxi-  dized completely to C0 2 by pyruvate dehydrogenase and the TCA cycle. Complete  aerobic oxidation of glucose to C0 2 can generate approximately 30 to 32 moles  of ATP per mole of glucose.   When cells have a limited supply of oxygen (e.g., kidney medulla), or few or  no mitrochondria (e.g., the red cell), or greatly increased demands for ATP  (e.g., skeletal muscle during high-intensity exercise), they rely on anaerobic  glycolysis for generation of ATP. In anaerobic glycolysis, lactate dehydroge-  nase oxidizes the NADH generated from glycolysis by reducing pyruvate to  lactate (Fig. 22.2). Because 0 2 is not required to reoxidize the NADH, the  pathway is referred to as anaerobic. The energy yield from anaerobic glycolysis  (2 moles of ATP per mole of glucose) is much lower than the yield from aerobic  oxidation. The lactate (lactic acid) is released into the blood. Under pathologic  conditions that cause hypoxia, tissues may generate enough lactic acid to  cause lactic acidemia.   In each cell, glycolysis is regulated to ensure that ATP homeostasis is  maintained, without using more glucose than necessary. In most cell types,  hexokinase (HK), the first enzyme of glycolysis, is inhibited by glucose  6-phosphate (see Fig. 22.1). Thus, glucose is not taken up and phosphorylated  by a cell unless glucose-6-P enters a metabolic pathway, such as glycolysis or  glycogen synthesis. The control of glucose-6-P entry into glycolysis occurs at  phosphofructokinase-l(PFK-l), the rate-limiting enzyme of the pathway.  PFK-1 is allosterically inhibited by ATP and allosterically activated by AMP.  AMP increases in the cytosol as ATP is hydrolyzed by energy -re quiring  reactions.    For glucose 6-phosphate and other  sugar phosphoesters, the phos-  phate group will be denoted with   Glycolysis has functions in addition to ATP production. For example, in liver  and adipose tissue, this pathway generates pyruvate as a precursor for fatty acid  biosynthesis. Glycolysis also provides precursors for the synthesis of compounds  such as amino acids and 5 -carbon sugar phosphates.   Fig. 22.2. Anaerobic glycolysis (shown in  blue). The conversion of glucose to lactate  generates 2 ATP from substrate-level phospho-  rylation. Because there is no net generation of  NADH, there is no need for 0 2 , and, thus, the  pathway is anaerobic.   • Lopa Fusor is a 68-year-old woman who is admitted to the hospital  flP ) emergency room with very low blood pressure (80/40 mm Hg) caused   " by an acute hemorrhage from a previously diagnosed ulcer of the stom-  ach. Lopa's bleeding stomach ulcer has reduced her effective blood volume  severely enough to compromise her ability to perfuse (deliver blood to) her tis-  sues. She is, therefore, a "low perfuser." She is also known to have chronic  obstructive pulmonary disease (COPD) as a result of 42 years of smoking two  packs of cigarettes per day. Her respiratory rate is rapid and labored, her skin is  cold and clammy, and her lips are slightly blue (cyanotic). She appears anxious  and moderately confused.   As appropriate emergency measures are taken to stabilize her and elevate her  blood pressure, blood is sent for immediate blood typing and cross-matching, so  that blood transfusions can be started. A battery of laboratory tests are ordered,  including venous hemoglobin, hematocrit, and lactate levels, and arterial blood pH,  partial pressures of oxygen (p0 2 ) and carbon dioxide (pC0 2 ), bicarbonate, and oxy-  gen saturation. Results show that the hemorrhaging and COPD have resulted in  hypoxemia, with decreased oxygen delivery to her tissues, and both a respiratory  and metabolic acidosis.   Otto Shape, a 26-year-old medical student, had gained weight during his  first sedentary year in medical school. During his second year, he began  watching his diet, jogging for an hour 4 times each week, and playing ten-  nis twice a week. He has decided to compete in a 5 -km race. To prepare for the race,  he begins training with wind sprints, bouts of alternately running and walking.   Ivan Applebod is a 56-year-old morbidly obese accountant (see Chapters  1-3). He decided to see his dentist because he felt excruciating pain in his  teeth when he ate ice cream. He really likes sweets and keeps hard candy  in his pocket. The dentist noted from Mr. Applebod 's history that he had numerous  cavities as a child in his baby teeth. At this visit, the dentist found cavities in two of  Mr. Applebod 's teeth.   The hematocrit (the percentage of  \m ) the volume of blood occupied by  *S packed red blood cells) and hemo-  globin content (g hemoglobin in 100 ml_  blood) are measured to determine whether  the oxygen-carrying capacity of the blood is  adequate. They can be decreased by condi-  tions that interfere with erythropoiesis (syn-  thesis of red blood cells in bone marrow),  such as iron deficiency. They also can be  decreased during chronic bleeding, but not  during immediate acute hemorrhage, if  interstitial fluid replaces the lost blood vol-  ume and dilutes out the red blood cells. The  pC0 2 and p0 2 are the partial pressures of  C0 2 and 0 2 in the blood. The p0 2 and oxy-  gen saturation determine whether adequate  oxygen is available for tissues. Measure-  ment of the pC0 2 and bicarbonate can dis-  tinguish between a metabolic and a respira-  tory acidosis (see Chapter 4).   Glycolysis is one of the principle pathways for generating ATP in cells and is  present in all cell types. The central role of glycolysis in fuel metabolism is  related to its ability to generate ATP with, and without, oxygen. The oxidation of  glucose to pyruvate generates ATP from substrate-level phosphorylation (the  transfer of phosphate from high-energy intermediates of the pathway to ADP) and  NADH. Subsequently, the pyruvate may be oxidized to C0 2 in the TCA cycle and  ATP generated from electron transfer to oxygen in oxidative phosphorylation.  However, if the pyruvate and NADH from glycolysis are converted to lactate  (anaerobic glycolysis), ATP can be generated in the absence of oxygen, via  substrate-level phosphorylation.   Glucose is readily available from our diet, internal glycogen stores, and the  blood. Carbohydrate provides 50% or more of the calories in most diets, and glu-  cose is the major carbohydrate. Other dietary sugars, such as fructose and galactose,  are oxidized by conversion to intermediates of glycolysis. Glucose is stored in cells  as glycogen, which can provide an internal source of fuel for glycolysis in emer-  gency situations (e.g., decreased supply of fuels and oxygen during ischemia, a low  blood flow). Insulin and other hormones maintain blood glucose at a constant level  (glucose homeostasis), thereby ensuring that glucose is always available to cells that  depend on glycolysis for generation of ATP.   In addition to serving as an anaerobic and aerobic source of ATP, glycolysis is an  anabolic pathway that provides biosynthetic precursors. For example, in liver and  adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosyn-  thesis. Glycolysis also provides precursors for the synthesis of compounds such as  amino acids and ribose-5 -phosphate, the precursor of nucleotides. The integration  of glycolysis with other anabolic pathways is discussed in Chapter 36.   After a high-carbohydrate meal,  glucose is the major fuel for almost  all tissues. Exceptions include  intestinal mucosal cells, which transport glu-  cose from the gut into the blood, and cells in  the proximal convoluted tubule of the kid-  ney, which return glucose from the renal fil-  trate to the blood. During fasting, the brain  continues to oxidize glucose because it has a  limited capacity for the oxidation of fatty  acids or other fuels. Cells also continue to  use glucose for the portion of their ATP gen-  eration that must be met by anaerobic gly-  colysis, due to either a limited oxygen sup-  ply or a limited capacity for oxidative  phosphorylation (e.g., the red blood cell).   The glycolytic pathway, which cleaves 1 mole of glucose to 2 moles of the 3 -car-  bon compound pyruvate, consists of a preparative phase and an ATP-generating  phase. In the initial preparative phase of glycolysis, glucose is phosphorylated   Hexokinases, other kinases, and  many other enzymes that catalyze  reactions involving the hydrolysis  of ATP require Mg 2+ . The Mg 2+ forms a com-  plex with the phosphate groups of ATP.  Kinases also require K + .    twice by ATP and cleaved into two triose phosphates (Fig. 22.3). The ATP expen-  diture in the beginning of the preparative phase is sometimes called "priming the  pump," because this initial utilization of 2 moles of ATP/ mole of glucose results  in the production of 4 moles of ATP/mole of glucose in the ATP-generating  phase.   In the ATP-generating phase, glyceraldehyde 3 -phosphate (a triose phosphate) is  oxidized by NAD + and phosphorylated using inorganic phosphate. The high-  energy phosphate bond generated in this step is transferred to ADP to form ATP. The  remaining phosphate is also rearranged to form another high-energy phosphate  bond that is transferred to ADP. Because there were 2 moles of triose phosphate  formed, the yield from the ATP-generating phase is 4 ATP and 2 NADH. The result  is a net yield of 2 moles of ATP, 2 moles of NADH, and 2 moles of pyruvate per  mole of glucose.   Glucose metabolism begins with transfer of a phosphate from ATP to glucose to  form glucose-6-P (Fig. 22.4). Phosphorylation of glucose commits it to metabolism  within the cell because glucose-6-P cannot be transported back across the plasma  membrane. The phosphorylation reaction is irreversible under physiologic condi-  tions because the reaction has a high negative AG 0 '. Phosphorylation does not,  however, commit glucose to glycolysis.   Glucose-6-P is a branchpoint in carbohydrate metabolism. It is a precursor for  almost every pathway that uses glucose, including glycolysis, the pentose phosphate  pathway, and glycogen synthesis. From the opposite point of view, it also can be  generated from other pathways of carbohydrate metabolism, such as glycogenoly-  sis (breakdown of glycogen), the pentose phosphate pathway, and gluconeogenesis  (the synthesis of glucose from non-carbohydrate sources).   Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a  family of tissue- specific isoenzymes that differ in their kinetic properties. The  isoenzyme found in liver and p cells of the pancreas has a much higher K m than  other hexokinases and is called glucokinase. In many cells, some of the hexokinase  is bound to porins in the outer mitochondrial membrane (voltage-dependent anion  channels; see Chapter 21), which gives these enzymes first access to newly synthe-  sized ATP as it exits the mitochondria.   In the remainder of the preparative phase of glycolysis, glucose-6-P is isomerized  to fructose 6-phosphate (fructose-6-P), again phosphorylated, and subsequently  cleaved into two 3 -carbon fragments (Fig 22.5). The isomerization, which positions  a keto group next to carbon 3, is essential for the subsequent cleavage of the bond  between carbons 3 and 4.   The next step of glycolysis, phosphorylation of fructose-6-P to fructose 1,6-  bisphosphate (fructose- 1,6-bisP) by phosphofructokinase- 1 (PFK-1), is generally  considered the first committed step of the pathway. This phosphorylation requires  ATP and is thermodynamically and kinetically irreversible. Therefore, PFK-1 irrev-  ocably commits glucose to the glycolytic pathway. PFK- 1 is a regulated enzyme in  cells, and its regulation controls the entry of glucose into glycolysis. Like hexoki-  nase, it exists as tissue-specific isoenzymes whose regulatory properties match vari-  ations in the role of glycolysis in different tissues.   Fructose- 1,6-bisP is cleaved into two phosphorylated 3 -carbon compounds  (triose phosphates) by aldolase (see Fig. 22.5). Dihydroxyacetone phosphate  (DHAP) is isomerized to glyceraldehyde 3 -phosphate (glyceraldehyde- 3 -P), which  is a triose phosphate. Thus, for every mole of glucose entering glycolysis, 2 moles  of glyceraldehyde- 3 -P continue through the pathway.   Fig. 22.5. Reactions of glycolysis. High-energy phosphates are shown in blue.   Aldolase is named for the mechanism of the forward reaction, which is an aldol cleavage, and the mechanism of the  reverse reaction, which is an aldol condensation. The enzyme exists as tissue-specific isoenzymes, which all catalyze the  cleavage of fructose 1,6-bisphosphate but differ in their specificities for fructose 1-P. The enzyme uses a lysine residue at  the active site to form a covalent bond with the substrate during the course of the reaction. Inability to form this covalent linkage  inactivates the enzyme.   Kinases transfer a phosphate from  ATP to another compound. Hexoki-  nase transfers a phosphate to glu-  cose or another hexose to form a hexose  phosphate. 3-Phosphoglycerate kinase is  named for the reaction that is the reverse of  glycolysis, transfer of phosphate from ATP  to 3-phosphoglycerate to form 1,3-bisphos-  phoglycerate. Pyruvate kinase is also named  for the reverse reaction (phosphorylation of  pyruvate by ATP), although this direction  does not occur under physiologic condi-  tions.   The confusion experienced by  Lopa Fusor in the emergency room  is caused by an inadequate deliv-  ery of oxygen to the brain. Neurons have  very high ATP requirements, and most of  this ATP is provided by aerobic oxidation of  glucose to pyruvate in glycolysis, and pyru-  vate oxidation to C0 2 in the TCA cycle. The  brain has little or no capacity to oxidize fatty  acids, and, therefore, its glucose consump-  tion is high (approximately 125-150 g/day in  the adult). Its oxygen demands are also high.  If cerebral oxygen supply were completely  interrupted, the brain would last only 10 sec-  onds. The only reason consciousness lasts  longer during anoxia or asphyxia is that  there is still some oxygen in the lungs and in  circulating blood. A decrease of blood flow  to approximately Vi of the normal rate  results in a loss of consciousness.   In the next part of the glycolytic pathway, glyceraldehyde-3-P is oxidized and phos-  phorylated so that subsequent intermediates of glycolysis can donate phosphate to  ADP to generate ATP. The first reaction in this sequence, catalyzed by glyceralde-  hyde-3-P dehydrogenase, is really the key to the pathway (see Fig. 22.5). This  enzyme oxidizes the aldehyde group of glyceraldehyde-3-P to an enzyme-bound  carboxyl group and transfers the electrons to NAD + to form NADH. The oxidation  step is dependent on a cysteine residue at the active site of the enzyme, which forms  a high-energy thioester bond during the course of the reaction. The high-energy  intermediate immediately accepts an inorganic phosphate to form the high-energy  acyl phosphate bond in 1,3-bisphosphoglycerate, releasing the product from the  cysteine residue on the enzyme. This high-energy phosphate bond is the start of sub-  strate-level phosphorylation (the formation of a high-energy phosphate bond where  none previously existed, without the utilization of oxygen).   In the next reaction, the phosphate in this bond is transferred to ADP to form ATP  by 3-phosphoglycerate kinase. The energy of the acyl phosphate bond is high  enough (—13 kcal/mole) so that transfer to ADP is an energetically favorable  process. 3-phosphoglycerate is also a product of this reaction.   To transfer the remaining low-energy phosphoester on 3-phosphoglycerate to  ADP, it must be converted into a high-energy bond. This conversion is accom-  plished by moving the phosphate to the second carbon (forming 2-phosphoglycer-  ate) and then removing water to form phosphoenolpyruvate (PEP). The enolphos-  phate bond is a high-energy bond (its hydrolysis releases approximately 14  kcal/mole of energy), so the transfer of phosphate to ADP by pyruvate kinase is  energetically favorable (see Fig. 22.5). This final reaction converts PEP to pyruvate.   Glucose + 2NAD + + 2P { + 2 ADP -> 2Pyruvate + 2NADH + 4H + + 2ATP + 2H 2 0   The pathway occurs with an overall negative AG 0 of approximately -22 kcal.  Therefore, it cannot be reversed without the expenditure of energy.   The NADH produced from glycolysis must be continuously reoxidized back to  NAD + to provide an electron acceptor for the glyceraldehyde-3-P dehydrogenase  reaction and prevent product inhibition. Without oxidation of this NADH, glycoly-  sis cannot continue. There are two alternate routes for oxidation of cytosolic NADH  (Fig. 22.6). One route is aerobic, involving shuttles that transfer reducing equiva-  lents across the mitochondrial membrane and ultimately to the electron transport  chain and oxygen (see Fig. 22. 6A). The other route is anaerobic (without the use of  oxygen). In anaerobic glycolysis, NADH is reoxidized in the cytosol by lactate  dehydrogenase, which reduces pyruvate to lactate (see Fig. 22. 6B).   The fate of pyruvate depends on the route used for NADH oxidation. If NADH  is reoxidized in a shuttle system, pyruvate can be used for other pathways, one of  which is oxidation to acetyl-CoA and entry into the TCA cycle for complete oxida-  tion. Alternatively, in anaerobic glycolysis, pyruvate is reduced to lactate and  diverted away from other potential pathways. Thus, the use of the shuttle systems  allows for more ATP to be generated than by anaerobic glycolysis by both oxidiz-  ing the cytoplasmically derived NADH in the electron transport chain and by allow-  ing pyruvate to be oxidized completely to C0 2 .   The reason that shuttles are required for the oxidation of cytosolic NADH by the  electron transport chain is that the inner mitochondrial membrane is impermeable   Fig. 22.6. Alternate fates of pyruvate. A. The pyruvate produced by glycolysis enters mitochondria and is oxidized to C0 2 and H 2 0. The reduc-  ing equivalents in NADH enter mitochondria via a shuttle system. B. Pyruvate is reduced to lactate in the cytosol, thereby using the reducing  equivalents in NADH.   to NADH, and no transport protein exists that can directly translocate NADH across  this membrane. Consequently, NADH is reoxidized to NAD + in the cytosol by a  reaction that transfers the electrons to DHAP in the glycerol 3 -phosphate (glycerol-  3-P) shuttle and oxaloacetate in the malate-aspartate shuttle. The NAD + that is  formed in the cytosol returns to glycolysis while glycerol-3-P or malate carry the  reducing equivalents that are ultimately transferred across the inner mitochondrial  membrane. Thus, these shuttles transfer electrons and not NADH per se.   The glycerol 3-phosphate shuttle is the major shuttle in most tissues. In this shuttle,  cytosolic NAD + is regenerated by cytoplasmic glycerol 3-phosphate dehydrogenase,  which transfers electrons from NADH to DHAP to form glycerol 3-phosphate (Fig.  22.7). Glycerol 3-phosphate then diffuses through the outer mitochondrial mem-  brane to the inner mitochondrial membrane, where the electrons are donated to a  membrane-bound flavin adenive dinucleofide (FAD)-containing glycerophosphate  dehydrogenase. This enzyme, like succinate dehydrogenase, ultimately donates elec-  trons to CoQ, resulting in an energy yield of approximately 1.5 ATP from oxidative  phosphorylation. Dihydroxyacetone phosphate returns to the cytosol to continue the  shuttle. The sum of the reactions in this shuttle system is simply:   Many tissues contain both the glycerol-3-P shuttle and the malate-aspartate shuttle.  In the malate-aspartate shuttle (Fig. 22.8), cytosolic NAD + is regenerated by  cytosolic malate dehydrogenase, which transfers electrons from NADH to cytoso-  lic oxaloacetate to form malate. Malate is transported across the inner mitochondr-  ial membrane by a specific translocase, which exchanges malate for a-ketoglu-  tarate. In the matrix, malate is oxidized back to oxaloacetate by mitochondrial  malate dehydrogenase, and NADH is generated. This NADH can donate electrons  to the electron transport chain with generation of approximately 2.5 moles of ATP  per mole of NADH. The newly formed oxaloacetate cannot pass back through the  inner mitochondrial membrane under physiologic conditions, so aspartate is used to   Fig. 22.7. Glycerol 3-phosphate shuttle.  Because NAD + and NADH cannot cross the  mitochondrial membrane, shuttles transfer the  reducing equivalents into mitochondria. Dihy-  droxyacetone phosphate (DHAP) is reduced to  glycerol-3-P by cytosolic glycerol 3-P dehy-  drogenase, using cytosolic NADH produced in  glycolysis. Glycerol-3-P then reacts in the  inner mitochondrial membrane with mitochon-  drial glycerol-3-P dehydrogenase, which trans-  fers the electrons to FAD and regenerates  DHAP, which returns to the cytosol. The elec-  tron transport chain transfers the electrons to  0 2 , which generates approximately 1.5 ATP  for each FAD(2H) that is oxidized.   Fig. 22.8. Malate-aspartate shuttle. NADH produced by glycolysis reduces oxaloacetate (OAA) to malate, which crosses the mitochondrial  membrane and is reoxidized to OAA. The mitochondrial NADH donates electrons to the electron transport chain, with 2.5 ATPs generated for  each NADH. To complete the shuttle, oxaloacetate must return to the cytosol, although it cannot be directly transported on a translocase. Instead,  it is transaminated to aspartate, which is then transported out to the cytosol, where it is transaminated back to oxaloacetate. The translocators  exchange compounds in such a way that the shuttle is completely balanced. TA = transamination reaction. a-KG = a-ketoglutarate.   Fig. 22.9. Lactate dehydrogenase reaction.  Pyruvate, which may be produced by glycoly-  sis, is reduced to lactate. The reaction, which  occurs in the cytosol, requires NADH and is  catalyzed by lactate dehydrogenase. This reac-  tion is readily reversible.   return the oxaloacetate carbon skeleton to the cytosol. In the matrix, transamination  reactions transfer an amino group to oxaloacetate to form aspartate, which is trans-  ported out to the cytosol (using an aspartate/glutamate exchange translocase) and  converted back to oxaloacetate through another transamination reaction. The sum of  all the reactions of this shuttle system is simply:   When the oxidative capacity of a cell is limited (e.g., the red blood cell, which has  no mitochondria), the pyruvate and NADH produced from glycolysis cannot be oxi-  dized aerobically. The NADH is therefore oxidized to NAD + in the cytosol by  reduction of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase  (LDH) (Fig. 22.9). The net reaction for anaerobic glycolysis is:   Glucose + 2 ADP + 2 P i -> 2 Lactate + 2 ATP + 2 H 2 0 + 2 H +   In both aerobic and anaerobic glycolysis, each mole of glucose generates 2 moles  of ATP, 2 of NADH and 2 of pyruvate. The energy yield from anaerobic glycolysis  (glucose to 2 lactate) is only 2 moles of ATP per mole of glucose, as the NADH is  recycled to NAD + by reducing pyruvate to lactate. Neither the NADH nor pyruvate  produced is thus used for further energy generation. However, when oxygen is  available, and cytosolic NADH can be oxidized via a shuttle system, pyruvate can  also enter the mitochondria and be completely oxidized to C0 2 via PDH and the  TCA cycle. The oxidation of pyruvate via this route generates roughly 12.5 moles  of ATP per mole of pyruvate. If the cytosolic NADH is oxidized by the glycerol 3-P  shuttle, approximately 1.5 moles of ATP are produced per NADH. If, instead, the  NADH is oxidized by the malate-aspartate shuttle, approximately 2.5 moles are  produced. Thus, the two NADH molecules produced during glycolysis can lead to  3 to 5 molecules of ATP being produced, depending on which shuttle system is used  to transfer the reducing equivalents. Because each pyruvate produced can give rise  to 12.5 molecules of ATP, altogether 30 to 32 molecules of ATP can be produced  from one mole of glucose oxidized to carbon dioxide.   In response to the hypoxemia caused by Lopa Fusor's COPD, she has increased hypoxia-inducible factor-1 (HIF-1) in her  tissues. HIF-1 is a gene transcription factor found in tissues throughout the body (including brain, heart, kidney, lung, liver,  pancreas, skeletal muscle, and white blood cells) that plays a homeostatic role in coordinating tissue responses to hypoxia.  Each tissue will respond with a subset of the following changes. HIF-1 increases transcription of the genes for many of the glycolytic  enzymes, including PFK-1, enolase, phosphoglycerate kinase, and lactate dehydrogenase. HIF-1 also increases synthesis of a  number of proteins that enhance oxygen delivery to tissues, including erythropoietin, which increases the generation of red blood  cells in bone marrow; vascular endothelial growth factor, which regulates angiogenesis (formation of blood vessels); and inducible  nitric oxide synthase, which synthesizes nitric oxide, a vasodilator. As a consequence, Mrs. Fusor was able to maintain hematocrit  and hemoglobin levels that were on the high side of the normal range, and her tissues had an increased capacity for anaerobic  glycolysis.    To produce the same amount of ATP per unit time from anaerobic glycolysis as  from the complete aerobic oxidation of glucose to C0 2 , anaerobic glycolysis must  occur approximately 15 times faster, and use approximately 15 times more glucose.  Cells achieve this high rate of glycolysis by expressing high levels of glycolytic  enzymes. In certain skeletal muscles and in most cells during hypoxic crises, high  rates of glycolysis are associated with rapid degradation of internal glycogen stores  to supply the required glucose-6-P.   Anaerobic glycolysis results in acid production in the form of H + . Glycolysis  forms pyruvic acid, which is reduced to lactic acid. At an intracellular pH of  7.35, lactic acid dissociates to form the carboxylate anion, lactate, and H + (the  pKa for lactic acid is 3.85). Lactate and the H + are both transported out of the  cell into interstitial fluid by a transporter on the plasma membrane and eventu-  ally diffuse into the blood. If the amount of lactate generated exceeds the buffer-  ing capacity of the blood, the pH drops below the normal range, resulting in lac-  ticacidosis (see Chapter 4).   Many tissues, including red and white blood cells, the kidney medulla, the tissues  of the eye, and skeletal muscles, rely on anaerobic glycolysis for at least a portion  of their ATP requirements (Table 22.1). Tissues (or cells) that are heavily depend-  ent on anaerobic glycolysis usually have a low ATP demand, high levels of gly-  colytic enzymes, and few capillaries, such that oxygen must diffuse over a greater  distance to reach target cells. The lack of mitochondria, or the increased rate of gly-  colysis, is often related to some aspect of cell function. For example, the mature red  blood cell has no mitochondria because oxidative metabolism might interfere with  its function in transporting oxygen bound to hemoglobin. Some of the lactic acid  generated by anaerobic glycolysis in skin is secreted in sweat, where it acts as an  antibacterial agent. Many large tumors use anaerobic glycolysis for ATP produc-  tion, and lack capillaries in their core.   In tissues with some mitochondria, both aerobic and anaerobic glycolysis occur  simultaneously. The relative proportion of the two pathways depends on the mito-  chondrial oxidative capacity of the tissue and its oxygen supply and may vary  between cell types within the same tissue because of cell distance from the capil-  laries. When a cell's energy demand exceeds the capacity of the rate of the electron  transport chain and oxidative phosphorylation to produce ATP, glycolysis is acti-  vated, and the increased NADH/NAD + ratio will direct excess pyruvate into lactate.  Because under these conditions pyruvate dehydrogenase, the TCA cycle, and the  electron transport chain are operating as fast as they can, anaerobic glycolysis is  meeting the need for additional ATP.    The dental caries in Ivan Apple-  bod's mouth were caused princi-  pally by the low pH generated from  lactic acid production by oral bacteria. Below  a pH of 5.5, decalcification of tooth enamel  and dentine occurs. Lactobacilli and S.  mutans are major contributors to this  process because almost all of their energy is  derived from the conversion of glucose or  fructose to lactic acid, and they are able to  grow well at the low pH generated by this  process. Mr. Applebod's dentist explained  that bacteria in his dental plaque could con-  vert all the sugar in his candy into acid in  less than 20 minutes. The acid is buffered by  bicarbonate and other buffers in saliva, but  saliva production decreases in the evening.  Thus, the acid could dissolve the hydroxyap-  atite in his tooth enamel during the night.   Table 22.1. Major Tissue Sites of  Lactate Production in a Resting Man.  An average 70-kg man consumes  about 300 g of carbohydrate per day.   ®ln the complete oxidation of pyru-  vate to carbon dioxide, four steps  generate NADH (pyruvate dehydro-  genase, isocitrate dehydrogenase, a-ketoglu-  tarate dehydrogenase, and malate dehydro-  genase). One step generates FAD(2H)  (succinate dehydrogenase), and one substrate  level phosphorylation (succinate thiokinase).  Thus, because each NADH generates 2.5  ATPs, the overall contribution by NADH is 10  ATP molecules. The FAD(2H) generates an  additional 1.5 ATP, and the substrate-level  phosphorylation provides one more. There-  fore, 10 + 1.5 + 1 = 12.5 molecules of ATP.    The tissues of the eye are also par-  tially dependent on anaerobic gly-  colysis.   The eye contains cells that transmit or focus  light, and these cells cannot, therefore, be  filled with opaque structures such as mito-  chondria, or densely packed capillary beds.  The corneal epithelium generates most of its  ATP aerobically from its few mitochondria  but still metabolizes some glucose anaerobi-  cally. Oxygen is supplied by diffusion from  the air. The lens of the eye is composed of  fibers that must remain birefringent to trans-  mit and focus light, so mitochondria are  nearly absent. The small amount of ATP  required (principally for ion balance) can  readily be generated from anaerobic glycol-  ysis even though the energy yield is low. The  lens is able to pick up glucose and release  lactate into the vitreous body and aqueous  humor. It does not need oxygen and has no  use for capillaries.    Lactate dehydrogenase (LDH) is a  tetramer composed of A subunits  (also called M for skeletal muscle   form) and B subunits (also called H for  heart). Different tissues produce different  amounts of the two subunits, which then  combine randomly to form five different  tetramers (M 4 , MsH-,, M 2 H 2 , M-,!-^, and H 4 ).  These isoenzymes differ only slightly in their  properties, with the kinetic properties of the  M 4 form facilitating conversion of pyruvate  to lactate in skeletal muscle and the H 4 form  facilitating conversion of lactate to pyruvate  in the heart.   Lactate released from cells undergoing anaerobic glycolysis is taken up by other tis-  sues (primarily the liver, heart, and skeletal muscle) and oxidized back to pyruvate.  In the liver, the pyruvate is used to synthesize glucose (gluconeogenesis), which is  returned to the blood. The cycling of lactate and glucose between peripheral tissues  and liver is called the Cori cycle (Fig. 22.10).   In many other tissues, lactate is oxidized to pyruvate, which is then oxidized to C0 2  in the TCA cycle. Although the equilibrium of the lactate dehydrogenase reaction  favors lactate production, flux occurs in the opposite direction if NADH is being rap-  idly oxidized in the electron transport chain (or being used for gluconeogenesis):   Lactate + NAD + -> Pyruvate + NADH + H +   The heart, with its huge mitochondrial content and oxidative capacity, is able to  use lactate released from other tissues as a fuel. During an exercise such as bicycle  riding, lactate released into the blood from skeletal muscles in the leg might be used  by resting skeletal muscles in the arm. In the brain, glial cells and astrocytes pro-  duce lactate, which is used by neurons or released into the blood.   Glycolysis, in addition to providing ATP, generates precursors for biosynthetic path-  ways (Fig. 22.11). Intermediates of the pathway can be converted to ribose 5-  phosphate, the sugar incorporated into nucleotides such as ATP. Other sugars, such  as UDP-glucose, mannose, and sialic acid, are also formed from intermediates of  glycolysis. Serine is synthesized from 3 -phosphogly cerate, and alanine from pyru-  vate. The backbone of triacylglycerols, glycerol 3 -phosphate, is derived from dihy-  droxyacetone phosphate in the glycolytic pathway.   The liver is the major site of biosynthetic reactions in the body. In addition to  those pathways mentioned previously, the liver synthesizes fatty acids from the  pyruvate generated by glycolysis. It also synthesizes glucose from lactate, glycerol  3 -phosphate, and amino acids in the gluconeogenic pathway, which is principally a  reversal of glycolysis. Consequently, in liver, many of the glycolytic enzymes exist  as isoenzymes with properties suited for these functions.   The bisphosphoglycerate shunt is a "side reaction" of the glycolytic pathway in  which 1,3-bis-phosphoglycerate is converted to 2,3-bis-phosphoglycerate (2,3-  BPG). Red blood cells form 2,3-BPG to serve as an allosteric inhibitor of oxygen  binding to heme (see Chapter 44). 2,3-BPG reenters the glycolytic pathway via  dephosphorylation to 3 -phosphogly cerate. 2,3-BPG also functions as a coenzyme in  the conversion of 3 -phosphogly cerate to 2-phosphoglycerate by the glycolytic    Fig. 22.10. Cori cycle. Glucose, produced in the liver by gluconeogenesis, is converted by  glycolysis in muscle, red blood cells, and many other cells, to lactate. Lactate returns to the  liver and is reconverted to glucose by gluconeogenesis.   Fig. 22.11. Biosynthetic functions of glycolysis. Compounds formed from intermediates of  glycolysis are shown in blue. These pathways are discussed in subsequent chapters of the  book. Dotted lines indicate that more than one step is required for the conversion shown in  the figure.   enzyme phosphoglyceromutase. Because 2,3 -BPG is not depleted by its role in this  catalytic process, most cells need only very small amounts.   One of the major functions of glycolysis is the generation of ATP, and, therefore,  the pathway is regulated to maintain ATP homeostasis in all cells. Phosphofruc-  tokinase-1 (PFK-1) and pyruvate dehydrogenase (PDH), which links glycolysis  and the TCA cycle, are both major regulatory sites that respond to feedback indi-  cators of the rate of ATP utilization (Fig. 22.12). The supply of glucose-6-P for  glycolysis is tissue dependent and can be regulated at the steps of glucose trans-  port into cells, glycogenolysis (the degradation of glycogen to form glucose), or  the rate of glucose phosphorylation by hexokinase isoenzymes. Other regulatory  mechanisms integrate the ATP-generating role of glycolysis with its anabolic  roles.   All of the regulatory enzymes of glycolysis exist as tissue-specific isoenzymes,  which alter the regulation of the pathway to match variations in conditions and  needs in different tissues. For example, in the liver, an isoenzyme of pyruvate kinase  introduces an additional regulatory site in glycolysis that contributes to the inhibi-  tion of glycolysis when the reverse pathway, gluconeogenesis, is activated.   The AMP levels within the cytosol provide a better indicator of the rate of ATP uti-  lization than the ATP concentration itself (Fig. 22.13). The concentration of AMP   Fig. 22.13. Changes in ATP, ADP, and AMP  concentrations in skeletal muscle during exer-  cise. The concentration of ATP decreases by  only approximately 20% during exercise, and  the concentration of ADP rises. The concentra-  tion of AMP, produced by the adenylate kinase  reaction, increases manyfold and serves as a  sensitive indicator of decreasing ATP levels.    Fig. 22.12. Major sites of regulation in the glycolytic pathway. Hexokinase and phos-  phofructokinase-1 are the major regulatory enzymes in skeletal muscle. The activity of  pyruvate dehydrogenase in the mitochondrion determines whether pyruvate is converted  to lactate or to acetyl CoA. The regulation shown for pyruvate kinase only occurs for the  liver (L) isoenzyme.   in the cytosol is determined by the equilibrium position of the adenylate kinase  reaction.   The equilibrium is such that hydrolysis of ATP to ADP in energy-requiring reac-  tions increases both the ADP and AMP contents of the cytosol. However, ATP is  present in much higher quantities than AMP or ADP, so that a small decrease of  ATP concentration in the cytosol causes a much larger percentage increase in the  small AMP pool. In skeletal muscles, for instance, ATP levels are approximately 5  mM and decrease by no more than 20% during strenuous exercise (see Fig. 22.13).  At the same time, ADP levels may increase by 50%, and AMP levels, which are in   the micromolar range, increase by 300%. AMP activates a number of metabolic  pathways, including glycolysis, glycogenolysis, and fatty acid oxidation (particu-  larly in muscle tissues), to ensure that ATP homeostasis is maintained.   Hexokinases exist as tissue-specific isoenzymes whose regulatory properties reflect  the role of glycolysis in different tissues. In most tissues, hexokinase is a low-K m  enzyme with a high affinity for glucose (see Chapter 9). It is inhibited by physio-  logic concentrations of its product, glucose-6-P (see Fig. 22.12). If glucose-6-P  does not enter glycolysis or another pathway, it accumulates and decreases the  activity of hexokinase. In the liver, the isoenzyme glucokinase is a high-K m enzyme  that is not readily inhibited by glucose-6-P. Thus, glycolysis can continue in liver  even when energy levels are high so that anabolic pathways, such as the synthesis  of the major energy storage compounds, glycogen and fatty acids, can occur.   Phosphofructokinase-1 (PFK-1) is the rate-limiting enzyme of glycolysis and controls  the rate of glucose-6-P entry into glycolysis in most tissues. PFK-1 is an allosteric  enzyme that has a total of six binding sites: two are for substrates (Mg-ATP and fruc-  tose-6-P) and four are allosteric regulatory sites (see Fig. 22.12). The allosteric regula-  tory sites occupy a physically different domain on the enzyme than the catalytic site.  When an allosteric effector binds, it changes the conformation at the active site and  may activate or inhibit the enzyme (see also Chapter 9). The allosteric sites for PFK-1  include an inhibitory site for MgATP, an inhibitory site for citrate and other anions, an  allosteric activation site for AMP, and an allosteric activation site for fructose 2,6-bis-  phosphate (fructose-2,6-bisP) and other bisphosphates. Several different tissue-specific  isoforms of PFK-1 are affected in different ways by the concentration of these sub-  strates and allosteric effectors, but all contain these four allosteric sites.   ATP binds to two different sites on the enzyme, the substrate binding site and an  allosteric inhibitory site. Under physiologic conditions in the cell, the ATP concen-  tration is usually high enough to saturate the substrate binding site and inhibit the  enzyme by binding to the ATP allosteric site. This effect of ATP is opposed by AMP,  which binds to a separate allosteric activator site (Figure 22.14). For most of the  PFK-1 isoenzymes, the binding of AMP increases the affinity of the enzyme for  fructose 6-P (e.g., shifts the kinetic curve to the left). Thus, increases in AMP con-  centration can greatly increase the rate of the enzyme (see Fig. 22.14), particularly  when fructose-6-P concentrations are low.   Fig. 22.14. Regulation of PFK-1 by AMP,  ATP and fructose-2,6-bisP. A. AMP and fruc-  tose 2,6-bisphosphate activate PFK-1. B. ATP  increases the rate of the reaction at low con-  centrations, but allosterically inhibits the  enzyme at high concentrations.   Fructose-2,6-bisP is also an allosteric activator of PFK-1 that opposes the ATP inhi-  bition. Its effect on the rate of activity of PFK- 1 is qualitatively similar to that of  AMP, but it has a separate binding site. Fructose-2,6-bisP is NOT an intermediate   Otto Shape has started high-intensity exercise that will increase the production of lactate in his exercising skeletal muscles. In  skeletal muscles, the amount of aerobic versus anaerobic glycolysis that occurs varies with intensity of the exercise, with dura-  tion of the exercise, with the type of skeletal muscle fiber involved, and with the level of training. Human skeletal muscles are  usually combinations of type I fibers (called fast glycolytic fibers, or white muscle fibers) and type Mb fibers (called slow oxidative fibers,  or red muscle fibers). The designation of fast or slow refers to their rate of shortening, which is determined by the isoenzyme of myosin  ATPase present. Compared with glycolytic fibers, oxidative fibers have a higher content of mitochondria and myoglobin, which gives them  a red color. The gastrocnemius, a muscle in the leg used for running, has a high content of type Mb fibers. However, these fibers will still  produce lactate during sprints when the ATP demand exceeds their oxidative capacity.   PFK-1 exists as a group of tissue-  specific isoenzymes whose regula-  tory features match the role of gly-  colysis in different tissues. Three different  types of PFK-1 isoenzyme subunits exist: M  (muscle), L (liver), and C. The three subunits  show variable expression in different tis-  sues, with some tissues having more than  one type. For example, mature human mus-  cle expresses only the M subunit, the liver  expresses principally the L subunit, and ery-  throcytes express both the M and the L sub-  units. The C subunit is present in highest lev-  els in platelets, placenta, kidney, and  fibroblasts but is relatively common to most  tissues. Both the M and L subunits are sensi-  tive to AMP and ATP regulation, but the C  subunits are much less so. Active PFK-1 is a  tetramer, composed of four subunits. Within  muscle, the M4 form predominates but  within tissues that express multiple isoen-  zymes of PFK-1 heterotetramers can form  that have full activity.   Under ischemic conditions, AMP  levels within the heart rapidly  increase because of the lack of ATP  production via oxidative phosphorylation.  The increase in AMP levels activates an  AMP-dependent protein kinase (protein  kinase B), which phosphorylates the heart  isoenzyme of PFK-2 to activate its kinase  activity. This results in increased levels of  fructose-2,6-bisP, which activates PFK-1  along with AMP such that the rate of glycol-  ysis can increase to compensate for the lack  of ATP production via aerobic means.   During Cora Nari's myocardial  infarction (see Chapter 20), her  heart had a limited supply of oxy-  gen and blood-borne fuels. The absence of  oxygen for oxidative phosphorylation would  decrease the levels of ATP and increase  those of AMP, an activator of PFK-1 and the  AMP-dependent protein kinase, resulting in  a compensatory increase of anaerobic gly-  colysis and lactate production. However,  obstruction of a vessel leading to her heart  would decrease lactate removal, resulting in  a decrease of intracellular pH. Under these  conditions, at very low pH levels, glycolysis  is inhibited and unable to compensate for  the lack of oxidative phosphorylation.    of glycolysis but is synthesized by an enzyme that phosphorylates fructose 6-phos-  phate at the 2 position. The enzyme is therefore named phosphofructokinase-2  (PFK-2); it is a bifunctional enzyme with two separate domains, a kinase domain  and a phosphatase domain. At the kinase domain, fructose-6-P is phosphorylated to  fructose-2,6-bisP and at the phosphatase domain, fructose-2,6-bisP is hydrolyzed  back to fructose-6-P. PFK-2 is regulated through changes in the ratio of activity of  the two domains. For example, in skeletal muscles, high concentrations of fructose-  6-P activate the kinase and inhibit the phosphatase, thereby increasing the concen-  tration of fructose-2,6-bisP and activating glycolysis.   PFK-2 also can be regulated through phosphorylation by serine/threonine pro-  tein kinases. The liver isoenzyme contains a phosphorylation site near the amino  terminal that decreases the activity of the kinase and increases the phosphatase  activity. This site is phosphorylated by the cAMP-dependent protein kinase (protein  kinase A) and is responsible for decreased levels of liver fructose-2,6-bisP during  fasting conditions (as modulated by circulating glucagon levels, which is discussed  in detail in Chapters 26 and 31). The cardiac isoenzyme contains a phosphorylation  site near the carboxy terminal that can be phosphorylated in response to adrenergic  activators of contraction (such as norepinephrine) and by increased AMP levels.  Phosphorylation at this site increases the kinase activity and increases fructose-2,  6-bisP levels, thereby contributing to the activation of glycolysis.   The function of the citrate-anion allosteric site is to integrate glycolysis with other  pathways. For example, the inhibition of PFK-1 by citrate may play a role in  decreasing glycolytic flux in the heart during the oxidation of fatty acids.   Pyruvate kinase exists as tissue-specific isoenzymes. The form present in brain and  muscle contains no allosteric sites, and pyruvate kinase does not contribute to the  regulation of glycolysis in these tissues. However, the liver isoenzyme can be inhib-  ited through phosphorylation by the cAMP-dependent protein kinase, and by a  number of allosteric effectors that contribute to the inhibition of glycolysis during  fasting conditions. These allosteric effectors include activation by fructose- 1,6-bisP,  which ties the rate of pyruvate kinase to that of PFK- 1 , and inhibition by ATP, which  signifies high energy levels.   Pyruvate dehydrogenase is also regulated principally by the rate of ATP utilization  (see Chapter 20) through rapid phosphorylation to an inactive form. Thus, in a nor-  mal respiring cell, with an adequate supply of 0 2 , glycolysis and the TCA cycle are  activated together, and glucose can be completely oxidized to C0 2 . However, when  tissues do not have an adequate supply of 0 2 to meet their ATP demands, the  increased NADH/NAD + ratio inhibits pyruvate dehydrogenase, but AMP activates  glycolysis. A proportion of the pyruvate will then be reduced to lactate to allow gly-  colysis to continue.   Lactate production is a normal part of metabolism. In the absence of disease, ele-  vated lactate levels in the blood are associated with anaerobic glycolysis during  exercise. In lactic acidosis, lactic acid accumulates in blood to levels that signifi-  cantly affect the pH (lactate levels greater than 5 mM and a decrease of blood pH  below 7.2).   Lactic acidosis generally results from a greatly increased NADH/NAD + ratio in  tissues (Fig. 22. 15). The increased NADH concentration prevents pyruvate oxida-  tion in the TCA cycle and directs pyruvate to lactate. To compensate for the  decreased ATP production from oxidative metabolism, PFK-1, and, therefore, the  entire glycolytic pathway is activated. For example, consumption of high amounts  of alcohol, which is rapidly oxidized in the liver and increases NADH levels, can  result in a lactic acidosis. Hypoxia in any tissue increases lactate production as cells  attempt to compensate for a lack of 0 2 for oxidative phosphorylation.   A number of other problems that interfere either with the electron transport chain  or pyruvate oxidation in the TCA cycle result in lactic acidemia (see Fig. 22. 15). For  example, OXPHOS diseases (inherited deficiencies in subunits of complexes in the  electron transport chain, such as MERFF) increase the NADH/NAD + ratio and   Lopa Fusor had a decreased arterial p0 2 and elevated arterial pC0 2 caused by  underperfusion of her lungs. The elevated C0 2 content resulted in an increase  of H 2 C0 3 and acidity of the blood (see Chapter 4). The decreased 0 2 delivery to  tissues resulted in increased lactate production from anaerobic glycolysis, and an eleva-  tion of serum lactate to 10 times normal levels. The reduction in her arterial pH to 7.18  (reference range, 7.35-7.45) resulted, therefore, from both a mild respiratory acidosis  (elevated pC0 2 ) and a more profound metabolic acidosis (elevated serum lactate level).    Lactate and pyruvate are in equilib-  rium in the cell, and the ratio of lac-  tate to pyruvate reflects the  NADH/NAD + ratio. Both acids are released  into blood, and the normal ratio of lactate to  pyruvate in blood is approximately 25:1.  This ratio can provide a useful clinical diag-  nostic tool. Because lactic acidemia can be  the result of a number of problems, such as  hypoxia, MERFF, thiamine deficiency, and  pyruvate dehdyrogenase deficiency, under  which of these conditions would you expect  the lactate/pyruvate ratio in blood to be  much greater than normal?    Anoxia, ischemia, cyanide, CO  poisoning and other interruptions of  the ET chain prevent electron flow  and ATP synthesis, so glycolysis  operates anaerobically to produce  ATP, and lactate is formed   Genetic defects in proteins encoded  by mtDNA (some subunits of Complexes  I, III, IV and F 0 F-|-ATPase) decrease  electron transport and ATP synthesis,  so glycolysis operates anaerobically  to produce ATP, and lactate is formed   y^-^v Hypoxia and inherited deficiencies  a\ # ) of subunits in the electron trans-  port chain impair NADH oxidation,  resulting in a higher NADH/NAD + ratio in  the cell, and, therefore, a higher lactate/pyru-  vate ratio in blood. In contrast, conditions  that cause lactic acidemia as a result of  defects in the enzymes of pyruvate metabo-  lism (thiamine deficiency or pyruvate dehy-  drogenase deficiency) would increase both  pyruvate and lactate in the blood and have  little effect on the ratio.    Fig. 22.16. General structure of dextran. Glu-  cosyl residues are linked by a- 1,3, a- 1,6, and  some a- 1,4 bonds.   inhibit PDH (see Chapter 21). Impaired PDH activity from an inherited deficiency  of Ej (the decarboxylase subunit of the complex), or from severe thiamine defi-  ciency, increases blood lactate levels (see Chapter 20). Pyruvate carboxylase defi-  ciency also can result in lactic acidosis (see Chapter 20), because of an accumula-  tion of pyruvate.   Lactic acidosis can also result from inhibition of lactate utilization in gluconeoge-  nesis (e.g., hereditary fructose intolerance, which is due to a defective aldolase gene).  If other pathways that use glucose-6-P are blocked, glucose-6-P can be shunted into  glycolysis and lactate production (e.g., glucose 6-phosphatase deficiency).   Lopa Fusor was admitted to he hospital with severe hypotension caused  by an acute hemorrhage. Her plasma lactic acid level was elevated and her  arterial pH was low. The underlying mechanism for Ms. Fusor 's derange-  ment in acid-base balance is a severe reduction in the amount of oxygen delivered  to her tissues for cellular respiration (hypoxemia). Several concurrent processes  contributed to this oxygen lack. The first was her severely reduced blood pressure  caused by a brisk hemorrhage from a bleeding gastric ulcer. The blood loss led to  hypoperfusion and, therefore, reduced delivery of oxygen to her tissues. The  marked reduction in the number of red blood cells in her circulation caused by  blood loss further compromised oxygen delivery. The preexisting chronic obstruc-  tive pulmonary disease (COPD) added to her hypoxemia by decreasing her ventila-  tion, and, therefore, the transfer of oxygen to her blood (low p0 2 ). In addition, her  COPD led to retention of carbon dioxide (high pC0 2 ), which caused a respiratory  acidosis because the retained C0 2 interacted with water to form carbonic acid  (H 2 C0 3 ), which dissociates to H + and bicarbonate.   In skeletal muscles, lactate production occurs when the need for ATP  exceeds the capacity of the mitochondria for oxidative phosphorylation.  Thus, increased lactate production accompanies an increased rate of the  TCA cycle. The extent to which skeletal muscles use aerobic versus anaerobic gly-  colysis to supply ATP varies with the intensity of exercise. At low-intensity exer-  cise, the rate of ATP utilization is lower, and fibers can generate this ATP from  oxidative phosphorylation, with the complete oxidation of glucose to C0 2 . How-  ever, when Otto Shape sprints, a high-intensity exercise, the ATP demand exceeds  the rate at which the electron transport chain and TCA cycle can generate ATP  from oxidative phosphorylation. The increased AMP level signals the need for  additional ATP and stimulates PFK-1. The NADH/NAD + ratio directs the increase  in pyruvate production toward lactate. The fall in pH causes muscle fatigue and  pain. As he trains, the amount of mitochondria and myoglobin will increase in his  skeletal muscle fibers, and these fibers will rely less on anaerobic glycolysis.   Ivan Applebod had two sites of dental caries: one on a smooth surface  and one in a fissure. The decreased pH resulting from lactic acid produc-  tion by lactobacilli, which grow anaerobically within the fissure, is a major  cause of fissure caries. Streptococs mutans (S. mutans) plays a major role in smooth  surface caries because it secretes dextran, an insoluble polysaccharide, which forms  the base for plaque. S. mutans contains dextran- sucrase, a glucosyltransferase that  transfers glucosyl units from dietary sucrose (the glucose-fructose disaccharide in  sugar and sweets) to form the a(l— »6) and a(l— >3) linkages between the glucosyl  units in dextran (Fig. 22.16). Dextran- sucrase is specific for sucrose and does not  catalyze the polymerization of free glucose, or glucose from other disaccharides or  polysaccharides. Thus sucrose is responsible for the cariogenic potential of candy.  The sticky water-insoluble dextran mediates the attachment of S. mutans and other   bacteria to the tooth surface. This also keeps the acids produced from these bacte-  ria close to the enamel surface. Fructose from sucrose is converted to intermediates  of glycolysis and is rapidly metabolized to lactic acid. Other bacteria present in the  plaque produce different acids from anaerobic metabolism, such as acetic acid and  formic acid. The decrease in pH that results initiates demineralization of the  hydroxy apatite of the tooth enamel. Ivan Applebod's caries in his baby teeth could  have been caused by sucking on bottles containing fruit juice. The sugar in fruit  juice is also sucrose, and babies who fall asleep with a bottle of fruit juice in their  mouth may develop caries. Rapid decay of these baby teeth can harm the develop-  ment of their permanent teeth.   How is the first high-energy bond created in the glycolytic pathway?  This is the work of the glyceraldehyde 3 -phosphate dehydrogenase reac-  tion, which converts glyceraldehyde-3-P to 1,3 bisphosphgly cerate. This  reaction can be considered to be two separate half reactions, the first being the  oxidation of glyceraldehyde-3-P to 3-phosphoglycerate, and the second the addi-  tion of inorganic phosphate to 3-phosphoglycerate to produce 1,3 bisphospho-  glycerate. The AG 0 ' for the first reaction is approximately —12 kcal/mole; for  the second reaction, it is approximately +12 kcal/mole. Thus, although the first  half reaction is extremely favorable, the second half reaction is unfavorable and  would not proceed under cellular conditions. So how does the enzyme help this  reaction to proceed? This is accomplished through the enzyme forming a cova-  lent bond with the substrate, using an essential cysteine residue at the active site  to form a high-energy thioester linkage during the course of the reaction   Fig. 22.17. Mechanism of the glyceraldehyde 3-phosphate dehydrogenase reaction. 1. The enzyme forms a covalent linkage with the substrate,  using a cysteine group at the active site. The enzyme also contains bound NAD + close to the active site. 2. The substrate is oxidized, forming a  high-energy thioester linkage (in blue), and NADH. 3. NADH has a low affinity for the enzyme and is replaced by a new molecule of NAD + .  4. Inorganic phosphate attacks the thioester linkage, releasing the product 1,3 bisphosphoglycerate, and regenerating the active enzyme in a form  ready to initiate another reaction.   (Fig. 22.17). Thus, the energy that would be released as heat in the oxidation of  glyceraldehyde-3-P to 3-phosphoglycerate is conserved in the thioester linkage  that is formed (such that the AG 0 ' of the formation of the thioester intermediate  from glyceraldehyde-3-P is close to zero). Then, replacement of the sulfur with  inorganic phosphate to form the final product, 1,3 bisphosphoglycerate, is rela-  tively straightforward, as the AG 0 ' for that conversion is also close to zero, and  the acylphosphate bond retains the energy from the oxidation of the aldehyde.  This is one example of how covalent catalysis by an enzyme can result in the  conservation of energy between different bond types.   Cole AS, Eastoe JE. Biochemistry and Oral Biology. 2nd Ed. Boston: Butterworth, 1988:490-519.  Robinson BH. Lacticacidemia: Disorders of pyruvate carboxylase and pyruvate dehydrogenase. In:   Scriver CR, Beudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited   1. A major role of glycolysis is which of the following?   2. Starting with glyceraldehyde 3-phosphate and synthesizing one molecule of pyruvate, the net yield of ATP and NADH would  be which of the following?   3. When glycogen is degraded, glucose 1 -phosphate is formed. Glucose 1 -phosphate can then be isomerized to glucose 6-phos-  phate. Starting with glucose 1 -phosphate, and ending with 2 molecules of pyruvate, what is the net yield of glycolysis, in terms  of ATP and NADH formed?   4. Which of the following statements correctly describes an aspect of glycolysis?   (B) 2 ATP are used in the beginning of the pathway.   (D) One pyruvate and three C0 2 are formed from the oxidation of one glucose molecule.   (E) The reactions take place in the matrix of the mitochondria.   5. How many moles of ATP are generated by the complete aerobic oxidation of 1 mole of glucose to 6 moles of C0 2 ?   Fatty acids are a major fuel for humans and supply our energy needs between  meals and during periods of increased demand, such as exercise. During  overnight fasting, fatty acids become the major fuel for cardiac muscle, skeletal  muscle, and liver The liver converts fatty acids to ketone bodies (acetoacetate and  (3-hydroxybutyrate), which also serve as major fuels for tissues (e.g., the gut). The  brain, which does not have a significant capacity for fatty acid oxidation, can use  ketone bodies as a fuel during prolonged fasting.   The route of metabolism for a fatty acid depends somewhat on its chain length.  Fatty acids are generally classified as very-long-chain length fatty acids ( greater  than C20 ), long-chain fatty acids (C12-C20), medium-chain fatty acids  (C6-C12), and short-chain fatty acids (C4).   ATP is generated from oxidation of fatty acids in the pathway of  fi-oxidation. Between meals and during overnight fasting, long-chain fatty  acids are released from adipose tissue triacy I glycerols. They circulate through  blood bound to albumin (Fig. 23.1). In cells, they are converted to fatty acyl  Co A derivatives by acyl Co A synthetases. The activated acyl group is  transported into the mitochondrial matrix bound to carnitine, where fatty acyl  Co A is regenerated. In the pathway of oxidation, the fatty acyl group is  sequentially oxidized to yield FAD(2H), NADH, and acetyl Co A. Subsequent  oxidation of NADH and FAD(2H) in the electron transport chain, and oxidation  of acetyl Co A to C0 2 in the TCA cycle, generates ATP from oxidative  phosphorylation .   Many fatty acids have structures that require variations of this basic pattern.  Long- chain fatty acids that are unsaturated fatty acids generally require addi-  tional isomerization and oxidation-reduction reactions to rearrange their double  bonds during ^-oxidation. Metabolism of water-soluble medium- chain-length  fatty acids does not require carnitine and occurs only in liver. Odd- chain-length  fatty acids undergo fi-oxidation to the terminal three-carbon propionyl Co A,  which enters the TCA cycle as succinyl Co A.   Fatty acids that do not readily undergo mitochondrial /3 -oxidation are  oxidized first by alternate routes that convert them to more suitable substrates  or to urinary excretion products. Excess fatty acids may undergo microsomal  ^-oxidation, which converts them to dicarboxylic acids that appear in urine.  Very -long- chain fatty acids (both straight chain and branched fatty acids such  as phytanic acid) are whittled down to size in peroxisomes. Peroxisomal  a- and (3-oxidiation generates hydrogen peroxide (H 2 0 2 ), NADH, acetyl  CoA, or propionyl CoA and a short- to medium-chain-length acyl CoA. The  acyl CoA products are transferred to mitochondria to complete their  metabolism.   In the liver, much of the acetyl CoA generated from fatty acid oxidation is con-  verted to the ketone bodies, acetoacetate and fi-hydroxybutyrate, which enter the  blood (see Fig. 23.1 ). In other tissues, these ketone bodies are converted to acetyl   Fig. 23.1. Overview of mitochondrial long-chain fatty acid metabolism. (1) Fatty acid bind-  ing proteins (FaBP) transport fatty acids across the plasma membrane and bind them in the  cytosol. (2) Fatty acyl CoA synthetase activates fatty acids to fatty acyl CoAs. (3) Carnitine  transports the activated fatty acyl group into mitochondria. (4) p-oxidation generates NADH,  FAD(2H), and acetyl CoA (5) In the liver, acetyl CoA is converted to ketone bodies   CoA, which is oxidized in the TCA cycle. The liver synthesizes ketone bodies but  cannot use them as a fuel.   The rate of fatty acid oxidation is linked to the rate of NADH, FAD(2H), and  acetyl CoA oxidation, and, thus, to the rate of oxidative phosphorylation and ATP  utilization. Additional regulation occurs through malonyl CoA, which inhibits for-  mation of the fatty acyl carnitine derivatives. Fatty acids and ketone bodies are  used as a fuel when their level increases in the blood, which is determined by  hormonal regulation of adipose tissue lipolysis.   Otto Shape was disappointed that he did not place in his 5 -km race and  has decided that short-distance running is probably not right for him. After  careful consideration, he decides to train for the marathon by running 12  miles three times per week. He is now 13 pounds over his ideal weight, and he plans  on losing this weight while studying for his Pharmacology finals. He considers a  variety of dietary supplements to increase his endurance and selects one containing  carnitine, CoQ, pantothenate, riboflavin, and creatine.   The liver transaminases measured  \% ) in the blood are aspartate amino-  *S transferase (AST), which was for-  merly called serum glutamate-oxaloacetate  transaminase (SGOT), and alanine amino-  transferase (ALT), which was formerly called  serum glutamate pyruvate transaminase  (SGPT). Elevation of liver enzymes reflects  damage of the liver plasma membrane.   • Lofata Burne is a 16-year-old girl. Since age 14 months she has experi-  flP ) enced recurrent episodes of profound fatigue associated with vomiting and   " increased perspiration, which required hospitalization. These episodes  occurred only if she fasted for more than 8 hours. Because her mother gave her food  late at night and woke her early in the morning for breakfast, Lofata 's physical and  mental development had progressed normally.   On the day of admission for this episode, Lofata had missed breakfast, and by  noon she was extremely fatigued, nauseated, sweaty, and limp. She was unable to  hold any food in her stomach and was rushed to the hospital, where an infusion of  glucose was started intravenously. Her symptoms responded dramatically to this  therapy.   Her initial serum glucose level was low at 38 mg/dL (reference range for fasting  serum glucose levels = 70-100). Her blood urea nitrogen (BUN) level was slightly  elevated at 26 mg/dL (reference range = 8-25) as a result of vomiting, which led  to a degree of dehydration. Her blood levels of liver transaminases were slightly ele-  vated, although her liver was not palpably enlarged. Despite elevated levels of free  fatty acids (4.3 mM) in the blood, blood ketone bodies were below normal.   • Di Abietes, a 27-year-old woman with type 1 diabetes mellitus, had been  flP ) admitted to the hospital in a ketoacidotic coma a year ago (see Chapter 4).   " She had been feeling drowsy and had been vomiting for 24 hours before  that admission. At the time of admission, she was clinically dehydrated, her blood  pressure was low, and her breathing was deep and rapid (Kussmaul breathing). Her  pulse was rapid, and her breath had the odor of acetone. Her arterial blood pH was  7.08 (reference range, 7.36-7.44), and her blood ketone body levels were 15 mM  (normal is approximately 0.2 mM for a person on a normal diet).   0 © During Otto's distance running (a  ( /~oA ) moderate-intensity exercise), dec-  ™ reases in insulin and increases in  insulin counterregulatory hormones, such as  epinephrine and norepinephrine, increase adi-  pose tissue lipolysis. Thus, his muscles are  being provided with a supply of fatty acids in  the blood that they can use as a fuel.   0 o Lofata Burne developed symptoms  ( /^OA ) during fasting, when adipose tis-  ™ sue lipolysis was elevated. Under  these circumstances, muscle tissue, liver,  and many other tissues are oxidizing fatty  acids as a fuel. After overnight fasting,  approximately 60 to 70% of our energy  supply is derived from the oxidation of fatty  acids.   The fatty acids oxidized as fuels are principally long-chain fatty acids released from  adipose tissue triacylglycerol stores between meals, during overnight fasting, and  during periods of increased fuel demand (e.g., during exercise). Adipose tissue tri-  acylglycerols are derived from two sources; dietary lipids and triacylglycerols  synthesized in the liver. The major fatty acids oxidized are the long-chain fatty  acids, palmitate, oleate, and stearate, because they are highest in dietary lipids and  are also synthesized in the human.   Between meals, a decreased insulin level and increased levels of insulin counter-  regulatory hormones (e.g., glucagon) activate lipolysis, and free fatty acids are  transported to tissues bound to serum albumin. Within tissues, energy is derived  from oxidation of fatty acids to acetyl CoA in the pathway of p-oxidation. Most of  the enzymes involved in fatty acid oxidation are present as 2-3 isoenzymes, which  have different but overlapping specificities for the chain length of the fatty acid.  Metabolism of unsaturated fatty acids, odd-chain-length fatty acids, and medium-  chain-length fatty acids requires variations of this basic pattern. The acetyl CoA  produced from fatty acid oxidation is principally oxidized in the TCA cycle or  converted to ketone bodies in the liver.   Fat constitutes approximately 38% of the calories in the average North American  diet. Of this, more than 95% of the calories are present as triacylglycerols (3 fatty  acids esterified to a glycerol backbone). During ingestion and absorption, dietary  triacylglycerols are broken down into their constituents and then reassembled for  transport to adipose tissue in chylomicrons (see Chapter 2). Thus, the fatty acid  composition of adipose triacylglycerols varies with the type of food consumed.   The most common dietary fatty acids are the saturated long-chain fatty acids  palmitate (CI 6) and stearate (CI 8), the monounsaturated fatty acid oleate (CI 8:1),  and the polyunsaturated essential fatty acid, linoleate (CI 8:2) (To review fatty acid  nomenclature, consult Chapter 5). Animal fat contains principally saturated and  monounsaturated long-chain fatty acids, whereas vegetable oils contain linoleate  and some longer-chain and polyunsaturated fatty acids. They also contain smaller  amounts of branched-chain and odd-chain-length fatty acids. Medium-chain-length  fatty acids are present principally in dairy fat (e.g., milk and butter), maternal milk,  and vegetable oils.   Adipose tissue triacylglycerols also contain fatty acids synthesized in the liver,  principally from excess calories ingested as glucose. The pathway of fatty acid syn-  thesis generates palmitate, which can be elongated to form stearate, and unsaturated  to form oleate. These fatty acids are assembled into triacylglycerols and transported  to adipose tissue as the lipoprotein VLDL (very-low-density lipoprotein).   Long-chain fatty acids are hydrophobic and water insoluble. In addition, they are  toxic to cells because they can disrupt the hydrophobic bonding between amino acid  side chains in proteins. Consequently, they are transported in the blood and in cells  bound to proteins.   During fasting and other conditions of metabolic need, long-chain fatty acids are  released from adipose tissue triacylglycerols by lipases. They travel in the blood  bound in the hydrophobic binding pocket of albumin, the major serum protein (see  Fig. 23.1).   Fatty acids enter cells both by a saturable transport process and by diffusion  through the lipid plasma membrane. A fatty acid binding protein in the plasma  membrane facilitates transport. An additional fatty acid binding protein binds the  fatty acid intracellularly and may facilitate its transport to the mitochondrion. The  free fatty acid concentration in cells is, therefore, extremely low.   Fatty acids must be activated to acyl CoA derivatives before they can participate in  p-oxidation and other metabolic pathways (Fig. 23.2). The process of activation  involves an acyl CoA synthetase (also called a thiokinase) that uses ATP energy to  form the fatty acyl CoA thioester bond. In this reaction, the /3 bond of ATP is  cleaved to form a fatty acyl AMP intermediate and pyrophosphate (PPi). Subse-  quent cleavage of PPi helps to drive the reaction.   The acyl CoA synthetase that activates long-chain fatty acids, 12 to 20 carbons  in length, is present in three locations in the cell: the endoplasmic reticulum, outer  mitochondrial membranes, and peroxisomal membranes (Table 23.1). This enzyme  has no activity toward C22 or longer fatty acids, and little activity below C12. In  contrast, the synthetase for activation of very-long-chain fatty acids is present in  peroxisomes, and the medium-chain-length fatty acid activating enzyme is present  only in the mitochondrial matrix of liver and kidney cells.   Fatty acyl CoA formation, like the phosphorylation of glucose, is a prerequisite to  metabolism of the fatty acid in the cell (Fig. 23.3). The multiple locations of the long-  chain acyl CoA synthetase reflects the location of different metabolic routes taken by  fatty acyl CoA derivatives in the cell (e.g., triacylglycerol and phospholipid synthesis   Fig. 23.3. Major metabolic routes for long-  chain fatty acyl Co As. Fatty acids are acti-  vated to acyl CoA compounds for degradation  in mitochondrial p-oxidation, or incorporation  into triacylglycerols or membrane lipids.  When p-oxidation is blocked through an  inherited enzyme deficiency, or metabolic reg-  ulation, excess fatty acids are diverted into tri-  acylglycerol synthesis.   O-P-O-P - O - P - O H Adenosine  I I *\   Fig. 23.2. Activation of a fatty acid by a fatty acyl CoA synthetase. The fatty acid is acti-  vated by reacting with ATP to form a high-energy fatty acyl AMP and pyrophosphate. The  AMP is then exchanged for CoA. Pyrophosphate is cleaved by a pyrophosphatase.   Table 23.1. Chain-Length Specificity of Fatty Acid Activation and Oxidation Enzymes   Enzyme present in membranes of ER, mitochondria, and peroxisomes to facilitate different  metabolic routes of acyl CoAs.   Exists as many variants, present only in mitochondrial matrix of kidney and liver. Also involved  in xenobiotic metabolism.   Although maximum activity is for fatty acids 12-16 carbons long, it also acts on many smaller  acyl CoA derivatives   High level in skeletal muscle and heart to facilitate use of acetate as a fuel   Members of same enzyme family, which also includes acyl CoA dehydrogenases for  carbon skeleton of branched-chain amino acids.   Complex of long-chain enoyl hydratase, acyl CoA dehydrogenase and a thiolase with  broad specificity. Most active with longer chains.   in the endoplasmic reticulum, oxidation and plasmalogen synthesis in the peroxisome,  and p-oxidation in mitochondria). In the liver and some other tissues, fatty acids that  are not being used for energy generation are re-incorporated (re-esterified) into  triacylglycerols.   Carnitine serves as the carrier that transports activated long chain fatty acyl groups  across the inner mitochondrial membrane (Fig. 23.4). Carnitine acyl transferases are  able to reversibly transfer an activated fatty acyl group from CoA to the hydroxyl  group of carnitine to form an acylcarnitine ester. The reaction is reversible, so that  the fatty acyl CoA derivative can be regenerated from the carnitine ester.   Carnitine :palmitoyltransf erase I (CPTI; also called carnitine acyltransf erase I,  CATI), the enzyme that transfers long-chain fatty acyl groups from CoA to carni-  tine, is located on the outer mitochondrial membrane (Fig. 23.5). Fatty acylcarnitine  crosses the inner mitochondrial membrane with the aid of a translocase. The fatty  acyl group is transferred back to CoA by a second enzyme, carnitine :palmitoyl-  transf erase II (CPTII or CATII). The carnitine released in this reaction returns to the  cytosolic side of the mitochondrial membrane by the same translocase that brings  fatty acylcarnitine to the matrix side. Long-chain fatty acyl CoA, now located  within the mitochondrial matrix, is a substrate for (3 -oxidation.   Carnitine is obtained from the diet or synthesized from the side chain of lysine  by a pathway that begins in skeletal muscle, and is completed in the liver. The  reactions use S-adenosylmethionine to donate methyl groups, and vitamin C  (ascorbic acid) is also required for these reactions. Skeletal muscles have a    A number of inherited diseases in  the metabolism of carnitine or acyl-  carnitines have been described.  These include defects in the following  enzymes or systems: the transporter for car-  nitine uptake into muscle; CPT I; carnitine-  acylcarnitine translocase; and CPTII. Classi-  cal CPTII deficiency, the most common of  these diseases, is characterized by adoles-  cent to adult onset of recurrent episodes of  acute myoglobinuria precipitated by pro-  longed exercise or fasting. During these  episodes, the patient is weak, and may be  somewhat hypoglycemic with diminished  ketosis (hypoketosis), but metabolic decom-  pensation is not severe. Lipid deposits are  found in skeletal muscles. CPK levels, and  long-chain acylcarnitines are elevated in the  blood. CPTII levels in fibroblasts are approx-  imately 25% of normal. The remaining CPTII  activity probably accounts for the mild effect  on liver metabolism. In contrast, when CPTII  deficiency has presented in infants, CPT II  levels are below 10% of normal, the hypo-  glycemia and hypoketosis are severe,  hepatomegaly occurs from the triacylglyc-  erol deposits, and cardiomyopathy is also  present.   Fig. 23.5. Transport of long-chain fatty acids into mitochondria. The fatty acyl CoA crosses  the outer mitochondrial membrane. Carnitine palmitoyl transferase I in the outer mitochon-  drial membrane transfers the fatty acyl group to carnitine and releases CoASH. The fatty acyl  carnitine is translocated into the mitochondrial matrix as carnitine moves out. Carnitine  palmitoyl transferase II on the inner mitochondrial membrane transfers the fatty acyl group  back to CoASH, to form fatty acyl CoA in the matrix.   Fig. 23.4. Structure of fatty acylcarnitine.  Carnitine: palmitoyl transferases catalyze the  reversible transfer of a long-chain fatty acyl  group from the fatty acyl CoA to the hydroxyl  group of carnitine. The atoms in the dashed  box originate from the fatty acyl CoA.   Otto Shape's power supplement  contains carnitine. However, his  body can synthesize enough carni-  tine to meet his needs, and his diet contains  carnitine. Carnitine deficiency has been  found only in infants fed a soy-based for-  mula that was not supplemented with carni-  tine. His other supplements likewise proba-  bly provide no benefit, but are designed to  facilitate fatty acid oxidation during exercise.  Riboflavin is the vitamin precursor of FAD,  which is required for acyl CoA dehydroge-  nases and ETFs. CoQ is synthesized in the  body, but it is the recipient in the electron  transport chain for electrons passed from  complexes I and II and the ETFs. Some  reports suggest that supplementation with  pantothenate, the precursor of CoA,  improves performance.   Fig. 23.6. Overview of (3 -oxidation. Oxida-  tion at the p-carbon is followed by cleavage of  the a — p bond, releasing acetyl CoA and a  fatty acyl CoA that is two carbons shorter than  the original. The carbons cleaved to form  acetyl CoA are shown in blue. Successive spi-  rals of (3 -oxidation completely cleave an even-  chain fatty acyl CoA to acetyl CoA.   high-affinity uptake system for carnitine, and most of the carnitine in the body is  stored in skeletal muscle.   The oxidation of fatty acids to acetyl CoA in the p-oxidation spiral conserves  energy as FAD(2H) and NADH. FAD(2H) and NADH are oxidized in the electron  transport chain, generating ATP from oxidative phosphorylation. Acetyl CoA is oxi-  dized in the TCA cycle or converted to ketone bodies.   The fatty acid p-oxidation pathway sequentially cleaves the fatty acyl group into 2-  carbon acetyl CoA units, beginning with the carboxyl end attached to CoA  (Fig. 23.6). Before cleavage, the p-carbon is oxidized to a keto group in two reac-  tions that generate NADH and FAD(2H); thus, the pathway is called p-oxidation.  As each acetyl group is released, the cycle of p-oxidation and cleavage begins  again, but each time the fatty acyl group is 2 carbons shorter.   There are four types of reactions in the p-oxidation pathway (Fig. 23.7). In the  first step, a double bond is formed between the p- and a-carbons by an acyl CoA  dehydrogenase that transfers electrons to FAD. The double bond is in the trans   P a ii  CH 3\y\/\/ CH 2 - CH 2 - CH 2 - C~ SCoA   CH 3 \y\y\/ CH 2 - CH - CH 2 - C ~ SCoA   CH 3\y\y\/ CH 2 - c- ch 2 - c - SCoA   Fig. 23.7. Steps of P-oxidation . The four steps are repeated until an even-chain fatty acid is  completely converted to acetyl CoA. The FAD(2H) and NADH are reoxidized by the electron  transport chain, producing ATP.   configuration (a A 2 '-trans double bond). In the next step, an OH from water is  added to the p -carbon, and an H from water is added to the p -carbon. The enzyme  is called an enoyl hydratase (hydratases add the elements of water, and "ene" in a  name denotes a double bond). In the third step of p -oxidation, the hydroxyl group  on the p -carbon is oxidized to a ketone by a hydroxy acyl Co A dehydrogenase. In  this reaction, as in the conversion of most alcohols to ketones, the electrons are  transferred to NAD + to form NADH. In the last reaction of the sequence, the bond  between the p- and a-carbons is cleaved by a reaction that attaches Co ASH to the  p -carbon, and acetyl Co A is released. This is a thiolytic reaction (lysis refers to  breakage of the bond, and thio refers to the sulfur), catalyzed by enzymes called  p-ketothiolases. The release of two carbons from the carboxyl end of the original  fatty acyl CoA produces acetyl CoA and a fatty acyl CoA that is two carbons  shorter than the original.   The shortened fatty acyl CoA repeats these four steps until all of its carbons  are converted to acetyl CoA. P-Oxidation is, thus, a spiral rather than a cycle. In  the last spiral, cleavage of the four-carbon fatty acyl CoA (butyryl CoA) pro-  duces two acetyl CoA. Thus, an even chain fatty acid such as palmitoyl CoA,  which has 16 carbons, is cleaved seven times, producing 7 FAD(2H), 7 NADH,  and 8 acetyl CoA.   Like the FAD in all flavoproteins, FAD(2H) bound to the acyl CoA dehydrogenases  is oxidized back to FAD without dissociating from the protein (Fig. 23.8). Electron  transfer flavoproteins (ETF) in the mitochondrial matrix accept electrons from the  enzyme-bound FAD(2H) and transfer these electrons to ETF-QO (electron transfer  flavoprotein -CoQ oxidoreductase) in the inner mitochondrial membrane. ETF-QO,  also a flavoprotein, transfers the electrons to CoQ in the electron transport chain.  Oxidative phosphorylation thus generates approximately 1.5 ATP for each  FAD(2H) produced in the p-oxidation spiral.   The total energy yield from the oxidation of 1 mole of palmityl CoA to 8 moles  of acetyl CoA is therefore 28 moles of ATP: 1.5 for each of the 7 FAD(2H), and 2.5  for each of the 7 NADH. To calculate the energy yield from oxidation of 1 mole of  palmitate, two ATP need to be subtracted from the total because two high-energy  phosphate bonds are cleaved when palmitate is activated to palmityl CoA.   The four reactions of p-oxidation are catalyzed by sets of enzymes that are each  specific for fatty acids with different chain lengths (see Table 23.1). The acyl  dehydrogenases, which catalyze the first step of the pathway, are part of an  enzyme family that have four different ranges of specificity. The subsequent steps  of the spiral use enzymes specific for long- or short-chain enoyl Co As. Although  these enzymes are structurally distinct, their specificity overlaps to some extent.   The p-oxidation spiral uses the  \ % ) same reaction types seen in the  *S JCA cycle when succinate is con-  verted to oxaloacetate.   Fig. 23.8. Transfer of electrons from acyl CoA  dehydrogenase to the electron transport chain.  Abbreviations: ETF, electron-transferring  flavoprotein; ETF-QO, electron-transferring  flavoprotein-Coenzyme Q oxidoreductase.   What is the total ATP yield for the  oxidation of 1 mole of palmitic acid  to carbon dioxide and water?    After reviewing Lofata Burne's previous hospital records, a specialist suspected that Lofata's medical problems were caused by  a disorder in fatty acid metabolism. A battery of tests showed that Lofata's blood contained elevated levels of several partially  oxidized medium-chain fatty acids, such as octanoic acid (8:0) and 4-decenoic acid (10:1, A4). A urine specimen showed an  increase in organic acid metabolites of medium-chain fatty acids containing 6 to 10 carbons, including medium-chain acylcarnitine deriv-  atives. The profile of acylcarnitine species in the urine was characteristic of a genetically determined medium-chain acyl CoA dehydroge-  nase (MCAD) deficiency. In this disease, long-chain fatty acids are metabolized by p-oxidation to a medium-chain-length acyl CoA, such  as octanoyl CoA. Because further oxidation of this compound is blocked in MCAD deficiency, the medium chain acyl group is transferred  back to carnitine. These acylcarnitines are water soluble and appear in blood and urine. The specific enzyme deficiency was demonstrated  in cultured fibroblasts from Lofata's skin as well as in her circulating monocytic leukocytes.   In LCAD deficiency, fatty acylcarnitines accumulate in the blood. Those containing 14 carbons predominate. However, these do not  appear in the urine.   Palmitic acid is 16 carbons long,  with no double bonds, so it  requires 7 oxidation spirals to be  completely converted to acetyl-CoA. After 7  spirals, there are 7 FAD(2H), 7 NADH, and 8  acetyl-CoA. Each NADH yields 2.5 ATP, each  FAD(2H) yields 1.5 ATP, and each acetyl-CoA  yields 10 ATP as it is processed around the  TCA cycle. This then yields 17.5 + 10.5  + 80.5 = 108 ATP. However, activation of  palmitic acid to palmityl-CoA requires two  high-energy bonds, so the net yield is 108  - 2, or 106 moles of ATP.   Linoleate, although high in the  diet, cannot be synthesized in the  human and is an essential fatty  acid. It is required for formation of arachido-  nate, which is present in plasma lipids, and  is used for eicosanoid synthesis. Therefore,  only a portion of the linoleate pool is rapidly  oxidized.    As the fatty acyl chains are shortened by consecutive cleavage of two acetyl units,  they are transferred from enzymes that act on longer chains to those that act on  shorter chains. Medium- or short-chain fatty acyl Co As that may be formed from  dietary fatty acids, or transferred from peroxisomes, enter the spiral at the enzyme  most active for fatty acids of their chain length   Approximately one half of the fatty acids in the human diet are unsaturated, con-  taining cis double bonds, with oleate (C18:l, A 9 ) and linoleate (18:2,A 912 ) being the  most common. In P-oxidation of saturated fatty acids, a trans double bond is cre-  ated between the 2nd and 3rd (a and p) carbons. For unsaturated fatty acids to  undergo the p-oxidation spiral, their cis double bonds must be isomerized to trans  double bonds that will end up between the 2nd and 3rd carbons during (3 -oxidation,  or the double bond must be reduced. The process is illustrated for the polyunsatu-  rated fatty acid linoleate in Fig. 23.9. Linoleate undergoes p-oxidation until one  double bond is between carbons 3 and 4 near the carboxyl end of the fatty acyl  chain, and the other is between carbons 6 and 7. An isomerase moves the double  bond from the 3,4 position so that it is trans and in the 2,3 position, and p-oxida-  tion continues. When a conjugated pair of double bonds is formed (two double  bonds separated by one single bond) at positions 2 and 4, an NADPH-dependent  reductase reduces the pair to one trans double bond at position 3. Then isomeriza-  tion and p-oxidation resume.   In oleate (C18:l, A 9 ), there is only one double bond between carbons 9 and 10.  It is handled by an isomerization reaction similar to that shown for the double bond  at position 9 of linoleate.   The medium-chain-length acyl CoA  \ \ ) synthetase has a broad range of  specificity for compounds of  approximately the same size that contain a  carboxyl group, such as drugs (salicylate,  from aspirin metabolism, and valproate,  which is used to treat epileptic seizures), or  benzoate, a common component of plants.  Once the drug acyl CoA is formed, the acyl  group is conjugated with glycine to form a  urinary excretion product. With certain dis-  orders of fatty acid oxidation, medium- and  short-chain fatty acylglycines may appear in  the urine, together with acylcarnitines or  dicarboxylic acids.   Fatty acids containing an odd number of carbon atoms undergo p-oxidation, pro-  ducing acetyl CoA, until the last spiral, when five carbons remain in the fatty acyl  CoA. In this case, cleavage by thiolase produces acetyl CoA and a three-carbon  fatty acyl CoA, propionyl CoA (Fig. 23.10). Carboxylation of propionyl CoA yields  methylmalonyl CoA, which is ultimately converted to succinyl CoA in a vitamin  B12-dependent reaction (Fig. 23.1 1). Propionyl CoA also arises from the oxidation  of branched chain amino acids.   The propionyl CoA to succinyl CoA pathway is a major anaplerotic route for  the TCA cycle and is used in the degradation of valine, isoleucine, and a number  of other compounds. In the liver, this route provides precursors of oxaloacetate,  which is converted to glucose. Thus, this small proportion of the odd-carbon-  number fatty acid chain can be converted to glucose. In contrast, the acetyl CoA  formed from P-oxidation of even-chain-number fatty acids in the liver either  enters the TCA cycle, where it is principally oxidized to C0 2 , or is converted to  ketone bodies.   Dietary medium-chain-length fatty acids are more water soluble than long-chain  fatty acids and are not stored in adipose triacylglyce. After a meal, they enter the  blood and pass into the portal vein to the liver. In the liver, they enter the mito-  chondrial matrix by the monocarboxylate transporter and are activated to acyl CoA  derivatives in the mitochondrial matrix (see Fig. 23.1). Medium-chain-length acyl  CoAs, like long-chain acyl CoAs, are oxidized to acetyl CoA via the p-oxidation  spiral. Medium-chain acyl CoAs also can arise from the peroxisomal oxidation  pathway.   One spiral of  P oxidation  and the first step  of the second spiral   Fig. 23.9. Oxidation of linoleate. After three spirals of p-oxidation (dashed lines), there is  now a 3,4 cis double bond and a 6,7 cis double bond. The 3,4 cis double bond is isomerized  to a 2,3-trans double bond, which is in the proper configuration for the normal enzymes to  act. One spiral of p-oxidation occurs, plus the first step of a second spiral. A reductase that  uses NADPH now converts these two double bonds (between carbons 2 and 3 and carbons 4  and 5) to one double bond between carbons 3 and 4 in a trans configuration. The isomerase  (which can act on double bonds that are in either the cis or the trans configuration) moves  this double bond to the 2,3-trans position, and p-oxidation can resume.   Fatty acids are used as fuels principally when they are released from adipose tissue  triacylglycerols in response to hormones that signal fasting or increased demand.  Many tissues, such as muscle and kidney, oxidize fatty acids completely to C0 2 and  H 2 0. In these tissues, the acetyl CoA produced by p -oxidation enters the TCA  cycle. The FAD(2H) and the NADH from P-oxidation and the TCA cycle are   Fig. 23.10. Formation of propionyl CoA from  odd-chain fatty acids. Successive spirals of  p-oxidation cleave each of the bonds marked  with dashed lines, producing acetyl CoA  except for the three carbons at the oo-end,  which produce propionyl CoA.   H H  I I /,  H-C-C-C  A " N ,  / C \ H  * // \ * ,   Fig. 23.11. Conversion of propionyl CoA to  succinyl CoA. Succinyl CoA, an intermediate  of the TCA cycle, can form malate, which can  be converted to glucose in the liver through the  process of gluconeogenesis. Certain amino  acids also form glucose by this route (see  Chapter 39).    As Otto Shape runs, his skeletal muscles increase their use of ATP and their  rate of fuel oxidation. Fatty acid oxidation is accelerated by the increased rate  of the electron transport chain. As ATP is used and AMP increases, an AMP-  dependent protein kinase acts to facilitate fuel utilization and maintain ATP homeosta-  sis. Phosphorylation of acetyl CoA carboxylase results in a decreased level of malonyl  CoA and increased activity of carnitine: palmitoyl CoA transferase I. At the same time,  AMP-dependent protein kinase facilitates the recruitment of glucose transporters into  the plasma membrane of skeletal muscle, thereby increasing the rate of glucose uptake.  AMP and hormonal signals also increase the supply of glucose 6-P from glycogenoly-  sis. Thus, his muscles are supplied with more fuel, and all the oxidative pathways are  accelerated.   reoxidized by the electron transport chain, and ATP is generated. The process of p-  oxidation is regulated by the cells' requirements for energy (i.e., by the levels of  ATP and NADH), because fatty acids cannot be oxidized any faster than NADH and  FAD(2H) are reoxidized in the electron transport chain.   Fatty acid oxidation also may be restricted by the mitochondrial Co ASH pool  size. Acetyl Co ASH units must enter the TCA cycle or another metabolic pathway  to regenerate CoASH required for formation of the fatty acyl CoA derivative from  fatty acyl carnitine.   An additional type of regulation occurs at carnitine :palmitoyltransferase I  (CPTI). Carnitine :palmitoy transferase I is inhibited by malonyl CoA, which is syn-  thesized in the cytosol of many tissues by acetyl CoA carboxylase (Fig. 23.12).  Acetyl CoA carboxylase is regulated by a number of different mechanisms, some  of which are tissue dependent. In skeletal muscles and liver, it is inhibited when  phosphorylated by protein kinase B, an AMP-dependent protein kinase. Thus, dur-  ing exercise when AMP levels increase, AMP-dependent protein kinase phosphory-  lates acetyl CoA carboxylase, which becomes inactive. Consequently, malonyl CoA  levels decrease, carnitine :palmitoy transferase I is activated, and the p -oxidation of  fatty acids is able to restore ATP homeostasis and decrease AMP levels. In liver, in  addition to the regulation by the AMP-dependent protein kinase acetyl CoA car-  boxylase is activated by insulin-dependent mechanisms, which promotes the con-  version of malonyl CoA to palmitate in the fatty acid synthesis pathway. Thus, in  the liver, malonyl CoA inhibition of CPTI prevents newly synthesized fatty acids  from being oxidized.   p -oxidation is strictly an aerobic pathway, dependent on oxygen, a good blood  supply, and adequate levels of mitochondria. Tissues that lack mitochondria, such   Fig. 23.12. Regulation of p-oxidation. (1) Hormones control the supply of fatty acids in the  blood. (2) Carnitine ipalmitoyl transferase I is inhibited by malonyl CoA, which is synthe-  sized by acetyl CoA carboxylase (ACC). AMP-PK is the AMP-dependent protein kinase.  (3) The rate of ATP utilization controls the rate of the electron transport chain, which regu-  lates the oxidative enzymes of p-oxidation and the TCA cycle.   as red blood cells, cannot oxidize fatty acids by p-oxidation. Fatty acids also do not  serve as a significant fuel for the brain. They are not used by adipocytes, whose  function is to store triacylglycerols to provide a fuel for other tissues. Those tissues  that do not use fatty acids as a fuel, or use them only to a limited extent, are able to  use ketone bodies instead.   Fatty acids that are not readily oxidized by the enzymes of p -oxidation enter alter-  nate pathways of oxidation, including peroxisomal p- and a-oxidation and micro-  somal w-oxidation. The function of these pathways is to convert as much as possi-  ble of the unusual fatty acids to compounds that can be used as fuels or biosynthetic  precursors, and to convert the remainder to compounds that can be excreted in bile  or urine. During prolonged fasting, fatty acids released from adipose triacylglyc-  erols may enter the co-oxidation or peroxisomal p-oxidation pathway, even though  they have a normal composition. These pathways not only use fatty acids, but they  act on xenobiotic carboxylic acids that are large hydrophobic molecules resembling  fatty acids.   Xenobiotic: a term used to cover all  \\ ) organic compounds that are for-  *r eign to an organism. This can also  include naturally occurring compounds that  are administered by alternate routes or at  unusual concentrations. Drugs can be con-  sidered xenobiotics.   A small proportion of our diet consists of very-long-chain fatty acids (20 or more  carbons) or branched-chain fatty acids arising from degradative products of chloro-  phyll. Very-long-chain fatty acid synthesis also occurs within the body, especially  in cells of the brain and nervous system, which incorporate them into the sphin-  golipids of myelin. These fatty acids are oxidized by peroxisomal p- and a-oxida-  tion pathways, which are essentially chain- shortening pathways.   Very-long-chain fatty acids of 24 to 26 carbons are oxidized exclusively in peroxi-  somes by a sequence of reactions similar to mitochondrial p -oxidation in that they  generate acetyl Co A and NADH. However, the peroxisomal oxidation of straight-  chain fatty acids stops when the chain reaches 4 to 6 carbons in length. Some of the  long-chain fatty acids also may be oxidized by this route.   The long-chain fatty acyl CoA synthetase is present in the peroxisomal mem-  brane, and the acyl CoA derivatives enter the peroxisome by a transporter that does  not require carnitine. The first enzyme of peroxisomal (3-oxidation is an oxidase,  which donates electrons directly to molecular oxygen and produces hydrogen per-  oxide (H 2 0 2 ) (Fig.23.13). (In contrast, the first enzyme of mitochondrial p-oxida-  tion is a dehydrogenase that contains FAD and transfers the electrons to the electron  transport chain via ETF.) Thus, the first enzyme of peroxisomal oxidation is not  linked to energy production. The three remaining steps of p -oxidation are catalyzed  by enoyl-CoA hydratase, hydroxyacyl CoA dehydrogenase, and thiolase, enzymes  with activities similar to those found in mitochondrial (3 -oxidation, but coded for by  different genes. Thus, one NADH and one acetyl CoA are generated for each turn  of the spiral. The peroxisomal p -oxidation spiral continues generating acetyl CoA  until a medium-chain acyl CoA, which may be as short as butyryl CoA, is produced  (Fig. 23.14).   Within the peroxisome, the acetyl groups can be transferred from CoA to carni-  tine by an acetylcarnitine transferase, or they can enter the cytosol. A similar reac-  tion converts medium-chain-length acyl CoAs and the short-chain butyryl CoA to  acyl carnitine derivatives. These acylcarnitines diffuse from the peroxisome to the  mitochondria, pass through the outer mitochondrial membrane, and are transported  through the inner mitochondrial membrane via the carnitine translocase system.    Fig. 23.13. Oxidation of fatty acids in peroxi-  somes. The first step of p-oxidation is cat-  alyzed by an FAD-containing oxidase. The  electrons are transferred from FAD(2H) to 0 2 ,  which is reduced to hydrogen peroxide (H 2 0 2 ).   A number of inherited deficiencies  of peroxisomal enzymes have been  described. Zellweger's syndrome,  which results from defective peroxisomal  biogenesis, leads to complex developmental  and metabolic phenotypes affecting princi-  pally the liver and the brain. One of the  metabolic characteristics of these diseases is  an elevation of C26:0, and C26:1 fatty acid  levels in plasma. Refsum's disease is caused  by a deficiency in a single peroxisomal  enzyme, the phytanoyl CoA hydroxylase that  carries out a-oxidation of phytanic acid.  Symptoms include retinitis pigmentosa,  cerebellar ataxia, and chronic polyneuropa-  thy. Because phytanic acid is obtained solely  from the diet, placing patients on a low-  phytanic acid diet has resulted in marked  improvement.    Fig. 23.14. Chain- shortening by peroxisomal p-oxidation. Abbreviations: VLCFA, very-long-chain fatty acyl; VLACS, very-long-chain acyl-  CoA synthetase; MCFA, medium-chain fatty acyl; SCFA, short-chain fatty acyl; CAT, carnitine :acety transferase; COT, carnitine :octanoy trans-  ferase; CAC: carnitine :acylcarnitine carrier; CPT1, carnitine: palmitoyltransferase 1; CPT2, carnitine: palmityltransferase 2; OMM, outer mito-  chondrial membrane; IMM, inner mitochondrial membrane. Very-long-chain fatty acyl CoAs and some long-chain fatty acyl CoAs are oxidized  in peroxisomes through n cycles of p-oxidation to the stage of a short- to medium-chain fatty acyl CoA. These short to medium fatty acyl CoAs  are converted to carnitine derivatives by COT or CAT in the peroxisomes. In the mitochondria, SCFA-carnitine are converted back to acyl CoA  derivatives by either CPT2 or CAT.   Fig. 23.15. Oxidation of phytanic acid. A per-  oxisomal a-hydroxylase oxidizes the a-car-  bon, and its subsequent oxidation to a carboxyl  group releases the carboxyl carbon as C0 2 .  Subsequent spirals of peroxisomal (3 -oxidation  alternately release propionyl and acetyl CoA.  At a chain length of approximately 8 carbons,  the remaining branched fatty acid is trans-  ferred to mitochondria as a medium-chain  carnitine derivative.   They are converted back to acyl CoAs by carnitine: acy transferases appropriate for  their chain length and enter the normal pathways for (3 -oxidation and acetyl CoA  metabolism. The electrons from NADH and acetyl CoA can also pass from the per-  oxisome to the cytosol. The export of NADH-containing electrons occurs through  use of a shuttle system similar to those described for NADH electron transfer into  the mitochondria.   Peroxisomes are present in almost every cell type and contain many degradative  enzymes, in addition to fatty acyl CoA oxidase, that generate hydrogen peroxide.  H 2 0 2 can generate toxic free radicals. Thus, these enzymes are confined to peroxi-  somes, where the H 2 0 2 can be neutralized by the free radical defense enzyme, cata-  lase. Catalase converts H 2 0 2 to water and 0 2 .   Two of the most common branched-chain fatty acids in the diet are phytanic acid  and pristanic acid, which are degradation products of chlorophyll and thus are con-  sumed in green vegetables (Fig. 23. 15). Animals do not synthesize branched-chain  fatty acids. These two multi-methylated fatty acids are oxidized in peroxisomes to  the level of a branched C8 fatty acid, which is then transferred to mitochondria. The  pathway thus is similar to that for the oxidation of straight very-long-chain fatty  acids.   Phytanic acid, a multi-methylated C20 fatty acid, is first oxidized to pristanic  acid using the a-oxidation pathway (see Fig. 23. 15). Phytanic acid hydroxylase  introduces a hydroxyl group on the a-carbon, which is then oxidized to a carboxyl  group with release of the original carboxyl group as C0 2 . By shortening the fatty  acid by one carbon, the methyl groups will appear on the a-carbon rather than the   CHAPTER 23 / OXIDATION OF FATTY ACIDS AND KETONE BODIES 431   p -carbon during the p-oxidation spiral, and can no longer interfere with oxidation  of the p-carbon. Peroxisomal p-oxidation thus can proceed normally, releasing pro-  pionyl CoA and acetyl CoA with alternate turns of the spiral. When a medium chain  length of approximately eight carbons is reached, the fatty acid is transferred to the  mitochondrion as a carnitine derivative, and p -oxidation is resumed.   Fatty acids also may be oxidized at the oo-carbon of the chain (the terminal methyl  group) by enzymes in the endoplasmic reticulum (Fig. 23.16). The co-methyl group  is first oxidized to an alcohol by an enzyme that uses cytochrome P450, molecular  oxygen, and NADPH. Dehydrogenases convert the alcohol group to a carboxylic  acid. The dicarboxylic acids produced by co-oxidation can undergo P-oxidation,  forming compounds with 6 to 10 carbons that are water-soluble. Such compounds  may then enter blood, be oxidized as medium-chain fatty acids, or be excreted in  urine as medium-chain dicarboxylic acids.   The pathways of peroxisomal a and p-oxidation, and microsomal o> oxidation,  are not feedback regulated. These pathways function to decrease levels of water-  insoluble fatty acids or of xenobiotic compounds with a fatty acid-like structure that  would become toxic to cells at high concentrations. Thus, their rate is regulated by  the availability of substrate.   Overall, fatty acids released from adipose triacylglycerols serve as the major fuel  for the body during fasting. These fatty acids are completely oxidized to C0 2 and  H 2 0 by some tissues. In the liver, much of the acetyl CoA generated from P-oxida-  tion of fatty acids is used for synthesis of the ketone bodies acetoacetate and p-  hydroxybutyrate, which enter the blood (Fig. 23.17). In skeletal muscles and other   Fig. 23.16. co-Oxidation of fatty acids con-  verts them to dicarboxylic acids.   Normally, w-oxidation is a minor  process. However, in conditions  that interfere with p-oxidation  (such as carnitine deficiency or deficiency in  an enzyme of p-oxidation), w-oxidation pro-  duces dicarboxylic acids in increased  amounts. These dicarboxylic acids are  excreted in the urine.   Lofata Burne was excreting dicarboxylic  acids in her urine, particularly adipic acid  (which has 6 carbons) and suberic acid  (which has 8 carbons).  -OOC— CH2— CH2— CH2— CH2— COO-  Adipic acid    Fig. 23.17. The ketone bodies, acetoacetate and p-hydroxybutyrate, are synthesized in the  liver. Their principle fate is conversion back to acetyl CoA and oxidation in the TCA cycle  in other tissues.   tissues, these ketone bodies are converted back to acetyl Co A, which is oxidized in  the TCA cycle with generation of ATP. An alternate fate of acetoacetate in tissues is  the formation of cytosolic acetyl CoA.   In the liver, ketone bodies are synthesized in the mitochondrial matrix from acetyl  CoA generated from fatty acid oxidation (Fig. 23.18). The thiolase reaction of fatty  acid oxidation, which converts acetoacetyl CoA to two molecules of acetyl CoA, is  a reversible reaction, although formation of acetoacetyl-CoA is not the favored  direction. It can, thus, when acetyl-CoA levels are high, generate acetoacetyl CoA   Fig. 23.18. Synthesis of the ketone bodies acetoacetate, p -hydroxybutyrate, and acetone.  The portion of HMG-CoA shown in blue is released as acetyl CoA, and the remainder of the  molecule forms acetoacetate. Acetoacetate is reduced to p -hydroxybutyrate or decarboxy-  lated to acetone. Note that the dehydrogenase that interconverts acetoacetate and  p -hydroxybutyrate is specific for the D-isomer. Thus, it differs from the dehydrogenases of  p-oxidation, which act on 3-hydroxy acyl CoA derivatives and is specific for the L-isomer.   for ketone body synthesis. The acetoacetyl Co A will react with acetyl Co A to pro-  duce 3-hydroxy-3-methylglutaryl Co A (HMG-CoA). The enzyme that catalyzes  this reaction is HMG-CoA synthase. In the next reaction of the pathway, HMG-CoA  lyase catalyzes the cleavage of HMG-CoA to form acetyl Co A and acetoacetate.   Acetoacetate can directly enter the blood or it can be reduced by p -hydroxy bu-  tyrate dehydrogenase to p-hydroxybutyrate, which enters the blood (see Fig.  23.18). This dehydrogenase reaction is readily reversible and interconverts these  two ketone bodies, which exist in an equilibrium ratio determined by the  NADH/NAD + ratio of the mitochondrial matrix. Under normal conditions, the ratio  of p-hydroxybutyrate to acetoacetate in the blood is approximately 1:1.   An alternate fate of acetoacetate is spontaneous decarboxylation, a nonenzy-  matic reaction that cleaves acetoacetate into C02 and acetone (see Fig. 23.18).  Because acetone is volatile, it is expired by the lungs. A small amount of acetone  may be further metabolized in the body.   Acetoacetate and p -hydroxy butyrate can be oxidized as fuels in most tissues,  including skeletal muscle, brain, certain cells of the kidney, and cells of the intes-  tinal mucosa. Cells transport both acetoacetate and p -hydroxy butyrate from the cir-  culating blood into the cytosol, and into the mitochondrial matrix. Here p-hydrox-  ybutyrate is oxidized back to acetoacetate by p-hydroxybutyrate dehydrogenase.  This reaction produces NADH. Subsequent steps convert acetoacetate to acetyl  CoA (Fig. 23.19).   In mitochondria, acetoacetate is activated to acetoacetyl CoA by succinyl  CoA: acetoacetate CoA transferase. As the name suggests, CoA is transferred from  succinyl CoA, a TCA cycle intermediate, to acetoacetate. Although the liver pro-  duces ketone bodies, it does not use them, because this thiotransf erase enzyme is  not present in sufficient quantity.   Acetoacetyl CoA is cleaved to two molecules of acetyl CoA by acetoacetyl CoA  thiolase, the same enzyme involved in P -oxidation. The principal fate of this acetyl  CoA is oxidation in the TCA cycle.   The energy yield from oxidation of acetoacetate is equivalent to the yield for  oxidation of 2 acetyl CoA in the TCA cycle (20 ATP) minus the energy for activation  of acetoacetate (1 ATP). The energy of activation is calculated at one high-energy phos-  phate bond, because succinyl CoA is normally converted to succinate in the TCA cycle,  with generation of one molecule of GTP (the energy equivalent of ATP). However,  when the high-energy thioester bond of succinyl CoA is transferred to acetoacetate,  succinate is produced without the generation of this GTP. Oxidation of p-hydroxybu-  tyrate generates one additional NADH. Therefore the net energy yield from one mole-  cule of p-hydroxybutyrate is approximately 21.5 molecules of ATP.   Although fatty acid oxidation is usually the major source of ketone bodies, they also  can be generated from the catabolism of certain amino acids: leucine, isoleucine,  lysine, tryptophan, phenylalanine, and tyrosine. These amino acids are called keto-  genic amino acids because their carbon skeleton is catabolized to acetyl CoA or ace-  toacetyl CoA, which may enter the pathway of ketone body synthesis in liver.  Leucine and isoleucine also form acetyl CoA and acetoacetyl CoA in other tissues,  as well as the liver.   Acetoacetate can be activated to acetoacetyl CoA in the cytosol by an enzyme  similar to the acyl CoA synthetases. This acetoacetyl CoA can be used directly in  cholesterol synthesis. It also can be cleaved to two molecules of acetyl CoA by a  cytosolic thiolase. Cytosolic acetyl CoA is required for processes such as acetyl-  choline synthesis in neuronal cells.   Fig. 23.19. Oxidation of ketone bodies. (3-  Hydroxybutyrate is oxidized to acetoacetate,  which is activated by accepting a CoA group  from succinyl CoA. Acetoacetyl CoA is  cleaved to two acetyl CoA, which enter the  TCA cycle and are oxidized.   Ketogenic diets, which are high-fat  \ \ ) diets with a 3:1 ratio of lipid to car-  *r bohydrate, are being used to  reduce the frequency of epileptic seizures in  children. The reason for its effectiveness in  the treatment of epilepsy is not known.  Ketogenic diets are also used to treat chil-  dren with pyruvate dehydrogenase defi-  ciency. Ketone bodies can be used as a fuel  by the brain in the absence of pyruvate  dehydrogenase. They also can provide a  source of cytosolic acetyl CoA for acetyl-  choline synthesis. They often contain  medium-chain triglycerides, which induce  ketosis more effectively than long-chain  triglycerides.   Children are more prone to ketosis  \\ ) than adults because their body   enters the fasting state more rap-  idly. Their bodies use more energy per unit  mass (because their muscle-to-adipose-  tissue ratio is higher), and liver glycogen  stores are depleted faster (the ratio of their  brain mass to liver mass is higher). In chil-  dren, blood ketone body levels reach 2 mM  in 24 hours; in adults, it takes more than 3  days to reach this level. Mild pediatric infec-  tions causing anorexia and vomiting are the  commonest cause of ketosis in children.  Mild ketosis is observed in children after  prolonged exercise, perhaps attributable to  an abrupt decrease in muscular use of fatty  acids liberated during exercise. The liver  then oxidizes these fatty acids and produces  ketone bodies.   IV. THE ROLE OF FATTY ACIDS AND KETONE BODIES  IN FUEL HOMEOSTASIS   Fatty acids are used as fuels whenever fatty acid levels are elevated in the blood, that  is, during fasting, starvation, as a result of a high-fat, low-carbohydrate diet, or dur-  ing long-term low- to mild-intensity exercise. Under these conditions, a decrease in  insulin and increased levels of glucagon, epinephrine, or other hormones stimulate  adipose tissue lipolysis. Fatty acids begin to increase in the blood approximately 3  to 4 hours after a meal and progressively increase with time of fasting up to approx-  imately 2 to 3 days (Fig. 23.20). In the liver, the rate of ketone body synthesis  increases as the supply of fatty acids increases. However, the blood level of ketone  bodies continues to increase, presumably because their utilization by skeletal mus-  cles decreases.   After 2 to 3 days of starvation, ketone bodies rise to a level in the blood that  enables them to enter brain cells, where they are oxidized, thereby reducing the  amount of glucose required by the brain. During prolonged fasting, they may sup-  ply as much as two thirds of the energy requirements of the brain. The reduction in  glucose requirements spares skeletal muscle protein, which is a major source of  amino acid precursors for hepatic glucose synthesis from gluconeogenesis.   As fatty acids increase in the blood, they are used by skeletal muscles and cer-  tain other tissues in preference to glucose. Fatty acid oxidation generates NADH  and FAD(2H) through both ^-oxidation and the TCA cycle, resulting in rela-  tively high NADH/NAD + ratios, acetyl CoA concentration, and ATP/ADP or  ATP/AMP levels. In skeletal muscles, AMP-dependent protein kinase (see Sec-  tion I.E.) adjusts the concentration of malonyl CoA so that CPT1 and p-oxida-  tion operate at a rate that is able to sustain ATP homeostasis. With adequate lev-  els of ATP obtained from fatty acid (or ketone body) oxidation, the rate of  glycolysis is decreased. The activity of the regulatory enzymes in glycolysis and  the TCA cycle (pyruvate dehydrogenase and PFK-1) are decreased by the  changes in concentration of their allosteric regulators (ADP, an activator of PDH,   Fig. 23.20. Levels of ketone bodies in the blood at various times during fasting. Glucose lev-  els remain relatively constant, as do levels of fatty acids. Ketone body levels, however,  increase markedly, rising to levels at which they can be used by the brain and other nervous  tissue. From Cahill GF Jr, Aoki TT. Med Times 1970;98:109.   decreases in concentration; NADH, and acetyl CoA, inhibitors of PDH, are  increased in concentration under these conditions; and ATP and citrate,  inhibitors of PFK-1, are increased in concentration). As a consequence, glucose-  6-P accumulates. Glucose-6-P inhibits hexokinase, thereby decreasing the rate of  entry of glucose into glycolysis, and its uptake from the blood. In skeletal mus-  cles, this pattern of fuel metabolism is facilitated by the decrease in insulin con-  centration (see Chapter 36). Preferential utilization of fatty acids does not, how-  ever, restrict the ability of glycolysis to respond to an increase in AMP or ADP  levels, such as might occur during exercise or oxygen limitation.   Skeletal muscles, the heart, the liver, and many other tissues use fatty acids as their  major fuel during fasting and other conditions that increase fatty acids in the blood.  However, a number of other tissues (or cell types), such as the brain, use ketone  bodies to a greater extent. For example, cells of the intestinal muscosa, which trans-  port fatty acids from the intestine to the blood, use ketone bodies and amino acids  during starvation, rather than fatty acids. Adipocytes, which store fatty acids in tri-  acylglycerols, do not use fatty acids as a fuel during fasting but can use ketone bod-  ies. Ketone bodies cross the placenta, and can be used by the fetus. Almost all tis-  sues and cell types, with the exception of liver and red blood cells, are able to use  ketone bodies as fuels.    The level of total ketone bodies in  Lofata Burne's blood greatly  exceeds normal fasting levels and  the mild ketosis produced during exercise. In  a person on a normal mealtime schedule,  total blood ketone bodies rarely exceed 0.2  mM. During prolonged fasting, they may  rise to 4 to 5 mM. Levels above 7 mM are  considered evidence of ketoacidosis,  because the acid produced must reach this  level to exceed the bicarbonate buffer sys-  tem in the blood and compensatory respira-  tion (Kussmaul's respiration) (see Chapter 4).   A number of events, in addition to the increased supply of fatty acids from adipose  triacylglycerols, promote hepatic ketone body synthesis during fasting. The  decreased insulin/glucagon ratio results in inhibition of acetyl CoA carboxylase and  decreased malonyl CoA levels, which activates CPTI, thereby allowing fatty acyl  CoA to enter the pathway of p-oxidation. (Fig. 23.21). When oxidation of fatty acyl  CoA to acetyl CoA generates enough NADH and FAD(2H) to supply the ATP needs  of the liver, acetyl CoA is diverted from the TCA cycle into ketogenesis and  oxaloacetate in the TCA cycle is diverted toward malate and into glucose synthesis  (gluconeogenesis). This pattern is regulated by the NADH/NAD + ratio, which is  relatively high during (3-oxidation. As the length of time of fasting continues,  increased transcription of the gene for mitochondrial HMG-CoA synthase facili-  tates high rates of ketone body production. Although the liver has been described as  "altruistic" because it provides ketone bodies for other tissues, it is simply getting  rid of fuel that it does not need.   ^ As Otto Shape runs, he increases the rate at which his muscles oxidize all  fuels. The increased rate of ATP utilization stimulates the electron trans-  port chain, which oxidizes NADH and FAD(2H) much faster, thereby  increasing the rate at which fatty acids are oxidized. During exercise, he also uses  muscle glycogen stores, which contribute glucose to glycolysis. In some of the  fibers, the glucose is used anaerobically, thereby producing lactate. Some of the lac-  tate will be used by his heart, and some will be taken up by the liver to be converted  to glucose. As he trains, he increases his mitochondrial capacity, as well as his oxy-  gen delivery, resulting in an increased ability to oxidize fatty acids and ketone bod-  ies. As he runs, he increases fatty acid release from adipose tissue triacylglycerols.  In the liver, fatty acids are being converted to ketone bodies, providing his muscles  with another fuel. As a consequence, he experiences mild ketosis after his 12-mile  run.   Red blood cells lack mitochondria,  which is the site of ketone body uti-  lization.   Fig. 23.21. Regulation of ketone body synthesis. (1) The supply of fatty acids is increased.   (2) The malonyl CoA inhibition of CPTI is lifted by inactivation of acetyl CoA carboxylase.   (3) p-Oxidation supplies NADH and FAD(2H), which are used by the electron transport  chain for oxidative phosphorylation. As ATP levels increase, less NADH is oxidized, and the  NADH/NAD + ratio is increased. (4) Oxaloacetate is converted into malate because of the  high NADH levels, and the malate enters the cytoplasm for gluconeogenesis,. (5) Acetyl CoA  is diverted from the TCA cycle into ketogenesis, in part because of low oxaloacetate levels,  which reduces the rate of the citrate synthase reaction.   More than 25 enzymes and specific  \ \ ) transport proteins participate in  «^ mitochondrial fatty acid metabo-  lism. At least 15 of these have been impli-  cated in inherited diseases in the human.    Recently, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency,  the cause of Lofata Burne's problems, has emerged as one of the most  common of the inborn errors of metabolism, with a carrier frequency rang-  ing from 1 in 40 in northern European populations to less than 1 in 100 in Asians.  Overall, the predicted disease frequency for MCAD deficiency is 1 in 15,000 per-  sons.   MCAD deficiency is an autosomal recessive disorder caused by the substitution  of a T for an A at position 985 of the MCAD gene. This mutation causes a lysine to  replace a glutamate residue in the protein, resulting in the production of an unsta-  ble dehydrogenase.   The most frequent manifestation of MCAD deficiency is intermittent hypoke-  totic hypoglycemia during fasting (low levels of ketone bodies and low levels of  glucose in the blood). Fatty acids normally would be oxidized to C0 2 and H 2 0  under these conditions. In MCAD deficiency, however, fatty acids are oxidized  only until they reach medium-chain length As a result, the body must rely to a  greater extent on oxidation of blood glucose to meet its energy needs.   However, hepatic gluconeogenesis appears to be impaired in MCAD. Inhibi-  tion of gluconeogenesis may be caused by the lack of hepatic fatty acid oxida-  tion to supply the energy required for gluconeogenesis, or by the accumulation  of unoxidized fatty acid metabolites that inhibit gluconeogenic enzymes. As a  consequence, liver glycogen stores are depleted more rapidly, and hypoglycemia  results. The decrease in hepatic fatty acid oxidation results in less acetyl CoA for  ketone body synthesis, and consequently a hypoketotic hypoglycemia develops.   Some of the symptoms once ascribed to hypoglycemia are now believed to be  caused by the accumulation of toxic fatty acid intermediates, especially in those   patients with only mild reductions in blood glucose levels. Lofata Burne's mild  elevation in the blood of liver transaminases may reflect an infiltration of her liver  cells with unoxidized medium-chain fatty acids.   The management of MCAD-deficient patients includes the intake of a relatively  high-carbohydrate diet and the avoidance of prolonged fasting.   Di Abietes, a 26-year-old woman with type 1 diabetes mellitus, was  admitted to the hospital in diabetic ketoacidosis. In this complication of  diabetes mellitus, an acute deficiency of insulin, coupled with a relative  excess of glucagon, results in a rapid mobilization of fuel stores from muscle  (amino acids) and adipose tissue (fatty acids). Some of the amino acids are con-  verted to glucose, and fatty acids are converted to ketones (acetoacetate, p-hydrox-  ybutyrate, and acetone). The high glucagon: insulin ratio promotes the hepatic pro-  duction of ketones. In response to the metabolic "stress," the levels of  insulin-antagonistic hormones, such as catecholamines, glucocorticoids, and  growth hormone, are increased in the blood. The insulin deficiency further reduces  the peripheral utilization of glucose and ketones. As a result of this interrelated dys-  metabolism, plasma glucose levels reach 500 mg/dL (27.8 mmol/L) or more (nor-  mal fasting levels are 70-100 mg/dL, or 3.9-5.5 mmol/L), and plasma ketones rise  to levels of 8 to 15 mmol/L or more (normal is in the range of 0.2-2 mmol/L,  depending on the fed state of the individual).   The increased glucose presented to the renal glomeruli induces an osmotic diure-  sis, which further depletes intravascular volume, further reducing the renal excre-  tion of hydrogen ions and glucose. As a result, the metabolic acidosis worsens, and  the hyperosmolarity of the blood increases, at times exceeding 330 mOsm/kg (nor-  mal is in the range of 285-295 mOsm/kg). The severity of the hyperosmolar state  correlates closely with the degree of central nervous system dysfunction and may  end in coma and even death if left untreated.   The unripe fruit of the akee tree produces a toxin, hypoglycin, which  causes a condition known as Jamaican vomiting sickness. The victims of  the toxin are usually unwary children who eat this unripe fruit and develop  a severe hypoglycemia, which is often fatal.   Although hypoglycin causes hypoglycemia, it acts by inhibiting an acyl CoA  dehydrogenase involved in (3 -oxidation that has specificity for short- and medium-  chain fatty acids. Because more glucose must be oxidized to compensate for the  decreased ability of fatty acids to serve as fuel, blood glucose levels may fall to  extremely low levels. Fatty acid levels, however, rise because of decreased p-  oxidation. As a result of the increased fatty acid levels, a-oxidation increases, and  dicarboxylic acids are excreted in the urine. The diminished capacity to oxidize  fatty acids in liver mitochondria results in decreased levels of acetyl CoA, the sub-  strate for ketone body synthesis.   Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to dia-  betes. Diabetes Metab Rev 1999;15:412-426.   Roe CR, Ding J. Mitochondrial fatty acid oxidation disorders. In: Scriver CR, Beudet AL, Sly WS, Valle  D, eds. The Metabolic and Molecular Bases of Inherited Disease, vol 1, 8th Ed. New York: McGraw-  Hill, 2001: 2297-2326.   Wanders JA, Jakobs C, Skjeldal OH. Refsum disease. In: Scriver CR, Beudet AL, Sly WS, Valle D, eds.  The Metabolic and Molecular Bases of Inherited Disease, vol 1, 8th Ed. New York: McGraw-Hill,  2001: 3303-3321.   Ronald JA, Tein I. Metabolic myopathies. Seminars in Pediatric Neurology 1996;3:59-98.   1. A lack of the enzyme ETF:CoQ oxidoreductase leads to death. This is due to which of the following reasons?   (C) The energy yield from ketone body utilization is dramatically reduced.   (D) The energy yield from fatty acid utilization is dramatically reduced.   2. The ATP yield from the complete oxidation of 1 mole of a C 18:0 fatty acid to carbon dioxide and water would be closest to  which ONE of the following?   3. The oxidation of fatty acids is best described by which of the following sets of reactions?   4. An individual with a deficiency of an enzyme in the pathway for carnitine synthesis is not eating adequate amounts of carni-  tine in the diet. Which of the following effects would you expect during fasting as compared with an individual with an ade-  quate intake and synthesis of carnitine?   (D) The levels of dicarboxylic acids in the blood would be increased.   (E) The levels of very-long-chain fatty acids in the blood would be increased.   5. At which one of the periods listed below will fatty acids be the major source of fuel for the tissues of the body?   0 2 is both essential to human life and toxic. We are dependent on 0 2 for oxida-  tion reactions in the pathways of adenosine triphosphate (ATP) generation, detox-  ification, and biosynthesis. However, when 0 2 accepts single electrons, it is trans-  formed into highly reactive oxygen radicals that damage cellular lipids, proteins,  and DNA. Damage by reactive oxygen radicals contributes to cellular death and  degeneration in a wide range of diseases (Table 24.1 ).   Radicals are compounds that contain a single electron, usually in an outside  orbital. Oxygen is a biradical, a molecule that has two unpaired electrons in  separate orbitals (Fig. 24.1). Through a number of enzymatic and nonenzymatic  processes that routinely occur in cells, 0 2 accepts single electrons to form  reactive oxygen species (ROS). ROS are highly reactive oxygen radicals, or com-  pounds that are readily converted in cells to these reactive radicals. The ROS  formed by reduction of 0 2 are the radical superoxide (0 2 ~), the nonradical  hydrogen peroxide (H 2 0 2 ), and the hydroxy I radical (OH* ).   ROS may be generated nonenzymatic ally, or enzymatically as accidental  byproducts or major products of reactions. Superoxide may be generated nonenzy-  matic ally from CoQ, or from metal- containing enzymes (e.g., cytochrome P450,  xanthine oxidase, and NADPH oxidase). The highly toxic hydroxy I radical is  formed nonenzymatic ally from superoxide in the presence ofFe 3+ or Cu + by the  Fenton reaction, and from hydrogen peroxide in the Haber-Weiss reaction.   Oxygen radicals and their derivatives can be deadly to cells. The hydroxyl rad-  ical causes oxidative damage to proteins and DNA. It also forms lipid peroxides  and malondialdehyde from membrane lipids containing polyunsaturated fatty  acids. In some cases, free radical damage is the direct cause of a disease state  (e.g., tissue damage initiated by exposure to ionizing radiation). In neurodegener-  ative diseases, such as Parkinson s disease, or in ischemia-rep erf usion injury,  ROS may perpetuate the cellular damage caused by another process.   Oxygen radicals are joined in their destructive damage by the free radical  nitric oxide (NO) and the reactive oxygen species hypochlorous acid (HOC I). NO   Fig 24.1. 0 2 is a biradical. It has two anti-  bonding electrons with parallel spins, denoted  by the parallel arrows. It has a tendency to  form toxic reactive oxygen species (ROS),  such as superoxide (0 2 ~), the nonradical  hydrogen peroxide (H 2 0 2 ), and the hydroxyl  radical (OH*).   Ischemia/reperfusion injury following stroke  Oxphos diseases (Mitochondrial DNA disorders)  Multiple sclerosis  Parkinson's disease   Fig 24.2. Oxidative stress. Oxidative stress  occurs when the rate of ROS and RNOS pro-  duction overbalances the rate of their removal  by cellular defense mechanisms. These  defense mechanisms include a number of  enzymes and antioxidants. Antioxidants usu-  ally react nonenzymatically with ROS.   combines with 0 2 or superoxide to form reactive nitrogen oxygen species  (RNOS), such as the nonradical peroxy nitrite or the radical nitrogen dioxide,  RNOS are present in the environment (e.g., cigarette smoke) and generated in  cells. During phagocytosis of invading microorganisms, cells of the immune sys-  tem produce 0 2 ~ , HOC I, and NO through the actions ofNADPH oxidase,  myeloperoxidase, and inducible nitric oxide synthase, respectively. In addition to  killing phagocytosed invading microorganisms, these toxic metabolites may dam-  age surrounding tissue components.   Cells protect themselves against damage by ROS and other radicals through  repair processes, compartmentalization of free radical production, defense  enzymes, and endogenous and exogenous antioxidants (free radical scavengers).  The defense enzyme superoxide dismutase (SOD) removes the superoxide free  radical. Catalase and glutathione peroxidase remove hydrogen peroxide and lipid  peroxides. Vitamin E, vitamin C, and plant flavonoids act as antioxidants.  Oxidative stress occurs when the rate of ROS generation exceeds the capacity of  the cell for their removal (Fig. 24.2).   The basal ganglia are part of a neu-  \ \ ) ronal feedback loop that modulates  *r and integrates the flow of informa-  tion from the cerebral cortex to the motor  neurons of the spinal cord. The neostriatum  is the major input structure from the cerebral  cortex. The substantia nigra pars compacta  consists of neurons that provide integrative  input to the neostriatum through pigmented  neurons that use dopamine as a neurotrans-  mitter (the nigrastriatal pathway). Integrated  information feeds back to the basal ganglia  and to the cerebral cortex to control volun-  tary movement. In Parkinson's disease, a  decrease in the amount of dopamine reach-  ing the basal ganglia results in the move-  ment disorder.   0 © In ventricular fibrillation, rapid pre-  I > r \A } mature beats from an irritative  ™ focus in ventricular muscle occur in  runs of varying duration. Persistent fibrilla-  tion compromises cardiac output, leading to  death. This arrythmia can result from severe  ischemia (lack of blood flow) in the ventricu-  lar muscle of the heart caused by clots form-  ing at the site of a ruptured atherosclerotic  plaque. However, Cora Nari's rapid beats  began during the infusion of TPA as the clot  was lysed. Thus, they probably resulted from  reperfusing a previously ischemic area of her  heart with oxygenated blood. This phenome-  non is known as ischemia-reperfusion injury,  and it is caused by cytotoxic ROS derived  from oxygen in the blood that reperfuses  previously hypoxic cells. Ischemic-reperfu-  sion injury also may occur when tissue oxy-  genation is interrupted during surgery or  transplantation.   • Two years ago, Les Dopaman (less dopamine), a 62-year-old man, noted  Hfl ) an increasing tremor of his right hand when sitting quietly (resting tremor).   " The tremor disappeared if he actively used this hand to do purposeful  movement. As this symptom progressed, he also complained of stiffness in his mus-  cles that slowed his movements (bradykinesia). His wife noticed a change in his  gait; he had begun taking short, shuffling steps and leaned forward as he walked  (postural imbalance). He often appeared to be staring ahead with a rather immobile  facial expression. She noted a tremor of his eyelids when he was asleep and,  recently, a tremor of his legs when he was at rest. Because of these progressive  symptoms and some subtle personality changes (anxiety and emotional lability),  she convinced Les to see their family doctor.   The doctor suspected that her patient probably had primary or idiopathic parkin-  sonism (Parkinson's disease) and referred Mr. Dopaman to a neurologist. In Parkin-  son's disease, neurons of the substantia nigra pars compacta, containing the pigment  melanin and the neurotransmitter dopamine, degenerate.   Cora Nari had done well since the successful lysis of blood clots in her  coronary arteries with the use of intravenous recombinant tissue plasmino-  gen activator (TPA)(see Chapters 19 and 21). This therapy had quickly  relieved the crushing chest pain (angina) she experienced when she won the lottery.  At her first office visit after discharge from the hospital, Cora's cardiologist told her  she had developed multiple premature contractions of the ventricular muscle of her  heart as the clots were being lysed. This process could have led to a life-threatening  arrhythmia known as ventricular fibrillation. However, Cora's arrhythmia  responded quickly to pharmacologic suppression and did not recur during the  remainder of her hospitalization.   The generation of reactive oxygen species from 0 2 in our cells is a natural everyday  occurrence. They are formed as accidental products of nonenzymatic and enzymatic   reactions. Occasionally, they are deliberately synthesized in enzyme-catalyzed  reactions. Ultraviolet radiation and pollutants in the air can increase formation of  toxic oxygen-containing compounds.   A radical, by definition, is a molecule that has a single unpaired electron in an  orbital. A free radical is a radical capable of independent existence. (Radicals  formed in an enzyme active site during a reaction, for example, are not considered  free radicals unless they can dissociate from the protein to interact with other mol-  ecules.) Radicals are highly reactive and initiate chain reactions by extracting an  electron from a neighboring molecule to complete their own orbitals. Although the  transition metals (e.g., Fe, Cu, and Mo) have single electrons in orbitals, they are  not usually considered free radicals because they are relatively stable, do not  initiate chain reactions, and are bound to proteins in the cell.   The oxygen atom is a biradical, which means it has two single electrons in dif-  ferent orbitals. These electrons cannot both travel in the same orbital because they  have parallel spins (spin in the same direction). Although oxygen is very reactive  from a thermodynamic standpoint, its single electrons cannot react rapidly with the  paired electrons found in the covalent bonds of organic molecules. As a conse-  quence, 0 2 reacts slowly through the acceptance of single electrons in reactions  that require a catalyst (such as a metal-containing enzyme).   0 2 is capable of accepting a total of four electrons, which reduces it to water  (Fig. 24.3). When 0 2 accepts one electron, superoxide is formed. Superoxide is still  a radical because it has one unpaired electron remaining. This reaction is not ther-  modynamically favorable and requires a moderately strong reducing agent that can  donate single electrons (e.g., CoQH- in the electron transport chain). When super-  oxide accepts an electron, it is reduced to hydrogen peroxide, which is not a radi-  cal. The hydroxyl radical is formed in the next one-electron reduction step in the  reduction sequence. Finally, acceptance of the last electron reduces the hydroxyl  radical to H 2 0.   Reactive oxygen species (ROS) are oxygen-containing compounds that are highly  reactive free radicals, or compounds readily converted to these oxygen free radi-  cals in the cell. The major oxygen metabolites produced by one-electron reduction  of oxygen (superoxide, hydrogen peroxide, and the hydroxyl radical) are classified  as ROS (Table 24.2).   Reactive free radicals extract electrons (usually as hydrogen atoms) from other  compounds to complete their own orbitals, thereby initiating free radical chain  reactions. The hydroxyl radical is probably the most potent of the ROS. It initiates  chain reactions that form lipid peroxides and organic radicals and adds directly to  compounds. The superoxide anion is also highly reactive, but has limited lipid sol-  ubility and cannot diffuse far. However, it can generate the more reactive hydroxyl  and hydroperoxy radicals by reacting nonenzymatically with hydrogen peroxide in  the Haber- Weiss reaction (Fig 24.4).   Hydrogen peroxide, although not actually a radical, is a weak oxidizing agent  that is classified as an ROS because it can generate the hydroxyl radical (OH«).  Transition metals, such as Fe 2+ or Cu + , catalyze formation of the hydroxyl radical  from hydrogen peroxide in the nonenzymatic Fenton reaction (see Fig. 24.4.).   The two unpaired electrons in oxy-  gen have the same (parallel) spin  and are called antibonding elec-  trons. In contrast, carbon-carbon and  carbon-hydrogen bonds each contain two  electrons, which have antiparallel spins and  form a thermodynamically stable pair. As a  consequence, 0 2 cannot readily oxidize a  covalent bond because one of its electrons  would have to flip its spin around to make  new pairs. The difficulty in changing spins is  called the spin restriction. Without the  spin restriction, organic life forms could not  have developed in the oxygen atmosphere  on earth because they would be sponta-  neously oxidized by 0 2 . Instead, 0 2 is con-  fined to slower one-electron reactions cat-  alyzed by metals (or metalloenzymes).   Fig 24.3. Reduction of oxygen by four one-  electron steps. The four one-electron reduction  steps for 0 2 progressively generate superoxide,  hydrogen peroxide, and the hydroxyl radical  plus water. Superoxide is sometimes written  0 2 - to better illustrate its single unpaired elec-  tron. H 2 0 2 , the half-reduced form of 0 2 , has  accepted two electrons and is, therefore, not an  oxygen radical.    To decrease occurrence of the Fenton reaction, accessibility to transition metals, such as Fe 2+ and Cu + , are highly restricted in  cells, or in the body as a whole. Events that release iron from cellular storage sites, such as a crushing injury, are associated with  increased free radical injury.   Table 24.2. Reactive Oxygen Species (ROS) and Reactive Nitrogen-Oxygen Species (RNOS)   Produced by the electron transport chain and at other sites. Cannot diffuse far from the site of origin.  Generates other ROS.   Not a free radical, but can generate free radicals by reaction with a transition metal (e.g., Fe 2+ ). Can diffuse  into and through cell membranes.   The most reactive species in attacking biologic molecules. Produced from H 2 0 2 in the Fenton reaction in the  presence of Fe 2+ or Cu + .   Organic free radicals (R denotes remainder of the compound.) Produced from ROH, RH (e.g., at the carbon  of a double bond in a fatty acid) or RSH by OH*' attack.   An organic peroxyl radical, such as occurs during lipid degradation (also denoted LOO)   Produced in neutrophils during the respiratory burst to destroy invading organisms. Toxicity is through  halogenation and oxidation reactions. Attacking species is OCI"   Oxygen with antiparallel spins. Produced at high oxygen tensions from absorption of l/v light. Decays so fast  that it is probably not a significant in vivo source of toxicity.   RNOS. A free radical produced endogenously by nitric oxide synthase. Binds to metal ions. Combines with 0 2  or other oxygen-containing radicals to produce additional RNOS.   RNOS. A strong oxidizing agent that is not a free radical. It can generate N0 2 (nitrogen dioxide), which  is a radical.   Fig 24.4. Generation of the hydroxyl radical  by the nonenzymatic Haber-Weiss and Fenton  reactions. In the simplified versions of these  reactions shown here, the transfer of single  electrons generates the hydroxyl radical. ROS  are shown in blue. In addition to Fe 2+ , Cu + and  many other metals can also serve as single-  electron donors in the Fenton reaction.   Because hydrogen peroxide is lipid soluble, it can diffuse through membranes and  generate OH« at localized Fe 2+ - or Cu + -containing sites, such as the mitochondria.  Hydrogen peroxide is also the precursor of hypochlorous acid (HOCI), a powerful  oxidizing agent that is produced endogenously and enzymatically by phagocytic  cells.   Organic radicals are generated when superoxide or the hydroxyl radical indis-  criminately extract electrons from other molecules. Organic peroxy radicals are  intermediates of chain reactions, such as lipid peroxidation. Other organic radicals,  such as the ethoxy radical, are intermediates of enzymatic reactions that escape into  solution (see Table 24.2).   An additional group of oxygen-containing radicals, termed RNOS, contain nitro-  gen as well as oxygen. These are derived principally from the free radical nitric  oxide (NO), which is produced endogenously by the enzyme nitric oxide synthase.  Nitric oxide combines with 0 2 or superoxide to produce additional RNOS.   C. Major Sources of Primary Reactive Oxygen  Species in the Cell   ROS are constantly being formed in the cell; approximately 3 to 5% of the oxy-  gen we consume is converted to oxygen free radicals. Some are produced as acci-  dental by-products of normal enzymatic reactions that escape from the active site  of metal-containing enzymes during oxidation reactions. Others, such as hydro-  gen peroxide, are physiologic products of oxidases in peroxisomes. Deliberate  production of toxic free radicals occurs in the inflammatory response. Drugs,  natural radiation, air pollutants, and other chemicals also can increase formation  of free radicals in cells.   One of the major sites of superoxide generation is Coenzyme Q (CoQ) in the mito-  chondrial electron transport chain (Fig. 24.5). The one-electron reduced form of  CoQ (CoQH«) is free within the membrane and can accidentally transfer an electron  to dissolved 0 2 , thereby forming superoxide. In contrast, when 0 2 binds to  cytochrome oxidase and accepts electrons, none of the 0 2 radical intermediates are  released from the enzyme, and no ROS are generated.   With insufficient oxygen, Cora Nari's ischemic heart muscle mitochondria  were unable to maintain cellular ATP levels, resulting in high intracellular Na +  and Ca 2+ levels. The reduced state of the electron carriers in the absence of  oxygen, and loss of mitochondrial ion gradients or membrane integrity, leads to  increased superoxide production once oxygen becomes available during reperfusion.  The damage can be self-perpetuating, especially if iron bound to components of the elec-  tron transport chain becomes available for the Fenton reaction, or the mitochondrial per-  meability transition is activated.   Most of the oxidases, peroxidases, and oxygenases in the cell bind 0 2 and transfer  single electrons to it via a metal. Free radical intermediates of these reactions may  be accidentally released before the reduction is complete.   Cytochrome P450 enzymes are a major source of free radicals "leaked" from  reactions.   Because these enzymes catalyze reactions in which single electrons are trans-  ferred to 0 2 and an organic substrate, the possibility of accidentally generating  and releasing free radical intermediates is high (see Chapters 19 and 25). Induc-  tion of P450 enzymes by alcohol, drugs, or chemical toxicants leads to increased  cellular injury. When substrates for cytochrome P450 enzymes are not present,  its potential for destructive damage is diminished by repression of gene tran-  scription.   Hydrogen peroxide and lipid peroxides are generated enzymatically as major  reaction products by a number of oxidases present in peroxisomes, mitochondria,  and the endoplasmic reticulum. For example, monoamine oxidase, which oxidatively  degrades the neurotransmitter dopamine, generates H 2 0 2 at the mitochondrial mem-  brane of certain neurons. Peroxisomal fatty acid oxidase generates H 2 0 2 rather than  FAD(2H) during the oxidation of very-long-chain fatty acids (see Chapter 23). Xan-  thine oxidase, an enzyme of purine degradation that can reduce 0 2 to 0 2 ~or H 2 0 2  in the cytosol, is thought to be a major contributor to ischemia-reperfusion injury,  especially in intestinal mucosal and endothelial cells. Lipid peroxides are also  formed enzymatically as intermediates in the pathways for synthesis of many  eicosanoids, including leukotrienes and prostaglandins.   Cosmic rays that continuously bombard the earth, radioactive chemicals, and x-  rays are forms of ionizing radiation. Ionizing radiation has a high enough energy  level that it can split water into the hydroxyl and hydrogen radicals, thus leading  to radiation damage to the skin, mutations, cancer, and cell death (Fig. 24.6). It  also may generate organic radicals through direct collision with organic cellular  components.   Production of ROS by xanthine oxidase in endothelial cells may be enhanced  during ischemia-reperfusion in Cora Nari's heart. In undamaged tissues, xan-  thine oxidase exists as a dehydrogenase that uses NAD + rather than 0 2 as an  electron acceptor in the pathway for degradation of purines (hypoxanthine xanthine  <-» uric acid (see Chapter 41). When 0 2 levels decrease, phosphorylation of ADP to ATP  decreases, and degradation of ADP and adenine through xanthine oxidase increases. In  the process, xanthine dehydrogenase is converted to an oxidase. As long as 0 2 levels are  below the high K m of the enzyme for 0 2 , little damage is done. However, during reperfu-  sion when 0 2 levels return to normal, xanthine oxidase generates H 2 0 2 and 0 2 ~ at the  site of injury.    Fig 24.5. Generation of superoxide by CoQ in  the electron transport chain. In the process of  transporting electrons to 0 2 , some of the elec-  trons escape when CoQH* accidentally inter-  acts with 0 2 to form superoxide. Fe-H repre-  sents the Fe-heme center of the cytochromes.   Carbon tetrachloride (CCI 4 ), which is  used as a solvent in the dry-cleaning  industry, is converted by  cytochrome P450 to a highly reactive free rad-  ical that has caused hepatocellular necrosis in  workers. When the enzyme-bound CCI 4  accepts an electron, it dissociates into CCI 3 -  and CI-. The CCI 3 - radical, which cannot con-  tinue through the P450 reaction sequence,  "leaks" from the enzyme active site and initi-  ates chain reactions in the surrounding  polyunsaturated lipids of the endoplasmic  reticulum. These reactions spread into the  plasma membrane and to proteins, eventually  resulting in cell swelling, accumulation of  lipids, and cell death.   Les Dopaman, who is in the early  stages of Parkinson's disease, is  treated with a monoamine oxidase  B inhibitor. Monoamine oxidase is a copper-  containing enzyme that inactivates dopamine  in neurons, producing H 2 0 2 . The drug was  originally administered to inhibit dopamine  degradation. However, current theory sug-  gests that the effectiveness of the drug is also  related to decrease of free radical formation  within the cells of the basal ganglia. The  dopaminergic neurons involved are particu-  larly susceptible to the cytotoxic effects of  ROS and RNOS that may arise from H 2 0 2 .   0 © The appearance of lipofuscin gran-  l /~OA J ules in many tissues increases dur-  ™ ing aging. The pigment lipofuscin  (from the Greek "lipos" for lipids and the  Latin "fuscus" for dark) consists of a hetero-  geneous mixture of cross-linked polymer-  ized lipids and protein formed by reactions  between amino acid residues and lipid per-  oxidation products, such as malondialde-  hyde. These cross-linked products are prob-  ably derived from peroxidatively damaged  cell organelles that were autophagocytized  by lysosomes but could not be digested.  When these dark pigments appear on the  skin of the hands in aged individuals, they  are referred to as "liver spots," a traditional  hallmark of aging. In Les Dopaman and  other patients with Parkinson's disease, lipo-  fuscin appears as Lewy bodies in degenerat-  ing neurons.   Evidence of protein damage shows up in  many diseases, particularly those associated  with aging. In patients with cataracts, pro-  teins in the lens of the eye exhibit free radi-  cal damage and contain methionine sulfox-  ide residues and tryptophan degradation  products.   Oxygen radicals produce cellular dysfunction by reacting with lipids, proteins, car-  bohydrates, and DNA to extract electrons (summarized in Fig. 24.7). Evidence of  free radical damage has been described in over 100 disease states. In some of these  diseases, free radical damage is the primary cause of the disease; in others, it  enhances complications of the disease.   Chain reactions that form lipid free radicals and lipid peroxides in membranes make  a major contribution to ROS-induced injury (Fig. 24.8). An initiator (such as a  hydroxyl radical produced locally in the Fenton reaction) begins the chain reaction.  It extracts a hydrogen atom, preferably from the double bond of a polyunsaturated  fatty acid in a membrane lipid. The chain reaction is propagated when 0 2 adds to  form lipid peroxyl radicals and lipid peroxides. Eventually lipid degradation occurs,  forming such products as malondialdehyde (from fatty acids with three or more  double bonds), and ethane and pentane (from the co-terminal carbons of 3 and 6  fatty acids, respectively). Malondialdehyde appears in the blood and urine and is  used as an indicator of free radical damage.   Peroxidation of lipid molecules invariably changes or damages lipid molecular  structure. In addition to the self-destructive nature of membrane lipid peroxidation,  the aldehydes that are formed can cross-link proteins. When the damaged lipids are  the constituents of biologic membranes, the cohesive lipid bilayer arrangement and  stable structural organization is disrupted (see Fig. 24.7). Disruption of mitochon-  drial membrane integrity may result in further free radical production.   Fig 24.7. Free radical-mediated cellular injury. Superoxide and the hydroxyl radical initiate  lipid peroxidation in the cellular, mitochondrial, nuclear, and endoplasmic reticulum membranes.  The increase in cellular permeability results in an influx of Ca 2 + , which causes further mito-  chondrial damage. The cysteine sulfhydryl groups and other amino acid residues on proteins are  oxidized and degraded. Nuclear and mitochondrial DNA can be oxidized, resulting in strand  breaks and other types of damage. RNOS (NO, N0 2 , and peroxynitrite) have similar effects.   In proteins, the amino acids proline, histidine, arginine, cysteine, and methionine are  particularity susceptible to hydroxyl radical attack and oxidative damage. As a conse-  quence of oxidative damage, the protein may fragment or residues cross-link with other  residues. Free radical attack on protein cysteine residues can result in cross-linking and  formation of aggregates that prevents their degradation. However, oxidative damage  increases the susceptibility of other proteins to proteolytic digestion.   Free radical attack and oxidation of the cytsteine sulfhydryl residues of the  tripeptide glutathione (7-glutamyl-cysteinyl-glycine; see section V.A.3.) increases  oxidative damage throughout the cell. Glutathione is a major component of cellular  defense against free radical injury, and its oxidation reduces its protective effects.   Oxygen-derived free radicals are also a major source of DNA damage. Approximately  20 types of oxidatively altered DNA molecules have been identified. The nonspecific  binding of Fe 2+ to DNA facilitates localized production of the hydroxyl radical, which  can cause base alterations in the DNA (Fig. 24.9). It also can attack the deoxyribose  backbone and cause strand breaks. This DNA damage can be repaired to some extent  by the cell (see Chapter 12), or minimized by apoptosis of the cell.   Nitric oxide (NO) is an oxygen-containing free radical which, like 0 2 , is both essen-  tial to life and toxic. NO has a single electron, and therefore binds to other com-  pounds containing single electrons, such as Fe 3+ . As a gas, it diffuses through the  cytosol and lipid membranes and into cells. At low concentrations, it functions  physiologically as a neurotransmitter and a hormone that causes vasodilation. How-  ever, at high concentrations, it combines with 0 2 or with superoxide to form  additional reactive and toxic species containing both nitrogen and oxygen (RNOS).  RNOS are involved in neurodegenerative diseases, such as Parkinson's disease, and  in chronic inflammatory diseases, such as rheumatoid arthritis.   At low concentrations, nitric oxide serves as a neurotransmitter or a hormone. It is  synthesized from arginine by nitric oxide synthases (Fig 24.10). As a gas, it is able  to diffuse through water and lipid membranes, and into target cells. In the target  cell, it exerts its physiologic effects by high-affinity binding to Fe-heme in the  enzyme guanylyl cyclase, thereby activating a signal transduction cascade. How-  ever, NO is rapidly inactivated by nonspecific binding to many molecules, and  therefore cells that produce NO need to be close to the target cells.   The body has three different tissue-specific isoforms of NO synthase, each  encoded by a different gene: neuronal nitric oxide synthase (nNOS, isoform I),  inducible nitric oxide synthase (iNOS, isoform II), and endothelial nitric oxide  synthase (eNOS, isoform III). nNOS and eNOS are tightly regulated by Ca 2+  concentration to produce the small amounts of NO required for its role as a  neurotransmitter and hormone. In contrast, iNOS is present in many cells of the  immune system and cell types with a similar lineage, such as macrophages and   Nitroglycerin, in tablet form, is often given to patients with coronary artery dis-  ease who experience ischemia-induced chest pain (angina). The nitroglycerin  decomposes in the blood, forming NO, a potent vasodilator, which increases  blood flow to the heart and relieves the angina.   Fig 24.8. Lipid peroxidation: a free radical  chain reaction. A. Lipid peroxidation is initi-  ated by a hydroxyl or other radical that extracts  a hydrogen atom from a polyunsaturated lipid  (LH), thereby forming a lipid radical (L*).  B. The free radical chain reaction is propa-  gated by reaction with 0 2 , forming the lipid  peroxy radical (LOO») and lipid peroxide  (LOOH). C. Rearrangements of the single  electron result in degradation of the lipid. Mal-  ondialdehyde, one of the compounds formed,  is soluble and appears in blood. D. The chain  reaction can be terminated by vitamin E and  other lipid- soluble antioxidants that donate  single electrons. Two subsequent reduction  steps form a stable, oxidized antioxidant.   Fig 24.9. Conversion of guanine to 8-hydrox-  yguanine by the hydroxy radical. The amount  of 8-hydroxyguanosine present in cells can be  used to estimate the amount of oxidative dam-  age they have sustained. The addition of the  hydroxyl group to guanine allows it to mispair  with T residues, leading to the creation of a  daughter molecule with an A-T base pair in  this position.   brain astroglia. This isoenzyme of nitric oxide synthase is regulated principally  by induction of gene transcription, and not by changes in Ca 2+ concentration. It  produces high and toxic levels of NO to assist in killing invading microorgan-  isms. It is these very high levels of NO that are associated with generation of  RNOS and NO toxicity.   The toxic actions of NO can be divided into two categories: direct toxic effects  resulting from binding to Fe-containing proteins, and indirect effects mediated by  compounds formed when NO combines with 0 2 or with superoxide to form RNOS.   NO, as a radical, exerts direct toxic effects by combining with Fe-containing com-  pounds that also have single electrons. Major destructive sites of attack include Fe-  S centers (e.g., electron transport chain complexes I-III, aconitase) and Fe-heme  proteins (e.g., hemoglobin and electron transport chain cytochromes). However,  there is usually little damage because NO is present in low concentrations and Fe-  heme compounds are present in excess capacity. NO can cause serious damage,  however, through direct inhibition of respiration in cells that are already compro-  mised through oxidative phosphorylation diseases or ischemia.   When present in very high concentrations (e.g., during inflammation), NO com-  bines nonenzymatically with superoxide to form peroxynitrite (ONOCT ), or with  0 2 to form N 2 0 3 (Fig. 24.11). Peroxynitrite, although not a free radical, is a strong   Fig 24.10. Nitric oxide synthase synthesizes  the free radical NO. Like cytochrome P450  enzymes, NO synthase uses Fe-heme, FAD,  and FMN to transfer single electrons from  NADPH to 0 9 .   Fig 24.11. Formation of RNOS from nitric oxide. RNOS are shown in blue. The type of  damage caused by each RNOS is shown in parentheses. Of all the nitrogen-oxygen-contain-  ing compounds shown, only nitrate is relatively nontoxic.   oxidizing agent that is stable and directly toxic. It can diffuse through the cell and  lipid membranes to interact with a wide range of targets, including protein methio-  nine and -SH groups (e.g., Fe-S centers in the electron transport chain). It also  breaks down to form additional RNOS, including the free radical nitrogen dioxide  (N0 2 ), an effective initiator of lipid peroxidation. Peroxynitrite products also nitrate  aromatic rings, forming compounds such as nitrotyrosine or nitroguanosine. N 2 0 3 ,  which can be derived either from N0 2 or nitrite, is the agent of nitrosative stress,  and nitrosylates sulfhydryl and similarily reactive groups in the cell. Nitrosylation  will usually interefere with the proper functioning of the protein or lipid that has  been modified. Thus, RNOS can do as much oxidative and free radical damage as  non-nitrogen-containing ROS, as well as nitrating and nitrosylating compounds.  The result is widespread and includes inhibition of a large number of enzymes;  mitochondrial lipid peroxidation; inhibition of the electron transport chain and  energy depletion; single- stranded or double- stranded breaks in DNA; and modifi-  cation of bases in DNA.   N0 2 is one of the toxic agents pres-  ent in smog, automobile exhaust,  gas ranges, pilot lights, cigarette  smoke, and smoke from forest fires or burn-  ing buildings.   In response to infectious agents and other stimuli, phagocytic cells of the immune  system (neutrophils, eosinophils, and monocytes/macrophages) exhibit a rapid con-  sumption of 0 2 called the respiratory burst. The respiratory burst is a major source  of superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid (HOC1),  and RNOS. The generation of free radicals is part of the human antimicrobial  defense system and is intended to destroy invading microorganisms, tumor cells,  and other cells targeted for removal.   The respiratory burst results from the activity of NADPH oxidase, which  catalyzes the transfer of an electron from NADPH to 0 2 to form superoxide  (Fig. 24.12). NADPH oxidase is assembled from cytosol and membranous pro-  teins recruited into the phagolysosome membrane as it surrounds an invading  microorganism.   Superoxide is released into the intramembranous space of the phagolysosome,  where it is generally converted to hydrogen peroxide and other ROS that are effec-  tive against bacteria and fungal pathogens. Hydrogen peroxide is formed by super-  oxide dismutase, which may come from the phagocytic cell or the invading  microorganism.   The formation of hypochlorous acid from H 2 0 2 is catalyzed by myeloperoxidase, a  heme-containing enzyme that is present only in phagocytic cells of the immune  system (predominantly neutrophils).   Myeloperoxidase contains two Fe heme-like centers, which give it the green  color seen in pus. Hypochlorous acid is a powerful toxin that destroys bacteria  within seconds through halogenation and oxidation reactions. It oxidizes many Fe  and S-containing groups (e.g., sulfhydryl groups, iron-sulfur centers, ferredoxin,  heme-proteins, methionine), oxidatively decarboxylates and deaminates proteins,  and cleaves peptide bonds. Aerobic bacteria under attack rapidly lose membrane   genetic defects in NADPH oxidase.  NADPH oxidase has four different subunits  (two in the cell membrane and two recruited  from the cytosol), and the genetic defect  may be in any of the genes that encode  these subunits. The membrane catalytic sub-  unit p of NADPH oxidase is a 91-kDa flavocy-  tochrome glycoprotein. It transfers electrons  from bound NADPH to FAD, which transfers  them to the Fe-heme components. The  membranous a-subunit (p22) is required for  stabilization. Two additional cytosolic pro-  teins (p47phox and p67phox) are also  required for assembly of the complex. Rac, a  monomeric GTPase in the Ras subfamily of  the Rho superfamily (see Chapter 9), is also  required for assembly. The 91-kDa subunit is  affected most often in X-linked chronic gran-  ulatomous disease, whereas the a-subunit is  affected in a rare autosomal recessive form.  The cytosolic subunits are affected most  often in patients with the autosomal reces-  sive form of granulomatous disease. In addi-  tion to their enhanced susceptibility to bac-  terial and fungal infections, these patients  suffer from an apparent dysregulation of  normal inflammatory responses.   During Cora Nari's ischemia  (decreased blood flow), the ability  of her heart to generate ATP from  oxidative phosphorylation was compro-  mised. The damage appeared to accelerate  when oxygen was first reintroduced (reper-  fused) into the tissue. During ischemia, CoQ  and the other single-electron components of  the electron transport chain become satu-  rated with electrons. When oxygen is rein-  troduced (reperfusion), electron donation to  0 2 to form superoxide is increased. The  increase of superoxide results in enhanced  formation of hydrogen peroxide and the  hydroxyl radical. Macrophages in the area to  clean up cell debris from ischemic injury  produce nitric oxide, which may further  damage mitochondria by generating RNOS  that attack Fe-S centers and cytochromes in  the electron transport chain membrane  lipids. Thus, the RNOS may increase the  infarct size.   Fig 24.12. Production of reactive oxygen species during the phagocytic respiratory burst by  activated neutrophils. (1) Activation of NADPH oxidase on the outer side of the plasma mem-  brane initiates the respiratory burst with the generation of superoxide. During phagocytosis,  the plasma membrane invaginates, so superoxide is released into the vacuole space. (2)  Superoxide (either spontaneously or enzymatically via superoxide dismutase [SOD]) gener-  ates H 2 0 2 . (3) Granules containing myeloperoxidase are secreted into the phagosome, where  myeloperoxidase generates HOC1 and other halides. (4) H 2 0 2 can also generate the hydroxyl  radical from the Fenton reaction. (5) Inducible nitric oxide synthase may be activated and  generate NO. (6) Nitric oxide combines with superoxide to form peroxy nitrite, which may  generate additional RNOS. The result is an attack on the membranes and other components  of phagocytosed cells, and eventual lysis. The whole process is referred to as the respiratory  burst because it lasts only 30 to 60 minutes and consumes 0 2 .   transport, possibly because of damage to ATP synthase or electron transport chain  components (which reside in the plasma membrane of bacteria).   When human neutrophils of the immune system are activated to produce NO,  NADPH oxidase is also activated. NO reacts rapidly with superoxide to generate  peroxynitrite, which forms additional RNOS. NO also may be released into the  surrounding medium, to combine with superoxide in target cells.   In a number of disease states, free radical release by neutrophils or macrophages  during an inflammation contributes to injury in the surrounding tissues. During  stroke or myocardial infarction, phagocytic cells that move into the ischemic area  to remove dead cells may increase the area and extent of damage. The self-  perpetuating mechanism of radical release by neutrophils during inflammation and  immune complex formation may explain some of the features of chronic inflam-  mation in patients with rheumatoid arthritis. As a result of free radical release, the  immunoglobulin G (IgG) proteins present in the synovial fluid are partially oxi-  dized, which improves their binding with the rheumatoid factor antibody. This  binding, in turn, stimulates the neutrophils to release more free radicals.   Our defenses against oxygen toxicity fall into the categories of antioxidant defense  enzymes, dietary and endogenous antioxidants (free radical scavengers), cellular  compartmentation, metal sequestration, and repair of damaged cellular components.  The antioxidant defense enzymes react with ROS and cellular products of free rad-  ical chain reactions to convert them to nontoxic products. Dietary antioxidants, such  as vitamin E and flavonoids, and endogenous antioxidants, such as urate, can   Fig 24.13 Compartmentation of free radical defenses. Various defenses against ROS are  found in the different subcellular compartments of the cell. The location of free radical  defense enzymes (shown in blue) matches the type and amount of ROS generated in each  subcellular compartment. The highest activities of these enzymes are found in the liver, adre-  nal gland, and kidney, where mitochondrial and peroxisomal contents are high, and  cytochrome P450 enzymes are found in abundance in the smooth ER. The enzymes super-  oxide dismutase (SOD) and glutathione peroxidase are present as isozymes in the different  compartments. Another form of compartmentation involves the sequestration of Fe, which is  stored as mobilizable Fe in ferritin. Excess Fe is stored in nonmobilizable hemosiderin  deposits. Glutathione (GSH) is a nonenzymatic antioxidant.   terminate free radical chain reactions. Defense through compartmentation refers to  separation of species and sites involved in ROS generation from the rest of the cell  (Fig. 24.13). For example, many of the enzymes that produce hydrogen peroxide are  sequestered in peroxisomes with a high content of antioxidant enzymes. Metals are  bound to a wide range of proteins within the blood and in cells, preventing their par-  ticipation in the Fenton reaction. Iron, for example, is tightly bound to its storage  protein, ferritin and cannot react with hydrogen peroxide. Repair mechanisms for  DNA, and for removal of oxidized fatty acids from membrane lipids, are available  to the cell. Oxidized amino acids on proteins are continuously repaired through pro-  tein degradation and resynthesis of new proteins.   The enzymatic defense against ROS includes superoxide dismutase, catalase, and  glutathione peroxidase.   Conversion of superoxide anion to hydrogen peroxide and 0 2 (dismutation) by  superoxide dismutase (SOD) is often called the primary defense against oxidative  stress because superoxide is such a strong initiator of chain reactions (Fig 24.14).  SOD exists as three isoenzyme forms, a Cu + -Zn 2+ form present in the cytosol, a  Mn 2+ form present in mitochondria, and a Cu + -Zn 2+ form found extracellularly.  The activity of Cu + -Zn 2+ SOD is increased by chemicals or conditions (such as  hyperbaric oxygen) that increase the production of superoxide.   Fig 24.14. Superoxide dismutase converts  superoxide to hydrogen peroxide, which is  nontoxic unless converted to other ROS.   In the body, iron and other metals  are sequestered from interaction  with ROS or 0 2 by their binding to  transport proteins (haptoglobin, hemoglo-  bin, transferrin, ceruloplasmin, and metal-  lothionein) in the blood, and to intracellular  storage proteins (ferritin, hemosiderin). Met-  als also are found bound to many enzymes,  particularly those that react with 0 2 . Usually,  these enzymes have reaction mechanisms  that minimize nonspecific single-electron  transfer from the metal to other compounds.   The intracellular form of the Cu +  -Zn 2+ superoxide dismutase is  encoded by the SOD1 gene. To  date, 58 mutations in this gene have been  discovered in individuals affected by familial  amyotrophic lateral sclerosis (Lou Gehrig's  disease). How a mutation in this gene leads  to the symptoms of this disease has yet to  be understood. It is important to note that  only 5 to 10% of the total cases of diagnosed  amyotrophic lateral sclerosis are caused by  the familial form.   Why does the cell need such a high  content of SOD in mitochondria?   Premature infants with low levels of lung surfactant (see Chapter 33) require oxygen therapy. The level of oxygen must be closely  monitored to prevent retinopathy and subsequent blindness (the retinopathy of prematurity) and to prevent bronchial pulmonary  dysplasia. The tendency for these complications to develop is enhanced by the possibility of low levels of SOD and vitamin E in  the premature infant.   Mitochondria are major sites for  generation of superoxide from the  interaction of CoQ and 0 2 . The Mn 2+  superoxide dismutase present in mitochon-  dria is not regulated through induction/repres-  sion of gene transcription, presumably  because the rate of superoxide generation is  always high. Mitochondria also have a high  content of glutathione and glutathione perox-  idase, and can thus convert H 2 0 2 to H 2 0 and  prevent lipid peroxidation.   Fig 24.15. Catalase reduces hydrogen perox-  ide. (ROS is shown in a blue box).   Selenium (Se) is present in human  proteins principally as selenocys-  teine (cysteine with the sulfur  group replaced by Se, abbreviated sec). This  amino acid functions in catalysis, and has  been found in 11 or more human enzymes,  including the four enzymes of the glu-  tathione peroxidase family. Selenium is sup-  plied in the diet as selenomethionine from  plants (methionine with the Se replacing the  sulfur), selenocysteine from animal foods,  and inorganic selenium. Se from all of these  sources can be converted to selenophos-  phate. Selenophosphate reacts with a  unique tRNA containing bound serine to  form a selenocysteine-tRNA, which incorpo-  rates selenocystiene into the appropriate  protein as it is being synthesized. Se home-  ostasis in the body is controlled principally  through regulation of its secretion as methy-  lated Se. The current dietary requirement is  approximately 70 |xg/day for adult males and  55 (jtg for females. Deficiency symptoms  reflect diminished antioxidant defenses and  include symptoms of vitamin E deficiency.   Hydrogen peroxide, once formed, must be reduced to water to prevent it from form-  ing the hydroxyl radical in the Fenton reaction or Haber- Weiss reactions (see Fig.  24.4) One of the enzymes capable of reducing hydrogen peroxide is catalase  (Fig. 24. 15). Catalase is found principally in peroxisomes, and to a lesser extent in  the cytosol and microsomal fraction of the cell. The highest activities are found in  tissues with a high peroxisomal content (kidney and liver). In cells of the immune  system, catalase serves to protect the cell against its own respiratory burst.   Glutathione (7-glutamylcysteinylglycine) is one of the body's principal means of  protecting against oxidative damage (see also Chapter 29). Glutathione is a tripep-  tide composed of glutamate, cysteine, and glycine, with the amino group of cys-  teine joined in peptide linkage to the 7-carboxyl group of glutamate (Fig. 24.16).  In reactions catalyzed by glutathione peroxidases, the reactive sulfhydryl groups  reduce hydrogen peroxide to water and lipid peroxides to nontoxic alcohols. In  these reactions, two glutathione molecules are oxidized to form a single molecule,  glutathione disulfide. The sulfhydryl groups are also oxidized in nonenzymatic  chain terminating reactions with organic radicals.   Glutathione peroxidases exist as a family of selenium enzymes with somewhat dif-  ferent properties and tissue locations. Within cells, they are found principally in the  cytosol and mitochondria, and are the major means for removing H 2 0 2 produced out-  side of peroxisomes. They contribute to our dietary requirement for selenium and  account for the protective effect of selenium in the prevention of free radical injury.   Once oxidized glutathione (GSSG) is formed, it must be reduced back to the  sulfhydryl form by glutathione reductase in a redox cycle (Fig. 24.17). Glutathione  reductase contains an FAD, and catalyzes transfer of electrons from NADPH to the  disulfide bond of GSSG. NADPH is, thus, essential for protection against free rad-  ical injury. The major source of NADPH for this reaction is the pentose phosphate  pathway (see Chapter 29).   Free radical scavengers convert free radicals to a nonradical nontoxic form in  nonenzymatic reactions. Most free radical scavengers are antioxidants, compounds   Fig 24.16. Glutathione peroxidase reduces hydrogen peroxide to water. A. The structure of  glutathione. The sulfhydryl group of glutathione, which is oxidized to a disulfide, is shown  in blue. B. Glutathione peroxidase transfer electrons from glutathione (GSH) to hydrogen  peroxide.   Fig 24.17. Glutathione redox cycle. Glutathione reductase regenerates reduced glutathione.  (ROS is shown in the blue box).   that neutralize free radicals by donating a hydrogen atom (with its one electron) to  the radical. Antioxidants, therefore, reduce free radicals and are themselves oxi-  dized in the reaction. Dietary free radical scavengers (e.g., vitamin E, ascorbic acid,  carotenoids, and flavonoids) as well as endogenously produced free radical scav-  engers (e.g., urate and melatonin) have a common structural feature, a conjugated  double bond system that may be an aromatic ring.   Vitamin E (a-tocopherol), the most widely distributed antioxidant in nature, is a  lipid- soluble antioxidant vitamin that functions principally to protect against  lipid peroxidation in membranes (see Fig. 24.13). Vitamin E comprises a num-  ber of tocopherols that differ in their methylation pattern. Among these, a-  tocopherol is the most potent antioxidant and present in the highest amount in  our diet (Fig. 24.18).   Vitamin E is an efficient antioxidant and nonenzymatic terminator of free radi-  cal chain reactions, and has little pro-oxidant activity. When Vitamin E donates an  electron to a lipid peroxy radical, it is converted to a free radical form that is stabi-  lized by resonance. If this free radical form were to act as a pro-oxidant and abstract  an electron from a polyunsaturated lipid, it would be oxidizing that lipid and actu-  ally propagate the free radical chain reaction. The chemistry of vitamin E is such  that it has a much greater tendency to donate a second electron and go to the fully  oxidized form.   Although ascorbate (vitamin C) is an oxidation-reduction coenzyme that functions  in collagen synthesis and other reactions, it also plays a role in free radical defense.  Reduced ascorbate can regenerate the reduced form of vitamin E through donating  electrons in a redox cycle (Fig. 24.19). It is water-soluble and circulates unbound in  blood and extracellular fluid, where it has access to the lipid-soluble vitamin E  present in membranes and lipoprotein particles.   Fig 24.18. Vitamin E (a-tocopherol) terminates  free radical lipid peroxidation by donating single  electrons to lipid peroxyl radicals (LOO») to  form the more stable lipid peroxide, LOOH. In  so doing, the a-tocopherol is converted to the  fully oxidized tocopheryl quinone.   Carotenoids is a term applied to p -carotene (the precursor of vitamin A) and simi-  lar compounds with functional oxygen-containing substituents on the rings, such as  zeaxanthin and lutein (Fig. 24.20). These compounds can exert antioxidant effects,  as well as quench singlet 0 2 (singlet oxygen is a highly reactive oxygen species in  which there are no unpaired electrons in the outer orbitals, but there is one orbital  that is completely empty). Epidemiologic studies have shown a correlation between  diets high in fruits and vegetables and health benefits, leading to the hypothesis  that carotenoids might slow the progression of cancer, atherosclerosis, and other  degenerative diseases by acting as chain-breaking antioxidants. However, in clinical   Vitamin E is found in the diet in the  \ \ ) lipid fractions of some vegetable oils  *S and in liver, egg yolks, and cereals. It  is absorbed together with lipids, and fat mal-  absorption results in symptomatic deficien-  cies. Vitamin E circulates in the blood in  lipoprotein particles. Its deficiency causes  neurologic symptoms, probably because the  polyunsaturated lipids in myelin and other  membranes of the nervous system are partic-  ularly sensitive to free radical injury.   Fig 24.19. L-Ascorbate (the reduced form) donates single electrons to free radicals or disulfides in two steps as it is oxidized to dehydro-L-ascor-  bic acid. Its principle role in free radical defense is probably regeneration of vitamin E. However, it also may react with superoxide, hydrogen  peroxide, hypochlorite, the hydroxyl and peroxyl radicals, and N0 2 .   Epidemiologic evidence suggests  \m J that individuals with a higher  *r intake of foods containing vitamin  E, p-carotene, and vitamin C have a some-  what lower risk of cancer and certain other  ROS-related diseases than do individuals on  diets deficient in these vitamins. However,  studies in which well-nourished populations  were given supplements of these antioxi-  dant vitamins found either no effects or  harmful effects compared with the beneficial  effects from eating foods containing a wide  variety of antioxidant compounds. Of the  pure chemical supplements tested, there is  evidence only for the efficacy of vitamin E. In  two clinical trials, p-carotene (or p-carotene  + vitamin A) was associated with a higher  incidence of lung cancer among smokers  and higher mortality rates. In one study, vita-  min E intake was associated with a higher  incidence of hemorrhagic stroke (possibly  because of vitamin K mimicry).    Fig 24.20. Carotenoids are compounds related in structure to p-carotene. Lutein and  zeathanthin (the macular carotenoids) are analogs containing hydroxyl groups.   trials, p-carotene supplements had either no effect or an undesirable effect. Its  ineffectiveness may be due to the pro-oxidant activity of the free radical form.   In contrast, epidemiologic studies relating the intake of lutein and zeoxanthin  with decreased incidence of age-related macular degeneration have received pro-  gressive support. These two carotenoids are concentrated in the macula (the central  portion of the retina) and are called the macular carotenoids.   Age-related macular degeneration (AMD) is the leading cause of blindness in  the United States among persons older than 50 years of age, and it affects 1.7  million people worldwide. In AMD, visual loss is related to oxidative damage to  the retinal pigment epithelium (RPE) and the choriocapillaris epithelium. The photore-  ceptor/retinal pigment complex is exposed to sunlight, it is bathed in near arterial levels  of oxygen, and the membranes contain high concentrations of polyunsaturated fatty  acids, all of which are conducive to oxidative damage. Lipofuscin granules, which accu-  mulate in the RPE throughout life, may serve as photosensitizers, initiating damage by  absorbing blue light and generating singlet oxygen that forms other radicals. Dark sun-  glasses are protective. Epidemiologic studies showed that the intake of lutein and  zeanthin in dark green leafy vegetables (e.g., spinach and collard greens) also may be  protective. Lutein and zeaxanthein accumulate in the macula and protect against free  radical damage by absorbing blue light and quenching singlet oxygen.   Flavonoids are a group of structurally similar compounds containing two spatially  separate aromatic rings that are found in red wine, green tea, chocolate, and other  plant-derived foods (Fig. 24.21). Flavonoids have been hypothesized to contribute  to our free radical defenses in a number of ways. Some flavonoids inhibit enzymes  responsible for superoxide anion production, such as xanthine oxidase. Others effi-  ciently chelate Fe and Cu, making it impossible for these metals to participate in the  Fenton reaction. They also may act as free radical scavengers by donating electrons  to superoxide or lipid peroxy radicals, or stabilize free radicals by complexing with  them.   It is difficult to tell how much dietary flavonoids contribute to our free radical  defense system; they have a high pro-oxidant activity and are poorly absorbed.  Nonetheless, we generally consume large amounts of flavonoids (approximately  800 mg/day), and there is evidence that they can contribute to the maintenance of  vitamin E as an antioxidant.   A number of compounds synthesized endogenously for other functions, or as uri-  nary excretion products, also function nonenzymatically as free radical antioxi-  dants. Uric acid is formed from the degradation of purines and is released into extra-  cellular fluids, including blood, saliva, and lung lining fluid (Fig. 24.22). Together  with protein thiols, it accounts for the major free radical trapping capacity of  plasma. It is particularly important in the upper airways, where there are few other  antioxidants. It can directly scavenge hydroxyl radicals, oxyheme oxidants formed  between the reaction of hemoglobin and peroxy radicals, and peroxyl radicals them-  selves. Having acted as a scavenger, uric acid produces a range of oxidation  products that are subsequently excreted.   Melatonin, which is a secretory product of the pineal gland, is a neurohor-  mone that functions in regulation of our circadian rhythm, light-dark signal  transduction, and sleep induction. In addition to these receptor-mediated func-  tions, it functions as a nonenzymatic free radical scavenger that donates an elec-  tron (as hydrogen) to "neutralize" free radicals. It also can react with ROS and  RNOS to form addition products, thereby undergoing suicidal transformations.  Its effectiveness is related to both its lack of pro-oxidant activity and its joint  hydrophilic/hydrophobic nature that allows it to pass through membranes and the  blood-brain barrier.   Fig 24.21. The flavonoid quercetin. All  flavonoids have the same ring structure, shown  in blue. They differ in ring substituents (=0,  -OH, and OCH 3 ). Quercetin is effective in Fe  chelation and antioxidant activity. It is widely  distributed in fruits (principally in the skins)  and in vegetables (e.g., onions).   Fig 24.23. A model for the role of ROS and  RNOS in neuronal degradation in Parkinson's  disease. 1. Dopamine levels are reduced by  monoamine oxidase, which generates H 2 0 2 .  2. Superoxide also can be produced by mito-  chondria, which SOD will convert to H 2 0 2 .  Iron levels increase, which allows the Fenton  reaction to proceed, generating hydroxyl radi-  cals. 3. NO, produced by inducible nitric oxide  synthase, reacts with superoxide to form  RNOS. 4. The RNOS and hydroxyl radical  lead to radical chain reactions that result in  lipid peroxidation, protein oxidation, the for-  mation of lipofuscin, and neuronal degenera-  tion. The end result is a reduced production  and release of dopamine, which leads to the  clinical symptoms observed.   Les Dopaman has "primary" parkinsonism. The pathogenesis of this  disease is not well established and may be multifactorial (Fig. 24.23). The  major clinical disturbances in Parkinson's disease are a result of dopamine  depletion in the neostriatum, resulting from degeneration of dopaminergic neurons  whose cell bodies reside in the substantia nigra pars compacta. The decrease in  dopamine production is the result of severe degeneration of these nigrostriatal neu-  rons. Although the agent that initiates the disease is unknown, a variety of studies  support a role for free radicals in Parkinson's disease. Within these neurons,  dopamine turnover is increased, dopamine levels are lower, glutathione is  decreased, and lipofuscin (Lewy bodies) is increased. Iron levels are higher, and fer-  ritin, the storage form of iron, is lower. Furthermore, the disease is mimicked by the  compound l-methyl-4-phenylpyridinium (MPP + ), an inhibitor of NADH dehydro-  genase that increases superoxide production in these neurons. Even so, it is not  known whether oxidative stress makes a primary or secondary contribution to the  disease process.   Drug therapy is based on the severity of the disease. In the early phases of the  disease, a monoamine oxidase B -inhibitor is used that inhibits dopamine degrada-  tion and decreases hydrogen peroxide formation. In later stages of the disease,  patients are treated with levodopa (L-dopa), a precursor of dopamine.   a Cora Nari experienced angina caused by severe ischemia in the ventric-  ) ular muscle of her heart. The ischemia was caused by clots that formed at  the site of atherosclerotic plaques within the lumen of the coronary arter-  ies. When TPA was infused to dissolve the clots, the ischemic area of her heart was  reperfused with oxygenated blood, resulting in ischemic-reperfusion injury. In her  case, the reperfusion injury resulted in ventricular fibrillation.   During ischemia, several events occur simultaneously in cardiomyocytes. A  decreased 0 2 supply results in decreased ATP generation from mitochondrial oxida-  tive phosphorylation and inhibition of cardiac muscle contraction. As a conse-  quence, cytosolic AMP concentration increases, activating anaerobic glycolysis and  lactic acid production. If ATP levels are inadequate to maintain Na + , K + -ATPase  activity, intracellular Na + increases, resulting in cellular swelling, a further increase  in H + concentration, and increases of cytosolic and subsequently mitochondrial  Ca 2+ levels. The decrease in ATP and increase in Ca 2+ may open the mitochondrial  permeability transition pore, resulting in permanent inhibition of oxidative phos-  phorylation. Damage to lipid membranes is further enhanced by  Ca 2+ activation of phospholipases.   Reperfusion with 0 2 allows recovery of oxidative phosphorylation, provided that  the mitochondrial membrane has maintained some integrity and the mitochondrial  transition pore can close. However, it also increases generation of free radicals. The  transfer of electrons from CoQ« to 0 2 to generate superoxide is increased. Endothe-  lial production of superoxide by xanthine oxidase also may increase. These radicals  may go on to form the hydroxyl radical, which can enhance the damage to compo-  nents of the electron transport chain and mitochondrial lipids, as well as activate the   Currently, an intense study of ischemic insults to a variety of animal organs is underway, in an effort to discover ways of pre-  KJ ] venting reperfusion injury. These include methods designed to increase endogenous antioxidant activity, to reduce the genera-  \^^ / tion of free radicals, and, finally, to develop exogenous antioxidants that, when administered before reperfusion, would prevent  its injurious effects. Each of these approaches has met with some success, but their clinical application awaits further refinement. With  the growing number of invasive procedures aimed at restoring arterial blood flow through partially obstructed coronary vessels, such as  clot lysis, balloon or laser angioplasty, and coronary artery bypass grafting, development of methods to prevent ischemia-reperfusion  injury will become increasingly urgent.   mitochondrial permeability transition. As macrophages move into the area to clean  up cellular debris, they may generate NO and superoxide, thus introducing perox-  ynitrite and other free radicals into the area. Depending on the route and timing  involved, the acute results may be cell death through necrosis, with slower cell  death through apoptosis in the surrounding tissue.   In Cora Nari's case, oxygen was restored before permanent impairment of  oxidative phosphorylation had occurred and the stage of irreversible injury was  reached. However, reintroduction of oxygen induced ventricular fibrillation, from  which she recovered.   ft Protection Against Ozone in Lung Lining Fluid The lung lin-  JL ) ing fluid, a thin fluid layer extending from the nasal cavity to the most dis-  tal lung alveoli, protects the epithelial cells lining our airways from ozone  and other pollutants. Although ozone is not a radical species, many of its toxic  effects are mediated through generation of the classical ROS, as well as generation  of aldehydes and ozonides. Polyunsaturated fatty acids represent the primary target  for ozone, and peroxidation of membrane lipids is the most important mechanism  of ozone-induced injury. However, ozone also oxidizes proteins.   The lung lining fluid has two phases; a gel-phase that traps microorganisms and  large particles, and a sol (soluble) phase containing a variety of ROS defense mech-  anisms that prevent pollutants from reaching the underlying lung epithelial cells  (Fig. 24.24). When the ozone level of inspired air is low, ozone is neutralized prin-  cipally by uric acid (UA) present in the fluid lining the nasal cavity. In the proximal  and distal regions of the respiratory tract, glutathione (GSH) and ascorbic acid  (AA), in addition to UA, react directly with ozone. Ozone that escapes this anti-  oxidant screen may react directly with proteins, lipids, and carbohydrates (CHO) to  generate secondary oxidants, such as lipid peroxides, that can initiate chain reac-  tions. A second layer of defense protects against these oxidation and peroxidation  products: p -tocopherol (vitamin E) and glutathione react directly with lipid radi-  cals; glutathione peroxidase reacts with hydrogen peroxide and lipid peroxides, and    Although most individuals are able  to protect against small amounts of  ozone in the atmosphere, even  slightly elevated ozone concentrations pro-  duce respiratory symptoms in 10 to 20% of  the healthy population.    Fig 24.24. Protection against ozone in the lung lining fluid. GSH, glutathione; AA, ascorbic acid (vitamin C); UA, uric acid; CHO, carbohy-  drate; a-TOC, vitamin E; GSH-Px, glutathione peroxidase; ED-SOD, extracellular superoxide dismutase; Neut, neutrophil.   extracellular superoxide dismutase (EC- SOD) converts superoxide to hydrogen per-  oxide. However, oxidative stress may still overwhelm even this extensive defense  network because ozone also promotes neutrophil migration into the lung lining  fluid. Once activated, the neutrophils (Neut) produce a second wave of ROS (super-  oxide, HOC1, and NO).   Gutteridge JMC, Halliwell B. Antioxidants in Nutrition, Health and Disease. Oxford: Oxford University  Press, 1994.   Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfu-   sion injury. Biochem Soc Symp 1999;66:181-203.  Mudway IS, Kelly FJ. Ozone and the lung: a sensitive issue. Mol Aspects Med 2000;21:1-48.  Pietta P-G. Flavonoids as antioxidants. J Nat Prod 2000;63:1035-1042.   Reiter RJ, Tan D-X, Wenbo A, Manchester LC, Karownik M, Calvo JR. Pharmacology and physiology  of melatonin in the reduction of oxidative stress in vivo. Biol Signals Recept 2000;9:160-171.   Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl  Acad Sci USA 1994;92:10771-10778.   Winkler BS, Boulton ME, Gottsch JD, Sternberg P. Oxidative damage and age-related macular degener-  ation. Molecular Vision 1999;5:32.   Zhang Y, Dawson, VL, Dawson, TM. Oxidative stress and genetics in the pathogenesis of Parkinson's  disease. Neurobiol Dis 2000;7:240-250.   1 . Which of the following vitamins or enzymes is unable to protect against free radical damage?   (A) 0 2 - + e" + 2H + yields H 2 0 2   (B) 2 0 2 - + 2H + yields H 2 0 2 + 0 2   (C) 0 2 - + HO+ H+ yields C0 2 + H 2 0   (D) H 2 0 2 + 0 2 yields 4 H 2 0   (E) 0 2 - + H 2 0 2 + H + yields 2 H 2 0 + 0 2   3. The mechanism of vitamin E as an antioxidant is best described by which of the following?   (A) Vitamin E binds to free radicals and sequesters them from the contents of the cell.   (D) Vitamin E forms a covalent bond with the radicals, thereby stabilizing the radical state.   4. An accumulation of hydrogen peroxide in a cellular compartment can be converted to dangerous radical forms in the presence  of which metal?   5. The level of oxidative damage to mitochondrial DNA is 10 times greater than that to nuclear DNA. This could be due, in part,  to which of the following?   (C) The nuclear membrane presents a barrier to reactive oxygen species.   Ethanol is a dietary fuel that is metabolized to acetate principally in the liver,  with the generation ofNADH. The principal route for metabolism of ethanol is  through hepatic alcohol dehydrogenases, which oxidize ethanol to acetaldehyde  in the cytosol (Fig. 25.1). Acetaldehyde is further oxidized by acetaldehyde dehy-  drogenases to acetate, principally in mitochondria. Acetaldehyde, which is toxic,  also may enter the blood. NADH produced by these reactions is used for adeno-  sine triphosphate (ATP) generation through oxidative phosphorylation. Most of  the acetate enters the blood and is taken up by skeletal muscles and other tissues,  where it is activated to acetyl Co A and is oxidized in the TCA cycle.   Approximately 10 to 20% of ingested ethanol is oxidized through a microsomal  oxidizing system (ME OS), comprising cytochrome P450 enzymes in the endoplas-  mic reticulum (especially CYP2E1). CYP2E1 has a high K m for ethanol and is  inducible by ethanol. Therefore, the proportion of ethanol metabolized through  this route is greater at high ethanol concentrations, and greater after chronic con-  sumption of ethanol.   Acute effects of alcohol ingestion arise principally from the generation of  NADH, which greatly increases the NADHINAD + ratio of the liver. As a conse-  quence, fatty acid oxidation is inhibited, and ketogenesis may occur. The elevated  NADHINAD + ratio may also cause lactic acidosis and inhibit gluconeogenesis.   Ethanol metabolism may result in alchohol-induced liver disease, including  hepatic steatosis (fatty liver), alcohol-induced hepatitis, and cirrhosis. The prin-  cipal toxic products of ethanol metabolism include acetaldehyde and free  radicals, Acetaldehyde forms adducts with proteins and other compounds. The  hydroxyethyl radical produced by MEOS and other radicals produced during    Fig. 25.1. The major route for metabolism of ethanol and use of acetate by the muscle. (ADH, alcohol dehydrogenase; ALDH, acetaldehyde  dehydrogenase; ACS, acetyl-CoA synthetase).   inflammation cause irreversible damage to the liver Many other tissues are  adversely affected by ethanol, acetaldehyde, or by the consequences of hepatic  dysmetabolism and injury. Genetic polymorphisms in the enzymes of ethanol  metabolism may be responsible for individual variations in the development of  alcoholism or the development of liver cirrhosis.   A dietary history for Ivan Applebod showed that he had continued his habit  of drinking scotch and soda each evening while watching TV, but he did not  add the ethanol calories to his dietary intake. He justifies this calculation on  the basis of a comment he heard on a radio program that calories from alcohol inges-  tion "don't count" because they are empty calories that do not cause weight gain.   Al Martini was found lying semiconscious at the bottom of the stairs by  his landlady when she returned from an overnight visit with friends. His  face had multiple bruises and his right forearm was grotesquely angulated.  Nonbloody dried vomitus stained his clothing. Mr. Martini was rushed by ambu-  lance to the emergency room at the nearest hospital. In addition to multiple bruises  and the compound fracture of his right forearm, he had deep and rapid (Kussmaul)  respirations and was moderately dehydrated.   Initial laboratory studies showed a relatively large anion gap of 34 mmol/L (ref-  erence range = 9-15 mmol/L). An arterial blood gas analysis confirmed the pres-  ence of a metabolic acidosis. Mr. Martini's blood alcohol level was only slightly  elevated. His serum glucose was 68 mg/dL (low normal).   Jean Ann Tonich, a 46-year-old commercial artist, recently lost her job  because of absenteeism. Her husband of 24 years had left her 10 months  earlier. She complains of loss of appetite, fatigue, muscle weakness, and  emotional depression. She has had occasional pain in the area of her liver, at times  accompanied by nausea and vomiting.   On physical examination she appears disheveled and pale. The physician notes  tenderness to light percussion over her liver and detects a small amount of ascites  (fluid within the peritoneal cavity around the abdominal organs). The lower edge of  her liver is palpable about 2 inches below the lower margin of her right rib cage,  suggesting liver enlargement, and feels somewhat more firm and nodular than nor-  mal. Jean Ann's spleen is not palpably enlarged. There is a suggestion of mild jaun-  dice. No obvious neurologic or cognitive abnormalities are present.   After detecting a hint of alcohol on Jean Ann's breath, the physician questions  her about possible alcohol abuse, which she denies. With more intensive question-  ing, however, Jean Ann admits that for the last 5 or 6 years she began drinking gin  on a daily basis (approximately 4-5 drinks, or 68-85 g ethanol) and eating infre-  quently. Laboratory tests showed that her serum ethanol level on the initial office  visit was 245 mg/dL (0.245%). A serum ethanol level above 150 mg/dL (0.15%) is  considered indicative of inebriation.   Ethanol is a small molecule that is both lipid and water soluble. It is, therefore, read-  ily absorbed from the intestine by passive diffusion. A small percentage of ingested  ethanol (0-5%) enters the gastric mucosal cells of the upper GI tract (tongue, mouth,    The anion gap is calculated by sub-  tracting the sum of the value for  serum chloride and for the serum  HC0 3 " content from the serum sodium con-  centration. If the gap is greater than normal,  it suggests that acids such as the ketone  bodies acetoacetate and p-hydroxybutyrate  are present in the blood in increased  amounts.   Jaundice is a yellow discoloration  involving the sclerae (the "whites'"  of the eyes) and skin. It is caused  by the deposition of bilirubin, a yellow  degradation product of heme. Bilirubin accu-  mulates in the blood under conditions of  liver injury, bile duct obstruction, and exces-  sive degradation of heme.   Jean Ann Tonich's admitted  ethanol consumption exceeds the  definition of moderate drinking.  Moderate drinking is now defined as not  more than two drinks per day for men, but  only one drink per day for women. A drink is  defined as 12 oz of regular beer, 5 oz of wine,  or 1.5 oz distilled spirits (80 proof).   Fig. 25.2. The pathway of ethanol metabolism  (ADH, alcohol dehydrogenase; ALDH,  acetaldehyde dehydrogenase).   Fig. 25.3. The reaction catalyzed by MEOS  (which includes CYP2E1) in the endoplasmic  reticulum.   esophagus, and stomach), where it is metabolized. The remainder enters the blood.  Of this, 85 to 98% is metabolized in the liver, and only 2 to 10% is excreted through  the lungs or kidneys.   The major route of ethanol metabolism in the liver is through liver alcohol dehy-  drogenase, a cytosolic enzyme that oxidizes ethanol to acetaldehyde with reduction  of NAD + to NADH (Fig. 25. 2). If it is not removed by metabolism, acetaldehyde  exerts toxic actions in the liver and can enter the blood and exert toxic effects in  other tissues.   Approximately 90% of the acetaldehyde that is generated is further metabolized to  acetate in the liver. The major enzyme involved is a low K m mitochondrial acetalde-  hyde dehydrogenase (ALDH), which oxidizes acetaldehyde to acetate with generation  of NADH (see Fig. 25.2). Acetate, which has no toxic effects, may be activated to  acetyl CoA in the liver (where it can enter either the TCA cycle or the pathway for fatty  acid synthesis). However, most of the acetate that is generated enters the blood and is  activated to acetyl CoA in skeletal muscles and other tissues (see Fig. 25.1). Acetate is  generally considered nontoxic and is a normal constituent of the diet.   The other principal route of ethanol oxidation in the liver is the microsomal  ethanol oxidizing system (MEOS), which also oxidizes ethanol to acetaldehyde  (Fig. 25.3). The principal microsomal enzyme involved is a cytochrome P450  mixed-function oxidase isozyme (CYP2E1), which uses NADPH as an additional  electron donor and 0 2 as an electron acceptor. This route accounts for only 10 to  20% of ethanol oxidation in a moderate drinker.   Each of the enzyme activities involved in ethanol metabolism (alcohol dehydro-  genase, acetaldehyde dehydrogenase, and CYP2E1) exist as a family of isoen-  zymes. Individual variations in the quantity of these isoenzymes influence a num-  ber of factors, such as the rate of ethanol clearance from the blood, the degree of  inebriation exhibited by an individual, and differences in individual susceptibility to  the development of alcohol-induced liver disease.   Alcohol dehydrogenase (ADH) exists as a family of isoenzymes with varying speci-  ficity for chain length of the alcohol substrate (Table 25.1). Ethanol is a small mol-  ecule that does not exhibit much in the way of unique structural characteristics and,  at high concentrations, is nonspecifically metabolized by many members of the  ADH family. The alcohol dehydrogenases that exhibit the highest specificity for  ethanol are the class I alcohol dehydrogenases. We have three genes for class I alco-  hol dehydrogenases, each of which exists as allelic variants (polymorphisms).   Present in highest levels in upper Gl tract, gingiva  and mouth, esophagus, down to the stomach. Not  present in liver.   K m of 0.05-4 mM for ethanol.  Active only with ethanol. High  tissue capacity.   Relatively inactive toward  ethanol. Active mainly toward  long-chain alcohols, and gj-OH  fatty acids.   K m of 28 mM. It is the most  active of medium-chain alcohol  DH toward retinal.   The class I alcohol dehydrogenases are present in high quantities in the liver, rep-  resenting approximately 3% of all soluble protein. These alcohol dehydrogenases,  commonly referred to collectively as liver alcohol dehydrogenase, have low K m s for  ethanol between 0.05 and 4 mM (high affinities). Thus, the liver is the major site of  ethanol metabolism and the major site at which the toxic metabolite acetaldehyde is  generated.   Although the class IV and class II enzymes make minor contributions to ethanol  metabolism, they may contribute to its toxic effects. Ethanol concentrations can be  quite high in the upper GI tract (e.g., beer is approximately 0.8 M ethanol), and  acetaldehyde generated here by class IV enzymes (gastric ADH) might contribute  to the risk for cancer associated with heavy drinking. Class II ADH genes are  expressed primarily in the liver and at lower levels in the lower gastrointestinal tract.   Acetaldehyde is oxidized to acetate, with the generation of NADH, by acetaldehyde  dehydrogenases (see Fig. 25.2). More than 80% of acetaldehyde oxidation in the  human liver is normally catalyzed by mitochondrial acetaldehyde dehydrogenase  (ALDH2), which has a high affinity for acetaldehyde and is highly specific. How-  ever, individuals with a common allelic variant of ALDH2 have a greatly decreased  capacity for acetaldehyde metabolism   Most of the remainder of acetaldehyde oxidation occurs through a cytosolic  acetaldehyde dehydrogenase (ALDH1). Additional aldehyde dehydrogenases act on  a variety of organic alcohols, toxins, and pollutants.   Metabolism of acetate requires activation to acetyl CoA by acetyl CoA synthetase  in a reaction similar to that catalyzed by fatty acyl CoA synthetases (Fig. 25.4). In  liver, the principle isoform of acetyl CoA synthetase (ACS I) is a cytosolic enzyme  that generates acetyl CoA for the cytosolic pathways of cholesterol and fatty acid  synthesis. Acetate entry into these pathways is under regulatory control by mecha-  nisms involving cholesterol or insulin. Thus, most of the acetate generated enters  the blood.   Acetate is taken up and oxidized by other tissues, notably heart and skeletal mus-  cle, which have a high concentration of the mitochondrial acetyl CoA synthetase  isoform (ACSII). This enzyme is present in the mitochondrial matrix. It therefore  generates acetyl CoA that can directly enter the TCA cycle and be oxidized to C0 2 .   Ethanol is also oxidized to acetaldehyde in the liver by the microsomal ethanol  oxidizing system, which comprises members of the cytochrome P450 superfam-  ily of enzymes. Ethanol and NADPH both donate electrons in the reaction, which  reduces 0 2 to 2H 2 0 (Fig. 25.5). The cytochrome P450 enzymes all have two   The accumulation of acetaldehyde causes nausea and vomiting, and, therefore,  inactive acetaldehyde dehydrogenases are associated with a distaste for alco-  holic beverages and protection against alcoholism. In one of the common  allelic variants of ALDH2 (ALDH2*2), a single substitution increases the K m for acetalde-  hyde 260-fold (lowers the affinity) and decreases the V max 10-fold, resulting in a very inac-  tive enzyme. Homozygosity for the ALDH2*2 allele affords absolute protection against  alcoholism; no individual with this genotype has been found among alcoholics. Alco-  holics are frequently treated with acetaldehyde dehydrogenase inhibitors (e.g., disulfi-  ram) to help them abstain from alcohol intake. Unfortunately, alcoholics who continue to  drink while taking this drug are exposed to the toxic effects of elevated acetaldehyde  levels.    The human has at least seven, and  possibly more, genes that code for  specific isoenzymes of medium-  chain-length alcohol dehydrogenases, the  major enzyme responsible for the oxidation  of ethanol to acetaldehyde in the human.  These different alcohol dehydrogenases  have an approximately 60 to 70% identity  and are assumed to have arisen from a com-  mon ancestral gene similar to the class III  isoenzyme many millions of years ago. The  class I alcohol dehydrogenases (ADH 1, ADH  2, and ADH 3) are all present in high concen-  tration in the liver, and have a relatively high  affinity and capacity for ethanol at low con-  centrations. (These properties are quantita-  tively reflected by their low K m , a parameter  discussed in Chapter 9). They have a 90 to  94% sequence identity and are able to form  both homo- and hetero-dimers, among  themselves (e.g., or However, none  of the ADHs can form dimers with an ADH  from another class. The three genes for class  I alcohol dehydrogenases are arranged in  tandem, head to tail, on chromosome 4. The  genes for the other classes of alcohol  dehydrogenase are also on chromosome 4  in nearby locations.   ADH 2 and ADH 3 are present as  functional polymorphisms that  differ in their properties. Genetic  polymorphisms for ADH partially account  for the observed differences in ethanol elim-  ination rates among various individuals or  populations. Although susceptibility to alco-  holism is a complex function of genetics and  socioeconomic factors, possession of the  ADH 2*2 allele, which encodes a relatively  fast ADH (high V max ), is associated with a  decreased susceptibility to alcoholism — pre-  sumably because of nausea and flushing  caused by acetaldehyde accumulation  (because the aldehyde dehydrogenase gene  cannot keep up with the amount of acetalde-  hyde produced). This particular allele has a  relatively high frequency in the East Asian  population and a low frequency among  white Europeans. In contrast, the ADH  2*1/2*1 genotype (homozygous for allele 1  of the ADH 2 gene) is a risk factor for the  development of Wernicke-Korsakoff syn-  drome, a neuropsychiatric syndrome com-  monly associated with alcoholism.   Fig. 25.5. General structure of cytochrome  P450 enzymes. 0 2 binds to the P450 Fe-heme in  the active site and is activated to a reactive form  by accepting electrons. The electrons are  donated by the cytochrome P450 reductase,  which contains an FAD plus an FMN or Fe-S  center to facilitate the transfer of single elec-  trons from NADPH to 0 2 . The P450 enzymes  involved in steroidogenesis have a somewhat  different structure. For CYP2E1, RH is ethanol  (CH 3 CH 2 OH) and ROH is acetaldehyde  (CH 3 COH).   CYP represents cytochrome P450.  KJ ) P450 is an Fe-heme similar to that   found in the cytochromes of the  electron transport chain ("P" denotes the  heme pjgment, and 450 is the wavelength of  visible light absorbed by the pigment). In  CYP2E1, the "2" refers to the gene family,  which comprises isoenzymes with greater  than 40% amino acid sequence identity. The  "E" refers to the subfamily, a grouping of  isoenzymes with greater than 55 to 60%  sequence identity, and the "1" refers to the  individual enzymes within this subfamily.   major catalytic protein components: an electron-donating reductase system that  transfers electrons from NADPH (cytochrome P450 reductase) and a cytochrome  P450. The cytochrome P450 protein contains the binding sites for 0 2 and the  substrate (e.g., ethanol) and carries out the reaction. The enzymes are present in  the endoplasmic reticulum, which on isolation from disrupted cells forms a  membrane fraction after centrifugation that was formerly called "microsomes"  by biochemists.   MEOS is part of the superfamily of cytochrome P450 enzymes, all of which cat-  alyze similar oxidative reactions. Within the superfamily, at least 10 distinct gene  families are found in mammals. More than 100 different cytochrome P450  isozymes exist within these 10 gene families. Each isoenzyme has a distinct classi-  fication according to its structural relationship with other isoenzymes. The isoen-  zyme that has the highest activity toward ethanol is called CYP2E1. A great deal of  overlapping specificity exists among the various P450 isoenzymes, and ethanol is  also oxidized by several other P450 isoenzymes. "MEOS" refers to the combined  ethanol oxidizing activity of all the P450 enzymes.   CYP2E1 has a much higher K m for ethanol than the class I alcohol dehydroge-  nases (11 mM [51 mg/dL] compared with 0.05-4 mM [0.23 to 18.4 mg/dL]). Thus,  a greater proportion of ingested ethanol is metabolized through CYP2E1 at high  levels of ethanol consumption than at low levels.   The P450 enzymes are inducible both by their most specific substrate and by sub-  strates for some of the other cytochrome P450 enzymes. Chronic consumption of  ethanol increases hepatic CYP2E1 levels approximately 5- to 10-fold. However, it  also causes a twofold to fourfold increase in some of the other P450s from the same  subfamily, from different subfamilies, and even from different gene families. The  endoplasmic reticulum undergoes proliferation, with a general increase in the con-  tent of microsomal enzymes, including those that are not directly involved in  ethanol metabolism.   The increase in CYP2E1 with ethanol consumption occurs through transcrip-  tional, post-transcriptional, and post-translational regulation. Increased levels of  mRNA, resulting from induction of gene transcription or stabilization of message,  are found in actively drinking patients. The protein is also stabilized against degra-  dation. In general, the mechanism for induction of P450 enzymes by their substrates  occurs through the binding of the substrate (or related compound) to an intracellu-  lar receptor protein, followed by binding of the activated receptor to a response ele-  ment in the target gene. Whether ethanol induction of CYP2E1 follows this general  pattern has not yet been shown.   Overlapping specificity in the catalytic activity of P450 enzymes and in their  Kj ) inducers is responsible for several types of drug interactions. For example, phe-  *r nobarbital, a barbiturate long used as a sleeping pill or for treatment of epilepsy,  is converted to an inactive metabolite by cytochrome P450 monooxygenases CYP2B1 and  CYP2B2. After treatment with phenobarbital, CYP2B2 is increased 50- to 100-fold. Individ-  uals who take phenobarbital for prolonged periods develop a drug tolerance as CYP2B2  is induced, and the drug is metabolized to an inactive metabolite more rapidly. Conse-  quently, these individuals use progressively higher doses of phenobarbital.   Ethanol is an inhibitor of the phenobarbital-oxidizing P450 system. When large  amounts of ethanol are consumed, the inactivation of phenobarbital is directly or indi-  rectly inhibited. Therefore, when high doses of phenobarbital and ethanol are consumed  at the same time, toxic levels of the barbiturate can accumulate in the blood.   Although induction of CYP2E1 increases ethanol clearance from the blood, it has  negative consequences. Acetaldehyde may be produced faster than it can be metab-  olized by acetaldehyde dehydrogenases, thereby increasing the risk of hepatic injury.  An increased amount of acetaldehyde can enter the blood and can damage other tis-  sues. In addition, cytochrome P450 enzymes are capable of generating free radicals,  which also may lead to increased hepatic injury and cirrhosis (see Chapter 24).   The routes and rates of ethanol oxidation vary from individual to individual. Dif-  ferences in ethanol metabolism may influence whether an individual becomes a  chronic alcoholic, develops alcohol-induced liver disease, or develops other dis-  eases associated with increased alcohol consumption (such as hepatocarcinogene-  sis, lung cancer, or breast cancer). Factors that determine the rate and route of  ethanol oxidation in individuals include:   • Genotype — Polymorphic forms of alcohol dehydrogenases and acetaldehyde  dehydrogenases can greatly affect the rate of ethanol oxidation and the accumu-  lation of acetaldehyde. CYP2E1 activity may vary as much as 20-fold between  individuals, partly because of differences in the inducibility of different allelic  variants.   • Drinking history — The level of gastric alcohol dehydrogenase (ADH) decreases  and CYP2E1 increases with the progression from a naive, to a moderate, and to  a heavy and chronic consumer of alcohol.   • Gender — Blood levels of ethanol after consuming a drink are normally higher  for women than for men, partly because of lower levels of gastric ADH activity  in women. After chronic consumption of ethanol, gastric ADH decreases in both  men and women, but the gender differences become even greater. Gender differ-  ences in blood alcohol levels also occur because women are normally smaller.  Furthermore, in females, alcohol is distributed in a 12% smaller water space  because a woman's body composition consists of more fat and less water than  that of a man.   • Quantity — The amount of ethanol an individual consumes over a small amount  of time determines its metabolic route. Small amounts of ethanol are metabo-  lized most efficiently through the low K m pathway of class I ADH and class II  ALDH. Little accumulation of NADH occurs to inhibit ethanol metabolism via  these dehydrogenases. However, when higher amounts of ethanol are consumed  in a short period, a disproportionately greater amount is metabolized through  MEOS. MEOS, which has a much higher K m for ethanol, functions principally  at high concentrations of ethanol. A higher activity of MEOS would be expected  to correlate with tendency to develop alcohol-induced liver disease, because both  acetaldehyde and free radical levels would be increased.   The ATP yield from ethanol oxidation to acetate varies with the route of ethanol  metabolism. If ethanol is oxidized by the major route of cytosolic ADH and mito-  chondrial ALDH, one cytosolic and one mitochondrial NADH are generated with a  maximum yield of 5 ATP. Oxidation of acetyl CoA in the TCA cycle and electron  transport chain leads to the generation of 10 high-energy phosphate bonds. How-  ever, activation of acetate to acetyl CoA requires two high-energy phosphate bonds  (one in the cleavage of ATP to AMP+ pyrophosphate and one in the cleavage of  pyrophosphate to phosphate), which must be subtracted. Thus the maximum total  energy yield is 13 moles of ATP per mole of ethanol.   In contrast, oxidation of ethanol to acetaldehyde by CYP2E1 consumes energy  in the form of NADPH, which is equivalent to 2.5 ATP. Thus, for every mole of   As blood ethanol concentration  rises above 18 mM (the legal intox-  ication limit is now defined as  0.08% in most states of the United States,  which is approximately 18 mM), the brain  and central nervous system are affected.  Induction of CYP2E1 increases the rate of  ethanol clearance from the blood, thereby  contributing to increased alcohol tolerance.  However, the apparent ability of a chronic  alcoholic to drink without appearing inebri-  ated is partly a learned behavior.   At Ivan Applebod's low level of  ethanol consumption, ethanol is  oxidized to acetate via ADH and  ALDH in the liver and the acetate is activated  to acetyl CoA and oxidized to C0 2 in skeletal  muscle and other tissues. The overall energy  yield of 13 ATP per ethanol molecule  accounts for the caloric value of ethanol,  approximately 7 Cal/g. However, chronic  consumption of substantial amounts of alco-  hol does not have the effect on body weight  expected from the caloric intake. This is  partly attributable to induction of MEOS,  resulting in a proportionately greater metab-  olism of ethanol through MEOS with its  lower energy yield (only approximately 8  ATP). In general, weight loss diets recom-  mend no, or low, alcohol consumption  because ethanol calories are "empty" in the  sense that alcoholic beverages are generally  low in vitamins, essential amino acids, and  other required nutrients, but not empty of  calories.   ethanol metabolized by this route, only a maximum of 8.0 moles of ATP can be gen-  erated (10 ATP from acetylCoA oxidation through the TCA cycle, minus 2 for  acetate activation; the NADH generated by aldehyde dehydrogenase is balanced by  the loss of NADPH in the MEOS step).   Alcohol-induced liver disease, a common and sometimes fatal consequence of  chronic ethanol abuse, may manifest itself in three forms: fatty liver, alcohol-induced  hepatitis, and cirrhosis. Each may occur alone, or they may be present in any combi-  nation in a given patient. Alcohol-induced cirrhosis is discovered in up to 9% of all  autopsies performed in the United States, with a peak incidence in patients 40 to 55  years of age.   However, ethanol ingestion also has acute effects on liver metabolism, including  inhibition of fatty acid oxidation and stimulation of triacylglycerol synthesis, lead-  ing to a fatty liver. It also can result in ketoacidosis or lactic acidosis and cause  hypoglycemia or hyperglycemia, depending on the dietary state. These effects are  considered reversible.   In contrast, acetaldehyde and free radicals generated from ethanol metabolism  can result in alcohol-induced hepatitis, a condition in which the liver is inflamed  and cells become necrotic and die. Diffuse damage to hepatocytes results in cirrho-  sis, characterized by fibrosis (scarring), disturbance of the normal architecture and  blood flow, loss of liver function and, ultimately, hepatic failure.   A. Acute Effects of Ethanol Arising from the Increased  NADH /NAD + Ratio   Many of the acute effects of ethanol ingestion arise from the increased  NADH/NAD + ratio in the liver (Fig. 25.6). At lower levels of ethanol intake, the rate  of ethanol oxidation is regulated by the supply of ethanol (usually determined by  how much ethanol we consume) and the rate at which NADH is reoxidized in the  electron transport chain. NADH is not a very effective product inhibitor of ADH or  ALDH, and there is no other feedback regulation by ATP, ADP, or AMP. As a con-  sequence, NADH generated in the cytosol and mitochondria tends to accumulate,  increasing the NADH/NAD + ratio to high levels (see Fig. 25.6, circle 1). The  increase is even greater as the mitochondria become damaged from acetaldehyde or  free radical injury.   ethanol. Thus, "happy hour" foods  (e.g., pizza, fried potato skins with sour  cream, nachos, and deep-fried peppers  stuffed with cream cheese and wrapped in  bacon) are exactly the wrong things to eat  while drinking. Steamed vegetables or sal-  ads with your beer would be much better for  your liver.   The high NADH/NAD + ratio generated from ethanol oxidation inhibits the oxida-  tion of fatty acids, which accumulate in the liver (see Fig. 25.6, circles 2 and 3)  These fatty acids are re-esterified into triacylglycerols by combining with glycerol  3 -P. The increased NADH/NAD + ratio increases the availability of glycerol 3-P by  promoting its synthesis from intermediates of glycolysis. The triacylglycerols are  incorporated into VLDL (very-low-density lipoproteins), which accumulate in the  liver and enter the blood, resulting in an ethanol-induced hyperlipidemia.   Although just a few drinks may result in hepatic fat accumulation, chronic con-  sumption of alcohol greatly enhances the development of a fatty liver. Re-esterifi-  cation of fatty acids into triacylglycerols by fatty acyl CoA transferases in the ER is  enhanced (see Fig. 25.6). Because the transferases are microsomal enzymes, they  are induced by ethanol consumption just as MEOS is induced. The result is a fatty  liver (hepatic steatosis).   The source of the fatty acids can be dietary fat, fatty acids synthesized in the  liver, or fatty acids released from adipose tissue stores. Adipose tissue lipolysis  increases after ethanol consumption, possibly because of a release of epinephrine.   Fig. 25.6. Acute effects of ethanol metabolism on lipid metabolism in the liver. (1) Metabolism of ethanol generates a high NADH/NAD +  ratio. (2) The high NADH/NAD + ratio inhibits fatty acid oxidation and the TCA cycle, resulting in accumulation of fatty acids. (3) Fatty  acids are re-esterified to glycerol 3-P by acy transferases in the endoplasmic reticulum. Glycerol 3-P levels are increased because a high  NADH/NAD + ratio favors its formation from dihydroxy acetone phosphate (an intermediate of glycolysis). Ethanol- stimulated increases of  endoplasmic reticulum enzymes also favors triacylglycerol formation. (4) NADH generated from ethanol oxidation can meet the require-  ments of the cell for ATP generation from oxidative phosphorylation. Thus, acetyl CoA oxidation in the TCA cycle is inhibited. (5) The  high NADH/NAD + ratio shifts oxaloacetate (OAA) toward malate, and acetyl CoA is directed into ketone body synthesis. Options 6-8 are  discussed in the text.   Fatty acids that are oxidized are converted to acetyl CoA and subsequently to ketone  bodies (acetoacetate and p-hydroxybutyrate). Enough NADH is generated from  oxidation of ethanol and fatty acids that there is no need to oxidize acetyl CoA in  the TCA cycle. The very high NADH/NAD + ratio shifts all of the oxaloacetate in  the TCA cycle to malate, leaving the oxaloacetate levels too low for citrate synthase  to synhesize citrate (see Fig. 25.6, circle 4). The acetyl CoA enters the pathway for  ketone body synthesis instead of the TCA cycle.   Although ketone bodies are being produced at a high rate, their metabolism in  other tissues is restricted by the supply of acetate, which is the preferred fuel. Thus,  the blood concentration of ketone bodies may be much higher than found under  normal fasting conditions.    Al Martini's admitting physician  suspected an alcohol-induced  ketoacidosis superimposed on a  starvation ketoacidosis. Tests showed that  his plasma free fatty acid level was elevated,  and his plasma (3-hydroxybutyrate level was  40 times the upper limit of normal. The  increased NADH/NAD" ratio from ethanol  consumption inhibited the TCA cycle and  shifted acetyl CoA from fatty acid oxidation  into the pathway of ketone body synthesis.   Another consequence of the very high NADH/NAD + ratio is that the balance in the  lactate dehydrogenase reaction is shifted toward lactate, resulting in a lacticacido-  sis (see Fig. 25.6, circle 6). The elevation of blood lactate may decrease excretion  of uric acid (see Fig. 25.6, circle 7) by the kidney. Consequently patients with gout  (which results from precipitated uric acid crystals in the joints) are advised not to  drink excessive amounts of ethanol. Increased degradation of purines also may  contribute to hyperuricemia.   The increased NADH/NAD + ratio also can cause hypoglycemia in a fasting indi-  vidual who has been drinking and is dependent on gluconeogenesis to maintain  blood glucose levels (Fig. 25.6, circles 6 and 8). Alanine and lactate are major glu-  coneogenic precursors that enter gluconeogenesis as pyruvate. The high  NADH/NAD + ratio shifts the lactate dehydrogenase equilibrium to lactate, so that  pyruvate formed from alanine is converted to lactate and cannot enter gluconeoge-  nesis. The high NADH/NAD + ratio also prevents other major gluconeogenic  precursors, such as oxaloacetate and glycerol, from entering the gluconeogenic  pathway.   In contrast, ethanol consumption with a meal may result in a transient hyper-  glycemia, possibly because the high NADH/NAD + ratio inhibits glycolysis at the  glyceraldehyde-3-P dehydrogenase step.   Many of the toxic effects of chronic ethanol consumption result from accumula-  tion of acetaldehyde, which is produced from ethanol both by alcohol dehydroge-  nases and MEOS. Acetaldehyde accumulates in the liver and is released into the  blood after heavy doses of ethanol (Fig. 25.7). It is highly reactive and binds cova-  lently to amino groups, sulfhydryl groups, nucleotides, and phospholipids to form  "adducts."   0 o The noncaloric effect of heavy and  ( /vA J chronic ethanol ingestion that led  Y Ivan Applebod to believe ethanol  has no calories may be partly attributable to  uncoupling of oxidative phosphorylation.  The hepatic mitochondria from tissues of  chronic alcoholics may be partially uncou-  pled and unable to maintain the transmem-  brane proton gradient necessary for normal  rates of ATP synthesis. Consequently, a  greater proportion of the energy in ethanol  would be converted to heat. Metabolic dis-  turbances such as the loss of ketone bodies  in urine, or futile cycling of glucose, also  might contribute to a dimished energy value  for ethanol.   One of the results of acetaldehyde-adduct formation with amino acids is a general  decrease in hepatic protein synthesis (see Fig. 25.7, circle 1). Calmodulin, ribonu-  clease, and tubulin are some of the proteins affected. Proteins in the heart and other  tissues also may be affected by acetaldehyde that appears in the blood.   As a consequence of forming acetaldehyde adducts of tubulin, there is a  diminished secretion of serum proteins and VLDL lipoproteins from the liver.  The liver synthesizes many blood proteins, including serum albumin, blood  coagulation factors, and transport proteins for vitamins, steroids, and iron. These  proteins accumulate in the liver, together with lipid. The accumulation of pro-  teins results in an influx of water (see Fig. 25.7, circle 6) within the hepatocytes  and a swelling of the liver that contributes to portal hypertension and a disrup-  tion of hepatic architecture.   Acetaldehyde adduct formation enhances free radical damage. Acetaldehyde binds  directly to glutathione and diminishes its ability to protect against H 2 0 2 and prevent  lipid peroxidation (see Fig. 25.7, circle 2). It also binds to free radical defense  enzymes.   Damage to mitochondria from acetaldehyde and free radicals perpetuates a  cycle of toxicity (see Fig. 25.7, circles 3 and 4). With chronic consumption of  ethanol, mitochondria become damaged, the rate of electron transport is inhib-  ited, and oxidative phosphorylation tends to become uncoupled. Fatty acid   Fig. 25.7. The development of alcohol-induced hepatitis. (1) Acetaldehyde adduct formation decreases protein synthesis and impairs protein  secretion. (2) Free radical injury results partly from acetaldehyde adduct formation with glutathione. (3) Induction of MEOS increases forma-  tion of free radicals, which leads to lipid peroxidation and cell damage. (4) Mitochondrial damage inhibits the electron transport chain, which  decreases acetaldehyde oxidation. (5) Microtubule damage increases VLDL and protein accumulation. (6) Cell damage leads to release of the  hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST).   oxidation is decreased even further, thereby enhancing lipid accumulation (see  Fig. 25.7, circle 5). The mitochondrial changes further impair mitochondrial  acetaldehyde oxidation, thereby initiating a cycle of progressively increasing  acetaldehyde damage.   Increased oxidative stress in the liver during chronic ethanol intoxication arises  from increased production of free radicals, principally by CYP2E1. FAD and FMN  in the reductase and heme in the cytochrome P450 system transfer single electrons,  thus operating through a mechanism that can generate free radicals. The hydrox-  y ethyl radical (CH 3 CH 2 0) is produced during ethanol metabolism and can be  released as a free radical. Induction of CYP2E1, as well as other cytochrome P450  enzymes, can increase the generation of free radicals from drug metabolism and  from the activation of toxins and carcinogens (see Fig. 25.7, circle 3). These effects  are enhanced by acetaldehyde-adduct damage.   Phospholipids, the major lipid in cellular membranes, are a primary target of per-  oxidation caused by free radical release. Peroxidation of lipids in the inner mito-  chondrial membrane may contribute to the inhibition of electron transport and  uncoupling of mitochondria, leading to inflammation and cellular necrosis. Induc-  tion of CYP2E1 and other P450 cytochromes also increases formation of other  radicals and the activation of hepatocarcinogens.   Because of the possibility of mild  alcoholic hepatitis and perhaps  chronic alcohol-induced cirrhosis,  the physician ordered liver function studies  on Jean Ann Tonich. The tests indicated an  alanine aminotransferase (ALT) level of 46  units/L (reference range = 5-30) and an  aspartate aminotransferase (AST) level of 98  units/L (reference range = 10-30). The con-  centration of these enzymes is high in hepa-  tocytes. When hepatocellular membranes  are damaged in any way, these enzymes are  released into the blood. Jean Ann Tonich's  serum alkaline phosphatase level was 151  units/L (reference range = 56-155 for an  adult female). The serum total bilirubin level  was 2.4 mg/dL (reference range = 0.2-1.0).  These tests show impaired capacity for nor-  mal liver function. Her blood hemoglobin  and hematocrit levels were slightly below  the normal range, consistent with a toxic  effect of ethanol on red blood cell produc-  tion by bone marrow. Serum folate, vitamin  B12 and iron levels were also slightly sup-  pressed. Folate is dependent on the liver for  its activation and recovery from the entero-  hepatic circulation. Vitamin B12 and iron are  dependent on the liver for synthesis of their  blood carrier proteins. Thus, Jean Ann  Tonich shows many of the consequences of  hepatic damage.   In liver fibrosis, disruption of the  normal liver architecture, including  sinusoids, impairs blood from the  portal vein. Increased portal vein pressure  (portal hypertension) causes capillaries to  anastomose (to meet and unite or run into  each other) and form thin-walled dilated  esophageal venous conduits known as  esophageal varices. When these burst, there  is hemorrhaging into the gastrointestinal  tract. The bleeding can be very profuse  because of the high venous pressure within  these varices in addition to the adverse  effect of impaired hepatic function on the  production of blood clotting proteins.   Liver injury is irreversible at the stage that hepatic cirrhosis develops. Initially the  liver may be enlarged, full of fat, crossed with collagen fibers (fibrosis), and have  nodules of regenerating hepatocytes ballooning between the fibers. As liver function  is lost, the liver becomes shrunken (Laennec's cirrhosis). During the development  of cirrhosis, many of the normal metabolic functions of the liver are lost, including  biosynthetic and detoxification pathways. Synthesis of blood proteins, including  blood coagulation factors and serum albumin, is decreased. The capacity to incor-  porate amino groups into urea is decreased, resulting in the accumulation of toxic  levels of ammonia in the blood. Conjugation and excretion of the yellow pigment  bilirubin (a product of heme degradation) is diminished, and bilirubin accumulates  in the blood. It is deposited in many tissues, including the skin and sclerae of the  eyes, causing the patient to become visibly yellow. Such a patient is said to be  jaundiced.   Ivan Applebod. When ethanol consumption is low (less than 15% of  the calories in the diet), it is efficiently used to produce ATP, thereby con-  tributing to Ivan Applebod's weight gain. However, in individuals with  chronic consumption of large amounts of ethanol, the caloric content of ethanol is  not converted to ATP as effectively. Some of the factors that may contribute to this  decreased efficiency include mitochondrial damage (inhibition of oxidative phos-  phorylation and uncoupling) resulting in the loss of calories as heat, increased  recycling of metabolites such as ketone bodies, and inhibition of the normal path-  ways of fatty acid and glucose oxidation. In addition, heavier drinkers metabolize  an increased amount of alcohol through MEOS, which generates less ATP.   Al Martini. Al Martini was suffering from acute effects of high ethanol  ingestion in the absence of food intake. Both heavy ethanol consumption  and low caloric intake increase adipose tissue lipolysis and elevate blood  fatty acids. As a consequence of his elevated hepatic NADH/NAD + ratio, acetyl  CoA produced from fatty acid oxidation was diverted from the TCA cycle into the  pathway of ketone body synthesis. Because his skeletal muscles were using acetate as  a fuel, ketone body utilization was diminished, resulting in ketoacidosis. Al Martini's  moderately low blood glucose level also suggests that his high hepatic NADH level  prevented pyruvate and glycerol from entering the gluconeogenic pathway. Pyru-  vate is diverted to lactate, which may have contributed to his metabolic acidosis and  anion gap.   Rehydration with intravenous fluids containing glucose and potassium was  initiated. His initial potassium was low, possibly secondary to vomiting. An  orthopedic surgeon was consulted regarding the compound fracture of his right  forearm.   Jean Ann Tonich. Jean Ann Tonich's signs and symptoms, as well as  her laboratory profile, were consistent with the presence of mild  reversible alcohol-induced hepatocellular inflammation (alcohol-induced  hepatitis) superimposed on a degree of irreversible scarring of liver tissues known  as chronic alcoholic (Laennec's) cirrhosis of the liver. The chronic inflammatory  process associated with long-term ethanol abuse in patients such as Jean Ann  Tonich is accompanied by increases in the levels of serum alanine aminotrans-  ferase (ALT) and aspartate aminotransferase (AST). Her elevated bilirubin and  alkaline phosphatase were consistent with hepatic damage. Her values for ALT and   AST were significantly below those seen in acute viral hepatitis. In addition, the  ratio of the absolute values for serum ALT and AST often differ in the two diseases,  tending to be greater than 1 in acute viral hepatitis and less than 1 in chronic alco-  hol-induced cirrhosis. The reason for the difference in ratio of enzyme activities  released is not understood, but a lower level of ALT in the serum may be attribut-  able to an alcohol-induced deficiency of pyridoxal phosphate. In addition, sero-  logic tests for viral hepatitis were nonreactive. Her serum folate, vitamin B 12, and  iron levels were also slightly suppressed, indicating impaired nutritional status.   Jean Ann Tonich was strongly cautioned to abstain from alcohol immediately  and to improve her nutritional status. In addition, Jean Ann was referred to the hos-  pital drug and alcohol rehabilitation unit for appropriate psychological therapy and  supportive social counseling. The physician also arranged for a follow-up office  visit in 2 weeks.   ft Fibrosis in Chronic Alcohol-Induced Liver Disease Fibrosis is  ) the excessive accumulation of connective tissue in parenchymal organs. In the  liver, it is a frequent event following a repeated or chronic insult of sufficient  intensity (such as chronic ethanol intoxication or infection by a hepatitis virus) to trig-  ger a "wound healing-like" reaction. Regardless of the insult, the events are similar: an  overproduction of extracellular matrix components occurs, with the tendency to  progress into sclerosis, accompanied by a degenerative alteration in the composition of  matrix components. (Table 25.2) Some individuals (fewer than 20% of those who  chronically consume alcohol) go on to develop cirrhosis.   The development of hepatic fibrosis after ethanol consumption is related to stimu-  lation of the mitogenic development of stellate (Ito) cells into myofibroblasts, and stim-  ulation of the production of collagen type I and fibronectin by these cells. The stellate  cells are perisinusoidal cells lodged in the space of Disse that produce extracellular  matrix protein. Normally the space of Disse contains basement membrane— like colla-  gen (collagen type IV) and laminin. As the stellate cells are activated, they change from  a resting cell filled with lipids and vitamin A to one that proliferates, loses its vitamin  A content, and secretes large quantities of extracellular matrix components.   One of the initial events in the activation and proliferation of stellate cells is the  activation of Kupffer cells, which are macrophages resident in the liver sinusoids   Although the full spectrum of alco-  \ \ ) hol-induced liver disease may be  present in a well-nourished individ-  ual, the presence of nutritional deficiencies  enhances the progression of the disease.  Ethanol creates nutritional deficiencies in a  number of different ways. The ingestion of  ethanol reduces the gastrointestinal absorp-  tion of foods containing essential nutrients,  including vitamins, essential fatty acids, and  essential amino acids. For example, ethanol  interferes with absorption of folate, thi-  amine, and other nutrients. Secondary mal-  absorption can occur through gastrointesti-  nal complications, pancreatic insufficiency,  and impaired hepatic metabolism or  impaired hepatic storage of nutrients, such  as vitamin A. Changes in the level of trans-  port proteins produced by the liver also  strongly affect nutrient status.   Distortion of sinusoidal architecture and parenchymal damage  Cirrhosis: End-stage process of liver fibrotic degeneration   Fig. 25.8. Proposed model for the development of hepatic fibrosis involving hepatocytes,  Kupffer cells, and stellate (Ito) cells. ROS, reactive oxygen species; NO, nitric oxide:  TGFpl, transforming growth factor pi.   Cytokines are proteins produced  rj ) by inflammatory cells that serve as   communicators with other cells.  Chemokines are even smaller proteins pro-  duced by inflammatory cells that promote  migration of other inflammatory cells (e.g.,  from the blood into the site of injury).   (Fig. 25. 8). The Kupffer cells are probably activated by a product of the damaged  hepatocytes, such as necrotic debris, iron, ROS, acetaldehyde, or aldehyde products  of lipid peroxidation. Kupffer cells also may produce acetaldehyde from ethanol  internally through their own MEOS pathway.   Activated Kupffer cells produce a number of products that contribute to activation  of stellate cells. They generate additional ROS through NADPH oxidase during the  oxidative burst and NOS through inducible NO synthase (see Chapter 24). In addition,  they secrete an impressive array of growth factors, such as cytokines, chemokines,  prostaglandins, and other reactive molecules. The cytokine transforming growth factor  pi (TGFpi), produced by both Kupffer cells and sinusoidal endothelial cells, is a  major player in the activation of stellate cells. Once activated, the stellate cells produce  collagen and proteases, leading to an enhanced fibrotic network within the liver.   Lieber CS. Medical disorders of alcoholism. New England J Med 1995;33:1058-1065.   Lieber CS. Cytochrome P-4502E1: Its physiological and pathological role. Physiol Rev   Poli G. Pathogenesis of liver fibrosis: Role of oxidative stress. Mol Aspects Med 2000;21:49-98.   1 . The fate of acetate, the product of ethanol metabolism, is which of the following?   (A) It is taken up by other tissues and activated to acetyl CoA.   (B) It is toxic to the tissues of the body and can lead to hepatic necrosis.   2. Which of the following would be expected to occur after acute alcohol ingestion?   3. A chronic alcoholic is in treatment for alcohol abuse. The drug disulfiram is prescribed for the patient. This drug deters the  consumption of alcohol by which of the following mechanisms?   (A) Inhibiting the absorption of ethanol so that an individual cannot become intoxicated, regardless of how much he drinks   (B) Inhibiting the conversion of ethanol to acetaldehyde, which would cause the excretion of unmetabolized ethanol   (C) Blocking the conversion of acetaldehyde to acetate, which causes the accumulation of acetaldehyde   (D) Activating the excessive metabolism of ethanol to acetate, which causes inebriation with consumption of a small amount  of alcohol   (E) Preventing the excretion of acetate, which causes nausea and vomiting   4. Induction of CYP2E1 would result in which of the following?   5. Which one of the following consequences of chronic alcohol consumption is irreversible?   Glucose is central to all of metabolism. It is the universal fuel for human  cells and the source of carbon for the synthesis of most other com-  pounds. Every human cell type uses glucose to obtain energy. The  release of insulin and glucagon by the pancreas aids in the body's use  and storage of glucose. Other dietary sugars (mainly fructose and  galactose) are converted to glucose or to intermediates of glucose   Glucose is the precursor for the synthesis of an array of other sugars required for  the production of specialized compounds, such as lactose, cell surface antigens,  nucleotides, or glycosaminoglycans. Glucose is also the fundamental precursor of  noncarbohydrate compounds; it can be converted to lipids (including fatty acids,  cholesterol, and steroid hormones), amino acids, and nucleic acids. Only those com-  pounds that are synthesized from vitamins, essential amino acids, and essential fatty  acids cannot be synthesized from glucose in humans.   More than 40% of the calories in the typical diet in the United States are obtained  from starch, sucrose, and lactose. These dietary carbohydrates are converted to glucose,  galactose, and fructose in the digestive tract (Fig. 1). Monosaccharides are absorbed  from the intestine, enter the blood, and travel to the tissues where they are metabolized.   After glucose is transported into cells, it is phosphorylated by a hexokinase to  form glucose 6-phosphate. Glucose 6-phosphate can then enter a number of meta-  bolic pathways. The three that are common to all cell types are glycolysis, the pen-  tose phosphate pathway, and glycogen synthesis (Fig. 2). In tissues, fructose and  galactose are converted to intermediates of glucose metabolism. Thus, the fate of  these sugars parallels that of glucose (Fig. 3).   The major fate of glucose 6-phosphate is oxidation via the pathway of glycoly-  sis (see Chapter 22), which provides a source of ATP for all cell types. Cells that  lack mitochondria cannot oxidize other fuels. They produce ATP from anaerobic  glycolysis (the conversion of glucose to lactic acid). Cells that contain mitochondria   Fig 1. Overview of carbohydrate digestion. The  major carbohydrates of the diet (starch, lactose,  and sucrose) are digested to produce monosac-  charides (glucose, fructose, and galactose),  which enter the blood.   Fig 4. Conversion of glucose to lactate or to  C0 2 . ETC = electron transport chain.   Fig 5. Conversion of glucose to amino acids  and to the glycerol and fatty acid (FA) moieties  of triacylglycerols (TG). OAA = oxaloacetate.   Fig 3. Overview of fructose and galactose metabolism. Fructose and galactose are converted  to intermediates of glucose metabolism.   oxidize glucose to C0 2 and H 2 0 via glycolysis and the TCA cycle (Fig. 4). Some  tissues, such as the brain, depend on the oxidation of glucose to C0 2 and H 2 0 for  energy because they have a limited capacity to use other fuels.   Glucose produces the intermediates of glycolysis and the TCA cycle that are  used for the synthesis of amino acids and both the glycerol and fatty acid moieties  of triacylglycerols (Fig. 5).   Another important fate of glucose 6-phosphate is oxidation via the pentose phos-  phate pathway, which generates NADPH. The reducing equivalents of NADPH are  used for biosynthetic reactions and for the prevention of oxidative damage to cells  (see Chapter 24). In this pathway, glucose is oxidatively decarboxylated to 5 -carbon  sugars (pentoses), which may reenter the glycolytic pathway. They also may be  used for nucleotide synthesis (Fig. 6). There are also non-oxidative reactions, which  can convert six- and five-carbon sugars.   Fig 6. Overview of the pentose phosphate pathway. The oxidative reactions generate both  NADPH and pentose phosphates. The non-oxidative reactions only generate pentose  phosphates.   Glucose 6-phosphate is also converted to UDP-glucose, which has many func-  tions in the cell (Fig. 7). The major fate of UDP-glucose is the synthesis of glyco-  gen, the storage polymer of glucose. Although most cells have glycogen to provide  emergency supplies of glucose, the largest stores are in muscle and liver. Muscle  glycogen is used to generate ATP during muscle contraction. Liver glycogen is used  to maintain blood glucose during fasting and during exercise or periods of enhanced  need. UDP-Glucose is also used for the formation of other sugars, and galactose and  glucose are interconverted while attached to UDP. UDP-Galactose is used for lac-  tose synthesis in the mammary gland. In the liver, UDP-glucose is oxidized to UDP-  glucuronate, which is used to convert bilirubin and other toxic compounds to glu-  curonides for excretion (see Fig. 7).   Nucleotide sugars are also used for the synthesis of proteoglycans, glycopro-  teins, and glycolipids (see Fig. 7). Proteoglycans are major carbohydrate compo-  nents of the extracellular matrix, cartilage, and extracellular fluids (such as the syn-  ovial fluid of joints), and they are discussed in more detail in Chapter 49. Most  extracellular proteins are glycoproteins, i.e., they contain covalently attached car-  bohydrates. For both cell membrane glycoproteins and glycolipids, the carbohy-  drate portion extends into the extracellular space.   All cells are continuously supplied with glucose under normal circumstances;  the body maintains a relatively narrow range of glucose concentration in the blood  (approximately 80-100 mg/dL) in spite of the changes in dietary supply and tissue  demand as we sleep and exercise. This process is called glucose homeostasis. Low  blood glucose levels (hypoglycemia) are prevented by a release of glucose from the  large glycogen stores in the liver (glycogenolysis); by synthesis of glucose from lac-  tate, glycerol, and amino acids in liver (gluconeogenesis) (Fig. 8); and to a limited  extent by a release of fatty acids from adipose tissue stores (lipolysis) to provide an  alternate fuel when glucose is in short supply. High blood glucose levels (hyper-  glycemia) are prevented both by the conversion of glucose to glycogen and by its  conversion to triacylglycerols in liver and adipose tissue. Thus, the pathways for  glucose utilization as a fuel cannot be considered as totally separate from pathways  involving amino acid and fatty acid metabolism (Fig. 9).    Fig 8. Production of blood glucose from  glycogen (by glycogenolysis) and from ala-  nine, lactate, and glycerol (by gluconeogene-  sis). PEP = phosphoenolpyruvate; OAA =  oxaloacetate.   Fig 9. Overview of the major pathways of glucose metabolism. Pathways for production of blood glucose are shown by  dashed lines. FA = fatty acids; TG = triacylglycerols; OAA = oxaloacetate; PEP = phosphoenolpyruvate; UDP-G =  UDP-glucose; DHAP = dihydroxyacetone phosphate.   Intertissue balance in the utilization and storage of glucose during fasting and  feeding is accomplished principally by the actions of the hormones of metabolic  homeostasis — insulin and glucagon (Fig. 10). However, Cortisol, epinephrine, nor-  epinephrine, and other hormones are also involved in intertissue adjustments of  supply and demand in response to changes of physiologic state.   Fig 10. Pathways regulated by the release of glucagon (in response to a lowering of blood  glucose levels) and insulin (released in response to an elevation of blood glucose levels).  Tissue-specific differences occur in the response to these hormones, as detailed in the subse-  quent chapters of this section.   All cells continuously use adenosine triphosphate (ATP) and require a constant  supply of fuels to provide energy for ATP generation. Insulin and glucagon are  the two major hormones that regulate fuel mobilization and storage. Their func-  tion is to ensure that cells have a constant source of glucose, fatty acids, and  amino acids for ATP generation and for cellular maintenance (Fig. 26.1).   Because most tissues are partially or totally dependent on glucose for ATP  generation and for production of precursors of other pathways, insulin and  glucagon maintain blood glucose levels near 80 to 100 mgldL (90 mgldL is the  same as 5 mM), despite the fact that carbohydrate intake varies considerably over  the course of a day. The maintenance of constant blood glucose levels (glucose  homeostasis) requires these two hormones to regulate carbohydrate, lipid, and  amino acid metabolism in accordance with the needs and capacities of individual  tissues. Basically, the dietary intake of all fuels in excess of immediate need is  stored, and the appropriate fuel is mobilized when a demand occurs. For example,  when dietary glucose is not available in sufficient quantities that all tissues can  use it, fatty acids are mobilized and made available to skeletal muscle for use as a  fuel (see Chapters 2 and 23), and the liver can convert fatty acids to ketone bod-  ies for use by the brain. Fatty acids spare glucose for use by the brain and other  glucose-dependent tissues (such as the red blood cell).   The concentrations of insulin and glucagon in the blood regulate fuel storage  and mobilization (Fig. 26.2). Insulin, released in response to carbohydrate inges-  tion, promotes glucose utilization as a fuel and glucose storage as fat and glyco-  gen. Insulin is also the major anabolic hormone of the body. It increases protein  synthesis and cell growth in addition to fuel storage. Blood insulin levels decrease  as glucose is taken up by tissues and used. Glucagon, the major counter-  regulatory hormone of insulin, is decreased in response to a carbohydrate meal  and elevated during fasting. Its concentration in the blood signals the absence of  dietary glucose, and it promotes glucose production via glycogenolysis ( glycogen  degradation) and glue oncogenesis (glucose synthesis from amino acids and other  none arbohydr ate precursors). Increased levels of glucagon relative to insulin also  stimulate the mobilization of fatty acids from adipose tissue. Epinephrine (the  fight or flight hormone) and Cortisol (a glucocorticoid released from the adrenal  cortex in response to fasting and chronic stress) have effects on fuel metabolism  that oppose those of insulin. These two hormones are therefore also considered  insulin counterregulatory hormones.   Insulin and glucagon are polypeptide hormones synthesized as prohormones in  the /3 and a cells, respectively, in the islets of Langerhans in the pancreas. Proin-  sulin is cleaved into mature insulin and C-peptide in vesicles and precipitated   Fig 26.2. Insulin and the insulin counterregu-  latory hormones. (A) Insulin promotes glucose  storage, as triglyceride (TG) or glycogen. (B)  Glucagon, epinephrine, and Cortisol promote  glucose release from the liver, activating  glycogenolysis and gluconeogenesis.   Fig 26.3. Cellular response to glucagon,  which is released from the pancreas in  response to a decrease in blood glucose levels.   with Zn 2+ . Insulin secretion is regulated principally by blood glucose levels.  Glucagon is also synthesized as a prohormone and cleaved into mature glucagon  within storage vesicles. Its release is regulated principally through suppression by  glucose and by insulin.   Glucagon exerts its effects on cells by binding to a receptor on the cell surface,  which stimulates the synthesis of the intracellular second messenger, cyclic adeno-  sine monophosphate (cAMP) (Fig. 26.3). cAMP activates protein kinase A, which  phosphorylates key regulatory enzymes, activating some and inhibiting others.  Changes of c AMP levels also induce or repress the synthesis of a number of  enzymes. Insulin promotes the dephosphorylation of these key enzymes.   Insulin binds to a receptor on the cell surface, but the postreceptor events that fol-  low differ from those stimulated by glucagon. Insulin binding activates both  autophosphorylation of the receptor and the phosphorylation of other enzymes by the  receptor s tyrosine kinase domain (see Chapter 11, section III.B.3). The complete  routes for signal transduction between this point and the final effects of insulin on  the regulatory enzymes of fuel metabolism have not yet been fully established.   Ann Sulin returned to her physician for her monthly office visit. She has  been seeing her physician for over a year because of obesity and elevated  blood glucose levels. She still weighed 198 lb, despite her insistence that  she had adhered strictly to her diet. Her blood glucose level at the time of the visit,  2 hours after lunch, was 180 mg/dL (reference range = 80-140).   Bea Selmass is a 46-year-old woman who 6 months earlier began noting  episodes of fatigue and confusion as she finished her daily pre-breakfast  jog. These episodes were occasionally accompanied by blurred vision and  an unusually urgent hunger. The ingestion of food relieved all of her symptoms  within 25 to 30 minutes. In the last month, these attacks have occurred more fre-  quently throughout the day, and she has learned to diminish their occurrence by eat-  ing between meals. As a result, she has recently gained 8 lb.   A random serum glucose level done at 4:30 pm during her first office visit was  subnormal at 46 mg/dL. Her physician, suspecting she had a form of fasting hypo-  glycemia, ordered a series of fasting serum glucose levels. In addition, he asked Bea  to keep a careful daily diary of all of the symptoms that she experienced when her  attacks were most severe.   Living cells require a constant source of fuels from which to derive ATP for the  maintenance of normal cell function and growth. Therefore, a balance must be  achieved between carbohydrate, fat, and protein intake, their storage when present  in excess of immediate need, and their mobilization and synthesis when in demand.  The balance between need and availability is referred to as metabolic homeostasis  (Fig. 26.4). The intertissue integration required for metabolic homeostasis is  achieved in three principal ways:   • The concentration of nutrients or metabolites in the blood affects the rate at  which they are used and stored in different tissues;   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   • Hormones carry messages to individual tissues about the physiologic state of the  body and nutrient supply or demand;   • The central nervous system uses neural signals to control tissue metabolism,  directly or through the release of hormones.   Insulin and glucagon are the two major hormones that regulate fuel storage and  mobilization (see Fig. 26.2). Insulin is the major anabolic hormone of the body. It  promotes the storage of fuels and the utilization of fuels for growth. Glucagon is the  major hormone of fuel mobilization. Other hormones, such as epinephrine, are  released as a response of the central nervous system to hypoglycemia, exercise, or  other types of physiologic stress. Epinephrine and other stress hormones also  increase the availability of fuels (Fig. 26.5).   The special role of glucose in metabolic homeostasis is dictated by the fact that  many tissues (e.g., the brain, red blood cells, the lens of the eye, the kidney medulla,  exercising skeletal muscle) are dependent on glycolysis for all or a portion of their  energy needs and require uninterrupted access to glucose on a second-to-second  basis to meet their rapid rate of ATP utilization. In the adult, a minimum of 190 g  glucose is required per day; approximately 150 g for the brain and 40 g for other  tissues. Significant decreases of blood glucose below 60 mg/dL limit glucose  metabolism in the brain and elicit hypoglycemic symptoms (as experienced by Bea  Selmass), presumably because the overall process of glucose flux through the  blood-brain barrier, into the interstitial fluid, and subsequently into the neuronal  cells, is slow at low blood glucose levels because of the K m values of the glucose  transporters required for this to occur (see Chapter 27)   The continuous movement of fuels into and out of storage depots is necessitated  by the high amounts of fuel required each day to meet the need for ATP. Disastrous  results would occur if even a day's supply of glucose, amino acids, and fatty acids  were left circulating in the blood. Glucose and amino acids would be at such high  concentrations that the hyperosmolar effect would cause progressively severe neu-  rologic deficits and even coma. The concentration of glucose and amino acids  would be above the renal tubular threshold for these substances (the maximal con-  centration in the blood at which the kidney can completely resorb metabolites), and  some of these compounds would be wasted as they spilled over into the urine.  Nonenzymatic glycosylation of proteins would increase at higher blood glucose   Fatty acids provide an example of  the influence that the level of a  compound in the blood has on its  own rate of metabolism. The concentration  of fatty acids in the blood is the major factor  determining whether skeletal muscles will  use fatty acids or glucose as a fuel (see  Chapter 23). In contrast, hormones are (by  definition) carriers of messages between tis-  sues. Insulin and glucagon, for example, are  two hormonal messengers that participate  in the regulation of fuel metabolism by car-  rying messages that reflect the timing and  composition of our dietary intake of fuels.  Epinephrine, however, is a flight-or-flight  hormone that signals an immediate need for  increased fuel availability. Its level is regu-  lated principally through the activation of  the sympathetic nervous system.   Fig 26.4. Metabolic homeostasis. The balance  between fuel availability and the needs of tis-  sues for different fuels is achieved by three  types of messages: the level of the fuel or  nutrients in the blood, the level of one of the  hormones of metabolic homeostasis, or nerve  impulses that affect tissue metabolism or the  release of hormones.   Hyperglycemia may cause a con-  stellation of symptoms such as  polyuria and subsequent polydip-  sia (increased thirst). The inability to move  glucose into cells necessitates the oxidation  of lipids as an alternative fuel. As a result  adipose stores are used, and the patient with  poorly controlled diabetes mellitus loses  weight in spite of a good appetite. Extremely  high levels of serum glucose can cause non-  ketotic hyperosmolar coma in patients with  type 2 diabetes mellitus. Such patients usu-  ally have sufficient insulin responsiveness to  block fatty acid release and ketone body for-  mation, but they are unable to significantly  stimulate glucose entry into peripheral tis-  sues. The severely elevated levels of glucose  in the blood compared with inside the cell  leads to an osmotic effect that causes water  to leave the cells and enter the blood.  Because of the osmotic diuretic effect of  hyperglycemia, the kidney produces more  urine, leading to dehydration, which in turn  may lead to even higher levels of blood glu-  cose. If dehydration becomes severe, further  cerebral dysfunction occurs and the patient  may become comatose. Chronic hyper-  glycemia also produces pathologic effects  through the nonenzymatic glycosylation of a  variety of proteins. Hemoglobin A (HbA),  one of the proteins that becomes glycosy-  lated, forms HbA 1c (see Chapter 7). Ann  Sulin's high levels of HbA 1c (14% of the total  HbA, compared with the reference range of  4.7-6.4%) indicate that her blood glucose  has been significantly elevated over the last  12 to 14 weeks, the half-life of hemoglobin in  the bloodstream.   All membrane and serum proteins  exposed to high levels of glucose in the  blood or interstitial fluid are candidates for  nonenzymatic glycosylation. This process  distorts protein structure and slows protein  degradation, which leads to an accumula-  tion of these products in various organs,  thereby adversely affecting organ function.  These events contribute to the long-term  microvascular and macrovascular complica-  tions of diabetes mellitus, which include dia-  betic retinopathy, nephropathy, and neu-  ropathy (microvascular), in addition to  coronary artery, cerebral artery, and periph-  eral artery insufficiency (macrovascular).    levels. Triacylglycerols circulate in cholesterol-containing lipoproteins, and the lev-  els of these lipoproteins would be chronically elevated, increasing the likelihood of  atherosclerotic vascular disease. Consequently, glucose and other fuels are continu-  ously moved in and out of storage depots as needed.   The hormones that contribute to metabolic homeostasis respond to changes in the  circulating levels of fuels that, in part, are determined by the timing and composi-  tion of our diet. Insulin and glucagon are considered the major hormones of meta-  bolic homeostasis because they continuously fluctuate in response to our daily eat-  ing pattern. They provide good examples of the basic concepts of hormonal  regulation. Certain features of the release and action of other insulin counterregula-  tory hormones, such as epinephrine, norepinephrine, and Cortisol, will be described  and compared with insulin and glucagon.   Insulin is the major anabolic hormone that promotes the storage of nutrients: glu-  cose storage as glycogen in liver and muscle, conversion of glucose to triacylglyc-  erols in liver and their storage in adipose tissue, and amino acid uptake and protein  synthesis in skeletal muscle (Fig. 26.6). It also increases the synthesis of albumin  and other blood proteins by the liver. Insulin promotes the utilization of glucose as  a fuel by stimulating its transport into muscle and adipose tissue. At the same time,  insulin acts to inhibit fuel mobilization.   Glucagon acts to maintain fuel availability in the absence of dietary glucose by  stimulating the release of glucose from liver glycogen (see Chapter 28), by stimulat-  ing gluconeogenesis from lactate, glycerol, and amino acids (see Chapter 31), and,  in conjunction with decreased insulin, by mobilizing fatty acids from adipose tria-  cylglycerols to provide an alternate source of fuel (see Chapter 23 and Fig. 26.7). Its  sites of action are principally the liver and adipose tissue; it has no influence on  skeletal muscle metabolism because muscle cells lack glucagon receptors.   The release of insulin from the beta cells of the pancreas is dictated primarily  by the blood glucose level. The highest levels of insulin occur approximately 30  to 45 minutes after a high-carbohydrate meal (Fig. 26.8). They return to basal  levels as the blood glucose concentration falls, approximately 120 minutes after  the meal. The release of glucagon from the alpha cells of the pancreas, con-  versely, is controlled principally through suppression by glucose and insulin.  Therefore, the lowest levels of glucagon occur after a high-carbohydrate meal.  Because all of the effects of glucagon are opposed by insulin, the simultaneous  stimulation of insulin release and suppression of glucagon secretion by a high-  carbohydrate meal provides an integrated control of carbohydrate, fat, and  protein metabolism.   Bea Selmass's studies confirmed that her fasting serum glucose levels were  below normal. She continued to experience the fatigue, confusion, and blurred  vision she had described on her first office visit. These symptoms are called  neuroglycopenic (neurologic symptoms resulting from an inadequate supply of glucose  to the brain for the generation of ATP).   Bea also noted the symptoms that are part of the adrenergic response to hypo-  glycemic stress. Stimulation of the sympathetic nervous system (because of the low lev-  els of glucose reaching the brain) results in the release of epinephrine, a stress hormone,  from the adrenal medulla. Elevated epinephrine levels cause tachycardia, palpitations,  anxiety, tremulousness, pallor, and sweating.   In addition to the symptoms described by Bea Selmass, individuals may experience  confusion, lightheadedness, headache, aberrant behavior, blurred vision, loss of con-  sciousness, or seizures.   Ms. Selmass's doctor explained that the general diagnosis of "fasting" hypoglycemia  was now established and that a specific cause for this disorder must be found.    CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES    Fig 26.6. Major sites of insulin action on fuel metabolism. + = stimulated by insulin;  — = inhibited by insulin.   Insulin and glucagon are not the only regulators of fuel metabolism. The inter-  tissue balance between the utilization and storage of glucose, fat, and protein is also  accomplished by the circulating levels of metabolites in the blood, by neuronal sig-  nals, and by the other hormones of metabolic homeostasis (epinephrine, norepi-  nephrine, Cortisol, and others) (Table 26.1). These hormones oppose the actions of  insulin by mobilizing fuels. Like glucagon, they are called insulin counterregulatory  hormones (Fig. 26.9). Of all these hormones, only insulin and glucagon are synthe-    Fig 26.7. Major sites of glucagon action in fuel metabolism. + = pathways stimulated by  glucagon; — = pathways inhibited by glucagon.   The message carried by glucagon  is that "glucose is gone"; i.e., the  current supply of glucose is inade-  quate to meet the immediate fuel require-  ments of the body.   • Stimulates fatty acid synthesis and  storage after a high-carbohydrate meal   Fig 26.8. Blood glucose, insulin, and glucagon  levels after a high-carbohydrate meal.   Fig 26.9. Major insulin counterregulatory hormones. The stress of a low blood glucose level  mediates the release of the major insulin counterregulatory hormones through neuronal sig-  nals. Hypoglycemia is one of the stress signals that stimulates the release of Cortisol, epi-  nephrine, and norepinephrine. Adrenocorticotropic hormone (ACTH) is released from the  pituitary and stimulates the release of Cortisol (a glucocorticoid) from the adrenal cortex.  Neuronal signals stimulate the release of epinephrine from the adrenal medulla and norepi-  nephrine from nerve endings. Neuronal signals also play a minor role in the release of  glucagon. Although norepinephrine has counterregulatory actions, it is not a major counter-  regulatory hormone.   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   sized and released in direct response to changing levels of fuels in the blood. The  release of Cortisol, epinephrine, and norepinephrine is mediated by neuronal sig-  nals. Rising levels of the insulin counterregulatory hormones in the blood, reflect,  for the most part, a current increase in the demand for fuel.   Insulin and glucagon are synthesized in different cell types of the endocrine pancreas,  which consists of microscopic clusters of small glands, the islets of Langerhans, scat-  tered among the cells of the exocrine pancreas. The a cells secrete glucagon, and the  p cells secrete insulin into the hepatic portal vein via the pancreatic veins.   Insulin is a polypeptide hormone. The active form of insulin is composed of two  polypeptide chains (the A-chain and the B -chain) linked by two interchain disulfide  bonds. The A-chain has an additional intrachain disulfide bond (Fig. 26.10).   Insulin, like many other polypeptide hormones, is synthesized as a preprohormone  that is converted in the rough endoplasmic reticulum (RER) to proinsulin. The "pre"  sequence, a short hydrophobic signal sequence at the N-terminal end, is cleaved as it  enters the lumen of the RER. Proinsulin folds into the proper conformation, and disul-  fide bonds are formed between the cysteine residues. It is then transported in  microvesicles to the Golgi complex. It leaves the Golgi complex in storage vesicles,  where a protease removes the C-peptide (a fragment with no hormonal activity) and  a few small remnants, resulting in the formation of biologically active insulin (see  Fig. 26.10). Zinc ions are also transported in these storage vesicles. Cleavage of the  C-peptide decreases the solubility of the resulting insulin, which then coprecipitates  with zinc. Exocytosis of the insulin storage vesicles from the cytosol of the p cell into  the blood is stimulated by rising levels of glucose in the blood bathing the p cells.   Glucose enters the p cell via specific glucose transporter proteins known as  GLUT2 (see Chapter 27). Glucose is phosphorylated through the action of glucok-  inase to form glucose 6-phosphate, which is metabolized through glycolysis, the  TCA cycle, and oxidative phosphorylation. These reactions result in an increase in  ATP levels within the p cell (circle 1 in Fig. 26.1 1). As the p cell [ATP]/[ADP] ratio  increases, the activity of a membrane-bound, ATP-dependent K + channel (K + ATP )  is inhibited (i.e., the channel is closed)(circle 2 in Fig. 26.11). The closing of this  channel leads to a membrane depolarization (circle 3, Fig. 26.11), which activates  a voltage-gated Ca 2+ channel that allows Ca 2+ to enter the p cell such that intracel-  lular Ca 2+ levels increase significantly (circle 4, Fig. 26.11). The increase in intra-  cellular Ca 2+ stimulates the fusion of insulin containing exocytotic vesicles with the  plasma membrane, resulting in insulin secretion (circle 5, Fig. 26.11). Thus, an  increase in glucose levels within the p cells initiates insulin release.   A form of diabetes known as MODY (maturity onset diabetes of the young)  results from mutations in either pancreatic glucokinase or specific nuclear tran-  scription factors. MODY type 2 is due to a glucokinase mutation that results in  an enzyme with reduced activity, either due to an elevated K m for glucose or a reduced  V max for the reaction. Because insulin release is dependent on normal glucose metabo-  lism within the (3 cell that yields a critical [ATP]/[ADP] ratio in the (3 cell, individuals with  this glucokinase mutation cannot significantly metabolize glucose unless glucose levels  are higher than normal. Thus, although these patients can release insulin, they do so at  higher than normal glucose levels, and are therefore almost always in a hyperglycemic  state. Interestingly, however, these patients are somewhat resistant to the long-term  complications of chronic hyperglycemia.    The message that insulin carries to  tissues is that glucose is plentiful  and it can be used as an immediate  fuel or can be converted to storage forms  such as triacylglycerol in adipocytes or  glycogen in liver and muscle.   Because insulin stimulates the uptake of  glucose into tissues where it may be immedi-  ately oxidized or stored for later oxidation, this  regulatory hormone lowers blood glucose lev-  els. Therefore, one of the possible causes of  Bea Selmass's hypoglycemia is an insulinoma,  a tumor that produces excessive insulin.   Whenever an endocrine gland con-  tinues to release its hormone in  spite of the presence of signals that  normally would suppress its secretion, this  persistent inappropriate release is said to be  "autonomous." Secretory neoplasms of  endocrine glands generally produce their  hormonal product autonomously in a  chronic fashion.   Autonomous hypersecretion of insulin  from a suspected pancreatic p-cell tumor (an  insulinoma) can be demonstrated in several  ways. The simplest test is to simultaneously  draw blood for the measurement of both glu-  cose and insulin at a time when the patient is  spontaneously experiencing the characteris-  tic adrenergic or neuroglycopenic symptoms  of hypoglycemia. During such a test, Bea  Selmass's glucose levels fell to 45 mg/dL  (normal = 80-100), and her ratio of insulin to  glucose was far higher than normal. The ele-  vated insulin levels markedly increased glu-  cose uptake by the peripheral tissues, result-  ing in a dramatic lowering of blood glucose  levels. In normal individuals, as blood glu-  cose levels drop, insulin levels also drop.   Di Abietes has type 1 diabetes melli-  tus, formerly known as insulin-  dependent diabetes mellitus (IDDM).  This metabolic disorder is usually caused by  antibody-mediated (autoimmune) destruction  of the p cells of the pancreas. Susceptibility to  type 1 diabetes mellitus is, in part, conferred  by a genetic defect in the human leukocyte  antigen (HLA) region of (3 cells which codes for  the major histocompatibility complex II (MHC  II). This protein presents an intracellular anti-  gen to the cell surface for "self-recognition" by  the cells involved in the immune response.  Because of this defective protein, a cell-medi-  ated immune response leads to varying  degrees of p cell destruction and eventually to  dependence on exogenous insulin administra-  tion to control the levels of glucose in the  blood.    Fig 26.10. Cleavage of proinsulin to insulin. Proinsulin is converted to insulin by proteolytic cleavage, which removes the C-peptide and a few  additional amino acid residues. Cleavage occurs at the arrows. From Murray RK, et al. Harper's Biochemistry, 23rd Ed. Stanford, CT: Appleton  &Lange, 1993:560.   The release of insulin occurs within minutes after the pancreas is exposed to a high  glucose concentration. The threshold for insulin release is approximately 80 mg  glucose/dL. Above 80 mg/dL, the rate of insulin release is not an all-or-nothing  response but is proportional to the glucose concentration up to approximately 300  mg/dL glucose. As insulin is secreted, the synthesis of new insulin molecules is  stimulated, so that secretion is maintained until blood glucose levels fall. Insulin is  rapidly removed from the circulation and degraded by the liver (and, to a lesser    Fig 26.11. Release of insulin by the (3-cells. Details are provided in the text.   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   b gut hormones that regulate fuel metabolism are discussed in Chapter 43.   extent, by kidney and skeletal muscle), so that blood insulin levels decrease rapidly  once the rate of secretion slows.   A number of factors other than the blood glucose concentration can modulate  insulin release (Table 26.2). The pancreatic islets are innervated by the autonomic  nervous system, including a branch of the vagus nerve. These neural signals help to  coordinate insulin release with the secretory signals initiated by the ingestion of  fuels. However, signals from the central nervous system are not required for insulin  secretion. Certain amino acids also can stimulate insulin secretion, although the  amount of insulin released during a high-protein meal is very much lower than that  released by a high-carbohydrate meal. Gastric inhibitory polypeptide (GIP, a gut hor-  mone released after the ingestion of food) also aids in the onset of insulin release.  Epinephrine, secreted in response to fasting, stress, trauma, and vigorous exercise,  decreases the release of insulin. Epinephrine release signals energy utilization, which  indicates that less insulin needs to be secreted, as insulin stimulates energy storage.   Glucagon, a polypeptide hormone, is synthesized in the a cells of the pancreas by  cleavage of the much larger preproglucagon, a 160-amino acid peptide. Like  insulin, preproglucagon is produced on the rough endoplasmic reticulum and is con-  verted to proglucagon as it enters the ER lumen. Proteolytic cleavage at various  sites produces the mature 29-amino acid glucagon (molecular weight 3,500) and  larger glucagon-containing fragments (named glucagon-like peptides 1 and 2).  Glucagon is rapidly metabolized, primarily in the liver and kidneys. Its plasma half-  life is only about 3 to 5 minutes.   Glucagon secretion is regulated principally by circulating levels of glucose and  insulin. Increasing levels of each inhibit glucagon release. Glucose probably has both  a direct suppressive effect on secretion of glucagon from the a cell as well as an indi-  rect effect, the latter being mediated by its ability to stimulate the release of insulin. The  direction of blood flow in the islets of the pancreas carries insulin from the (3 cells in  the center of the islets to the peripheral a cells, where it suppresses glucagon secretion.    Ann Sulin is taking a sulfonylurea  compound known as glipizide to  treat her diabetes. The sulfony-  lureas act on the K + ATP channels on the sur-  face of the pancreatic p cells. The binding of  the drug to these channels closes K + chan-  nels (as do elevated ATP levels), which, in  turn, increases Ca 2+ movement into the inte-  rior of the p cell. This influx of calcium mod-  ulates the interaction of the insulin storage  vesicles with the plasma membrane of the  P cell, resulting in the release of insulin into  the circulation.   Measurements of proinsulin and  the connecting peptide between  the a and (3 chains of insulin (C-  peptide) in Bea Selmass's blood during her  hospital fast provided confirmation that she  had an insulinoma. Insulin and C-peptide are  secreted in approximately equal proportions  from the p cell, but C-peptide is not cleared  from the blood as rapidly as insulin. There-  fore, it provides a reasonably accurate esti-  mate of the rate of insulin secretion. Plasma  C-peptide measurements are also potentially  useful in treating patients with diabetes mel-  litus because they provide a way to estimate  the degree of endogenous insulin secretion  in patients who are receiving exogenous  insulin, which lacks the C-peptide.    Patients with type 1 diabetes mellitus, such as Di Abietes, have almost unde-  tectable levels of insulin in their blood. Patients with type 2 diabetes mellitus,  such as Ann Sulin, conversely, have normal or even elevated levels of insulin  in their blood; however, the level of insulin in their blood is inappropriately low relative  to their elevated blood glucose concentration. In type 2 diabetes mellitus, skeletal mus-  cle, liver, and other tissues exhibit a resistance to the actions of insulin. As a result,  insulin has a smaller than normal effect on glucose and fat metabolism in such patients.  Levels of insulin in the blood must be higher than normal to maintain normal blood glu-  cose levels. In the early stages of type 2 diabetes mellitus, these compensatory adjust-  ments in insulin release may keep the blood glucose levels near the normal range. Over  time, as the p cells capacity to secrete high levels of insulin declines, blood glucose  levels increase, and exogenous insulin becomes necessary.   Amino acids induce both insulin  and glucagon secretion. Although  this may seem paradoxical, it actu-  ally makes good sense. Insulin release stim-  ulates amino acid uptake by tissues and  enhances protein synthesis. However,  because glucagon levels also increase in  response to a protein meal, gluconeogene-  sis is enhanced (at the expense of protein  synthesis), and the amino acids that are  taken up by the tissues serve as a substrate  for gluconeogenesis. The synthesis of glyco-  gen and triglycerides is also reduced when  glucagon levels rise in the blood.    In fasting subjects, the average  level of immunoreactive glucagon  in the blood is 75 pg/mL and does   not vary as much as insulin during the daily  fasting-feeding cycle. However, only 30 to  40% of the measured immunoreactive  glucagon is mature pancreatic glucagon.  The rest is composed of larger immunoreac-  tive fragments also produced in the pan-  creas or in the intestinal L cells.   The physiologic importance of  insulin's usual action of mediating  the suppressive effect of glucose  on glucagon secretion is apparent in  patients with types 1 and 2 diabetes mellitus.  Despite the presence of hyperglycemia,  glucagon levels in such patients initially  remain elevated (near fasting levels) either  because of the absence of insulin's suppres-  sive effect or because of the resistance of the  a cells to insulin's suppressive effect even in  the face of adequate insulin levels in type 2  patients. Thus, these patients have inappro-  priately high glucagon levels, leading to the  suggestion that diabetes mellitus is actually  a "bi-hormonal" disorder.   Certain hormones stimulate glucagon secretion. Among these are the cate-  cholamines (including epinephrine), Cortisol, and certain gastrointestinal (gut)  hormones (Table 26.3).   Many amino acids also stimulate glucagon release (Fig. 26.12). Thus, the high  levels of glucagon that would be expected in the fasting state do not decrease after  a high-protein meal. In fact, glucagon levels may increase, stimulating gluconeoge-  nesis in the absence of dietary glucose. The relative amounts of insulin and  glucagon in the blood after a mixed meal are dependent on the composition of the  meal, because glucose stimulates insulin release, and amino acids stimulate  glucagon release.   For a hormone to affect the flux of substrates through a metabolic pathway, it must  be able to change the rate at which that pathway proceeds by increasing or decreas-  ing the rate of the slowest step(s). Either directly or indirectly, hormones affect the    Fig 26.12. Release of insulin and glucagon in response to a high-protein meal. This figure  shows the increase in the release of insulin and glucagon into the blood after an overnight fast  followed by the ingestion of 100 g protein (equivalent to a slice of roast beef). Insulin levels  do not increase nearly as much as they do after a high-carbohydrate meal (see Fig. 26.8). The  levels of glucagon, however, significantly increase above those present in the fasting state.   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   activity of specific enzymes or transport proteins that regulate the flux through a  pathway. Thus, ultimately, the hormone must either cause the amount of the sub-  strate for the enzyme to increase (if substrate supply is a rate-limiting factor),  change the conformation at the active site by phosphorylating the enzyme, change  the concentration of an allosteric effector of the enzyme, or change the amount of  the protein by inducing or repressing its synthesis or by changing its turnover rate  or location. Insulin, glucagon, and other hormones use all of these regulatory mech-  anisms to regulate the rate of flux in metabolic pathways. The effects mediated by  phosphorylation or changes in the kinetic properties of an enzyme occur rapidly,  within minutes. In contrast, it may take hours for induction or repression of enzyme  synthesis to change the amount of an enzyme in the cell.   The details of hormone action were previously described in Chapter 1 1 and are  only summarized here.   A. Signal Transduction by Hormones That Bind to  Plasma Membrane Receptors   Hormones initiate their actions on target cells by binding to specific receptors or  binding proteins. In the case of polypeptide hormones (such as insulin and  glucagon), and catecholamines (epinephrine and norepinephrine), the action of the  hormone is mediated through binding to a specific receptor on the plasma mem-  brane (see Chapter 11, section III). The first message of the hormone is transmitted  to intracellular enzymes by the activated receptor and an intracellular second mes-  senger; the hormone does not need to enter the cell to exert its effects. (In contrast,  steroid hormones such as Cortisol and the thyroid hormone triiodothyronine [T 3 ]  enter the cytosol and eventually move into the cell nucleus to exert their effects.)   The mechanism by which the message carried by the hormone ultimately affects  the rate of the regulatory enzyme in the target cell is called signal transduction. The  three basic types of signal transduction for hormones binding to receptors on the  plasma membrane are (a) receptor coupling to adenylate cyclase which produces  cAMP, (b) receptor kinase activity, and (c) receptor coupling to hydrolysis of phos-  phatidylinositol bisphosphate (PIP 2 ). The hormones of metabolic homeostasis each  use one of these mechanisms to carry out their physiologic effect. In addition, some  hormones and neurotransmitters act through receptor coupling to gated ion channels  (previously described in Chapter 11).   Insulin initiates its action by binding to a receptor on the plasma membrane of  insulin's many target cells (see Fig. 11.13). The insulin receptor has two types of sub-  units, the a-subunits to which insulin binds, and the p-subunits, which span the mem-  brane and protrude into the cytosol. The cytosolic portion of the p-subunit has tyro-  sine kinase activity. On binding of insulin, the tyrosine kinase phosphorylates tyrosine  residues on the p-subunit (autophosphorylation) as well as on several other enzymes  within the cytosol. A principal substrate for phosphorylation by the receptor, insulin  receptor substrate (IRS-1), then recognizes and binds to various signal transduction  proteins in regions referred to as SH 2 domains. IRS-1 is involved in many of the phys-  iologic responses to insulin through complex mechanisms that are the subject of inten-  sive investigation. The basic tissue-specific cellular responses to insulin, however, can  be grouped into five major categories: (a) insulin reverses glucagon- stimulated phos-  phorylation, (b) insulin works through a phosphorylation cascade that stimulates the  phosphorylation of several enzymes, (c) insulin induces and represses the synthesis of  specific enzymes, (d) insulin acts as a growth factor and has a general stimulatory  effect on protein synthesis, and (e) insulin stimulates glucose and amino acid trans-  port into cells (see Fig. 10 of section introduction, p. 484).   During the "stress" of hypo-  glycemia, the autonomic nervous  system stimulates the pancreas to  secrete glucagon, which tends to restore the  serum glucose level to normal. The  increased activity of the adrenergic nervous  system (through epinephrine) also alerts a  patient, such as Bea Selmass, to the pres-  ence of increasingly severe hypoglycemia.  Hopefully, this will induce the patient to  ingest simple sugars or other carbohydrates,  which, in turn, will also increase glucose lev-  els in the blood. Bea Selmass gained 8 lb  before resection of her pancreatic insulin-  secreting adenoma through this mechanism.   A number of mechanisms have been proposed for the action of insulin in  reversing glucagon- stimulated phosphorylation of the enzymes of carbohydrate  metabolism. From the student's point of view, the ability of insulin to reverse  glucagon-stimulated phosphorylation occurs as if it were lowering cAMP  and stimulating phosphatases that could remove those phosphates added by pro-  tein kinase A. In reality, the mechanism is more complex and still not fully  understood.   mones that regulate fuel metabo-  lism. The specificity of the physiologic  response to each hormone results from the  presence of specific receptors for that hor-  mone in target tissues. For example,  glucagon activates glucose production from  glycogen in liver, but not in skeletal muscle  because glucagon receptors are present in  liver but absent in skeletal muscle. However,  skeletal muscle has adenylate cyclase,  cAMP, and protein kinase A, which can be  activated by epinephrine binding to the p 2  receptors in the membrane of muscle cells.  Liver cells also have epinephrine receptors.   Phosphodiesterase is inhibited by  jfl W 9 ) methylxanthines, a class of com-   pounds that includes caffeine.  Would the effect of a methylxanthine on fuel  metabolism be similar to fasting orto a high-  carbohydrate meal?   The pathway for signal transduction by glucagon is one common to a number of  hormones; the glucagon receptor is coupled to adenylate cyclase and cAMP  production (see Fig. 11.11). Glucagon, through G proteins, activates the membrane-  bound adenylate cyclase, increasing the synthesis of the intracellular second mes-  senger 3',5'-cyclic AMP (cAMP) (see Fig. 9.10). cAMP activates protein kinase A  (cAMP-dependent protein kinase), which changes the activity of enzymes by phos-  phorylating them at specific serine residues. Phosphorylation activates some  enzymes and inhibits others.   The G proteins, which couple the glucagon receptor to adenylate cyclase, are  proteins in the plasma membrane that bind guanosine triphosphate (GTP) and have  dissociable subunits that interact with both the receptor and adenylate cyclase. In  the absence of glucagon, the stimulatory G s protein complex binds guanosine  diphosphate (GDP) but cannot bind to the unoccupied receptor or adenylate cyclase  (see Fig. 1 1.17). Once glucagon binds to the receptor, the receptor also binds the G s  complex, which then releases GDP and binds GTP. The a-subunit then dissociates  from the p^-subunits and binds to adenylate cyclase, thereby activating it. As the  GTP on the a-subunit is hydrolyzed to GDP, the subunit dissociates and recom-  plexes with the p- and 7-subunits. Only continued occupancy of the glucagon  receptor can keep adenylate cyclase active.   Although glucagon works by activating adenylate cyclase, a few hormones  inhibit adenylate cyclase. In this case, the inhibitory G protein complex is called a  G t complex.   cAMP is very rapidly degraded to AMP by a membrane-bound phosphodi-  esterase. The concentration of cAMP is thus very low in the cell so that changes in  its concentration can occur rapidly in response to changes in the rate of synthesis.  The amount of cAMP present at any time is a direct reflection of hormone binding  and the activity of adenylate cyclase. It is not affected by ATP, ADP, or AMP levels  in the cell.   cAMP transmits the hormone signal to the cell by activating protein kinase A  (cAMP-dependent protein kinase). As cAMP binds to the regulatory subunits of  protein kinase A, these subunits dissociate from the catalytic subunits, which are  thereby activated (see Figs. 9.9 and 9.11). Activated protein kinase A phosphory-  lates serine residues of key regulatory enzymes in the pathways of carbohydrate and  fat metabolism. Some enzymes are activated and others are inhibited by this change  in phosphorylation state. The message of the hormone is terminated by the action  of semispecific protein phosphatases that remove phosphate groups from the  enzymes. The activity of the protein phosphatases is also controlled through hor-  monal regulation.   Changes in the phosphorylation state of proteins that bind to cAMP response ele-  ments (CREs) in the promoter region of genes contribute to the regulation of gene  transcription by a number of cAMP-coupled hormones (see Chapter 16). For  instance, cAMP response element binding protein (CREB) is directly phosphory-  lated by protein kinase A, a step essential for the initiation of transcription. Phos-  phorylation at other sites on CREB, by a variety of kinases, also may play a role in  regulating transcription.   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   The mechanism for signal transduction by glucagon illustrates some of the  important principles of hormonal signaling mechanisms. The first principle is  that specificity of action in tissues is conferred by the receptor on a target cell  for glucagon. In general, the major actions of glucagon occur in liver, adipose  tissue, and certain cells of the kidney that contain glucagon receptors. The sec-  ond principle is that signal transduction involves amplification of the first mes-  sage. Glucagon and other hormones are present in the blood in very low con-  centrations. However, these minute concentrations of hormone are adequate to  initiate a cellular response because the binding of one molecule of glucagon to  one receptor ultimately activates many protein kinase A molecules, each of  which phosphorylates hundreds of downstream enzymes. The third principle  involves integration of metabolic responses. For instance, the glucagon-stimu-  lated phosphorylation of enzymes simultaneously activates glycogen degrada-  tion, inhibits glycogen synthesis, and inhibits glycolysis in the liver (see Fig. 10  in section introduction, p. 484.). The fourth principle involves augmentation and  antagonism of signals. An example of augmentation involves the actions of  glucagon and epinephrine (which is released during exercise). Although these  hormones bind to different receptors, each can increase cAMP and stimulate  glycogen degradation. A fifth principle is that of rapid signal termination. In the  case of glucagon, both the termination of the G s protein activation and the rapid  degradation of cAMP contribute to signal termination.   B. Signal Transduction by Cortisol and Other Hormones  That Interact with Intracellular Receptors   Signal transduction by the glucocorticoid Cortisol and other steroids and by thyroid  hormone involves hormone binding to intracellular (cytosolic) receptors or binding  proteins, after which this hormone-binding protein complex moves into the  nucleus, where it interacts with chromatin. This interaction changes the rate of gene  transcription in the target cells (see Chapter 16). The cellular responses to these hor-  mones will continue as long as the target cell is exposed to the specific hormones.  Thus, disorders that cause a chronic excess in their secretion will result in an  equally persistent influence on fuel metabolism. For example, chronic stress such as  that seen in prolonged sepsis may lead to varying degrees of glucose intolerance if  high levels of epinephrine and Cortisol persist.   The effects of Cortisol on gene transcription are usually synergistic to those of  certain other hormones. For instance, the rates of gene transcription for some of the  enzymes in the pathway for glucose synthesis from amino acids (gluconeogenesis)  are induced by glucagon as well as by Cortisol.    Inhibition of phosphodiesterase by  methylxanthine would increase  cAMP and have the same effects  on fuel metabolism as would an increase of  glucagon and epinephrine, in the fasted  state. Increased fuel mobilization would  occur through glycogenolysis (the release of  glucose from glycogen), and through lipoly-  sis (the release of fatty acids from triacyl-  glycerols).   Protein kinases are a class of  KJ ) enzymes that change the activity of  *S other enzymes by phosphorylating  them at serine or tyrosine residues and are  referred to as serine or tyrosine kinases,  respectively. Serine kinases also phosphory-  late some enzymes at threonine residues.  Protein kinase A (a serine kinase) phospho-  rylates many of the enzymes in the path-  ways of fuel metabolism.   Epinephrine and norepinephrine are catecholamines (Fig. 26.13). They can act as  neurotransmitters or as hormones. A neurotransmitter allows a neural signal to be  transmitted across the juncture or synapse between the nerve terminal of a proximal  nerve axon and the cell body of a distal neuron. A hormone, conversely, is released  into the blood and travels in the circulation to interact with specific receptors on the  plasma membrane or cytosol of the target organ. The general effect of these cate-  cholamines is to prepare us for fight or flight. Under these acutely stressful circum-  stances, these "stress" hormones increase fuel mobilization, cardiac output, blood  flow, etc., which enable us to meet these stresses. The catecholamines bind to adren-  ergic receptors (the term adrenergic refers to nerve cells or fibers that are part of the  involuntary or autonomic nervous system, a system that employs norepinephrine as  a neurotransmitter).   Fig 26.13. Structure of epinephrine and nor-  epinephrine. Epinephrine and norepinephrine  are synthesized from tyrosine and act as both  hormones and neurotransmitters. They are cat-  echolamines, the term catechol referring to a  ring structure containing two hydroxy 1 groups.   Q o Ann O'Rexia, to stay thin, fre-  ( /~0A j quently fasts for prolonged periods,  ^ but jogs every morning (see Chap-  ter 2). The release of epinephrine and norep-  inephrine and the increase of glucagon and  fall of insulin during her exercise provide  coordinated and augmented signals that  stimulate the release of fuels above the fast-  ing levels. Fuel mobilization will occur, of  course, only as long as she has fuel stored as  triacylglycerols.   Q o Ann Sulin, a patient with type 2 dia-  j /~0A ) betes mellitus, is experiencing  ™ insulin resistance. Her levels of cir-  culating insulin are normal to high, although  inappropriately low for her elevated level of  blood glucose. However, her insulin target  cells, such as muscle and fat, do not respond  as those of a nondiabetic subject would to this  level of insulin. For most type 2 patients, the  site of insulin resistance is subsequent to  binding of insulin to its receptor; i.e., the num-  ber of receptors and their affinity for insulin is  near normal. However, the binding of insulin  at these receptors does not elicit most of the  normal intracellular effects of insulin dis-  cussed earlier. Consequently, there is little  stimulation of glucose metabolism and stor-  age after a high-carbohydrate meal and little  inhibition of hepatic gluconeogenesis.   There are nine different types of adrenergic receptors: a 1A , a 1B a 1D a 2A , a 2B a 2C  P 1? p 2 , and p 3 Only the three p and a r receptors are discussed here. The three p  receptors work through the adenylate cyclase-cAMP system, activating a G s pro-  tein, which activates adenylate cyclase, and eventually protein kinase A. The p!  receptor is the major adrenergic receptor in the human heart and is primarily stim-  ulated by norepinephrine. On activation, the fi r receptor increases the rate of mus-  cle contraction, in part because of PKA-mediated phosphorylation of phospholam-  ban (see Chapter 47). The p 2 receptor is present in liver, skeletal muscle, and other  tissues and is involved in the mobilization of fuels (such as the release of glucose  through glycogenolysis). It also mediates vascular, bronchial, and uterine smooth  muscle contraction. Epinephrine is a much more potent agonist for this receptor  than norepinephrine, whose major action is neurotransmission. The p 3 receptor is  found predominantly in adipose tissue and to a lesser extent in skeletal muscle.  Activation of this receptor stimulates fatty acid oxidation and thermogenesis, and  agonists for this receptor may prove to be beneficial weight loss agents. The a r  receptors, which are postsynaptic receptors, mediate vascular and smooth muscle  contraction. They work through the phosphatidylinositol bisphosphate system (see  Chapter 11, section III.B.2) through activation of a G q protein, and phospholipase  C-p. This receptor also mediates glycogenolysis in liver.   ^ Di Abietes has type 1 diabetes mellitus (formally designated insulin-  ) dependent diabetes mellitus, IDDM) whereas Ann Sulin has type 2 diabetes   S mellitus (formally called non-insulin-dependent diabetes mellitus). Although  the pathogenesis differs for these major forms of diabetes mellitus, both cause varying  degrees of hyperglycemia. In type 1, the pancreatic p cells are gradually destroyed by  antibodies directed at a variety of proteins within the p cells. As insulin secretory capac-  ity by the p cells gradually diminishes below a critical level, the symptoms of chronic  hyperglycemia develop rapidly. In type 2 diabetes mellitus, these symptoms develop  more subtly and gradually over the course of months or years. Eighty-five percent or  more of type 2 patients are obese and, like Ivan Applebod, have a high waist-hip ratio  with regard to adipose tissue disposition. This abnormal distribution of fat in the vis-  ceral (peri-intestinal) adipocytes is associated with reduced sensitivity of fat cells, mus-  cle cells, and liver cells to the actions of insulin outlined above. This insulin resistance  can be diminished through weight loss, specifically in the visceral depots.   a Bea Selmass underwent a high-resolution ultrasonographic (ultra-  ) sound) study of her upper abdomen, which showed a 2.6-cm mass in the  ^—•^ midportion of her pancreas. With this finding, her physicians decided that  further noninvasive studies would not be necessary before surgical exploration of  her upper abdomen was performed. At the time of surgery, a yellow- white 2. 8 -cm  mass consisting primarily of insulin-rich P cells was resected from her pancreas. No  cytologic changes of malignancy were seen on cytologic examination of the surgi-  cal specimen, and no gross evidence of malignant behavior by the tumor (such as  local metastases) was found. Bea had an uneventful postoperative recovery and no  longer experienced the signs and symptoms of insulin-induced hypoglycemia.   One of the important cellular responses to insulin is the reversal of  glucagon- stimulated phosphorylation of enzymes. Mechanisms proposed  for this action include the inhibition of adenylate cyclase, a reduction of   CHAPTER 26 / BASIC CONCEPTS IN THE REGULATION OF FUEL METABOLISM BY INSULIN, GLUCAGON, AND OTHER HORMONES   cAMP levels, the stimulation of phosphodiesterase, the production of a specific pro-  tein (insulin factor), the release of a second messenger from a bound glycosylated  phosphatidylinositol, and the phosphorylation of enzymes at a site that antagonizes  protein kinase A phosphorylation. Not all of these physiologic actions of insulin  occur in each of the insulin- sensitive organs of the body.   Insulin is also able to antagonize the actions of glucagon at the level of specific  induction or repression of key regulatory enzymes of carbohydrate metabolism. For  instance, the rate of synthesis of mRNA for phosphoenolpyruvate carboxykinase, a  key enzyme of the gluconeogenic pathway, is increased severalfold by glucagon  (via cAMP) and decreased by insulin. Thus, all of the effects of glucagon, even the  induction of certain enzymes, can be reversed by insulin. This antagonism is exerted  through an insulin- sensitive hormone response element (IRE) in the promoter  region of the genes. Insulin causes repression of the synthesis of enzymes that are  induced by glucagon.   The general stimulation of protein synthesis by insulin (its mitogenic or growth-  promoting effect) appears to occur through both a general increase in rates of  mRNA translation for a broad spectrum of structural proteins. These actions result  from a phosphorylation cascade initiated by autophosphorylation of the insulin  receptor and ending in the phosphorylation of subunits of proteins that bind to and  inhibit eukaryotic protein synthesis initiation factors (elFs). When phosphorylated,  the inhibitory proteins are released from the elFs, allowing translation of mRNA to  be stimulated. In this respect, the actions of insulin are similar to those of hormones  that act as growth factors and also have receptors with tyrosine kinase activity.   In addition to signal transduction, activation of the insulin receptor mediates the  internalization of receptor-bound insulin molecules increasing their subsequent  degradation. Although unoccupied receptors can be internalized and eventually  recycled to the plasma membrane, the receptor can be irreversibly degraded after  prolonged occupation by insulin. The result of this process, referred to as receptor  downregulation, is an attenuation of the insulin signal. The physiologic importance  of receptor internalization on insulin sensitivity is poorly understood but could  eventually lead to chronic hyperglycemia.   Kahn SE, Porte D Jr. p-cell dysfunction in type 2 diabetes. In: Scriver CR, Beaudet AL, Sly WS,  Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease, 8th Ed. New York: McGraw-  Hill, 2001:1407-1431.   • O'Brien C. Missing link in insulin's path to protein production. Science 1994;266:542-543.  Saltiel AR, Pessin JE. Insulin signalling pathways in time and space. Trends Cell Biol.  2002;12:65-71.   • Stride A, Hattersley AT. Different genes, different diabetes: lessons from maturity-onset diabetes of  the young. Ann Med 2002;34:207-216.   A patient with type I diabetes mellitus takes an insulin injection before eating dinner but then gets distracted and does not eat.  Approximately 3 hours later, the patient becomes shaky, sweaty, and confused. These symptoms have occurred because of  which of the following?   2. Caffeine is a potent inhibitor of the enzyme cAMP phosphodiesterase. Which of the following consequences would you expect  to occur in the liver after drinking two cups of strong expresso coffee?   3. Assume that an increase in blood glucose concentration from 5 to 10 mM would result in insulin release by the pancreas. A  mutation in pancreatic glucokinase can lead to MODY because of which of the following within the pancreatic p-cell?   4. Which one of the following organs has the highest demand for glucose as a fuel?   5. Glucagon release does not alter muscle metabolism because of which of the following?   Carbohydrates are the largest source of dietary calories for most of the world's  population. The major carbohydrates in the American diet are starch, lactose,  and sucrose. The starches amylose and amylopectin are polysaccharides com-  posed of hundreds to millions of glucosyl units linked together through a-1,4 and  a-1 ,6 glycosidic bonds (Fig. 27.1). Lactose is a disaccharide composed of glu-  cose and galactose, linked together through a (3-1,4 glycosidic bond. Sucrose is  a disaccharide composed of glucose and fructose, linked through an a-1 ,2 glyco-  sidic bond. The digestive processes convert all of these dietary carbohydrates to  their constituent monosaccharides by hydrolyzing glycosidic bonds between the  sugars.   The digestion of starch begins in the mouth (Fig. 27.2). The salivary gland  releases a-amylase, which converts starch to smaller polysaccharides called  a-dextrins. Salivary a-amylase is inactivated by the acidity of the stomach (HCl).  Pancreatic a-amylase and bicarbonate are secreted by the exocrine pancreas into  the lumen of the small intestine, where bicarbonate neutralizes the gastric secre-  tions. Pancreatic a-amylase continues the digestion of a-dextrins , converting them  to di sac char ides (maltose), trisaccharides (maltotriose), and oligosaccharides  called limit dextrins. Limit dextrins usually contain four to nine glucosyl residues  and an isomaltose branch (two glucosyl residues attached through an a-1,6  glycosidic bond).   The digestion of the disaccharides lactose and sucrose, as well as further  digestion of maltose, maltotriose and limit dextrins, occurs through disacchari-  dases attached to the membrane surface of the brush border (microvilli) of intes-  tinal epithelial cells. Glucoamylase hydrolyzes the a-1,4 bonds of dextrins. The  sucrase-isomaltase complex hydrolyzes sucrose, most of maltose, and almost all  of the isomaltose formed by glucoamylase from limit dextrins. Lactase-  glycosylceramidase (/3-glycosidase) hydrolyzes the /3 -glycosidic bonds in lactose  and glycolipids. A fourth disaccharidase complex, trehalase, hydrolyzes the bond  (an a-1,1 glycosidic bond) between two glucosyl units in the sugar trehalose. The  monosaccharides produced by these hydrolases (glucose, fructose, and galactose)  are then transported into the intestinal epithelial cells.   Fig. 27.1. The structures of common dietary carbohydrates. For disaccharides and greater,  the sugars are linked through glycosidic bonds between the anomeric carbon of one sugar and  a hydroxyl group on another sugar. The glycosidic bond may be either a or |3, depending on  its position above or below the plane of the sugar containing the anomeric carbon, (see Chap-  ter 5, Section II. A, to review terms used in the description of sugars). The starch amylose is  a polysaccharide of glucose residues linked with a-1,4 glycosidic bonds. Amylopectin is  amylase with the addition of a-1,6 glycosidic branchpoints. Dietary sugars may be mono-  saccharides (single sugar residues), disaccharides (two sugar residues), oligosaccharides  (several sugar residues) or polysaccharides (hundreds of sugar residues).   A common malabsorption syn-  drome, lactose intolerance, is char-  acterized by nausea, diarrhea, and  flatulence after ingesting dairy products or  other foods containing lactose. One of the  causes of lactose intolerance is a low level of  lactase, which decreases after infancy in  most of the world's population (nonpersis-  tant lactase or adult hypolactasia). However,  lactase activity remains high in some popu-  lations (persistent lactase), including North-  western Europeans and their descendants.   Fig. 27.2. Overview of carbohydrate digestion. Digestion of the carbohydrates occurs first,  followed by absorption of monosaccharides. Subsequent metabolic reactions occur after the  sugars are absorbed.   Dietary fiber, composed principally of polysaccharides, cannot be digested by  human enzymes in the intestinal tract. In the colon, dietary fiber and other nondi-  gested carbohydrates may be converted to gases (H 2 , C0 2 , and methane) and short-  chain fatty acids (principally acetic acid, propionic acid, and butyric acid) by bacte-  ria in the colon.   Glucose, galactose, and fructose formed by the digestive enzymes are trans-  ported into the absorptive epithelial cells of the small intestine by protein-medi-  ated Na + -dependent active transport and facilitative diffusion. Monosaccha-  rides are transported from these cells into the blood and circulate to the liver  and peripheral tissues, where they are taken up by facilitative transporters.  Facilitative transport of glucose across epithelial cells and other cell membranes  is mediated by a family of tissue -specific glucose transport proteins (GLUT  I-V). The type of transporter found in each cell reflects the role of glucose  metabolism in that cell.   Deria Voider is a 20-year-old exchange student from Nigeria who has  noted gastrointestinal bloating, abdominal cramps, and intermittent diar-  rhea ever since arriving in the United States 6 months earlier. A careful  history shows that these symptoms occur most commonly about 45 minutes to 1  hour after eating breakfast but may occur after other meals as well. Dairy products,  not a part of Deria 's diet in Nigeria, were identified as the probable offending agent  because her gastrointestinal symptoms disappeared when milk and milk products  were eliminated from her diet.   Ann Sulin's fasting and postprandial blood glucose levels are frequently  above the normal range in spite of good compliance with insulin therapy.  Her physician has referred her to a dietician skilled in training diabetic  patients in the successful application of an appropriate American Diabetes  Association diet. As part of the program, Ms. Sulin is asked to incorporate foods  containing fiber into her diet, such as whole grains (e.g., wheat, oats, corn), legumes  (e.g., peas, beans, lentils), tubers (e.g., potatoes, peanuts), and fruits.   Nona Melos (no sweets) is a 7-month-old baby girl, the second child born  to unrelated parents. Her mother had a healthy, full-term pregnancy, and  Nona's birth weight was normal. She did not respond well to breastfeeding  and was changed entirely to a formula based on cow's milk at 4 weeks. Between 7  and 12 weeks of age, she was admitted to the hospital twice with a history of  screaming after feeding but was discharged after observation without a specific  diagnosis. Elimination of cow's milk from her diet did not relieve her symptoms;  Nona's mother reported that the screaming bouts were worse after Nona drank juice  and that Nona frequently had gas and a distended abdomen. At 7 months she was  still thriving (weight above 97th percentile) with no abnormal findings on physical  examination. A stool sample was taken.   Carbohydrates are the largest source of calories in the average American diet and  usually constitute 40 to 45% of our caloric intake. The plant starches amylopectin  and amylose, which are present in grains, tubers, and vegetables, constitute approx-  imately 50 to 60% of the carbohydrate calories consumed. These starches are poly-  saccharides, containing 10,000 to 1 million glucosyl units. In amylose, the glucosyl  residues form a straight chain linked via a- 1,4 glycosidic bonds; in amylopectin, the  a- 1,4 chains contain branches connected via a- 1,6 glycosidic bonds (see Fig. 27.1).  The other major sugar found in fruits and vegetables is sucrose, a disaccharide of  glucose and fructose (see Fig. 27.1). Sucrose and small amounts of the monosac-  charides glucose and fructose are the major natural sweeteners found in fruit, honey,  and vegetables. Dietary fiber, that portion of the diet that cannot be digested by  human enzymes of the intestinal tract, is also composed principally of plant poly-  saccharides and a polymer called lignan.   Most foods derived from animals, such as meat or fish, contain very little carbo-  hydrate except for small amounts of glycogen (which has a structure similar to amy-  lopectin) and glycolipids. The major dietary carbohydrate of animal origin is lac-  tose, a disaccharide composed of glucose and galactose found exclusively in milk  and milk products (see Fig. 27.1).   The dietary sugar in fruit juice and  other sweets is sucrose, a disac-  charide composed of glucose and  fructose joined through their anomeric car-  bons. Nona Melos' symptoms of pain and  abdominal distension are caused by an  inability to digest sucrose or absorb fruc-  tose, which are converted to gas by colonic  bacteria. "Melos" is Latin for sweets, and  her name means "no sweets." Nona Melos's  stool sample had a pH of 5 and gave a posi-  tive test for sugar. The possibility of carbo-  hydrate malabsorption was considered, and  a hydrogen breath test was recommended.   Sweeteners, in the form of sucrose  and high-fructose corn syrup  (starch, partly hydrolyzed and iso-  merized to fructose), also appear in the diet  as additives to processed foods. On average,  a person in the United States consumes 65  lb added sucrose and 40 lb high-fructose  corn syrup solids per year.   ing weight without having to exer-  cise or reduce your daily caloric intake.  Starch blockers were based on a protein  found in beans, which blocked the action of  amylase. Thus, as the advertisements pro-  claimed, one could eat a large amount of  starch during a meal, and as long as you  took the starch blocker, the starch would  pass through the digestive track without  being metabolized. Unfortunately, this was  too good to be true, and starch blockers  were never shown to be effective in aiding  weight loss. This was probably because of a  combination of factors, such as inactivation  of the inhibitor by the low pH in the stom-  ach, and an excess of amylase activity as  compared with the amount of starch blocker  ingested. Recently this issue has been revis-  ited, as a starch blocker from wheat has  been developed that may work as adver-  tised, although much more work is required  to determine whether this amylase inhibitor  will be safe and effective in humans.   Although all cells require glucose for metabolic functions, neither glucose nor other  sugars are specifically required in the diet. Glucose can be synthesized from many  amino acids found in dietary protein. Fructose, galactose, xylose, and all the other sug-  ars required for metabolic processes in the human can be synthesized from glucose.   In the digestive tract, dietary polysaccharides and disaccharides are converted to  monosaccharides by glycosidases, enzymes that hydrolyze the glycosidic bonds  between the sugars. All of these enzymes exhibit some specificity for the sugar, the  glycosidic bond (a or (3), and the number of saccharide units in the chain. The mono-  saccharides formed by glycosidases are transported across the intestinal mucosal  cells into the interstitial fluid and subsequently enter the bloodstream. Undigested  carbohydrates enter the colon, where they may be fermented by bacteria.   The digestion of starch (amylopectin and amylose) begins in the mouth, where  chewing mixes the food with saliva. The salivary glands secrete approximately 1  liter of liquid per day into the mouth, containing salivary a-amylase and other com-  ponents. a-Amylase is an endoglucosidase, which means that it hydrolyzes internal  a- 1,4 bonds between glucosyl residues at random intervals in the polysaccharide  chains (Fig. 27.3). The shortened polysaccharide chains that are formed are called  a-dextrins. Salivary a-amylase may be largely inactivated by the acidity of the  stomach contents, which contain HC1 secreted by the peptic cells.   The acidic gastric juice enters the duodenum, the upper part of the small intes-  tine, where digestion continues. Secretions from the exocrine pancreas (approxi-  mately 1.5 liters/day) flow down the pancreatic duct and also enter the duodenum.  These secretions contain bicarbonate (HC0 3 ~), which neutralizes the acidic pH of  stomach contents, and digestive enzymes, including pancreatic a-amylase.   Pancreatic a-amylase continues to hydrolyze the starches and glycogen, forming  the disaccharide maltose, the trisaccharide maltotriose, and oligosaccharides. These  oligosaccharides, called limit dextrins, are usually four to nine glucosyl units long and  contain one or more a- 1,6 branches. The two glucosyl residues that contain the a- 1,6  glycosidic bond will eventually become the disaccharide isomaltose, but a-amylase  does not cleave these branched oligosaccharides all the way down to isomaltose.   a-Amylase has no activity toward sugar containing polymers other than glucose  linked by a- 1,4 bonds. a-Amylase displays no activity toward the a- 1,6- bond at  branchpoints and has little activity for the a- 1,4 bond at the nonreducing end of a chain.   The dietary disaccharides lactose and sucrose, as well as the products of starch diges-  tion, are converted to monosaccharides by glycosidases attached to the membrane in  the brush-border of absorptive cells (Fig. 27.4). The different glycosidase activities  are found in four glycoproteins: glucoamylase, the sucrase-maltase complex, the  smaller glycoprotein trehalase, and lactase-glucosylceramidase (Table 27.1). These  glycosidases are collectively called the small intestinal disaccharidases, although  glucoamylase is really an oligosaccharidase.   Glucoamylase and the sucrase-isomaltase complex have similar structures and  exhibit a great deal of sequence homogeneity (Fig. 27.5). A membrane- spanning  domain near the N-terminal attaches the protein to the luminal membrane. The long  polypeptide chain forms two globular domains, each with a catalytic site. In glu-  coamylase, the two catalytic sites have similar activities, with only small differences  in substrate specificity. The protein is heavily glycosylated with oligosaccharides  that protect it from digestive proteases.   Glucoamylase is an exoglucosidase that is specific for the a- 1,4 bonds between  glucosyl residues (Fig. 27.6). It begins at the nonreducing end of a polysaccharide  or limit dextrin, and sequentially hydrolyzes the bonds to release glucose monosac-  charides. It will digest a limit dextrin down to isomaltose, the glucosyl disaccharide  with an a-l,6-branch, that is subsequently hydrolyzed principally by the isomaltase  activity in the sucrase-isomaltase complex.   The structure of the sucrase-isomaltase complex is very similar to that of glu-  coamylase, and these two proteins have a high degree of sequence homology. How-  ever, after the single polypeptide chain of sucrase-isomaltase is inserted through the  membrane and the protein protrudes into the intestinal lumen, an intestinal protease  clips it into two separate subunits that remain attached to each other. Each subunit  has a catalytic site that differs in substrate specificity from the other through non-  covalent interactions. The sucrase-maltase site accounts for approximately 100% of  the intestine's ability to hydrolyze sucrose in addition to maltase activity; the iso-  maltase-maltase site accounts for almost all of the intestine's ability to hydrolyze  a-l,6-bonds (Fig. 27.7), in addition to maltase activity. Together, these sites account  for approximately 80% of the maltase activity of the small intestine. The remainder  of the maltase activity is found in the glucoamylase complex.   Amylase activity in the gut is abun-  dant and is not normally rate limit-  ing for the process of digestion.  Alcohol-induced pancreatitis or surgical  removal of part of the pancreas can decrease  pancreatic secretion. Pancreatic exocrine  secretion into the intestine also can be  decreased through cystic fibrosis, in which  mucus blocks the pancreatic duct, which  eventually degenerates. However, pancreatic  exocrine secretion can be decreased to 10% of  normal and still not affect the rate of starch  digestion, because amylases are secreted in  the saliva and pancreatic fluid in excessive  amounts. In contrast, protein and fat digestion  is more strongly affected in cystic fibrosis.    Can the glycosidic bonds of the structure  shown above be hydrolyzed by a-amylose?   Individuals with genetic defi-  ciencies of the sucrase-isomal-  tase complex show symptoms  of sucrose intolerance but are able to  digest normal amounts of starch in a  meal, without problems. The maltase  activity in the glucoamylase complex, and  residual activity in the sucrase-isomaltase  complex (which is normally present in  excess of need) is apparently sufficient to  digest normal amounts of dietary starch.   Which of the bonds in the structure above  are hydrolyzed by the sucrase-isomaltase  complex? Which by glucoamylase?    Table 27.1. The Different Forms of the Brush Border Glycosidases  Complex Catalytic Sites Principal Activities   ^^-^ No. This polysaccharide is cellu-  ^\ • J lose, which contains (3-1 ,4 glyco-  side bonds. Pancreatic and sali-  vary a-amylase cleave only a-1,4  bonds between glucosyl units.   ® Bonds (1) and (3) would first be  hydrolyzed by glucoamylase. Bond  (2) would require isomaltase.  Bonds (4) and (5) could then be hydrolyzed  by the sucrase-isomaltase complex, or by  the glucoamylase complex, all of which can  convert maltotriose and maltose to glucose.   Split a-1,4 glycosidic bonds between  glucosyl units, beginning sequentially  with the residue at the tail end  (nonreducing end) of the chain. This is an  exoglycosidase. Substrates include  amylase, amylopectin, glycogen and  maltose.   Same as above, but with slightly different  specificity and affinities for the  substrates   Splits a-1, 6 bonds in a number of limit  dextrins, as well as the a-1,4 bonds  in maltose and maltotriose.   Splits 0-glycosidic bonds between glucose  or galactose and hydrophobic residues,  such as the glycolipids glucosylceramide  and galactosylceramide   Splits the 0-1,4 bond between glucose and  galactose. To a lesser extent also splits  the 0-1,4 bond between some  cellulose disaccharides.   Splits bond in trehalose, which is 2 glucosyl  units linked a-1,1 through their anomeric  carbons.   Trehalase is only half as long as the other disaccharidases and has only one catalytic  site. It hydrolyzes the glycosidic bond in trehalose, a disaccharide composed of two  glucosyl units linked by an a-bond between their anomeric carbons (Fig. 27.8). Tre-  halose, which is found in insects, algae, mushrooms, and other fungi, is not cur-  rently a major dietary component in the United States. However, unwitting con-  sumption of trehalose can cause nausea, vomiting, and other symptoms of severe  gastrointestinal distress if consumed by an individual deficient in the enzyme. Tre-  halase deficiency was discovered when a woman became very sick after eating  mushrooms and was initially thought to have a-amanitin poisoning.   The p-glycosidase complex is another large glycoprotein found in the brush border  that has two catalytic sites extending in the lumen of the intestine. However, its pri-  mary structure is very different from the other enzymes, and it is attached to the  membrane through its carboxyl end by a phosphatidylglycan anchor (see Fig. 10.7).  The lactase catalytic site hydrolyzes the p-bond connecting glucose and galactose  in lactose (a p-galactosidase activity; Fig. 27.9). The major activity of the other cat-  alytic site in humans is the p-bond between glucose or galactose and ceramide in  glycolipids (this catalytic site is sometimes called phlorizin hydrolase, named for its  ability to hydrolyze an artificial substrate).   The production of maltose, maltotriose, and limit dextrins by pancreatic a-amylase  occurs in the duodenum, the most proximal portion of the small intestine.  Sucrase-isomaltase activity is highest in the jejunum, where the enzymes can  hydrolyze sucrose and the products of starch digestion. (3-Glycosidase activity is  also highest in the jejenum. Glucoamylase activity progressively increases along  the length of the small intestine, and its activity is highest in the ileum. Thus, it   Fig. 27.5. The major portion of the sucrase-isomaltase complex, containing the catalytic sites,  protrudes from the absorptive cells into the lumen of the intestine. Other domains of the protein  form a connecting segment (stalk), and an anchoring segment that extends through the mem-  brane into the cell. The complex is synthesized as a single polypeptide chain that is split into its  two enzyme subunits extracellularly. Each subunit is a domain with a catalytic site (sucrase-  maltase) and isomaltase-maltase sites. In spite of their maltase activity, these catalytic sites are  often called just sucrase and isomaltase.   Fig. 27.6. Glucoamylase activity. Glucoamy-  lase is an a- 1,4 exoglycosidase, which initiates  cleavage at the nonreducing end of the sugar.  Thus, for malotriose, the bond labeled 1 will  be hydrolyzed first, which frees up the bond at  position 2 to be the next one hydrolyzed.   Fig. 27.7. Isomaltase activity. Arrows indicate  the a- 1,6 bonds that are cleaved.   Fig. 27.9. Lactase activity. Lactase is a p-galactosidase. It cleaves the p-galactoside lactose,  the major sugar in milk, forming galactose and glucose.   Fig. 27.8. Trehalose. This disaccharide con-  tains two glucose moieties linked by an unusual  bond that joins their anomeric carbons. It is  cleaved by trehalase.    presents a final opportunity for digestion of starch oligomers that have escaped  amylase and disaccharidase activities at the more proximal regions of the intestine.   Not all of the starch ingested as part of foods is normally digested in the small intestine  (Fig. 27.10). Starches high in amylose, or less well hydrated (e.g., starch in dried beans),  are resistant to digestion and enter the colon. Dietary fiber and undigested sugars also  enter the colon. Here colonic bacteria rapidly metabolize the saccharides, forming gases,  short-chain fatty acids, and lactate. The major short-chain fatty acids formed are acetic  acid (two carbon), propionic acid (three carbon), and butyric acid (four carbon). The  short-chain fatty acids are absorbed by the colonic mucosal cells and can provide a sub-  stantial source of energy for these cells. The major gases formed are hydrogen gas (H 2 ),  carbon dioxide (C0 2 ), and methane (CH 4 ). These gases are released through the colon,  resulting in flatulence, or in the breath. Incomplete products of digestion in the intestines  increase the retention of water in the colon, resulting in diarrhea.   Fig. 27.10. Some indigestible carbohydrates. These compounds are components of dietary fiber.   Nona Melos was given a hydrogen breath test, a test measuring the amount of hydrogen gas released after consuming a test dose  of sugar. The association of Nona Melos's symptoms with her ingestion of fruit juices suggests that she might have a problem result-  ing from a low sucrase activity or an inability to absorb fructose. Her ability to thrive and her adequate weight gain suggest that any  deficiencies of the sucrase-isomaltase complex must be partial and do not result in a functionally important reduction in maltase activity (mal-  tase activity is also present in the glucoamylase complex). Her urine tested negative for sugar, suggesting the problem is in digestion or absorp-  tion, because only sugars that are absorbed and enter the blood can be found in urine. The basis of the hydrogen breath test is that if a sugar  is not absorbed, it is metabolized in the intestinal lumen by bacteria that produce various gases, including hydrogen. The test is often accom-  panied by measurements of the amount of sugar appearing in the blood or feces, and acidity of the feces.   Beans, peas, soybeans, and other leguminous plants contain oligosaccharides  with (1,6)-linked galactose residues that cannot be hydrolyzed for absorption,  including sucrose with 1, 2, or 3 galactose residues attached (see Fig. 27.10).  What is the fate of these polysaccharides in the intestine?   Lactose intolerance refers to a condition of pain, nausea, and flatulence after the  ingestion of foods containing lactose, most notably dairy products. Although it is  often caused by low levels of lactase, it also can be caused by intestinal injury  (defined below).   Lactase activity increases in the human from about 6 to 8 weeks of gestation, and it  rises during the late gestational period (27-32 weeks) through full term. It remains  high for about 1 month after birth and then begins to decline. For most of the  world's population, lactase activity decreases to adult levels at approximately 5 to 7  years of age. Adult levels are less than 10% of that present in infants. These popu-  lations have adult hypolactasia (formerly called adult lactase deficiency) and exhibit  the lactase nonpersistence phenotype. In people who are derived mainly from west-  ern Northern Europeans, and milk-dependent Nomadic tribes of Saharan Africa, the  levels of lactase remain at, or only slightly below, infant levels throughout adult-  hood (lactase persistence phenotype). Thus, adult hypolactasia is the normal  condition for most of the world's population. (Table 27.2).   Intestinal diseases that injure the absorptive cells of the intestinal villi diminish lac-  tase activity along the intestine, producing a condition known as secondary lactase  deficiency. Kwashiorkor (protein malnutrition), colitis, gastroenteritis, tropical and   Lactose intolerance can either be the result of a primary deficiency of lactase  production in the small bowel (as is the case for Deria Voider) or it can be sec-  ondary to an injury to the intestinal mucosa, where lactase is normally pro-  duced. The lactose that is not absorbed is converted by colonic bacteria to lactic acid,  methane gas (CH 4 ), and H 2 gas (see figure on left). The osmotic effect of the lactose and  lactic acid in the bowel lumen is responsible for the diarrhea often seen as part of this  syndrome. Similar symptoms can result from sensitivity to milk proteins (milk intoler-  ance) or from the malabsorption of other dietary sugars.   In adults suspected of having a lactase deficiency, the diagnosis is usually made  inferentially when avoidance of all dairy products results in relief of symptoms and a  rechallenge with these foods reproduces the characteristic syndrome. If the results of  these measures are equivocal, however, the malabsorption of lactose can be more  specifically determined by measuring the H 2 content of the patient's breath after a test  dose of lactose has been consumed.   Deria Voider's symptoms did not appear if she took tablets containing lactase when  she ate dairy products.   Watery  diarrhea  (1 liter extracellular liquid lost  per 9 grams of lactose in  1 glass of milk)   ® These sugars are not digested well  by the human intestine but form  good sources of energy for the bac-  teria of the gut. These bacteria convert the  sugars to H 2 , lactic acid and short-chain fatty  acids. The amount of gas released after a meal  containing beans is especially notorious.   Reproduced with permission from Annu Rev Med 1990;41:145. © 1990 by Annual Reviews, Inc.   nontropical sprue, and excessive alcohol consumption fall into this category. These  diseases also affect other disaccharidases, but sucrase, maltase, isomaltase, and  glucoamylase activities are usually present at such excessive levels that there are  no pathologic effects. Lactase is usually the first activity lost and the last to  recover.   Dietary fiber is the portion of the diet resistant to digestion by human digestive  enzymes. It consists principally of plant materials that are polysaccharide deriva-  tives and lignan (see Fig. 27. 10). The components of fiber are often divided into the  categories of soluble and insoluble fiber, according to their ability to dissolve in  water. Insoluble fiber consists of three major categories; cellulose, hemicellulose,  and lignins. Soluble fiber categories include pectins, mucilages, and gums  (Table 27.3). Although human enzymes cannot digest fiber, the bacterial flora in the  normal human gut may metabolize the more soluble dietary fibers to gases and  short-chain fatty acids, much as they do undigested starch and sugars. Some of these   Whole wheat flour, unprocessed bran, cabbage, peas,  green beans, wax beans, broccoli, brussel sprouts,  cucumber with skin, green peppers, apples, carrots   Oatmeal, dried beans, cauliflower, green beans, cabbage,  carrots, dried peas, potatoes, strawberries   fatty acids may be absorbed and used by the colonic epithelial cells of the gut, and  some may travel to the liver through the hepatic portal vein. We may obtain as much  as 10% of our total calories from compounds produced by bacterial digestion of  substances in our digestive tract.   In 2002, the Committee on Dietary Reference Intakes issued new guidelines for  fiber ingestion; anywhere from 19 to 38 g/day, depending on age and sex of the indi-  vidual. No distinction was made between soluble and insoluble fibers. Adult males  between the ages of 14 and 50 years require 38 grams of fiber per day. Females from  ages 4 to 8 years require 25 g/day; from ages 9 to 16 years, 26 g/day; and from ages  19 to 30, 25 g/day. These numbers are increased during pregnancy and lactation.  One beneficial effect of fiber is seen in diverticular disease, in which sacs or  pouches may develop in the colon because of a weakening of the muscle and sub-  mucosal structures. Fiber is thought to "soften" the stool, thereby reducing pressure  on the colonic wall and enhancing expulsion of feces.   Certain types of soluble fiber have been associated with disease prevention. For  example, pectins may lower blood cholesterol levels by binding bile acids, p-glucan  (obtained from oats) has also been shown, in some studies, to reduce cholesterol lev-  els through a reduction in bile acid resorption in the intestine (see Chapter 34).  Pectins also may have a beneficial effect in the diet of individuals with diabetes mel-  litus by slowing the rate of absorption of simple sugars and preventing high blood  glucose levels after meals. However, each of the beneficial effects which have been  related to "fiber" are relatively specific for the type of fiber, and the physical form of  food which contains the fiber. This factor, along with many others, has made it diffi-  cult to obtain conclusive results from studies of the effects of fiber on human health.   Once the carbohydrates have been split into monosaccharides, the sugars are trans-  ported across the intestinal epithelial cells and into the blood for distribution to all tis-  sues. Not all complex carbohydrates are digested at the same rate within the intestine,  and some carbohydrate sources lead to a near-immediate rise in blood glucose levels  after ingestion, whereas others slowly raise blood glucose levels over an extended  period after ingestion. The glycemic index of a food is an indication of how rapidly  blood glucose levels rise after consumption. Glucose and maltose have the highest  glycemic indices (142, with white bread defined as an index of 100). Table 27.4 indi-  cates the glycemic index for a variety of food types. Although there is no need to  memorize this table, note that cornflakes and potatoes have high glycemic indices,  whereas yogurt and skim milk have particularly low glycemic indices.   Glucose is transported through the absorptive cells of the intestine by facilitated dif-  fusion and by Na + -dependent facilitated transport. (See Chapter 10 for a description  of transport mechanisms.) Glucose, therefore, enters the absorptive cells by binding   The dietician explained to Ann Sulin the rationale for a person with diabetes to  watch their diet. It is important for Ann to add a variety of fibers to her diet. The  gel-forming, water-retaining pectins and gums delay gastric emptying and retard  the rate of absorption of disaccharides and monosaccharides, thus reducing the rate at  which blood glucose levels rise. The glycemic index of foods also needs to be considered  for appropriate maintenance of blood glucose levels in diabetic patients. Consumption of  a low glycemic index diet results in a lower rise in blood glucose levels after eating, which  can be more easily controlled by exogenous insulin. For example, Ms. Sulin is advised to  eat pasta and rice (glycemic index of 67 and 65, respectively) instead of potatoes (glycemic  index of 80-120, depending on the method of preparation), and to incorporate breakfast  cereals composed of wheat bran, barley, and oats into her morning routine.   Pectins are found in fruits, such as  apples. Could this be the basis for  the saying "An apple a day keeps  the doctor away"?   Carrageenan is a type of fiber  derived from seaweed. It is com-  posed of sulfated galactose and  galacturonic acid derivatives (see Fig. 27.10).  The negatively charged sulfate groups form  hydrogen bonds with water and convert the  polysaccharide into a gel-like substance. It is  added to many foods, such as ice cream and  McDonald's McLean burger.   The glycemic response to ingested  foods depends not only on the  glycemic index of the foods, but  also on the fiber and fat content of the food,  as well as its method of preparation. Highly  glycemic carbohydrates can be consumed  before and after exercise, as their metabo-  lism results in a rapid entry of glucose into  the blood, where it is then immediately  available for muscle use. Low glycemic car-  bohydrates enter the circulation slowly and  can be used to best advantage if consumed  before exercise, such that as exercise pro-  gresses glucose is slowly being absorbed  from the intestine into the circulation, where  it can be used to maintain blood glucose  levels during the exercise period.    Fig. 27.11. Facilitative transport. Transport of  glucose occurs without rotation of the glucose  molecule. Multiple groups on the protein bind  the hydroxyl groups of glucose and close  behind it as it is released into the cell (i.e., the  transporter acts like a "gated pore"). O = out-  side; I = inside.   The glucose molecule is extremely  polar and cannot diffuse through  the hydrophobic phospholipid  bilayer of the cell membrane. Each hydroxyl  group of the glucose molecule forms at least  two hydrogen bonds with water molecules,  and random movement would require  energy to dislodge the polar hydroxyl  groups from their hydrogen bonds and to  disrupt the Van der Waals' forces between  the hydrocarbon tails of the fatty acids in the  membrane phospholipid.   ^The epithelial cells of the kidney,  which reabsorb glucose into the  blood, have Na + -dependent glu-  cose transporters similar to those of intes-  tinal epithelial cells. They are thus also able  to transport glucose against its concentra-  tion gradient. Other types of cells use mainly  facilitative glucose transporters that carry  glucose down its concentration gradient.    Table 27.4 Glycemic Index of Selected Foods, with Values Adjusted to White  Bread of 100   to transport proteins, membrane-spanning proteins that bind the glucose molecule on  one side of the membrane and release it on the opposite side (Fig. 27.1 1). Two types  of glucose transport proteins are present in the intestinal absorptive cells: the Na + -  dependent glucose transporters and the facilitative glucose transporters (Fig. 27.12).   Na + -dependent glucose transporters, which are located on the luminal side of the  absorptive cells, enable these cells to concentrate glucose from the intestinal lumen.  A low intracellular Na + concentration is maintained by a Na + ,K + -ATPase on the  serosal (blood) side of the cell that uses the energy from ATP cleavage to pump Na +  out of the cell into the blood. Thus, the transport of glucose from a low concentra-  tion in the lumen to a high concentration in the cell is promoted by the cotransport  of Na + from a high concentration in the lumen to a low concentration in the cell  (secondary active transport).   Facilitative glucose transporters, which do not bind Na + , are located on the serosal  side of the cells. Glucose moves via the facilitative transporters from the high con-  centration inside the cell to the lower concentration in the blood without the expen-  diture of energy. In addition to the Na + -dependent glucose transporters, facilitative  transporters for glucose also exist on the luminal side of the absorptive cells. The  various types of facilitative glucose transporters found in the plasma membranes of  cells (referred to as GLUT 1 to GLUT 5), are described in Table 27.5. One common  structural theme to these proteins is that they all contain 12 membrane- spanning  domains. Note that the sodium-linked transporter on the luminal side of the  intestinal epithelial cell is not a member of the GLUT family.   Fig. 27.12. Na + -dependent and facilitative transporters in the intestinal epithelial cells. Both glucose and  fructose are transported by the facilitated glucose transporters on the luminal and serosal sides of the  absorptive cells. Glucose and galactose are transported by the Na + -glucose cotransporters on the luminal  (mucosal) side of the absorptive cells.   Table 27.5. Properties of the GLUT 1-GLUT 5 Isoforms of the Glucose  Transport Proteins   Expressed in cell types with barrier functions;  a high-affinity glucose transport system   A high capacity, low affinity transporter.  May be used as the glucose sensor in the  pancreas.   Insulin-sensitive transporter. In the presence  of insulin the number of GLUT 4  transporters increases on the cell surface.  A high-affinity system   Genetic techniques have identified additional GLUT transporters (GLUT 7-12), but the role of these  transporters has not yet been fully described.   Galactose is absorbed through the same mechanisms as glucose. It enters the  absorptive cells on the luminal side via the Na + -dependent glucose transporters and  facilitative glucose transporters and is transported through the serosal side on the  facilitative glucose transporters.   Fructose both enters and leaves absorptive epithelial cells by facilitated diffu-  sion, apparently via transport proteins that are part of the GLUT family. The trans-  porter on the luminal side has been identified as GLUT 5. Although this transporter  can transport glucose, it has a much higher activity with fructose (see Fig. 27.12).  Other fructose transport proteins also may be present. For reasons as yet unknown,  fructose is absorbed at a much more rapid rate when it is ingested as sucrose than  when it is ingested as a monosaccharide.   The erythrocyte (red blood cell) is  Kj ] an example of a tissue in which glu-  cose transport is not rate-limiting.  Although the glucose transporter (GLUT 1)  has a K m of 1 to 7 mM, it is present in  extremely high concentrations, constituting  approximately 5% of all membrane proteins.  Consequently, as the blood glucose levels  fall from a postprandial level of 140 mg/dL  (7.5 mM) to the normal fasting level of 80  mg/dL (4.5 mM), or even the hypoglycemic  level of 40 mg/dL (2.2 mM), the supply of  glucose is still adequate for the rates at  which glycolysis and the pentose phosphate  pathway operate.   The properties of the GLUT transport proteins differ between tissues, reflecting  the function of glucose metabolism in each tissue. In most cell types, the rate of  glucose transport across the cell membrane is not rate-limiting for glucose metab-  olism. This is because the isoform of transporter present in these cell types has a  relatively low K m for glucose (that is, a low concentration of glucose will result  in half the maximal rate of glucose transport) or is present in relatively high con-  centration in the cell membrane so that the intracellular glucose concentration  reflects that in the blood. Because the hexokinase isozyme present in these cells  has an even lower K m for glucose (0.05-0.10 mM), variations in blood glucose  levels do not affect the intracellular rate of glucose phosphorylation. However, in  several tissues, the rate of transport becomes rate limiting when the serum level  of glucose is low or when low levels of insulin signal the absence of dietary  glucose.   In the liver, the K m for the glucose transporter (GLUT 2) is relatively high  compared with that of other tissues, probably 15 mM or above. This is in keeping  with the liver's role as the organ that maintains blood glucose levels. As such, the  liver will only convert glucose into other energy storage molecules when the  blood glucose levels are high, such as the time immediately after ingestion of a  meal. In muscle and adipose tissue, the transport of glucose is greatly stimulated  by insulin. The mechanism involves the recruitment of glucose transporters  (specifically, GLUT 4) from intracellular vesicles into the plasma membrane (Fig.  27.13). In adipose tissue, the stimulation of glucose transport across the plasma  membrane by insulin increases its availability for the synthesis of fatty acids and  glycerol from the glycolytic pathway. In skeletal muscle, the stimulation of glu-  cose transport by insulin increases its availability for glycolysis and glycogen  synthesis.   A hypoglycemic response is elicited by a decrease of blood glucose concentration to  some point between 18 and 54 mg/dL (1 and 3 mM). The hypoglycemic response is  a result of a decreased supply of glucose to the brain and starts with light-headedness  and dizziness and may progress to coma. The slow rate of transport of glucose  through the blood-brain barrier (from the blood into the cerebrospinal fluid) at low  levels of glucose is thought to be responsible for this neuroglycopenic response. Glu-  cose transport from the cerebrospinal fluid across the plasma membranes of neurons  is rapid and is not rate limiting for ATP generation from glycolysis.   Fig. 27.14. Glucose transport through the capillary endothelium in neural and  nonneural tissues. Characteristics of transport in each type of tissue are listed by  numbers that refer to the numbers in the drawing. G = glucose.   In the brain, the endothelial cells of the capillaries have extremely tight  junctions, and glucose must pass from the blood into the extracellular cerebrospinal  fluid by GLUT 1 transporters in the endothelial cell membranes (Fig. 27.14), and  then through the basement membrane. Measurements of the overall process of glu-  cose transport from the blood into the brain (mediated by GLUT 3 on neural cells)  show a K m app of 7 to 11 mM, and a maximal velocity not much greater than the rate  of glucose utilization by the brain. Thus, decreases of blood glucose below the fast-  ing level of 80 to 90 mg/dL (approximately 5 mM) are likely to significantly affect  the rate of glucose metabolism in the brain, because of reduced glucose transport  into the brain.   Fig. 27.13. Stimulation by insulin of glucose  transport into muscle and adipose cells. Bind-  ing of insulin to its cell membrane receptor  causes vesicles containing glucose transport  proteins to move from inside the cell to the cell  membrane.   One of five Americans experiences some form of gastrointestinal discom-  fort from 30 minutes to 12 hours after ingesting lactose-rich foods. Most  become symptomatic when they consume more than 25 g lactose at one  time (e.g., 1 pint of milk or its equivalent). Deria Voider 's symptoms were caused  by her "new" diet in this country, which included a glass of milk in addition to the  milk she used on her cereal with breakfast each morning.   Management of lactose intolerance includes a reduction or avoidance of lactose-  containing foods depending on the severity of the deficiency of intestinal lactase.  Hard cheeses (cheddar, Swiss, Jarlsberg) are low in lactose and may be tolerated by  patients with only moderate lactase deficiency. Yogurt with "live and active cultures"  printed on the package contain bacteria that release free lactases when the bacteria  are lysed by gastric acid and proteolytic enzymes. The free lactases then digest the   lactose. Commercially available milk products that have been hydrolyzed with a  lactase enzyme provide a 70% reduction in total lactose content, which may be ade-  quate to prevent digestive symptoms in mildly affected patients. Tablets and capsules  containing lactase are also available and should be taken one-half hour before meals.   Many adults who have a lactase deficiency develop the ability to ingest small  amounts of lactose in dairy products without experiencing symptoms. This adapta-  tion probably involves an increase in the population of colonic bacteria that can  cleave lactose and not a recovery or induction of human lactase synthesis. For many  individuals, dairy products are the major dietary source of calcium, and their com-  plete elimination from the diet can lead to osteoporosis.   Lactose, however, is used as a "filler" or carrying agent in more than 1,000 pre-  scription and over-the-counter drugs in this country. People with lactose intolerance  often unwittingly ingest lactose with their medications.   ^ Poorly controlled diabetic patients such as Ann Sulin frequently have ele-  ) vations in serum glucose levels (hyperglycemia). This is often attributable  to a lack of circulating, active insulin, which will stimulate glucose uptake  (through the recruitment of GLUT 4 transporters from the endoplasmic reticulum to  the plasma membrane) by the peripheral tissues (heart, muscle, and adipose tissue).  Without uptake by these tissues, glucose tends to accumulate within the blood-  stream, leading to hyperglycemia.   The large amount of H 2 produced on fructose ingestion suggested that  Nona Melos's problem was one of a deficiency in fructose transport into  the absorptive cells of the intestinal villi. If fructose were being absorbed  properly, the fructose would not have traveled to the colonic bacteria, which metab-  olized the fructose to generate the hydrogen gas. To confirm the diagnosis, a jeju-  nal biopsy was taken; lactase, sucrase, maltase, and trehalase activities were normal  in the jejunal cells. The tissue was also tested for the enzymes of fructose metabo-  lism; these were in the normal range as well. Although Nona had no sugar in her  urine, malabsorption of disaccharides can result in their appearance in the urine if  damage to the intestinal mucosal cells allows their passage into the interstitial fluid.  When Nona was placed on a diet free of fruit juices and other foods containing fruc-  tose, she did well and could tolerate small amounts of pure sucrose.   More than 50% of the adult population are estimated to be unable to absorb fruc-  tose in high doses (50 g), and more than 10% cannot completely absorb 25 g  fructose. These individuals, like those with other disorders of fructose metabolism,  must avoid fruits and other foods containing high concentrations of fructose.   Cholera is an acute watery diarrheal disorder caused by the water-borne,  Gram-negative bacterium Vibrio cholerae. It is a disease of antiquity;  descriptions of epidemics of the disease date to before 500 bc. During epi-  demics, the infection is spread by large numbers of vibrio that enter water sources  from the voluminous liquid stools and contaminate the environment, particularly in  areas of extreme poverty where plumbing and modern waste-disposal systems are  primitive or nonexistent.   After being ingested, the V. cholerae organisms attach to the brush border of the  intestinal epithelium and secrete an exotoxin that binds irreversibly to a specific  chemical receptor (G M1 ganglioside) on the cell surface. This exotoxin catalyzes an  ADP-ribosylation reaction that increases adenylate cyclase activity and thus cAMP  levels in the enterocyte. As a result, the normal absorption of sodium, anions, and  water from the gut lumen into the intestinal cell is markedly diminished. The  exotoxin also stimulates the crypt cells to secrete chloride, accompanied by cations   and water, from the bloodstream into the lumen of the gut. The resulting loss of  solute-rich diarrheal fluid may, in severe cases, exceed 1 liter/hour, leading to rapid  dehydration and even death.   The therapeutic approach to cholera takes advantage of the fact that the Na + -  dependent transporters for glucose and amino acids are not affected by the cholera  exotoxin. As a result, coadministration of glucose and Na + by mouth results in the  uptake of glucose and Na + , accompanied by chloride and water, thereby partially  correcting the ion deficits and fluid loss. Amino acids and small peptides are also  adsorbed by Na + -dependent cotransport involving transport proteins distinct from  the Na + -dependent glucose transporters. Therefore, addition of protein to the glu-  cose-sodium replacement solution enhances its effectiveness and markedly  decreases the severity of the diarrhea. Adjunctive antibiotic therapy also shortens  the diarrheal phase of cholera but does not decrease the need for the oral  replacement therapy outlined earlier.   Bell GJ, Burant CF, Takeda J, Gould GW. Structure and function of mammalian facilitative sugar trans-  porters. J Biol Chem 1993;278:19161-19164.   Brown GK. Glucose transporters: structure, function and consequences of deficiency. J Inherit Metab Dis  2000;23:237-246.   Linder MC, ed. Nutrition and metabolism of carbohydrates. In: Nutritional Biochemistry and Metabo-  lism with Clinical Applications, 2nd Ed. New York: Elsevier, 1991:21-50.   Semenza G, Auricchio S, Mantei, N. Small-intestinal disaccharidases. In: Scriver CR, Beaudet AL, Sly  WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 8th Ed. New York:  McGraw-Hill, 2001:1623-1650.   1 . The facilitative transporter most responsible for transporting fructose from the blood into cells is which of the following?   2. An alcoholic patient developed a pancreatitis that affected his exocrine pancreatic function. He exhibited discomfort after eat-  ing a high-carbohydrate meal. The patient most likely had a reduced ability to digest which of the following?   3. A type I diabetic neglects to take his insulin injections while on a weekend vacation. Cells of which tissue would be most  greatly affected by this mistake?   4. After digestion of a piece of cake that contains flour, milk, and sucrose as its primary ingredients, the major carbohydrate  products entering the blood are which of the following?   5. A patient has a genetic defect that causes intestinal epithelial cells to produce disaccharidases of much lower activity than nor-  mal. Compared with a normal person, after eating a bowl of milk and oatmeal sweetened with table sugar, this patient will  exhibit higher levels of which of the following?   Glycogen is the storage form of glucose found in most types of cells. It is com-  posed of glucosyl units linked by ol-1,4 glycosidic bonds, with ol-1,6 branches  occurring roughly every 8 to 10 glucosyl units (Fig. 28.1). The liver and skeletal  muscle contain the largest glycogen stores.   The formation of glycogen from glucose is an energy-requiring pathway that  begins, like most of glucose metabolism, with the phosphorylation of glucose to  glucose 6-phosphate. Glycogen synthesis from glucose 6-phosphate involves the  formation of uridine diphosphate glucose (UDP-glucose) and the transfer of glu-  cosyl units from UDP-glucose to the ends of the glycogen chains by the enzyme  glycogen synthase. Once the chains reach approximately 11 glucosyl units, a  branching enzyme moves six to eight units to form an a (1 ,6) branch.   Glycogenolysis, the pathway for glycogen degradation, is not the reverse of the  biosynthetic pathway. The degradative enzyme glycogen phosphorylase removes  glucosyl units one at a time from the ends of the glycogen chains, converting them  to glucose 1 -phosphate without re synthesizing UDP-glucose or UTP. A debranch-  ing enzyme removes the glucosyl residues near each branchpoint.   Liver glycogen serves as a source of blood glucose. To generate glucose, the  glucose 1 -phosphate produced from glycogen degradation is converted to   Glycogen degradation is a phospho-  \m ) rolysis reaction (breaking of a bond  *S using a phosphate ion as a nucle-  ophile). Enzymes that catalyze phosphorolysis  reactions are named phosphorylase. Because  more than one type of phosphorylase exists,  the substrate usually is included in the name of  the enzyme, such as glycogen phosphorylase  or purine nucleoside phosphorylase.   Fig. 28.1. Glycogen structure. Glycogen is composed of glucosyl units linked by a-l,4-glycosidic bonds and a-l,6-glycosidic  bonds. The branches occur more frequently in the center of the molecule, and less frequently in the periphery. The anomeric car-  bon that is not attached to another glucosyl residue (the reducing end) is attached to the protein glycogenin by a glycosidic bond.   glucose 6-phosphate. Glucose 6-phosphatase , an enzyme found only in liver  and kidney, converts glucose 6-phosphate to free glucose, which then enters the  blood.   Glycogen synthesis and degradation are regulated in liver by hormonal  changes that signal the need for blood glucose (see Chapter 26). The body main-  tains fasting blood glucose levels at approximately 80 mgldL to ensure that the  brain and other tissues that are dependent on glucose for the generation of adeno-  sine triphosphate (ATP) have a continuous supply. The lack of dietary glucose,  signaled by a decrease of the insulin/ 'glucagon ratio, activates liver glycogenoly-  sis and inhibits glycogen synthesis. Epinephrine, which signals an increased uti-  lization of blood glucose and other fuels for exercise or emergency situations, also  activates liver glycogenolysis. The hormones that regulate liver glycogen metabo-  lism work principally through changes in the phosphorylation state of glycogen  synthase in the bio synthetic pathway and glycogen phosphorylase in the degrada-  tive pathway.   In skeletal muscle, glycogen supplies glucose 6-phosphate for ATP synthesis in  the glycolytic pathway. Muscle glycogen phosphorylase is stimulated during exer-  cise by the increase of adenosine monophosphate (AMP), an allosteric activator  of the enzyme, and also by phosphorylation. The phosphorylation is stimulated by  calcium released during contraction, and by the " fight -or-flight" hormone epi-  nephrine. Glycogen synthesis is activated in resting muscles by the elevation of  insulin after carbohydrate ingestion.   The neonate must rapidly adapt to an intermittent fuel supply after birth. Once  the umbilical cord is clamped, the supply of glucose from the maternal circulation  is interrupted. The combined effect of epinephrine and glucagon on the liver  glycogen stores of the neonate rapidly restore glucose levels to normal.   The Apgar score is an objective  estimate of the overall condition of  the newborn, determined at both 1  and 5 minutes after birth. The best score is  10 (normal in all respects).   • A newborn baby girl, Getta Carbo, was born after a 38-week gestation.  IB Her mother, a 36-year-old woman, had moderate hypertension during the   " last trimester of pregnancy related to a recurrent urinary tract infection that  resulted in a severe loss of appetite and recurrent vomiting in the month preceding  delivery. Fetal bradycardia (slower than normal fetal heart rate) was detected with  each uterine contraction of labor, a sign of possible fetal distress.   At birth Getta was cyanotic (a bluish discoloration caused by a lack of adequate  oxygenation of tissues) and limp. She responded to several minutes of assisted ven-  tilation. Her Apgar score of 3 was low at 1 minute after birth, but improved to a  score of 7 at 5 minutes.   Physical examination in the nursery at 10 minutes showed a thin, malnourished  female newborn. Her body temperature was slightly low, her heart rate was rapid,  and her respiratory rate of 35 breaths/minute was elevated. Getta 's birth weight  was only 2,100 g, compared with a normal value of 3,300 g. Her length was 47  cm, and her head circumference was 33 cm (low normal). The laboratory reported  that Getta's serum glucose level when she was unresponsive was 14 mg/dL. A glu-  cose value below 40 mg/dL (2.5 mM) is considered to be abnormal in newborn  infants.   At 5 hours of age, she was apneic (not breathing) and unresponsive. Ventilatory  resuscitation was initiated and a cannula placed in the umbilical vein. Blood for a  glucose level was drawn through this cannula, and 5 mL of a 20% glucose solution  was injected. Getta slowly responded to this therapy.   Jim Bodie, a 19-year-old body builder, was rushed to the hospital emer-  gency room in a coma. One-half hour earlier, his mother had heard a loud  crashing sound in the basement where Jim had been lifting weights and  completing his daily workout on the treadmill. She found her son on the floor hav-  ing severe jerking movements of all muscles (a grand mal seizure).   In the emergency room, the doctors learned that despite the objections of his  family and friends, Jim regularly used androgens and other anabolic steroids in an  effort to bulk up his muscle mass.   On initial physical examination, he was comatose with occasional involuntary  jerking movements of his extremities. Foamy saliva dripped from his mouth. He  had bitten his tongue and had lost bowel and bladder control at the height of the  seizure.   The laboratory reported a serum glucose level of 18 mg/dL (extremely low).  The intravenous infusion of 5% glucose (5 g of glucose per 100 mL of solution),  which had been started earlier, was increased to 10%. In addition, 50 g glucose was  given over 30 seconds through the intravenous tubing.   Glycogen, the storage form of glucose, is a branched glucose polysaccharide com-  posed of chains of glucosyl units linked by a- 1,4 bonds with a- 1,6 branches every  8 to 10 residues (see Fig. 28.1). In a molecule of this highly branched structure,  only one glucosyl residue has an anomeric carbon that is not linked to another glu-  cose residue. This anomeric carbon at the beginning of the chain is attached to the  protein glycogenin. The other ends of the chains are called nonreducing ends (see  Chapter 5). The branched structure permits rapid degradation and rapid synthesis  of glycogen because enzymes can work on several chains simultaneously from the  multiple nonreducing ends.   Glycogen is present in tissues as polymers of very high molecular weight  (10 7 -10 8 ) collected together in glycogen particles. The enzymes involved in glyco-  gen synthesis and degradation, and some of the regulatory enzymes, are bound to  the surface of the glycogen particles.   Glycogen is found in most cell types, where it serves as a reservoir of glucosyl  units for ATP generation from glycolysis.   Glycogen is degraded mainly to glucose 1 -phosphate, which is converted to glu-  cose 6-phosphate. In skeletal muscle and other cell types, the glucose 6-phosphate  enters the glycolytic pathway (Fig. 28.2). Glycogen is an extremely important fuel  source for skeletal muscle when ATP demands are high and when glucose 6-phos-  phate is used rapidly in anaerobic glycolysis. In many other cell types, the small  glycogen reservoir serves a similar purpose; it is an emergency fuel source that  supplies glucose for the generation of ATP in the absence of oxygen or during  restricted blood flow. In general, glycogenolysis and glycolysis are activated  together in these cells.   Glycogen serves a very different purpose in liver than in skeletal muscle and other  tissues (see Fig. 28.2). Liver glycogen is the first and immediate source of glucose  for the maintenance of blood glucose levels. In the liver, the glucose 6-phosphate that  is generated from glycogen degradation is hydrolyzed to glucose by glucose 6-phos-  phatase, an enzyme present only in the liver and kidneys. Glycogen degradation thus  provides a readily mobilized source of blood glucose as dietary glucose decreases, or  as exercise increases the utilization of blood glucose by muscles.    Jim Boclie's treadmill exercise and  most other types of moderate exer-  cise involving whole body move-  ment (running, skiing, dancing, tennis)  increase the utilization of blood glucose and  other fuels by skeletal muscles. The blood  glucose is normally supplied by the stimula-  tion of liver glycogenolysis and gluconeoge-  nesis.    Fig. 28.2. Glycogenolysis in skeletal muscle  and liver. Glycogen stores serve different func-  tions in muscle cells and liver. In the muscle  and most other cell types, glycogen stores  serve as a fuel source for the generation of  ATP. In the liver, glycogen stores serve as a  source of blood glucose.   Regulation of glycogen synthesis  \ \ ) serves to prevent futile cycling and  waste of ATP. Futile cycling refers  to a situation in which a substrate is con-  verted to a product through one pathway,  and the product converted back to the sub-  strate through another pathway. Because  the biosynthetic pathway is energy-requir-  ing, futile cycling results in a waste of high-  energy phosphate bonds. Thus, glycogen  synthesis is activated when glycogen degra-  dation is inhibited, and vice versa.   The pathways of glycogenolysis and gluconeogenesis in the liver both supply blood  glucose, and, consequently, these two pathways are activated together by glucagon. Glu-  coneogenesis, the synthesis of glucose from amino acids and other gluconeogenic pre-  cursors (discussed in detail in Chapter 31), also forms glucose 6-phosphate, so that glu-  cose 6-phosphatase serves as a "gateway" to the blood for both pathways (see Fig. 28.2).   Glycogen synthesis, like almost all the pathways of glucose metabolism, begins  with the phosphorylation of glucose to glucose 6-phosphate by hexokinase or, in the  liver, glucokinase (Fig. 28.3). Glucose 6-phosphate is the precursor of glycolysis,  the pentose phosphate pathway, and of pathways for the synthesis of other sugars.  In the pathway for glycogen synthesis, glucose 6-phosphate is converted to glucose  1 -phosphate by phosphoglucomutase, a reversible reaction.   Glycogen is both formed from and degraded to glucose 1 -phosphate, but the  biosynthetic and degradative pathways are separate and involve different enzymes  (see Fig. 28.3). The biosynthetic pathway is an energy-requiring pathway; high-  energy phosphate from UTP is used to activate the glucosyl residues to UDP-  glucose (Fig. 28.4). In the degradative pathway, the glycosidic bonds between the  glucosyl residues in glycogen are simply cleaved by the addition of phosphate to  produce glucose 1 -phosphate (or water to produce free glucose), and UDP-glucose  is not resynthesized. The existence of separate pathways for the formation and  degradation of important compounds is a common theme in metabolism. Because   Fig. 28.3. Scheme of glycogen synthesis and degradation. SI. Glucose 6-phosphate is formed  from glucose by hexokinase in most cells, and glucokinase in the liver. It is a metabolic branch-  point for the pathways of glycolysis, the pentose phosphate pathway, and glycogen synthesis.  S2. UDP-Glucose (UDP-G) is synthesized from glucose 1 -phosphate. UDP-Glucose is the  branchpoint for glycogen synthesis and other pathways requiring the addition of carbohydrate  units. S3. Glycogen synthesis is catalyzed by glycogen synthase and the branching enzyme.  Dl. Glycogen degradation is catalyzed by glycogen phosphorylase and a debrancher enzyme.  D2. Glucose 6-phosphatase in the liver generates free glucose from glucose 6-phosphate.   Fig. 28.4. Formation of UDP-glucose. The high-energy phosphate bond of UTP provides the energy for the formation of a high-energy bond in  UDP-glucose. Pyrophosphate (PPj), released by the reaction, is cleaved to 2 Pj.   the synthesis and degradation pathways use different enzymes, one can be activated  while the other is inhibited.   Glycogen synthesis requires the formation of a-l,4-glycosidic bonds to link glucosyl  residues in long chains and the formation of an a- 1,6 branch every 8 to 10 residues (Fig.  28.5). Most of glycogen synthesis occurs through the lengthening of the polysaccharide  chains of a preexisting glycogen molecule (a glycogen primer) in which the reducing  end of the glycogen is attached to the protein glycogenin. To lengthen the glycogen  chains, glucosyl residues are added from UDP-glucose to the nonreducing ends of the  chain by glycogen synthase. The anomeric carbon of each glucosyl residue is attached  in an a- 1,4 bond to the hydroxyl on carbon 4 of the terminal glucosyl residue. When the  chain reaches 1 1 residues in length, a 6- to 8-residue piece is cleaved by amylo-4:6-  transferase and reattached to a glucosyl unit by an a- 1,6 bond. Both chains continue to  lengthen until they are long enough to produce two new branches. This process contin-  ues, producing highly branched molecules. Glycogen synthase, the enzyme that attaches  the glucosyl residues in 1,4-bonds, is the regulated step in the pathway.   The synthesis of new glycogen primer molecules also occurs. Glycogenin, the  protein to which glycogen is attached, glycosylates itself (autoglycosylation) by  attaching the glucosyl residue of UDP-glucose to the hydroxyl side chain of a ser-  ine residue in the protein. The protein then extends the carbohydrate chain (using  UDP-glucose as the substrate) until the glucosyl chain is long enough to serve as a  substrate for glycogen synthase.   Glycogen is degraded by two enzymes, glycogen phosphorylase and the debrancher  enzyme (Fig. 28.6). The enzyme glycogen phosphorylase starts at the end of a chain  and successively cleaves glucosyl residues by adding phosphate to the terminal gly-  cosidic bond, thereby releasing glucose 1 -phosphate. However, glycogen phospho-  rylase cannot act on the glycosidic bonds of the four glucosyl residues closest to a  branchpoint because the branching chain sterically hinders a proper fit into the cat-  alytic site of the enzyme. The debrancher enzyme, which catalyzes the removal of  the four residues closest to the branchpoint, has two catalytic activities: it acts as a  transferase and as an a 1,6-glucosidase. As a transferase, the debrancher first  removes a unit containing three glucose residues, and adds it to the end of a longer  chain by an a- 1,4 bond. The one glucosyl residue remaining at the 1,6-branch is  hydrolyzed by the amylo- 1 ,6-glucosidase activity of the debrancher, resulting in the  release of free glucose. Thus, one glucose and approximately 7 to 9 glucose 1 -phos-  phate residues are released for every branchpoint.   ol Glucose residue #1 Glucose residue  Oj linked oc-1 ,4 #J linked oc-1 ,6   Branching of glycogen serves two  \\ ) major roles; increased sites for  *r synthesis and degradation, and  enhancing the solubility of the molecule.   A genetic defect of lysosomal glu-  cosidase, called type II glycogen  storage disease, leads to the accu-  mulation of glycogen particles in large,  membrane-enclosed residual bodies, which  disrupt the function of liver and muscle cells.  Children with this disease usually die of  heart failure at a few months of age.   A series of inborn errors of metab-  ■ B* olism, the glycogen storage dis-  eases, result from deficiencies in  the enzymes of glycogenolysis (see Table  28.1). Muscle glycogen phosphorylase, the  key regulatory enzyme of glycogen degrada-  tion, is genetically different from liver glyco-  gen phosphorylase, and thus a person may  have a defect in one and not the other. Why  do you think that a genetic deficiency in  muscle glycogen phosphorylase (McArdle's  disease) is a mere inconvenience, whereas a  deficiency of liver glycogen phosphorylase  (Hers' disease) can be lethal?   Some degradation of glycogen also occurs within lysosomes when glycogen  particles become surrounded by membranes that then fuse with the lysosomal mem-  branes. A lysosomal glucosidase hydrolyzes this glycogen to glucose.   The regulation of glycogen synthesis in different tissues matches the function of  glycogen in each tissue. Liver glycogen serves principally for the support of  blood glucose during fasting or during extreme need (e.g., exercise), and the  degradative and biosynthetic pathways are regulated principally by changes in   Enlarged liver and kidney,  growth failure, fasting  hypoglycemia, acidosis,  lipemia, thrombocyte dys-  function. Hypoglycemia is  the most severe.   Infantile form: early-onset  progressive muscle hypo-  tonia, cardiac failure, death  before 2 years; juvenile  form: later-onset myopa-  thy with variable cardiac  involvement, adultform:  limb-girdle muscular dys-  trophy-like features. Glyco-  gen deposits accumulate  in lysosomes.   Hepatosplenomegaly;  symptoms may arise from  a hepatic reaction to the  presence of a foreign  body (glycogen with long  outer branches). Usually  fatal.   Exercise-induced muscular  pain, cramps, and pro-  gressive weakness,  sometimes with  myoglobinuria   Reproduced with permission, from Annu Rev Nutr 1993; 13:85. © 1993 by Annual Reviews, Inc.  a All of these diseases but type 0 are characterized by increased glycogen deposits.  b FTT = failure to thrive   c Glucose 6-phosphatase is composed of several subunits that also transport glucose, glucose 6-phos-  phate, phosphate, and pyrophosphate across the endoplasmic reticulum membranes. Therefore, there are  several subtypes of this disease, corresponding to defects in the different subunits.  d There are several subtypes of this disease, corresponding to different mutations and patterns of inheritance.   Blood: Epinephrine t  Tissue: cAMP t  Ca 2 + -calmodulin t   Glycogen synthesis i  Glucose transport i  Glycogen synthesis t  Glucose transport t  Glycogen synthesis I  Glycogen degradation t  Glycolysis t   a t = increased compared with other physiologic states; I = decreased compared with other physiologic states.   the insulin/glucagon ratio and by blood glucose levels, which reflect the avail-  ability of dietary glucose (Table 28.2). Degradation of liver glycogen is also acti-  vated by epinephrine, which is released in response to exercise, hypoglycemia,  or other stress situations in which there is an immediate demand for blood glu-  cose. In contrast, in skeletal muscles, glycogen is a reservoir of glucosyl units for  the generation of ATP from glycolysis and glucose oxidation. As a consequence,  muscle glycogenolysis is regulated principally by AMP, which signals a lack of  ATP, and by Ca 2+ released during contraction. Epinephrine, which is released in  response to exercise and other stress situations, also activates skeletal muscle  glycogenolysis. The glycogen stores of resting muscle decrease very little during  fasting.   Liver glycogen is synthesized after a carbohydrate meal when blood glucose levels  are elevated, and degraded as blood glucose levels decrease. When an individual  eats a carbohydrate-containing meal, blood glucose levels immediately increase,  insulin levels increase, and glucagon levels decrease (see Fig. 26.8). The increase of  blood glucose levels and the rise of the insulin/glucagon ratio inhibit glycogen  degradation and stimulate glycogen synthesis. The immediate increased transport of  glucose into peripheral tissues, and storage of blood glucose as glycogen, helps to  bring circulating blood glucose levels back to the normal 80- to 100-mg/dL range  of the fasted state. As the length of time after a carbohydrate-containing meal  increases, insulin levels decrease, and glucagon levels increase. The fall of the  insulin/glucagon ratio results in inhibition of the biosynthetic pathway and   Muscle glycogen is used within the muscle to support exercise. Thus, an indi-  vidual with McArdle's disease (type V glycogen storage disease) experiences no  other symptoms but unusual fatigue and muscle cramps during exercise. These  symptoms may be accompanied by myoglobinuria and release of muscle creatine kinase  into the blood.   Liver glycogen is the first reservoir for the support of blood glucose levels, and a defi-  ciency in glycogen phosphorylase or any of the other enzymes of liver glycogen degra-  dation can result in fasting hypoglycemia. The hypoglycemia is usually mild because  patients can still synthesize glucose from gluconeogenesis (see Table 28.1).   Maternal blood glucose readily  crosses the placenta to enter the fetal  circulation. During the last 9 or 10  weeks of gestation, glycogen formed from  maternal glucose is deposited in the fetal liver  under the influence of the insulin-dominated  hormonal milieu of that period. At birth, mater-  nal glucose supplies cease, causing a tempo-  rary physiologic drop in glucose levels in the  newborn's blood, even in normal healthy  infants. This drop serves as one of the signals  for glucagon release from the newborn's pan-  creas, which, in turn, stimulates glycogenoly-  sis. As a result, the glucose levels in the new-  born return to normal.   Healthy full-term babies have adequate  stores of liver glycogen to survive short (12  hours) periods of caloric deprivation provided  other aspects of fuel metabolism are normal.  Because Getta Carbo's mother was markedly  anorexic during the critical period when the  fetal liver is normally synthesizing glycogen  from glucose supplied in the maternal blood,  Getta's liver glycogen stores were below nor-  mal. Thus, because fetal glycogen is the major  source of fuel for the newborn in the early  hours of life, Getta became profoundly hypo-  glycemic within 5 hours of birth because of  her low levels of stored carbohydrate.   Plasma glucose levels in the neonate. The  normal range of blood glucose levels in the  neonate lies between the two black lines.  The stippled blue area represents the range  of hypoglycemia in the neonate that should  be treated. Treatment of neonates with  blood glucose levels that fall within the  dashed blue box, the zone of clinical uncer-  tainty, is controversial. The units of plasma  glucose are given in millimoles/L Both mil-  ligrams/dL (milligrams/100 ml_) and mil-  limoles/L are used clinically for the values of  blood glucose: 80 mg/dL glucose is equiva-  lent to 5 mmol/L (5 mM). From Mehta A.  Arch Dis Child 1994;70:F54.   A patient was diagnosed as an  ■ B* infant with type III glycogen storage  disease, a deficiency of debrancher  enzyme (see Table 28.1). The patient had  hepatomegaly (an enlarged liver) and experi-  enced bouts of mild hypoglycemia. To diag-  nose the disease, glycogen was obtained from  the patient's liver by biopsy after the patient  had fasted overnight and compared with nor-  mal glycogen. The glycogen samples were  treated with a preparation of commercial  glycogen phosphorylase and commerical  debrancher enzyme. The amounts of glucose  1-phosphate and glucose produced in the  assay were then measured. The ratio of glu-  cose 1-phosphate to glucose for the normal  glycogen sample was 9:1, and the ratio for the  patient was 3:1. Can you explain these results?   Table 28.3. Effect of Fasting on Liver Glycogen Content in the Human   activation of the degradative pathway. As a result, liver glycogen is rapidly degraded  to glucose, which is released into the blood.   Although glycogenolysis and gluconeogenesis are activated together by the same  regulatory mechanisms, glycogenolysis responds more rapidly, with a greater out-  pouring of glucose. A substantial proportion of liver glycogen is degraded within  the first few hours after eating (Table 28.3). The rate of glycogenolysis is fairly con-  stant for the first 22 hours, but in a prolonged fast the rate decreases significantly as  the liver glycogen supplies dwindle. Liver glycogen stores are, therefore, a rapidly  rebuilt and degraded store of glucose, ever responsive to small and rapid changes of  blood glucose levels.   Both glycogen phosphorylase and glycogen synthase will be covalently modified to  regulate their activity (Fig. 28.7). When activated by covalent modification, glyco-  gen phosphorylase is referred to as glycogen phosphorylase a (remember a for  active); when the covalent modification is removed, and the enzyme is inactive, it is  referred to as glycogen phosphorylase b. Glycogen synthase, when not covalently  modified is active, and can be designated glycogen synthase a or glycogen synthase  I (the I stands for independent of modifiers for activity). When glycogen synthase  is covalently modified, it is inactive, in the form of glycogen synthase b or glyco-  gen synthase D (for dependent on a modifier for activity).   Insulin and glucagon regulate liver glycogen metabolism by changing the phosphory-  lation state of glycogen phosphorylase in the degradative pathway and glycogen syn-  thase in the biosynthetic pathway. An increase of glucagon and decrease of insulin  during the fasting state initiates a cAMP-directed phosphorylation cascade, which  results in the phosphorylation of glycogen phosphorylase to an active enzyme, and the   Fig. 28.7. The conversion of active and inactive forms of glycogen phosphorylase (A) and  glycogen synthase (B). Note how the nomenclature changes depending on the phosphoryla-  tion and activity state of the enzyme.   phosphorylation of glycogen synthase to an inactive enzyme (Fig. 28.8). As a conse-  quence, glycogen degradation is stimulated, and glycogen synthesis is inhibited.   3. GLUCAGON ACTIVATES A PHOSPHORYLATION CASCADE THAT  CONVERTS GLYCOGEN PHOSPHORYLASE b TO GLYCOGEN  PHOSPHORYLASE a   Glucagon regulates glycogen metabolism through its intracellular second mes-  senger cAMP and protein kinase A (see Chapter 26). Glucagon, by binding to its  cell membrane receptor, transmits a signal through G proteins that activates  adenylate cyclase, causing cAMP levels to increase (see Fig. 28.8). cAMP binds  to the regulatory subunits of protein kinase A, which dissociate from the catalytic  subunits. The catalytic subunits of protein kinase A are activated by the dissocia-  tion and phosphorylate the enzyme phosphorylase kinase, activating it. Phospho-  rylase kinase is the protein kinase that converts the inactive liver glycogen  phosphorylase b conformer to the active glycogen phosphorylase a conformer  by transferring a phosphate from ATP to a specific serine residue on the   ®With a deficiency of debrancher  enzyme, but normal levels of  glycogen phosphorylase, the  glycogen chains of the patient could be  degraded in vivo only to within 4 residues of  the branchpoint. When the glycogen sam-  ples were treated with the commercial  preparation containing normal enzymes,  one glucose residue was released for each  a-1,6 branch. However, in the patient's  glycogen sample, with the short outer  branches, three glucose 1-phosphates and  one glucose residue were obtained for each  a-1,6 branch. Normal glycogen has 8-10 glu-  cosyl residues per branch, and thus gives a  ratio of approximately 9 moles of glucose  1-phosphate to 1 mole of glucose.   Fig. 28.8. Regulation of glycogen synthesis and degradation in the liver. 1 . Glucagon binding to the glucagon receptor or epinephrine binding  to a (3 receptor in the liver activates adenylate cyclase, via G proteins, which synthesizes cAMP from ATP. 2. cAMP binds to protein kinase A  (cAMP-dependent protein kinase), thereby activating the catalytic subunits. 3. Protein kinase A activates phosphorylase kinase by phosphoryla-  tion. 4. Phosphorylase kinase adds a phosphate to specific serine residues on glycogen phosphorylase b, thereby converting it to the active glyco-  gen phosphorylase a. 5. Protein kinase A also phosphorylates glycogen synthase, thereby decreasing its activity. 6. As a result of the inhibition  of glycogen synthase and the activation of glycogen phosphorylase, glycogen is degraded to glucose 1-phosphate. The blue dashed lines denote  reactions that are decreased in the livers of fasting individuals.   To remember whether a particular  enzyme has been activated or  inhibited by cAMP-dependent  phosphorylation, consider whether it makes  sense for the enzyme to be active or inhib-  ited under fasting conditions (In a PHast,  PHosphorylate).   Most of the enzymes that are regu-  \\ lated by phosphorylation have mul-  tiple phosphorylation sites. Glyco-  gen phosphorylase, which has only one  serine per subunit, and can be phosphory-  lated only by phosphorylase kinase, is the  exception. For some enzymes, the phospho-  rylation sites are antagonistic, and phospho-  rylation initiated by one hormone counter-  acts the effects of other hormones. For other  enzymes, the phosphorylation sites are syn-  ergistic, and phosphorylation at one site  stimulated by one hormone can act synergis-  tically with phosphorylation at another site.    Most of the enzymes that are regu-  lated by phosphorylation also can  be converted to the active confor-   mation by allosteric effectors. Glycogen syn-  thase b, the less active form of glycogen syn-  thase, can be activated by the accumulation  of glucose 6-phosphate above physiologic  levels. The activation of glycogen synthase  by glucose 6-phosphate may be important in  individuals with glucose 6-phosphatase defi-  ciency, a disorder known as type I or von  Gierke's glycogen storage disease. When  glucose 6-phosphate produced from gluco-  neogenesis accumulates in the liver, it acti-  vates glycogen synthesis even though the  individual may be hypoglycemic and have  low insulin levels. Glucose 1-phosphate is  also elevated, resulting in the inhibition of  glycogen phosphorylase. As a consequence,  large glycogen deposits accumulate, and  hepatomegaly occurs.    phosphorylase subunits. As a result of the activation of glycogen phosphorylase,  glycogenolysis is stimulated.   When glycogen degradation is activated by the c AMP- stimulated phosphorylation  cascade, glycogen synthesis is simultaneously inhibited. The enzyme glycogen syn-  thase is also phosphorylated by protein kinase A, but this phosphorylation results in  a less active form, glycogen synthase b.   The phosphorylation of glycogen synthase is far more complex than glycogen  phosphorylase. Glycogen synthase has multiple phosphorylation sites and is acted  on by up to 10 different protein kinases. Phosphorylation by protein kinase A does  not, by itself, inactivate glycogen synthase. Instead, phosphorylation by protein  kinase A facilitates the subsequent addition of phosphate groups by other kinases,  and these inactivate the enzyme. A term that has been applied to changes of activ-  ity resulting from multiple phosphorylation is hierarchical or synergistic phospho-  rylation-the phosphorylation of one site makes another site more reactive and eas-  ier to phosphorylate by a different protein kinase   At the same time that protein kinase A and phosphorylase kinase are adding  phosphate groups to enzymes, the protein phosphatases that remove this phos-  phate are inhibited. Protein phosphatases remove the phosphate groups, bound to  serine or other residues of enzymes, by hydrolysis. Hepatic protein phosphatase-  1 (hepatic PP-1), one of the major protein phosphatases involved in glycogen  metabolism, removes phosphate groups from phosphorylase kinase, glycogen  phosphorylase, and glycogen synthase. During fasting, hepatic PP-1 is inacti-  vated by a number of mechanisms. One is dissociation from the glycogen parti-  cle, such that the substrates are no longer available to the phosphatase. A second  is the binding of inhibitor proteins, such as the protein called inhibitor- 1, which,  when phosphorylated by a glucagon (or epinephrine) -directed mechanism, binds  to and inhibits phosphatase action. Insulin indirectly activates hepatic PP-1  through its own signal transduction cascade initiated at the insulin receptor  tyrosine kinase.   Insulin is antagonistic to glucagon in the degradation and synthesis of glycogen.  The glucose level in the blood is the signal controlling the secretion of insulin and  glucagon. Glucose stimulates insulin release and suppresses glucagon release; one  increases while the other decreases after a high carbohydrate meal. However,  insulin levels in the blood change to a greater degree with the fasting-feeding cycle  than the glucagon levels, and thus insulin is considered the principal regulator of  glycogen synthesis and degradation. The role of insulin in glycogen metabolism is  often overlooked because the mechanisms by which insulin reverses all of the  effects of glucagon on individual metabolic enzymes is still under investigation. In  addition to the activation of hepatic PP-1 through the insulin receptor tyrosine  kinase phosphorylation cascade, insulin may activate the phosphodiesterase that  converts cAMP to AMP, thereby decreasing cAMP levels and inactivating protein  kinase A. Regardless of the mechanisms involved, insulin is able to reverse all of  the effects of glucagon and is the most important hormonal regulator of blood  glucose levels.   When an individual eats a high-carbohydrate meal, glycogen degradation immedi-  ately stops. Although the changes in insulin and glucagon levels are relatively rapid  (10-15 minutes), the direct inhibitory effect of rising glucose levels on glycogen  degradation is even more rapid. Glucose, as an allosteric effector, inhibits liver  glycogen phosphorylase a by stimulating dephosphorylation of this enzyme. As  insulin levels rise and glucagon levels fall, cAMP levels decrease and protein kinase  A reassociates with its inhibitory subunits and becomes inactive. The protein phos-  phatases are activated, and phosphorylase a and glycogen synthase b are dephos-  phorylated. The collective result of these effects is rapid inhibition of glycogen  degradation, and rapid activation of glycogen synthesis.   Epinephrine, the 4 'fight-or- flight" hormone, is released from the adrenal medulla in  response to neural signals reflecting an increased demand for glucose. To flee from  a dangerous situation, skeletal muscles use increased amounts of blood glucose to  generate ATP. As a result, liver glycogenolysis must be stimulated. In the liver,  epinephrine stimulates glycogenolysis through two different types of receptors, the  a- and p -agonist receptors.   Epinephrine, acting at the p -receptors, transmits a signal through G proteins to  adenylate cyclase, which increases cAMP and activates protein kinase A. Hence,  regulation of glycogen degradation and synthesis in liver by epinephrine and  glucagon are similar (see Fig. 28.8).    An inability of liver and muscle to  store glucose as glycogen con-  tributes to the hyperglycemia in  patients, such as Di Abietes, with type 1 dia-  betes mellitus and in patients, such as Ann  Sulin, with type 2 diabetes mellitus. The  absence of insulin in type 1 diabetes mellitus  patients and the high levels of glucagon result  in decreased activity of glycogen synthase.  Glycogen synthesis in skeletal muscles of type  1 patients is also limited by the lack of insulin-  stimulated glucose transport. Insulin resist-  ance in type 2 patients has the same effect.   An injection of insulin suppresses gluc-  agon release and alters the insulin/glucagon  ratio. The result is rapid uptake of glucose  into skeletal muscle and rapid conversion of  glucose to glycogen in skeletal muscle and  liver.    In the neonate, the release of epi-  nephrine during labor and birth nor-  mally contributes to restoring blood  glucose levels. Unfortunately, Getta Carbo  did not have adequate liver glycogen stores  to support a rise in her blood glucose levels.   Epinephrine also binds to a-receptors in the liver. This binding activates  glycogenolysis and inhibits glycogen synthesis principally by increasing the Ca 2+  levels in the liver. The effects of epinephrine at the a-agonist receptor are mediated  by the phosphatidylinositol bisphosphate (PIP 2 )-Ca 2+ signal transduction system,  one of the principal intracellular second messenger systems employed by many  hormones (Fig. 28.9) (see Chapter 11).   In the PIP 2 -Ca 2+ signal transduction system, the signal is transferred from the  epinephrine receptor to membrane-bound phospholipase C by G proteins. Phos-  pholipase C hydrolyzes PIP 2 to form diacylglycerol (DAG) and inositol trisphos-  phate (IP 3 ). IP 3 stimulates the release of Ca 2+ from the endoplasmic reticulum.  Ca 2+ and DAG activate protein kinase C. The amount of calcium bound to one of  the calcium-binding proteins, calmodulin, is also increased.   Calcium/calmodulin associates as a subunit with a number of enzymes and mod-  ifies their activities. It binds to inactive phosphorylase kinase, thereby partially acti-  vating this enzyme. (The fully activated enzyme is both bound to the  calcium/calmodulin subunit and phosphorylated.) Phosphorylase kinase then phos-  phorylates glycogen phosphorylase b, thereby activating glycogen degradation.  Calcium/calmodulin is also a modifier protein that activates one of the glycogen  synthase kinases (calcium/calmodulin synthase kinase). Protein kinase C, cal-  cium/calmodulin synthase kinase, and phosphorylase kinase all phosphorylate  glycogen synthase at different serine residues on the enzyme, thereby inhibiting  glycogen synthase and thus glycogen synthesis.   Fig. 28.9. Regulation of glycogen synthesis and degradation by epinephrine and Ca 2+ . 1 . The effect of  epinephrine binding to a-agonist receptors in liver transmits a signal via G proteins to phospholipase C,  which hydrolyzes PIP 2 to DAG and IP 3 . 2. IP 3 stimulates the release of Ca 2 + from the endoplasmic retic-  ulum. 3. Ca 2+ binds to the modifier protein calmodulin, which activates calmodulin-dependent protein  kinase and phosphorylase kinase. Both Ca 2 + and DAG activate protein kinase C. 4. These three kinases  phosphorylate glycogen synthase at different sites and decrease its activity. 5. Phosphorylase kinase phos-  phorylates glycogen phosphorylase b to the active form. It therefore activates glycogenolysis as well as  inhibiting glycogen synthesis.   The effect of epinephrine in the liver, therefore, enhances or is synergistic with the  effects of glucagon. Epinephrine release during bouts of hypoglycemia or during exer-  cise can stimulate hepatic glycogenolysis and inhibit glycogen synthesis very rapidly.   The regulation of glycogenolysis in skeletal muscle is related to the availability  of ATP for muscular contraction. Skeletal muscle glycogen produces glucose   Jim Bodie gradually regained consciousness with continued infusions of high-  concentration glucose titrated to keep his serum glucose level between 120 and  160 mg/dL. Although he remained somnolent and moderately confused over  the next 12 hours, he was eventually able to tell his physicians that he had self-injected  approximately 80 units of regular (short-acting) insulin every 6 hours while eating a high-  carbohydrate diet for the last 2 days preceding his seizure. Normal subjects under basal  conditions secrete an average of 40 units of insulin daily. He had last injected insulin just  before exercising. An article in a body-building magazine that he had recently read cited  the anabolic effects of insulin on increasing muscle mass. He had purchased the insulin  and necessary syringes from the same underground drug source from whom he regu-  larly bought his anabolic steroids.   Normally, muscle glycogenolysis supplies the glucose required for the kinds of high-  intensity exercise that require anaerobic glycolysis, such as weight-lifting. Jim's treadmill  exercise also uses blood glucose, which is supplied by liver glycogenolysis. The high  serum insulin levels, resulting from the injection he gave himself just before his workout,  activated both glucose transport into skeletal muscle and glycogen synthesis, while inhibit-  ing glycogen degradation. His exercise, which would continue to use blood glucose, could  normally be supported by breakdown of liver glycogen. However, glycogen synthesis in his  liver was activated, and glycogen degradation was inhibited by the insulin injection.   1 -phosphate and a small amount of free glucose. Glucose 1 -phosphate is converted to  glucose 6-phosphate, which is committed to the glycolytic pathway; the absence of  glucose 6-phosphatase in skeletal muscle prevents conversion of the glucosyl units  from glycogen to blood glucose. Skeletal muscle glycogen is therefore degraded only  when the demand for ATP generation from glycolysis is high. The highest demands  occur during anaerobic glycolysis, which requires more moles of glucose for each  ATP produced than oxidation of glucose to C0 2 (see Chapter 22). Anaerobic glycol-  ysis occurs in tissues that have fewer mitochondria, a higher content of glycolytic  enzymes, and higher levels of glycogen, or fast-twitch glycolytic fibers. It occurs most  frequently at the onset of exercise-before vasodilation occurs to bring in blood-borne  fuels. The regulation of skeletal muscle glycogen degradation therefore must respond  very rapidly to the need for ATP, indicated by the increase in AMP.   The regulation of skeletal muscle glycogen synthesis and degradation differs  from that in liver in several important respects: (a) glucagon has no effect on mus-  cle, and thus glycogen levels in muscle do not vary with the fasting/feeding state;  (b) AMP is an allosteric activator of the muscle isozyme of glycogen phosphory-  lase, but not liver glycogen phosphorylase (Fig. 28.10); (c) the effects of Ca 2+ in  muscle result principally from the release of Ca 2+ from the sarcoplasmic reticulum  after neural stimulation, and not from epinephrine-stimulated uptake; (d) glucose is  not a physiologic inhibitor of glycogen phosphorylase a in muscle; (e) glycogen is  a stronger feedback inhibitor of muscle glycogen synthase than of liver glycogen  synthase, resulting in a smaller amount of stored glycogen per gram weight of mus-  cle tissue. However, the effects of epinephrine-stimulated phosphorylation by pro-  tein kinase A on skeletal muscle glycogen degradation and glycogen synthesis are  similar to those occurring in liver (see Fig. 28.8).   Muscle glycogen phosphorylase is a genetically distinct isoenzyme of liver glyco-  gen phosphorylase and contains an amino acid sequence that has a purine nucleotide   Fig. 28.10. Activation of muscle glycogen phosphorylase during exercise. Glycogenolysis in  skeletal muscle is initiated by muscle contraction, neural impulses, and epinephrine. 1. AMP  produced from the degradation of ATP during muscular contraction allosterically activates  glycogen phosphorylase b. 2. The neural impulses that initiate contraction release Ca 2+ from  the sarcoplasmic reticulum. The Ca 2+ binds to calmodulin, which is a modifier protein that  activates phosphorylase kinase. 3. Phosphorylase kinase is also activated through phospho-  rylation by protein kinase A. The formation of cAMP and the resultant activation of protein  kinase A are initiated by the binding of epinephrine to plasma membrane receptors.   binding site. When AMP binds to this site, it changes the conformation at the cat-  alytic site to a structure very similar to that in the phosphorylated enzyme (see  Fig. 9.9). Thus, hydrolysis of ATP to ADP and the consequent increase of AMP gen-  erated by adenylate kinase during muscular contraction can directly stimulate  glycogenolysis to provide fuel for the glycolytic pathway. AMP also stimulates gly-  colysis by activating phosphofructokinase-1, so this one effector activates both  glycogenolysis and glycolysis. The activation of the calcium/calmodulin subunit of  phosphorylase kinase by the Ca 2+ released from the sarcoplasmic reticulum during  muscle contraction also provides a direct and rapid means of stimulating glycogen  degradation.   ^ Getta Carbo's hypoglycemia illustrates the importance of glycogen  ) stores in the neonate. At birth, the fetus must make two major adjustments  in the way fuels are used: it must adapt to using a greater variety of fuels  than were available in utero, and it must adjust to intermittent feeding. In utero, the  fetus receives a relatively constant supply of glucose from the maternal circulation  through the placenta, producing a level of glucose in the fetus that approximates  75% of maternal blood levels. With regard to the hormonal regulation of fuel uti-  lization in utero, fetal tissues function in an environment dominated by insulin,  which promotes growth. During the last 10 weeks of gestation, this hormonal milieu  leads to glycogen formation and storage. At birth, the infant's diet changes to one  containing greater amounts of fat and lactose (galactose and glucose in equal ratio),  presented at intervals rather than in a constant fashion. At the same time, the  neonate's need for glucose will be relatively larger than that of the adult because the  newborn's ratio of brain to liver weight is greater. Thus, the infant has even greater  difficulty in maintaining glucose homeostasis than the adult.   At the moment that the umbilical cord is clamped, the normal neonate is faced with  a metabolic problem: the high insulin levels of late fetal existence must be quickly  reversed to prevent hypoglycemia. This reversal is accomplished through the secretion  of the counterregulatory hormones epinephrine and glucagon. Glucagon release is  triggered by the normal decline of blood glucose after birth. The neural response that  stimulates the release of both glucagon and epinephrine is activated by the anoxia,  cord clamping, and tactile stimulation that are part of a normal delivery. These  responses have been referred to as the "normal sensor function" of the neonate.   Within 3 to 4 hours of birth, these counterregulatory hormones reestablish nor-  mal serum glucose levels in the newborn's blood through their glycogenolytic and  gluconeogenic actions. The failure of Getta 's normal "sensor function" was partly  the result of maternal malnutrition, which resulted in an inadequate deposition of  glycogen in Getta 's liver before birth. The consequence was a serious degree of  postnatal hypoglycemia.   The ability to maintain glucose homeostasis during the first few days of life also  depends on the activation of gluconeogenesis and the mobilization of fatty acids.  Fatty acid oxidation in the liver not only promotes gluconeogenesis (see Chapter  31) but generates ketone bodies. The neonatal brain has an enhanced capacity to use  ketone bodies relative to that of infants (fourfold) and adults (40-fold). This ability  is consistent with the relatively high fat content of breast milk.   ^ Jim Bodie attempted to build up his muscle mass with androgens and with  ) insulin. The anabolic (nitrogen-retaining) effects of androgens on skeletal  muscle cells enhance muscle mass by increasing amino acid flux into muscle  and by stimulating protein synthesis. Exogenous insulin has the potential to increase  muscle mass by similar actions and also by increasing the content of muscle glycogen.   The most serious side effect of exogenous insulin administration is the develop-  ment of severe hypoglycemia, such as occurred in Jim Bodie. The immediate  adverse effect relates to an inadequate flow of fuel (glucose) to the metabolizing  brain. When hypoglycemia is extreme, the patient may suffer a seizure and, if the  hypoglycemia worsens, may lapse into a coma and die. If untreated, irreversible  brain damage occurs in those who survive.    The regulatory effect of insulin is frequently described as one of activating  protein phosphatases. The effects of insulin on the regulation of hepatic  and skeletal PP- 1 are complex and not yet fully understood.   PP-1 is targeted to glycogen particles by four tissue- specific targeting factors:  G M is found in striated muscle; G L is found in liver; PTG (protein targeting to glyco-  gen) is found in almost all tissues; and R6 is also found in most tissues. The target-  ing factors bind to PP- 1 and glycogen and localize the PP- 1 to the glycogen parti-  cles, where the enzyme will be physically close to the regulated enzymes of  glycogen metabolism, phosphorylase kinase, glycogen phosphorylase, and glyco-  gen synthase. Regulation of the phosphatase will involve complex interactions  between the target enzymes, the targeting subunit, the phosphatase, and protein  inhibitor I. The interactions are also tissue specific in the case of G M and G L .   A simplistic view of hepatic PP-1 regulation is as follows. PP-1 is bound to G L  and the glycogen particle. Glycogen phosphorylase a binds to the complex, and in  so doing alters the conformation of PP-1, rendering it inactive. When glucose lev-  els rise in the blood (for example, after eating a meal), the glucose is transported  into the liver cells via GLUT 2 transporters, and the intracellular glucose level  increases. Glucose can bind to glycogen phosphorylase a, which relieves the inhi-  bition of PP-1, and glycogen phosphorylase a will be converted to glycogen phos-  phorylase b by active PP- 1 . Additionally, as the intracellular glucose is converted to  glucose 6-phosphate by glucokinase, the increase in glucose-6-P levels activates  PP-1 to dephosphorylate glycogen synthase, thereby activating the glycogen syn-  thesizing enzyme. The complicated view of hepatic PP-1 regulation also must take  into account the PTG-PP- 1 interactions (PTG is also expressed in the liver) and the  kinases that are activated by either insulin or glucagon/epinephrine, which lead to  alterations in glycogen metabolizing enzyme activities.   In contrast to hepatic regulation, muscle regulation of PP-1 activity via G M is  directly responsive to phosphorylation by kinases. A phosphorylation event that  appears to be critical is that of ser-67 in G M . Phosphorylation of ser-67 by the cAMP-  dependent protein kinase leads to a dissociation of PP- 1 from G M , and, therefore, the  phosphatase is removed from its substrates and cannot reverse the phosphorylation  of the target enzymes. If ser-67 is altered to a threonine, the phosphorylation at that  site is blocked, and PP-1 does not dissociate from G M . This indicates the importance  of the phosphorylation event in regulating PP-1 action in the muscle.   Future work will be needed before a complete understanding of how insulin  reverses glucagon/epinephrine stimulation of glycogenolysis is obtained.   Chen YT. Glycogen storage diseases. In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds. The Meta-  bolic and Molecular Bases of Inherited Disease, vol I, 8th Ed. New York: McGraw-Hill,  2001:1521-1551.   Parker PH, Ballew M, Greene HL. Nutritional management of glycogen storage disease. Annu Rev Nutr  1993;13:83-109.   Skurat AV, Roach PJ. Regulation of glycogen synthesis. In: LeRoith D, Taylor SI, Olefsky JM, eds. Dia-  betes Mellitus: A Fundamental and Clinical Text, 2 nd Ed. New York: Lippincott, Williams & Wilkins,  2000:251-264.    1 . The degradation of glycogen normally produces which of the following?   2. A patient has large deposits of liver glycogen, which, after an overnight fast, had shorter than normal branches. This abnor-  mality could be caused by a defective form of which of the following proteins or activities?   3. An adolescent patient with a deficiency of muscle phosphorylase was examined while exercising her forearm by squeez-  ing a rubber ball. Compared with a normal person performing the same exercise, this patient would exhibit which of the   (B) Have increased glucose levels in blood drawn from her forearm   (C) Have decreased lactate levels in blood drawn from her forearm   (D) Have lower levels of glycogen in biopsy specimens from her forearm muscle   4. In a glucose tolerance test, an individual in the basal metabolic state ingests a large amount of glucose. If the individual is nor-  mal, this ingestion should result in which of the following?   (B) An increased ratio of glycogen phosphorylase a to glycogen phosphorylase b in the liver   (C) An increased rate of lactate formation by red blood cells   (D) An inhibition of protein phosphatase I activity in the liver   5. Consider a type 1 diabetic who has neglected to take insulin for the past 72 hours and has not eaten much as well. Which  of the following best describes the activity level of hepatic enzymes involved in glycogen metabolism under these   29 Pathways of Sugar Metabolism:  Pentose Phosphate Pathway,  Fructose, and Galactose  Metabolism   Fig. 29.1. Fructose. The sugar fructose is  found in the diet as the free sugar in foods such  as honey or as a component of the disaccharide  sucrose in fruits and sweets. It also can be syn-  thesized from glucose via the polyol pathway.  In the lens of the eye, the polyol pathway con-  tributes to the formation of cataracts. Fructose  is metabolized by conversion to intermediates  of glycolysis.   Enzymes can generally use either  NADPH or NADH, but not both.  Reactions requiring the input of  electrons as hydride ions are usually cat-  alyzed by enzymes specific for NADPH.   Glucose is at the center of carbohydrate metabolism and is the major dietary  sugar. Other sugars in the diet are converted to intermediates of glucose metabo-  lism, and their fates parallel that of glucose. When carbohydrates other than glu-  cose are required for the synthesis of diverse compounds such as lactose, glyco-  proteins, or glycolipids, they are synthesized from glucose.   Fructose, the second most common sugar in the adult diet, is ingested princi-  pally as the monosaccharide or as part of sucrose (Fig. 29.1 ). It is metabolized  principally in the liver (and to a lesser extent in the small intestine and kidney) by  phosphorylation at the 1 -position to form fructose 1-P , followed by conversion to  intermediates of the glycolytic pathway. The major products of its metabolism in  liver are, therefore, the same as glucose ( including lactate, blood glucose, and  glycogen). Essential fructosuria (fructokinase deficiency) and hereditary fruc-  tose intolerance ( a deficiency of the fructose 1 -phosphate cleavage by aldolase B )  are inherited disorders of fructose metabolism.   Fructose synthesis from glucose in the polyol pathway occurs in seminal vesi-  cles and other tissues. Aldose reductase converts glucose to the sugar alcohol  sorbitol (a polyol), which is then oxidized to fructose. In the lens of the eye, ele-  vated levels of sorbitol in diabetes mellitus may contribute to cataract formation.   Galactose is ingested principally as lactose, which is converted to galactose  and glucose in the intestine. Galactose is converted to glucose principally in the  liver. It is phosphorylated to galactose 1 -phosphate by galactokinase and acti-  vated to a UDP -sugar by galactosyl uridylyltransf erase. The metabolic pathway  subsequently generates glucose 1 -phosphate. Classical galactosemia, a deficiency  of galactosyl uridylyltransf erase , results in the accumulation of galactose 1 -phos-  phate in the liver and the inhibition of hepatic glycogen metabolism and other  pathways that require UDP sugars. Cataracts can occur from accumulation of  galactose in the blood, which is converted to galactitol ( the sugar alcohol of  galactose) in the lens of the eye.   The pentose phosphate pathway consists of both oxidative and nonoxidative  components (Fig. 29.2). In the oxidative pathway, glucose 6-phosphate is oxidized  to ribulose 5-phosphate, C0 2 , and NADPH. Ribulose 5-phosphate, a pentose, can  be converted to ribose 5-phosphate for nucleotide biosynthesis. The NADPH is  used for reductive pathways, such as fatty acid biosynthesis, detoxification of  drugs by monooxygenases , and the glutathione defense system against injury by  reactive oxygen species (ROS).   In the nonoxidative phase of the pathway, ribulose 5-phosphate is converted to  ribose 5-phosphate and to intermediates of the glycolytic pathway. This portion of   Fig. 29.2. Overview of the pentose phosphate pathway. The pentose phosphate pathway gen-  erates NADPH for reactions requiring reducing equivalents (electrons) or ribose 5-phosphate  for nucleotide biosynthesis. Glucose 6-phosphate is a substrate for both the pentose phos-  phate pathway and glycolysis. The 5-carbon sugar intermediates of the pentose phosphate  pathway are reversibly interconverted to intermediates of glycolysis. The portion of glycoly-  sis that is not part of the pentose phosphate pathway is shown in blue.    v The pentose phosphate pathway is  i ) also called the hexose monophos-   phate shunt (HMP shunt). It shunts  hexoses from glycolysis, forming pentoses,  which may be reconverted to glycolytic  intermediates.   the pathway is reversible; therefore, ribose 5-phosphate can also be formed from  intermediates of glycolysis. One of the enzymes involved in these sugar intercon-  versions, transketolase , uses thiamine pyrophosphate as a coenzyme.   The sugars produced by the pentose phosphate pathway enter glycolysis as  fructose 6-phosphate and glyceraldehyde 3-phosphate, and their further metabo-  lism in the glycolytic pathway generates NADH, adenosine triphsphate (ATP), and  pyruvate. The overall equation for the conversion of glucose 6-phosphate to fruc-  tose 6-phosphate and glyceraldehyde 3-phosphate through both the oxidative and  nonoxidative reactions of the pentose phosphate pathway is:   3 glucose-6-P + 6 NADP + ->3C0 2 + 6 NADPH + 6 H + + 2 fructose-6-P   Candice Sucher is an 18-year-old girl who presented to her physician for  a precollege physical examination. While taking her medical history, the  doctor learned that she carefully avoided eating all fruits and any foods that  contained table sugar. She related that, from a very early age, she had learned that  these foods caused severe weakness and symptoms suggestive of low blood sugar,  such as tremulousness and sweating. Her medical history also indicated that her  mother had described her as having been a very irritable baby who often cried inces-  santly, especially after meals, and vomited frequently. At these times, Candice 's    CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   abdomen had become distended, and she became drowsy and apathetic. Her mother  had intuitively eliminated certain foods from Candice's diet, after which the sever-  ity and frequency of these symptoms diminished.   Erin Galway is a 3 -week-old female infant who began vomiting 3 days  after birth, usually within 30 minutes after breastfeeding. Her abdomen  became distended at these times, and she became irritable and cried  frequently. When her mother noted that the whites of Erin's eyes were yellow, she  took her to a pediatrician. The doctor agreed that Erin was slightly jaundiced. He  also noted an enlargement of her liver and questioned the possibility of early  cataract formation in the lenses of Erin's eyes. He ordered liver and kidney func-  tion tests and did two separate dipstick urine tests in his office, one designed to  measure only glucose in the urine and the other capable of detecting any of the  reducing sugars.   Al Martini developed a fever of 101. 5°F on the second day of his hospi-  talization for acute alcoholism. He had a cough productive of gray sputum.  A chest x-ray showed right lower lobe pneumonia. A stain of his sputum  showed many small pleomorphic Gram-negative bacilli. Sputum was sent for cul-  ture and a determination of which antibiotics would be effective in treating the  causative organism (sensitivity testing). Because his landlady stated that he had an  allergy to penicillin, he was started on a course of the antibiotic combination of  trimethoprim and sulfamethoxazole (TMP/sulfa). To his landlady's knowledge, he  had never been treated with a sulfa drug previously.   On the third day of therapy with TMP/sulfa for his pneumonia, Al Martini was  slightly jaundiced. His hemoglobin level had fallen by 3.5 g/dL from the value on  admission, and his urine was red-brown because of the presence of free hemo-  globin. Mr. Martini had apparently suffered acute hemolysis (lysis or destruction  of some of his red blood cells) induced by his infection and exposure to the sulfa  drug.   Fructose is found in the diet as a component of sucrose in fruit, as a free sugar in  honey, and in high-fructose corn syrup (see Fig. 29.1). Fructose enters epithelial  cells and other types of cells by facilitated diffusion on the GLUT V transporter. It  is metabolized to intermediates of glycolysis. Problems with fructose absorption  and metabolism are relatively more common than with other sugars.   Fructose is metabolized by conversion to glyceraldehyde-3-P and dihydroxy ace-  tone phosphate, which are intermediates of glycolysis (Fig. 29.3). The steps par-  allel those of glycolysis. The first step in the metabolism of fructose, as with  glucose, is phosphorylation. Fructokinase, the major kinase involved, phospho-  rylates fructose in the 1 -position. Fructokinase has a high V max , and rapidly phos-  phorylates fructose as it enters the cell. The fructose 1 -phosphate formed is not  an intermediate of glycolysis but rather is cleaved by aldolase B to dihydroxy-  acetone phosphate (an intermediate of glycolysis) and glyceraldehyde. Glycer-  aldehyde is then phosphorylated to glyceraldehyde- 3 -P by triose kinase. Dihy-  droxy acetone phosphate and glyceraldehyde 3 -phosphate are intermediates of  the glycolytic pathway and can proceed through it to pyruvate, the TCA cycle,  and fatty acid synthesis. Alternately, these intermediates can also be converted to  glucose by gluconeogenesis. In other words, the fate of fructose parallels that of  glucose.    When individuals with defects of  aldolase B ingest fructose, the  extremely high levels of fructose 1-  phosphate that accumulate in the liver and  kidney cause a number of adverse effects.  Hypoglycemia results from inhibition of  glycogenolysis and gluconeogenesis. Glyco-  gen phosphorylase (and possibly phospho-  glucomutase and other enzymes of glycogen  metabolism) are inhibited by the accumu-  lated fructose 1-phosphate. Aldolase B is  required for glucose synthesis from glycer-  aldehyde 3-phosphate and dihydroxyacetone  phosphate, and its low activity in aldolase  B-deficient individuals is further decreased  by the accumulated fructose 1-phosphate.  The inhibition of gluconeogenesis results in  lactic acidosis.   The accumulation of fructose 1-phos-  phate also substantially depletes the phos-  phate pools. The fructokinase reaction uses  ATP at a rapid rate such that the mitochon-  dria regenerate ATP rapidly, which leads to a  drop in free phosphate levels. The low levels  of phosphate release inhibition of AMP  deaminase, which converts AMP to inosine  monophosphate (IMP). The nitrogenous base  of IMP (hypoxanthine) is degraded to uric  acid. The lack of phosphate and depletion of  adenine nucleotides lead to a loss of ATP, fur-  ther contributing to the inhibition of biosyn-  thetic pathways, including gluconeogenesis.   Essential fructosuria is a rare and  ■ benign genetic disorder caused by   \3 a deficiency of the enzyme fructok-  inase. Why is this disease benign, when a  deficiency of aldolase B (hereditary fructose  intolerance) can be fatal? Could Candice  Sucher have essential fructosuria?   Fig. 29.3. Fructose metabolism. The pathway for the conversion of fructose to dihydroxy-  acetone phosphate and glyceraldehyde 3-phosphate is shown in blue. These two compounds  are intermediates of glycolysis and are converted in the liver principally to glucose, glyco-  gen, or fatty acids. In the liver, aldolase B cleaves both fructose 1 -phosphate in the pathway  for fructose metabolism, and fructose 1,6-bisphosphate in the pathway for glycolysis.   The metabolism of fructose occurs principally in the liver and to a lesser extent in  the small intestinal mucosa and proximal epithelium of the renal tubule, because  these tissues have both fructokinase and aldolase B. Aldolase exists as several iso-  forms: aldolases A, B, C, and fetal aldolase. Although all of these aldolase isoforms  can cleave fructose 1,6-bisphosphate, the intermediate of glycolysis, only aldolase B  can also cleave fructose 1 -phosphate. Aldolase A, present in muscle and most other  tissues, and aldolase C, present in brain, have almost no ability to cleave fructose  1 -phosphate. Fetal aldolase, present in the liver before birth, is similar to aldolase C.   Aldolase B is the rate-limiting enzyme of fructose metabolism, although it is not  a rate-limiting enzyme of glycolysis. It has a much lower affinity for fructose  1-phosphate than fructose 1,6-bisphosphate (although the k cat is the same) and is  very slow at physiologic levels of fructose 1-phosphate. As a consequence, after  ingesting a high dose of fructose, normal individuals accumulate fructose 1-phos-  phate in the liver while it is slowly converted to glycolytic intermediates. Individu-  als with hereditary fructose intolerance (a deficiency of aldolase B) accumulate  much higher amounts of fructose 1-phosphate in their livers.   Other tissues also have the capacity to metabolize fructose but do so much more  slowly. The hexokinase isoforms present in muscle, adipose tissue, and other tissues  can convert fructose to fructose 6-phosphate, but react so much more efficiently  with glucose. As a result, fructose phosphorylation is very slow in the presence of  physiologic levels of intracellular glucose and glucose 6-phosphate.   Fructose can be synthesized from glucose in the polyol pathway. The polyol  pathway is named for the first step of the pathway in which sugars are reduced  to the sugar alcohol by the enzyme aldose reductase (Fig. 29.4) Glucose is  reduced to the sugar alcohol sorbitol, and sorbitol is then oxidized to fructose.   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   This pathway is present in seminal vesicles, which synthesize fructose for the  seminal fluid. Spermatozoa use fructose as a major fuel source while in the sem-  inal fluid and then switch to glucose once in the female reproductive tract. Uti-  lization of fructose is thought to prevent acrosomal breakdown of the plasma  membrane (and consequent activation) while the spermatozoa are still in the  seminal fluid.   The polyol pathway is present in many tissues, but its function in all tissues is  not understood. Aldose reductase is relatively nonspecific, and its major function  may be the metabolism of an aldehyde sugar other than glucose. The activity of this  enzyme can lead to major problems in the lens of the eye, where it is responsible  for the production of sorbitol from glucose and galactitol from galactose. When the  concentration of glucose or galactose is elevated in the blood, their respective sugar  alcohols are synthesized in the lens more rapidly than they are removed, resulting  in increased osmotic pressure within the lens.   Dietary galactose is metabolized principally by phosphorylation to galactose  1 -phosphate, and then conversion to UDP-galactose and glucose 1 -phosphate  (Fig. 29.5). The phosphorylation of galactose, again an important first step in the  pathway, is carried out by a specific kinase, galactokinase. The formation of  UDP-galactose is accomplished by attack of the phosphate oxygen on galactose  1 -phosphate on the a phosphate of UDP-glucose, releasing glucose 1 -phosphate  while forming UDP-galactose. The enzyme that catalyzes this reaction is galac-  tose 1-phosphate uridylyltransf erase. The UDP-galactose is then converted to  UDP-glucose by the reversible UDP-glucose epimerase (the configuration of the  hydroxyl group on carbon four is reversed in this reaction). The net result of this  sequence of reactions is that galactose is converted to glucose 1-phosphate, at the  expense of 1 high-energy bond of ATP. The sum of these reactions is indicated in  the equations that follow:    The accumulation of sorbitol in  muscle and nerve tissues may con-  tribute to the peripheral neuropa-  thy characteristic of patients with poorly  controlled diabetes mellitus. This is one of  the reasons it is so important for Di Abietes  (who has type 1 diabetes mellitus) and Ann  Sulin (who has type 2 diabetes mellitus) to  achieve good glycemic control.   The accumulation of sugars and  sugar alcohols in the lens of patients  with hyperglycemia (e.g., diabetes  mellitus) results in the formation of cataracts.  Glucose levels are elevated and increase the  synthesis of sorbitol and fructose. As a conse-  quence, a high osmotic pressure is created in  the lens. The high glucose and fructose levels  also result in nonenzymatic glycosylation of  lens proteins. The result of the increased  osmotic pressure and the glycosylation of the  lens protein is an opaque cloudiness of the  lens known as a cataract. Erin Galway seemed  to have an early cataract, probably caused by  the accumulation of galactose and its sugar  alcohol galactitol.   Fig. 29.5. Metabolism of galactose. Galactose is phosphorylated to galactose 1-phosphate by  galactokinase. Galactose 1-phosphate reacts with UDP-glucose to release glucose 1-phos-  phate. Galactose thus can be converted to blood glucose, enter glycolysis, or enter any of the  metabolic routes of glucose. In classical galactosemia, a deficiency of galactose 1-phosphate  uridylyltransferase (shown in grey) results in the accumulation of galactose 1-phosphate in tis-  sues and the appearance of galactose in the blood and urine. In nonclassical galactosemia, a  deficiency of galactokinase results in the accumulation of galactose.   ®ln essential fructosuria, fructose  cannot be converted to fructose 1-  phosphate. This condition is benign  because no toxic metabolites of fructose  accumulate in the liver, and the patient  remains nearly asymptomatic. Some of the  ingested fructose is slowly phosphorylated  by hexokinase in nonhepatic tissues and  metabolized by glycolysis, and some  appears in the urine. There is no renal thresh-  old for fructose; the appearance of fructose  in the urine (fructosuria) does not require a  high fructose concentration in the blood.   Hereditary fructose intolerance, con-  versely, results in the accumulation of fruc-  tose 1-phosphate and fructose. By inhibiting  glycogenolysis and gluconeogenesis, the  high levels of fructose 1-phosphate caused  the hypoglycemia that Candice Sucher expe-  rienced as an infant when she became apa-  thetic and drowsy, and as an adult when she  experienced sweating and tremulousness.   Erin Galway's urine was negative  for glucose when measured with  the glucose oxidase strip but was  positive for the presence of a reducing  sugar. The reducing sugar was identified as  galactose. Her liver function tests showed an  increase in serum bilirubin and in several  liver enzymes. Albumin was present in her  urine. These findings and the clinical history  increased her physician's suspicion that Erin  had classical galactosemia.   Classical galactosemia is caused by a  deficiency of galactose 1-phosphate uridylyl-  transferase. In this disease, galactose 1-phos-  phate accumulates in tissues, and galactose  is elevated in the blood and urine. This con-  dition differs from the rarer deficiency of  galactokinase (nonclassical galactosemia), in  which galactosemia and galactosuria occur  but galactose 1-phosphate is not formed.  Both enzyme defects result in cataracts from  galactitol formation by aldose reductase in  the polyol pathway. Aldose reductase has a  relatively high K m for galactose, approxi-  mately 12 to 20 mM, so that galactitol is  formed only in galactosemic patients who  have eaten galactose. Galactitol is not further  metabolized and diffuses out of the lens very  slowly. Thus, hypergalactosemia is even  more likely to cause cataracts than hyper-  glycemia. Erin Galway, although only 3  weeks old, appeared to have early cataracts  forming in the lens of her eyes.   One of the most serious problems of clas-  sical galactosemia is an irreversible mental  retardation. Realizing this problem, Erin Gal-  way's physician wanted to begin immediate  dietary therapy. A test that measures galac-  tose 1-phosphate uridylyltransferase in ery-  throcytes was ordered. The enzyme activity  was virtually absent, confirming the diagno-  sis of classical galactosemia.   The enzymes for galactose conversion to glucose 1-phosphate are present in  many tissues, including the adult erythrocyte, fibroblasts, and fetal tissues. The liver  has a high activity of these enzymes, and can convert dietary galactose to blood glu-  cose and glycogen. The fate of dietary galactose, like that of fructose, therefore, par-  allels that of glucose. The ability to metabolize galactose is even higher in infants  than in adults. Newborn infants ingest up to 1 g galactose per kg per feeding (as lac-  tose). Yet the rate of metabolism is so high that the blood level in the systemic  circulation is less than 3 mg/dL, and none of the galactose is lost in the urine.   The pentose phosphate pathway is essentially a scenic bypass route around the  first stage of glycolysis that generates NADPH and ribose-5-P (as well as other  pentose sugars). Glucose 6-phosphate is the common precursor for both path-  ways. The oxidative first stage of the pentose phosphate pathway generates two  moles of NADPH per glucose 6-phosphate oxidized. The second stage of the  pentose phosphate pathway generates ribose-5-P and converts unused intermedi-  ates to fructose-6-P and glyceraldehyde-3-P in the glycolytic pathway (see Fig.  29.2). All cells require NADPH for reductive detoxification, and most cells  require ribose-5-P for nucleotide synthesis. Consequently, the pathway is present  in all cells. The enzymes reside in the cytosol, as do the enzymes of glycolysis.   In the oxidative first phase of the pentose phosphate pathway, glucose 6-phosphate is  oxidatively decarboxylated to a pentose sugar, ribulose 5 -phosphate (Fig. 29.6). The  first enzyme of this pathway, glucose 6-phosphate dehydrogenase, oxidizes the alde-  hyde at CI and reduces NADP + to NADPH. The gluconolactone that is formed is rap-  idly hydrolyzed to 6-phosphogluconate, a sugar acid with a carboxylic acid group at  CI. The next oxidation step releases this carboxyl group as C0 2 , with the electrons  being transferred to NADP + . This reaction is mechanistically very similar to the one  catalyzed by isocitrate dehydrogenase in the TCA cycle. Thus, two moles of NADPH  per mole of glucose 6-phosphate are formed from this portion of the pathway.   NADPH, rather than NADH, is generally used in the cell for pathways that require the input of electrons for reductive reactions  \\ ) because the ratio of NADPH/NADP + is much greater than the NADH/NAD + ratio. The NADH generated from fuel oxidation is rap-  idly oxidized back to NAD + by NADH dehydrogenase in the electron transport chain, so the level of NADH is very low in the cell.  NADPH can be generated from a number of reactions in the liver and other tissues, but not the red blood cell. For example, in tissues  with mitochondria, an energy-requiring transhydrogenase located near the complexes of the electron transport chain can transfer reduc-  ing equivalents from NADH to NADP + to generate NADPH.   NADPH, however, cannot be directly oxidized by the electron transport chain, and the ratio of NADPH to NADP + in cells is greater than  one. The reduction potential of NADPH therefore can contribute to the energy needed for biosynthetic processes and provide a constant  source of reducing power for detoxification reactions.   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   To generate ribose 5-phosphate from the oxidative pathway, the ribulose 5-phos-  phate formed from the action of the two oxidative steps is isomerized to produce  ribose 5-phosphate (a ketose-to-aldose conversion, similar to fructose 6-phos-  phate being isomerized to glucose 6-phosphate; see section III.B.l below). The  ribose 5-phosphate can then enter the pathway for nucleotide synthesis, if needed,  or can be converted to glycolytic intermediates, as described below for the nonox-  idative phase of the pentose phosphate pathway. The pathway through which the  ribose 5-phosphate travels is determined by the needs of the cell at the time of its  synthesis.   The nonoxidative reactions of this pathway are reversible reactions that allow inter-  mediates of glycolysis (specifically glyceraldehyde-3-P and fructose-6-P) to be  converted to five-carbon sugars (such as ribose-5-P), and vice versa. The needs of  the cell will determine in which direction this pathway proceeds. If the cell has pro-  duced ribose-5-P, but does not need to synthesize nucleotides, then the ribose-5-P  will be converted to glycolytic intermediates. If the cell still requires NADPH, the  ribose-5-P will be converted back into glucose-6-P using nonoxidative reactions  (see below). And finally, if the cell already has a high level of NADPH, but needs  to produce nucleotides, the oxidative reactions of the pentose phosphate pathway  will be inhibited, and the glycolytic intermediates fructose-6-P and glyceraldehyde-  3-P will be used to produce the five carbon sugars using exclusively the nonoxida-  tive phase of the pentose phosphate pathway.   The nonoxidative portion of the pentose phosphate pathway consists of a series  of rearrangement and transfer reactions that first convert ribulose 5-phosphate to  ribose 5-phosphate and xylulose 5-phosphate, and then the ribose 5-phosphate  and xyulose 5-phosphate are converted to intermediates of the glycolytic path-  way. The enzymes involved are an epimerase, an isomerase, transketolase, and  transaldolase.   The epimerase and isomerase convert ribulose 5-phosphate to two other 5 -carbon  sugars (Fig. 29.7). The isomerase converts ribulose 5-phosphate to ribose 5-phos-  phate. The epimerase changes the stereochemical position of one hydroxy 1 group (at  carbon 3), converting ribulose 5-phosphate to xylulose 5-phosphate.   Transketolase transfers 2-carbon fragments of keto sugars (sugars with a keto  group at C2) to other sugars. Transketolase picks up a 2-carbon fragment from xylu-  lose 5-phosphate by cleaving the carbon-carbon bond between the keto group and  the adjacent carbon, thereby releasing glyceraldehyde 3-phosphate (Fig. 29.8). The  2-carbon fragment is covalently bound to thiamine pyrophosphate, which transfers   Xyulose 5-phosphate has recently been identified as an activator of protein phos-  | I ) phatase 2A (PP2A). PP2A removes phosphates from PFK-2 and from a transcription  *r factor that binds to carbohydrate response elements in promoters of genes such as  pyruvate kinase. The hydrolysis of the phosphates activates both proteins, such that xyulose  5-phosphate can regulate pathways relating to both carbohydrate and fat metabolism.   Fig. 29.6. Oxidative portion of the pentose phosphate pathway. Carbon 1 of glucose 6-phosphate is oxidized to an acid and then released as CO  in an oxidative decarboxylation reaction. Each oxidation step generates an NADPH.    Doctors suspected that the underlying factor in the destruction of Al Martini's  red blood cells was an X-linked defect in the gene that codes for glucose 6-  phosphate dehydrogenase. The red blood cell is dependent on this enzyme for  a source of NADPH to maintain reduced levels of glutathione, one of its major defenses  against oxidative stress (see Chapter 24). Glucose 6-phosphate dehydrogenase defi-  ciency is the most common known enzymopathy, and affects approximately 7% of the  world's population and about 2% of the U.S. population. Most glucose 6-phosphate  dehydrogenase-deficient individuals are asymptomatic but can undergo an episode of  hemolytic anemia if exposed to certain drugs, to certain types of infections, or if they  ingest fava beans. When questioned, Al Martini replied that he did not know what a fava  bean was and had no idea whether he was sensitive to them.   Fig. 29.8. Two-carbon unit transferred by trans-  ketolase. Transketolase cleaves the bond next to  the keto group and transfers the 2-carbon keto  fragment to an aldehyde. Thiamine pyrophos-  phate carries the 2-carbon fragment, forming a  covalent bond with the carbon of the keto group.   it to the aldehyde carbon of another sugar, forming a new ketose. The role of  thiamine-pyrophosphate here is thus very similar to its role in the oxidative decar-  boxylation of pyruvate and a-ketoglutarate (see Chapter 20, section LB). Two  reactions in the pentose phosphate pathway use transketolase; in the first, the 2-car-  bon keto fragment from xylulose 5-phosphate is transferred to ribose 5-phosphate  to form sedoheptulose 7-phosphate, and in the other, a 2-carbon keto fragment (usu-  ally derived from xyulose 5-phosphate) is transferred to erythrose 4-phosphate to  form fructose 6-phosphate.   Fig. 29.7. Ribulose 5-phosphate is epimerized (to xyulose 5-phosphate) and isomerized (to  ribose 5-phosphate).   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   Transaldolase transfers a 3 -carbon keto fragment from sedoheptulose 7-phos-  phate to glyceraldehyde 3 -phosphate to form erythrose 4-phosphate and fructose 6-  phosphate (Fig. 29.9). The aldol cleavage occurs between the two hydroxyl carbons  adjacent to the keto group (on carbons 3 and 4 of the sugar). This reaction is simi-  lar to the aldolase reaction in glycolysis, and the enzyme uses an active amino  group, from the side chain of lysine, to catalyze the reaction.   The net result of the metabolism of 3 moles of ribulose 5 -phosphate in the pen-  tose phosphate pathway is the formation of 2 moles of fructose 6-phosphate and  1 mole of glyceraldehyde 3 -phosphate, which then continue through the glycolytic  pathway with the production of NADH, ATP, and pyruvate. Because the pentose  phosphate pathway begins with glucose 6-phosphate, and feeds back into the   Fig. 29.9. Transaldolase transfers a 3 -carbon fragment that contains an alcohol group next to  a keto group.   The transketolase activity of red  blood cells is used to measure thi-  amine nutritional status and diag-  nose the presence of thiamine deficiency.  The activity of transketolase is measured in  the presence and absence of added thiamine  pyrophosphate. If the thiamine intake of a  patient is adequate, the addition of thiamine  pyrophosphate does not increase the activ-  ity of transketolase because it already  contains bound thiamine pyrophosphate. If  the patient is thiamine deficient, transketo-  lase activity will be low, and adding thiamine  pyrophosphate will greatly stimulate the  reaction. Al Martini was diagnosed in Chap-  ter 19 as having beriberi heart disease result-  ing from thiamine deficiency. The diagnosis  was based on laboratory tests confirming  the thiamine deficiency.   Fig. 29.10. A balanced sequence of reactions in the pentose phosphate pathway. The interconversion of sugars in the pentose phosphate path-  way results in conversion of 3 glucose 6-phosphate to 6 NADPH, 3 C0 2 , 2 fructose 6-phosphate, and one glyceraldehyde 3-phosphate.   glycolytic pathway, it is sometimes called the hexose monophosphate shunt (a shunt  or a pathway for glucose 6-phosphate). The reaction sequence starting from  glucose-6-P, and involving both the oxidative and nonoxidative phases of the path-  way, is shown in Figure 29.10.   The reactions catalyzed by the epimerase, isomerase, transketolase, and transal-  dolase are all reversible reactions under physiologic conditions. Thus, ribose  5 -phosphate required for purine and pyrimidine synthesis can be generated from  intermediates of the glycolytic pathway, as well as from the oxidative phase of the  pentose phosphate pathway. The sequence of reactions that generate ribose 5 -phos-  phate from intermediates of glycolysis is indicated below.   (4) 2 Xyulose-5-P Epimerase ^ 2 Rjbu | ose _ 5 _ p   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   C. Role of the Pentose Phosphate Pathway in the  Generation of NADPH   In general, the oxidative phase of the pentose phosphate pathway is the major  source of NADPH in cells. NADPH provides the reducing equivalents for biosyn-  thetic reactions and for oxidation-reduction reactions involved in protection against  the toxicity of ROS (see Chapter 24). The glutathione-mediated defense against  oxidative stress is common to all cell types (including the red blood cell), and the  requirement for NADPH to maintain levels of reduced glutathione probably  accounts for the universal distribution of the pentose phosphate pathway among dif-  ferent types of cells. Fig. 29.11 illustrates the importance of this pathway in main-  taining the membrane integrity of the red blood cell. NADPH is also used for ana-  bolic pathways, such as fatty acid synthesis, cholesterol synthesis, and fatty acid  chain elongation (Table 29.1). It is the source of reducing equivalents for  cytochrome P450 hydroxy lation of aromatic compounds, steroids, alcohols, and  drugs. The highest concentrations of glucose 6-phosphate dehydrogenase are found  in phagocytic cells, where NADPH oxidase uses NADPH to form superoxide from  molecular oxygen. The superoxide then generates hydrogen peroxide, which kills  the microorganisms taken up by the phagocytic cells (see Chapter 24).   The entry of glucose 6-phosphate into the pentose phosphate pathway is con-  trolled by the cellular concentration of NADPH. NADPH is a strong product  inhibitor of glucose 6-phosphate dehydrogenase, the first enzyme of the pathway.  As NADPH is oxidized in other pathways, the product inhibition of glucose 6-  phosphate dehydrogenase is relieved, and the rate of the enzyme is accelerated to  produce more NADPH.    How does the net energy yield  from the metabolism of 3 moles of  glucose 6-phosphate through the  pentose phosphate pathway to pyruvate  compare with the yield of 3 moles of glucose  6-phosphate through glycolysis?    Fig. 29.11. Hemolysis caused by reactive oxygen species. 1. Maintenance of the integrity of the erythrocyte membrane depends on its ability to  generate ATP and NADH from glycolysis. 2. NADPH is generated by the pentose phosphate pathway. 3. NADPH is used for the reduction of  oxidized glutathione to reduced glutathione. Glutathione is necessary for the removal of H 2 0 2 and lipid peroxides generated by reactive oxygen  species (ROS). 4. In the erythrocytes of healthy individuals, the continuous generation of superoxide ion from the nonenzymatic oxidation of  hemoglobin provides a source of reactive oxygen species. The glutathione defense system is compromised by glucose 6-phosphate dehydroge-  nase deficiency, infections, certain drugs, and the purine glycosides of fava beans. 5. As a consequence, Heinz bodies, aggregates of cross-linked  hemoglobin, form on the cell membranes and subject the cell to mechanical stress as it tries to go through small capillaries. The action of the  ROS on the cell membrane as well as mechanical stress from the lack of deformability result in hemolysis.   f^S^ The net energy yield from 3 moles  /\ # ) of glucose 6-phosphate metabo-  lized through the pentose phos-  phate pathway and then the last portion of  the glycolytic pathway is 6 moles of NADPH,  3 moles of C0 2 , 5 moles of NADH, 8 moles of  ATP, and 5 moles of pyruvate. In contrast, the  metabolism of 3 moles of glucose 6-phos-  phate through glycolysis is 6 moles of  NADH, 9 moles of ATP, and 6 moles of  pyruvate.   Table 29.2. Cellular Needs Dictate the Direction of the Pentose Phosphate  Pathway Reactions   Oxidative reactions produce NADPH; nonoxidative reactions  convert ribulose-5-P to glucose-6-P to produce more NADPH   Oxidative reactions produce NADPH and ribulose-5-P; the  isomerase converts ribulose-5-P to ribose-5-P   Only the nonoxidative reactions. High NADPH inhibits  glucose- 6-P dehydrogenase, so transketolase and  transaldolase will be used to convert fructose-6-P and  glyceraldehyde-3-P to ribose-5-P.   Both the oxidative and nonoxidative reactions are used. The  oxidative reactions generate NADPH and ribulose-5-P. The  nonoxidative reactions convert the ribulose-5-P to fructose-  6-P and glyceraldehyde-3-P, and glycolysis will convert these  intermediates to pyruvate.   In the liver, the synthesis of fatty acids from glucose is a major route of NADPH  reoxidation. The synthesis of liver glucose 6-phosphate dehydrogenase, like the key  enzymes of glycolysis and fatty acid synthesis, is induced by the increased  insulin/glucagon ratio after a high-carbohydrate meal. A summary of the possible  routes glucose-6-P may follow using the pentose phosphate pathway is presented in  Table 29.2.   ^\ Hereditary fructose intolerance (HFI) is caused by a low level of fructose  ) 1 -phosphate aldolase activity in aldolase B, an isozyme of fructose 1,6-bis-  phosphate aldolase that is also capable of cleaving fructose 1 -phosphate. In  patients of European descent, the most common defect is a single missense muta-  tion in exon 5 (G — > C), resulting in an amino acid substitution (Ala — > Pro). As a  result of this substitution, a catalytically impaired aldolase B is synthesized in abun-  dance. The exact prevalence of HFI in the United States is not established but is  approximately 1 per 15,000 to 25,000 population. The disease is transmitted by an  autosomal recessive inheritance pattern.   When affected patients such as Candice Sucher ingest fructose, fructose is con-  verted to fructose 1 -phosphate. Because of the deficiency of aldolase B, fructose 1-  phosphate cannot be further metabolized to dihydroxyacetone phosphate and glyc-  eraldehyde and accumulates in those tissues that have fructokinase (liver, kidney,  and small intestine). Fructose is detected in the urine with the reducing sugar test  (see Chapter 5). A DNA screening test (based on the generation of a new restriction  site by the mutation) now provides a safe method to confirm a diagnosis of  hereditary fructose intolerance.   In the infant and small child, the major symptoms include poor feeding, vomit-  ing, intestinal discomfort, and failure to thrive. The greater the ingestion of dietary  fructose, the more severe the clinical reaction. The result of prolonged ingestion of  fructose is ultrastructural changes in the liver and kidney resulting in hepatic and  renal failure. Hereditary fructose intolerance is usually a disease of infancy, because  adults with fructose intolerance who have survived avoid the ingestion of fruits,  table sugar, and other sweets.   ^ Erin Gal way has galactosemia, which is caused by a deficiency of  ) galactose 1 -phosphate uridylyltransferase; it is one of the most common  genetic diseases. Galactosemia is an autosomal recessive disorder of galac-  tose metabolism that occurs in about 1 in 60,000 newborns. Approximately two   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   thirds of the states in the United States screen newborns for this disease because fail-  ure to begin immediate treatment results in mental retardation. Failure to thrive is the  most common initial clinical symptom. Vomiting or diarrhea is found in most patients,  usually starting within a few days of milk ingestion. Signs of deranged liver function,  either jaundice or hepatomegaly, are present almost as frequently after the first week  of life. The jaundice of intrinsic liver disease may be accentuated by the severe hemol-  ysis in some patients. Cataracts have been observed within a few days of birth.   Management of patients requires elimination of galactose from the diet. Failure  to eliminate this sugar results in progressive liver failure and death. In infants,  artificial milk made from casein or soybean hydrolysate is used.   Al Martini's sputum culture sent on the second day of his admission  for acute alcoholism and pneumonia grew out Haemophilus influenzae.  This organism is sensitive to a variety of antibiotics, including  TMP/sulfa. Unfortunately, it appeared that Mr. Martini had suffered an acute  hemolysis (lysis or destruction of some of his red blood cells), probably induced  by exposure to the sulfa drug and his infection with H. influenzae. The hemo-  globin that escaped from the lysed red blood cells was filtered by his kidneys and  appeared in his urine.   By mechanisms that are not fully delineated, certain drugs (such as sulfa drugs  and antimalarials), a variety of infectious agents, and exposure to fava beans can  cause red blood cell destruction in individuals with a genetic deficiency of glu-  cose 6-phosphate dehydrogenase. Presumably, these patients cannot generate  enough reduced NADPH to defend against the ROS. Although erythrocytes lack  most of the other enzymatic sources of NADPH for the glutathione antioxidant  system, they do have the defense mechanisms provided by the antioxidant vita-  mins E and C and catalase. Thus, individuals who are not totally deficient in glu-  cose 6-phosphate dehydrogenase remain asymptomatic unless an additional  oxidative stress, such as an infection, generates additional oxygen radicals.   Some drugs, such as the antimalarial primaquine and the sulfonamide which Al  Martini is taking, affect the ability of red blood cells to defend against oxidative  stress. Fava beans, which look like fat string beans and are sometimes called broad  beans, contain the purine glycosides vicine and isouramil. These compounds react  with glutathione. It has been suggested that cellular levels of reduced glutathione  (GSH) decrease to such an extent that critical sulfhydryl groups in some key pro-  teins cannot be maintained in reduced form.   The highest prevalence rates for glucose 6-phosphate dehydrogenase deficiency  are found in tropical Africa and Asia, in some areas of the Middle East and the  Mediterranean, and in Papua New Guinea. The geographic distribution of this  deficiency is similar to that of sickle cell trait, and is probably also related to the rel-  ative resistance it confers against the malaria parasite.   Because the individuals with this deficiency are asymptomatic unless exposed to  an "oxidant challenge," the clinical course of the hemolytic anemia is usually self-  limited if the causative agent is removed. However, genetic polymorphism accounts  for a substantial variability in the severity of the disease. Severely affected patients  may have a chronic hemolytic anemia and other sequelae even without known expo-  sure to drugs, infection, and other causative factors. In such patients, neonatal jaun-  dice is also common and can be severe enough to cause death.   Before the metabolic toxicity of fructose was appreciated, substitution of  fructose for glucose in intravenous solutions, and of fructose for sucrose  in enteral tube feeding or diabetic diets, was frequently recommended.   (Enteral tube feeding refers to tubes placed into the gut; parenteral tube feeding  refers to tubes placed into the vein, feeding intravenously.) Administration of  intravenous fructose to patients with diabetes mellitus or other forms of insulin  resistance avoided the hyperglycemia found with intravenous glucose, possibly  because fructose metabolism in the liver bypasses the insulin-regulated step at  phosphofructokinase-1. However, because of the unregulated flow of fructose  through glycolysis, intravenous fructose feeding frequently resulted in lactic aci-  dosis (see Fig. 29.3). In addition, the fructokinase reaction is very rapid, and tis-  sues would become depleted of ATP and phosphate when large quantities of fruc-  tose were metabolized over a short period. This would lead to cell death. Fructose  is less toxic in the diet or in enteral feeding because of the relatively slow rate of  fructose absorption.   Hasler J, Lee S. Acute hemolytic anemia after ingestion of fava beans [letter]. Am J Emerg Med  1993;11:560-561.   Holton JB, Walter JH, Tyfield LA. Galactosemia. In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al.,  eds. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. New York: McGraw-Hill,  2001:1553-1587.   Luzatto L, Mehta A, Vuillamy T. Glucose 6-phosphate dehydrogenase deficiency. In: Scriver CR,  Beaudet AL, Valle D, Sly WS, et al, eds. The Metabolic and Molecular Bases of Inherited Disease.  8th Ed. New York: McGraw-Hill, 2001:4517-4553.   Sheetz MJ, King GL. Molecular understanding of hyperglycemic adverse effects for diabetic complica-  tions. JAMA 2002:2579-2588.   Shibuya A, Hirono A, Ishii S, Fujii, Miwa S. Hemolytic crisis after excessive ingestion of fava beans in  a male infant with G6PD Canton. Int J Hematol 1999:233-235.   Steinman B, Gitzelmann R, Van den Berghe G. Disorders of fructose metabolism. In: Scriver CR,  Beaudet AL, Valle D, Sly WS, et al, eds. The Metabolic and Molecular Bases of Inherited Disease.  8th Ed. New York: McGraw-Hill, 2001:1489-1520.   1. Hereditary fructose intolerance is a rare recessive genetic disease that is most commonly caused by a mutation in exon 5 of  the aldolase B gene. The mutation fortuitously creates a new Ahall recognition sequence. To test for the mutation, DNA was  extracted from a wife, husband, and their two children, Jack and Jill. The DNA for exon 5 of the aldolase B gene was ampli-  fied by polymerase chain reaction (PCR), treated with Ahall, subjected to electrophoresis on an agarose gel, and stained with  a dye that binds to DNA.   CHAPTER 29 / PATHWAYS OF SUGAR METABOLISM: PENTOSE PHOSPHATE PATHWAY, FRUCTOSE, AND GALACTOSE METABOLISM   Which of the following conclusions can be made from the data presented?   2. On examining the gel himself, the husband became concerned that he might not be the biologic father of one or both of the  children. From the pattern on the gel, you can reasonably conclude which of the following?   3. An alcoholic is brought to the Emergency Room for a hypoglycemic coma. Because alcoholics are frequently malnourished,  which of the following enzymes can be used to test for a thiamine deficiency?   4. Intravenous fructose feeding can lead to lactic acidosis caused by which of the following?   5. The polyol pathway of sorbitol production and the HMP shunt pathway are linked by which of the following?   (A) The HMP shunt produces 6-phosphogluconate, an intermediate in the polyol pathway.   (B) The HMP shunt produces NADPH, which is required for the polyol pathway.   (C) The HMP shunt produces ribitol, an intermediate of the polyol pathway.   Many of the pathways for inter conversion of sugars or the formation of sugar  derivatives use activated sugars attached to nucleotides. Both UDP * -glucose and  UDP -galactose are used for glycosyltransf erase reactions in many systems. Lac-  tose, for example, is synthesized from IJDP -galactose and glucose in the mam-  mary gland. IJDP -glucose also can be oxidized to form UDP -glucuronate , which  is used to form glucuronide derivatives of bilirubin and xenobiotic compounds.  Glucuronide derivatives are generally more readily excreted in urine or bile than  the parent compound.   In addition to serving as fuel, carbohydrates are often found in glycoproteins  (carbohydrate chains attached to proteins) and glycolipids (carbohydrate chains  attached to lipids). Nucleotide sugars are used to donate sugar residues for the  formation of the glycosidic bonds in both glycoproteins and glycolipids. These  carbohydrate groups have many different types of functions.   Glycoproteins contain short chains of carbohydrates (oligosaccharides) that  are usually branched. These oligosaccharides are generally composed of glucose,  galactose, and their amino derivatives. In addition, mannose, L-fucose, and  N-acetylneuraminic acid (NANA) are frequently present. The carbohydrate chains  grow by the sequential addition of sugars to a serine or threonine residue of the  protein. Nucleotide- sugars are the precursors. Branched carbohydrate chains also  may be attached to the amide nitrogen of asparagine in the protein. In this case,  the chains are synthesized on dolichol phosphate and subsequently transferred to  the protein. Glycoproteins are found in mucus, in the blood, in compartments  within the cell (such as lysosomes), in the extracellular matrix, and embedded in  the cell membrane with the carbohydrate portion extending into the extracellular  space.   Glycolipids belong to the class of sphingolipids. They are synthesized from  nuceotide- sugars that add monosaccharides sequentially to the hydroxy methyl  group of the lipid cer amide (related to sphingosine). They often contain branches  of N-acetylneuraminic acid produced from CMP -NANA. They are found in the cell  membrane with the carbohydrate portion extruding from the cell surface. These  carbohydrates, as well as some of the carbohydrates of glycoproteins, serve as  cell recognition factors.   To help support herself through medical school, Erna Nemdy works  evenings in a hospital blood bank. She is responsible for assuring that  compatible donor blood is available to patients needing blood transfusions.   Fig. 30.1. Metabolism of UDP-glucose. The activated glucose moiety of UDP-glucose can be  attached by a glycosidic bond to other sugars, as in glycogen or the sugar oligosaccharide and  polysaccharide side chains of proteoglycans, glycoproteins, and glycolipids. UDP-glucose also  can be oxidized to UDP-glucuronate, or epimerized to UDP-galactose, a precursor of lactose.   As part of her training, Erna has learned that the external surfaces of all blood cells  contain large numbers of antigenic determinants. These determinants are often gly-  coproteins or glycolipids that differ from one individual to another. As a result, all  blood transfusions expose the recipient to many foreign immunogens. Most of  these, fortunately, do not induce antibodies, or they induce antibodies that elicit lit-  tle or no immunologic response. For routine blood transfusions, therefore, tests are  performed only for the presence of antigens that determine whether the patient's  blood type is A, B, AB, or O.   • Jay Sakz's psychomotor development has become progressively more  nfl ) abnormal. At 2 years of age, he is obviously mentally retarded and   " nearly blind. His muscle weakness has progressed to the point that he  cannot sit up or even crawl. As the result of a weak cough reflex, he is unable  to clear his normal respiratory secretions and has had recurrent respiratory  infections.   Activated sugars attached to nucleotides are converted to other sugars, oxidized to  sugar acids, and joined to proteins, lipids, or other sugars through glycosidic  bonds.   UDP-glucose is an activated sugar nucleotide that is a precursor of glycogen and  lactose, UDP-glucuronate and glucuronides, and the carbohydrate chains in proteo-  glycans, glycoproteins, and glycolipids (Fig. 30.1). Both proteoglycans and gly-  cosaminoglycans are discussed further in Chapter 49. In the synthesis of many of  the carbohydrate portions of these compounds, a sugar is transferred from the  nucleotide sugar to an alcohol or other nucleophilic group to form a glycosidic bond  (Fig. 30.2). The use of UDP as a leaving group in this reaction provides the energy  for formation of the new bond. The enzymes that form glycosidic bonds are sugar  transferases (for example, glycogen synthase is a glucosyltransferase). Transferases  are also involved in the formation of the glycosidic bonds in bilirubin glucuronides,  proteoglycans, and lactose.   Fig. 30.2. Glycosyltransferases. These enzymes  transfer sugars from nucleotide sugars to nucle-  ophilic amino acid residues on proteins, such as  the hydroxyl group of serine or the amide group  of asparagine. Other transferases transfer spe-  cific sugars from a nucleotide sugar to a  hydroxyl group of other sugars. The bond  formed between the anomeric carbon of the  sugar and the nucleophilic group of another  compound is a glycosidic bond.   Fig. 30.4. Formation of glucuronate and glu-  curonides. A glycosidic bond is formed  between the anomeric hydroxyl of glucuronate  and the hydroxyl group of a nonpolar com-  pound. The negatively charged carboxyl group  of the glucuronate increases the water solubil-  ity and allows otherwise nonpolar compounds  to be excreted in the urine or bile.   High concentrations of galactose 1-  phosphate inhibit phosphogluco-  mutase, the enzyme that converts  glucose 6-phosphate to glucose 1-phos-  phate. How can this inhibition account for  the hypoglycemia and jaundice that  accompany galactose 1-phosphate uridylyl-  transferase deficiency?   Fig. 30.3. Metabolic routes of UDP-glucuronate. UDP-glucuronate is formed from UDP-  glucose (shown in black). Glucuronate from UDP-glucuronate is incorporated into gly-  cosaminoglycans, where certain of the glucuronate residues are converted to iduronate (see  Chapter 49). UDP-glucuronate is a precursor of UDP-xylose, another sugar residue incorpo-  rated into glycosaminoglycans. Glucuronate is also transferred to the carboxyl groups of  bilirubin or the alcohol groups of steroids, drugs, and xenobiotics to form glucuronides. The  "ide" in the name glucuronide denotes that these compounds are glycosides. Xenobiotics are  pharmacologically, endocrinologically, or toxicologically active substances not endoge-  nously produced and therefore foreign to an organism. Drugs are an example of a xenobiotic.   One of the major routes of UDP-glucose metabolism is the formation of UDP-  glucuronate, which serves as a precursor of other sugars and of glucuronides  (Fig. 30.3). Glucuronate is formed by the oxidation of the alcohol on C6 of glucose  to an acid (through two oxidation states) by an NAD + -dependent dehydrogenase  (Fig. 30.4). Glucuronate is also present in the diet and can be formed from the  degradation of inositol (the sugar alcohol that forms inositol trisphosphate (IP3), an  intracellular second messenger for many hormones).   The function of glucuronate in the excretion of bilirubin, drugs, xenobiotics, and  other compounds containing a hydroxyl group is to add negative charges and  increase their solubility. Bilirubin is a degradation product of heme that is formed in  the reticuloendothelial system and is only slightly soluble in plasma. It is transported  to the liver bound to albumin. In the liver, glucuronate residues are transferred from  UDP-glucuronate to two carboxyl groups on bilirubin, sequentially forming biliru-  bin monoglucuronide and bilirubin diglucuronide, the "conjugated" forms of biliru-  bin (Fig. 30.5). The more soluble bilirubin diglucuronide (as compared with uncon-  jugated bilirubin) is then actively transported into the bile for excretion.   Many xenobiotics, drugs, steroids, and other compounds with hydroxyl groups  and a low solubility in water are converted to glucuronides in a similar fashion by  glucuronyltransferases present in the endoplasmic reticulum and cytoplasm of the  liver and kidney (Table 30.1). This is one of the major conjugation pathways for  excretion of these compounds.   A failure of the liver to transport, store, or conjugate bilirubin results in the  accumulation of unconjugated bilirubin in the blood. Jaundice, the yellowish  tinge to the skin and the whites of the eyes (sclera) experienced by Erin Galway,  occurs when plasma becomes supersaturated with bilirubin (>2-2.5 mg/dL),  and the excess diffuses into tissues.    When bilirubin levels are measured in the blood, one can measure either indirect bilirubin (this is the nonconjugated form of  \\ ) bilirubin, which is bound to albumin), direct bilirubin (the conjugated, water-soluble form), or total bilirubin (the sum of the direct  v^^y and indirect levels). If total bilirubin levels are high, then a determination of direct and indirect bilirubin is needed to appropri-  ately determine a cause for the elevation of total bilirubin.   Glucuronate, once formed, can reenter the pathways of glucose metabolism  through reactions that eventually convert it to D-xylulose 5 -phosphate, an interme-  diate of the pentose phosphate pathway. In most mammals other than humans, an  intermediate of this pathway is the precursor of ascorbic acid (vitamin C). Humans,  however, are deficient in this pathway and cannot synthesize vitamin C.   Lactose is synthesized from UDP-galactose and glucose (Fig. 30.6). However,  galactose is not required in the diet for lactose synthesis because galactose can be  synthesized from glucose.   Galactose and glucose are epimers; they differ only in the stereochemical position of  one hydroxyl group. Thus, the formation of UDP-galactose from UDP-glucose is an  epimerization (Fig. 30.7). The epimerase does not actually transfer the hydroxyl group;  it oxidizes the hydroxyl to a ketone by transferring electrons to NAD + , and then  donates electrons back to re-form the alcohol group on the other side of the carbon.   Estrogen (female sex hormone)  Progesterone (steroid hormone)  Triiodothyronine (thyroid hormone)  Acetylaminofluorene (xenobiotic carcinogen)  Meprobamate (drug for sleep)  Morphine (painkiller)   ®6-Phosphogluconate is produced  by the first oxidative reaction in the  pentose phosphate pathway, in  which carbon 1 of glucose is oxidized to a  carboxylate. In contrast, glucuronic acid is  oxidized at carbon 6 to the carboxylate form.   Lactose is unique in that it is synthesized only in the mammary gland of the adult  for short periods during lactation. Lactose synthase, an enzyme present in the endo-  plasmic reticulum of the lactating mammary gland, catalyzes the last step in lactose  biosynthesis, the transfer of galactose from UDP-galactose to glucose (see  Fig. 30.6). Lactose synthase has two protein subunits, a galactosyltransferase and  a-lactalbumin. a-Lactalbumin is a modifier protein synthesized after parturition  (childbirth) in response to the hormone prolactin. This enzyme subunit lowers the  K m of the galactosyltransferase for glucose from 1,200 to 1 mM, thereby increasing  the rate of lactose synthesis. In the absence of a-lactalbumin, galactosyltransferase  transfers galactosyl units to glycoproteins.   Many (60%) full-term newborns develop jaundice, termed neonatal jaundice.  This is usually caused by an increased destruction of red blood cells after birth  (the fetus has an unusually large number of red blood cells) and an immature  bilirubin conjugating system in the liver. This leads to elevated levels of nonconjugated  bilirubin, which is deposited in hydrophobic (fat) environments. If bilirubin levels reach  a certain threshold at the age of 48 hours, the newborn is a candidate for phototherapy,  in which the child is placed under lamps that emit light between the wavelengths of 425  and 475 nm. Bilirubin absorbs this light, undergoes chemical changes, and becomes  more water soluble. Usually, within a week of birth, the newborn's liver can handle the  load generated from red blood cell turnover.   ®The inhibition of phosphoglucomutase results in hypoglycemia by interfering  with both the formation of UDP-glucose (the glycogen precursor) and the  degradation of glycogen back to glucose 6-phosphate. Ninety percent of glyco-  gen degradation leads to glucose 1-phosphate, which can only be converted to glucose  6-phosphate by phosphoglucomutase. When phosphoglucomutase activity is inhibited,  less glucose-6-P production occurs, and hence, less glucose is available for export. Thus,  the stored glycogen is only approximately 10% efficient in raising blood glucose levels,  and hypoglycemia results. UDP-glucose levels are reduced because glucose-1-P is  required to synthesize UDP-glucose, and in the absence of phosphoglucomutase activity  glucose-6-P cannot be converted to glucose-1-P. This prevents the formation of UDP-  glucuronate, which is necessary to convert bilirubin to the diglucuronide form for trans-  port into the bile. Bilirubin accumulates in tissues, giving them a yellow color (jaundice).   A pregnant woman who was  €Bj) extremely lactose intolerant asked  her physician if she would still be  able to breastfeed her infant since she could  not drink milk or dairy products. What advice  should she be given?   Fig. 30.5. Formation of bilirubin diglu-  curonide. A glycosidic bond is formed  between the anomeric hydroxyl of glucuronate  and the carboxylate groups of bilirubin. The  addition of the hydrophilic carbohydrate  group, and the negatively charged carboxyl  group of the glucuronate, increases the water  solubility of the conjugated bilirubin and  allows the otherwise insoluble bilirubin to be  excreted in the urine or bile.   Fig. 30.6. Lactose synthesis. Lactose is a dis-  accharide composed of galactose and glucose.  UDP-galactose for the synthesis of lactose in  the mammary gland is usually formed from the  epimerization of UDP-glucose. Lactose syn-  thase attaches the anomeric carbon of the  galactose to the C4 alcohol group of glucose to  form a glycosidic bond. Lactose synthase is  composed of a galactosyltransferase and a-  lactalbumin, which is a regulatory subunit.   Table 30.2. Some Sugar Nucleotides  That Are Precursors for Transferase  Reactions   Although the lactose in dairy prod-  •] ucts is a major source of galactose,   the ingestion of lactose is not  required for lactation. UDP-galactose in the  mammary gland is derived principally from  the epimerization of glucose. Dairy products  are, however, a major dietary source of Ca 2+ ,  so breastfeeding mothers need increased  Ca 2+ from another source.   Fig. 30.7. Epimerization of UDP-glucose to UDP-galactose. The epimerization of glucose to  galactose occurs on UDP-sugars. The epimerase uses NAD + to oxidize the alcohol to a ketone,  and then reduce the ketone back to an alcohol. The reaction is reversible; glucose being con-  verted to galactose forms galactose for lactose synthesis, and galactose being converted to glu-  cose is part of the pathway for the metabolism of dietary galactose.   The transferases that produce the oligosaccharide and polysaccharide side chains of  glycolipids and attach sugar residues to proteins are specific for the sugar moiety  and for the donating nucleotide (e.g., UDP, CMP, or GDP). Some of the sugar-  nucleotides used for glycoprotein, proteoglycan (see Chapter 49) and glycolipid  formation are listed in Table 30.2. They include the derivatives of glucose and  galactose that we have already discussed, as well as acetylated amino sugars and  derivatives of mannose. The reason for the large variety of sugars attached to pro-  teins and lipids is that they have relatively specific and different functions, such as  targeting a protein toward a membrane, providing recognition sites on the cell  surface for other cells, hormones, or viruses, or acting as lubricants or molecular  sieves (see Chapter 42).   The pathways for utilization and formation of many of these sugars are summa-  rized in Figure 30.8. Note that many of the steps are reversible, so that glucose and  other dietary sugars enter a common pool from which the diverse sugars can be  formed.   The amino sugars are all derived from glucosamine 6-phosphate. To synthesize  glucosamine 6-phosphate, an amino group is transferred from the amide of gluta-  mine to fructose 6-phosphate (Fig. 30.9). Amino sugars, such as glucosamine, can  then be N-acetylated by an acetyltransf erase.   Mannose is found in the diet in small amounts. Like galactose, it is an epimer of  glucose, and mannose and glucose are interconverted by epimerization reactions.  The interconversion can take place either at the level of fructose 6-phosphate to  mannose 6-phosphate, or at the level of the derivatized sugars (see Fig. 30.8).   N-Acetyltransferases are present in the endoplasmic reticulum and cytosol and  \ \ ) provide another means of chemically modifying sugars, metabolites, drugs,  and xenobiotic compounds. Individuals may vary greatly in their capacity for  acetylation reactions.   Fig. 30.8. Pathways for the interconversion of sugars. All of the different sugars found in glycosaminoglycans, gangliosides, and other com-  pounds in the body can be synthesized from glucose. Dietary glucose, fructose, galactose, mannose, and other sugars enter a common pool from  which other sugars are derived. The activated sugar is transferred from the nucleotide sugar, shown in blue boxes, to form a glycosidic bond with  another sugar or amino acid residue. The box next to each nucleotide sugar lists some of the compounds that contain the sugar. Iduronic acid,  in the upper right corner of the diagram, is formed only after glucuronic acid is incorporated into a glycosaminoglycan (which is discussed in  more detail in Chapter 49).   Fig. 30.9. The formation of Af-acetylglu-  cosamine 6-phosphate. The amino sugar is  formed by a transfer of the amino group from  the amide of glutamine to a carbon of the  sugar. The amino group is acetylated by the  transfer of an acetyl group from acetyl CoA.   Fig. 30.11. Structure of salivary mucin. The  sugars form hydrogen bonds with water. Sialic  acid provides a negatively charged carboxylate  group. The protein is extremely large, and the  negatively charged sialic acids extend the car-  bohydrate chains so the molecules occupy a  large space. All of the salivary glycoproteins  are O-linked. NANA is a sialic acid.   Fig. 30.10. An example of a branched glycoprotein. NANA = A^-acetylneuraminic acid;  Gal= galactose; GlcNAc = A^-acetylglucosamine; Man = mannose; Fuc = fucose.   N-Acetylmannosamine is the precursor of N-acetylneuraminic acid (NANA, a sialic  acid) and GDP-mannose is the precursor of GDP-fucose (see Fig. 30.8). The nega-  tive charge on NANA is obtained by the addition of a 3 -carbon carboxyl moiety  from phosphoenolpyruvate.   Glycoproteins contain short carbohydrate chains covalently linked to either ser-  ine/threonine or asparagine residues in the protein. These oligosaccharide chains are  often branched, and they do not contain repeating disaccharides (Fig. 30.10). Most  proteins in the blood are glycoproteins. They serve as hormones, antibodies,  enzymes (including those of the blood clotting cascade), and as structural compo-  nents of the extracellular matrix. Collagen contains galactosyl units and disaccha-  rides composed of galactosyl-glucose attached to hydroxylysine residues (see  Chapter 49). The secretions of mucus -producing cells, such as salivary mucin, are  glycoproteins (Fig. 30.11).   Although most glycoproteins are secreted from cells, some are segregated in  lysosomes, where they serve as the lysosomal enzymes that degrade various types  of cellular and extracellular material. Other glycoproteins are produced like secre-  tory proteins, but hydrophobic regions of the protein remain attached to the cell  membrane, and the carbohydrate portion extends into the extracellular space  (Fig. 30.12)(also see Chapter 15, section I). These glycoproteins serve as receptors  for compounds such as hormones, as transport proteins, and as cell attachment and  cell-cell recognition sites. Bacteria and viruses also bind to these sites.   The protein portion of glycoproteins is synthesized on the endoplasmic reticulum  (ER). The carbohydrate chains are attached to the protein in the lumen of the ER  and the Golgi complex. In some cases, the initial sugar is added to a serine or a  threonine residue in the protein, and the carbohydrate chain is extended by the  sequential addition of sugar residues to the nonreducing end. As seen previously  in Table 2, UDP-sugars are the precursors for the addition of four of the seven  sugars that are usually found in glycoproteins — glucose, galactose, TV-acetylglu-  cosamine, and ^/-acetylgalactosamine. GDP-sugars are the precursors for the  addition of mannose and L-fucose, and CMP-NANA is the precursor for NANA.   Fig. 30.12. Route from the Golgi complex to the final destination for lysosomal enzymes, cell  membrane proteins, and secreted proteins, which include glycoproteins and proteoglycans.   The enzyme that is deficient in l-cell  disease is a phosphotransferase  located in the Golgi apparatus.   Q--P-0-P-0- CH? - CH? - C - CH? - CH?-CH = C-CH   Fig. 30.13. Structure of dolichol phosphate. In humans, the isoprene unit (in brackets) is  repeated approximately 17 times (n = ~17).   Dolichol phosphate (Fig. 30.13) (which is synthesized from isoprene units, as dis-  cussed in Chapter 34) is involved in transferring branched sugar chains to the  amide nitrogen of asparagine residues. Sugars are removed and added as the gly-  coprotein moves from the ER through the Golgi complex (Fig. 30.14). As dis-  cussed in Chapter 10, the carbohydrate chain is used as a targeting marker for  lysosomal enzymes.   I-cell (inclusion cell) disease is a rare condition in which lysosomal enzymes  lack the mannosephosphate marker that targets them to lysosomes. Consequently,  lysosomal enzymes are secreted from the cells. Because lysosomes lack their nor-  mal complement of enzymes, undegraded molecules accumulate within membranes  inside these cells, forming inclusion bodies.   Glycolipids are derivatives of the lipid sphingosine. These sphingolipids include the  cerebrosides and the gangliosides (Fig. 30.15; see also Fig. 5.22). They contain  ceramide, with carbohydrate moieties attached to its hydroxymethyl group.   Glycolipids are involved in intercellular communication. Oligosaccharides of  identical composition are present in both the glycolipids and glycoproteins associ-  ated with the cell membrane, where they serve as cell recognition factors. For exam-  ple, carbohydrate residues in these oligosaccharides are the antigens of the ABO  blood group substances (Fig. 30.16).   The phosphotransferase has the unique  ability to recognize lysosomal proteins  because of their three-dimensional struc-  ture, such that they can all be appropriately  tagged for transport to the lysosomes.   By identifying the nature of anti-  genic determinants on the surface  of the donor's red blood cells, Erna  Nemdy is able to classify the donor's blood  as belonging to certain specific blood  groups. These antigenic determinants are  located in the oligosaccharides of the glyco-  proteins and glycolipids of the cell mem-  branes. The most important blood group in  humans is the ABO group, which comprises  two antigens, A and B. Individuals with the A  antigen on their cells belong to blood group  A. Those with B belong to group B, and  those with both A and B belong to group AB.  The absence of both the A and the B antigen  results in blood type O (see Fig. 30.16).    Fig. 30.14. Action of dolichol phosphate in transferring carbohydrate groups to proteins (A)  and processing of these carbohydrate groups (B). Transfer of the branched oligosaccharide  from dolichol phosphate to a protein in the lumen of the rough endoplasmic reticulum (RER)  (step 1) and processing of the oligosaccharide (steps 2-11). Steps 1 through 4 occur in the  RER. The glycoprotein is transferred in vesicles to the Golgi complex, where further modi-  fications of the oligosaccharides occur (steps 5-11). B modified with permission from  Kornfeld R, Kornfeld S. Annu Rev Biochem 1985;54:640. © 1985 by Annual Reviews, Inc.  P = phosphate.   Fig. 30.15. Structures of cerebrosides and  gangliosides. In these glycolipids, sugars are  attached to ceramide (shown below the glycol-  ipids). The boxed portion of ceramide is sphin-  gosine, from which the name sphingolipids is  derived.    Fig. 30.16. Structures of the blood group substances. Note that these structures are the same  except that type A has ^acetylgalactosamine (GalNAc) at the nonreducing end, type B has  galactose (Gal), and type O has neither. R is either a protein or the lipid ceramide. Each anti-  genic determinant is boxed. Fuc = fucose; GlcNAc = A^-acetylglucosamine; Gal = galactose.   The blood group substances are oligosaccharide components of glycolipids  and glycoproteins found in most cell membranes. Those located on red blood  cells have been studied extensively. A single genetic locus with two alleles  determines an individual's blood type. These genes encode glycosyltransferases  involved in the synthesis of the oligosaccharides of the blood group substances.   Most individuals can synthesize the H substance, an oligosaccharide that contains a  fucose linked to a galactose at the nonreducing end of the blood group substance (see Fig.  30.16). Type A individuals produce an ^-acetylgalactosamine transferase (encoded by the  A gene) that attaches ^-acetylgalactosamine to the galactose residue of the H substance.  Type B individuals produce a galactosyltransferase (encoded by the B gene) that links  galactose to the galactose residue of the H substance. Type AB individuals have both alle-  les and produce both transferases. Thus, some of the oligosaccharides of their blood group  substances contain ^-acetylgalactosamine and some contain galactose. Type O individu-  als produce a defective transferase, and, therefore, they do not attach either A/-acetyl-  galactosamine or galactose to the H substance. Thus, individuals of blood type O have only  the H substance.   Cerebrosides are synthesized from ceramide and UDP-glucose or UDP-galactose.  They contain a single sugar (a monosaccharide). Gangliosides contain oligosaccha-  rides produced from UDP-sugars and CMP-NANA, which is the precursor for the  A^-acetylneuraminic acid residues that branch from the linear chain. The synthesis  of the sphingolipids is described in more detail in Chapter 33.   Sphingolipids are produced in the Golgi complex. Their lipid component  becomes part of the membrane of the secretory vesicle that buds from the trans face  of the Golgi. After the vesicle membrane fuses with the cell membrane, the lipid  component of the glycolipid remains in the outer layer of the cell membrane, and  the carbohydrate component extends into the extracellular space.   Cholera toxin binds to the carbohy-  drate portion of the GM1 ganglio-  side to allow its catalytic subunit to  enter the cell.   0^ Erna Nemdy determined that a  •1 patient's blood type was AB. The  new surgical resident was eager to  give this patient a blood transfusion and,  because AB blood is rare and an adequate  amount was not available in the blood bank,  he requested type A blood. Should Erna give  him type A blood for his patient?   Jay Sakz has Tay-Sachs disease,  which belongs to a group of gan-  gliosidoses that include Fabry's  and Gaucher's diseases. They mainly affect  the brain, the skin, and the reticuloendothe-  lial system (e.g., liver and spleen). In these  diseases, complex lipids accumulate. Each  of these lipids contains a ceramide as part of  its structure (Table 30.3). The rate at which  the lipid is synthesized is normal. However,  the lysosomal enzyme required to degrade it  is not very active, either because it is made  in deficient quantities because of a mutation  in a gene that specifically codes for the  enzyme or because a critical protein  required to activate the enzyme is deficient.  Because the lipid cannot be degraded, it  accumulates and causes degeneration of the  affected tissues with progressive malfunc-  tion, such as the psychomotor deficits that  occur as a result of the central nervous sys-  tem involvement seen in most of these stor-  age diseases.   NeuAc, A/-acetylneuraminic acid; Cer, ceramide: Glc, glucose; Gal, galactose; Fuc, fucose : site of deficient  enzyme reaction.   ®The patient could safely receive  type A blood cells from another  person because he has both A and  B antigens on his own cells and does not  have antibodies in his serum to either type A  or B cells. However, he should not be given  type A serum (or type A whole blood)  because type A serum contains antibodies to  type B antigens, which are present on his  cells.   ^ During her stint in the hospital blood bank, Erna Nemdy learned that the  ) importance of the ABO blood group system in transfusion therapy is based  on two principles (Table 30.4). (a) Antibodies to A and to B antigens occur  naturally in the blood serum of persons whose red blood cell surfaces lack the cor-  responding antigen (i.e., individuals with A antigens on their red blood cells have B  antibodies in their serum and vice versa). These antibodies may arise as a result of  previous exposure to cross -reacting antigens in bacteria and foods or to blood trans-  fusions, (b) Antibodies to A and B are usually present in high titers and are capable  of activating the entire complement system. As a result, these antibodies may cause  intravascular destruction of a large number of incompatible red blood cells given  during a blood transfusion. Individuals with type AB blood have both A and B anti-  gens and do not produce antibodies to either. Hence, they are "universal" recipients.  They can safely receive red blood cells from individuals of A, B, AB, or O blood  type. (However, they cannot safely receive serum from these individuals because it  contains antibodies to A or B antigens.) Those with type O blood do not have either  antigen. They are "universal" donors; i.e., their red cells can safely be infused into  type A, B, O, or AB individuals. (However, their serum contains antibodies to both  A and B antigens and cannot safely be used.)   The second important red blood cell group is the Rh group. It is important  because one of its antigenic determinants, the D antigen, is a very potent immuno-  gen, stimulating the production of a large number of antibodies.   The unique carbohydrate composition of the glycoproteins that constitute the  antigenic determinants on red blood cells in part contributes to the relative immuno-  genicity of the A, B, and Rh (D) red blood cell groups in human blood.   Tay-Sachs disease, the problem afflicting Jay Sakz, is an autosomal reces-  sive disorder that is rare in the general population (1 in 300,000 births), but  its prevalence in Jews of Eastern European extraction (who make up 90%  of the Jewish population in the United States) is much higher (1 in 3,600 births).  One in 28 Ashkenazi Jews carries this defective gene. Its presence can be discov-  ered by measuring the tissue level of the protein produced by the gene (hex-  osaminidase A) or by recombinant DNA techniques. Skin fibroblasts of concerned  couples planning a family are frequently used for these tests.   Carriers of the affected gene have a reduced but functional level of this enzyme  that normally hydrolyzes a specific bond between an Af-acetyl-D-galactosamine and  a D-galactose residue in the polar head of the ganglioside.   No effective therapy is available. Enzyme replacement has met with little success  because of the difficulties in getting the enzyme across the blood-brain barrier.   Hexosaminidase A, the enzyme defective in Tay-Sachs disease, is actually  composed of two subunits, an a and a p chain. The exact stoichiometry of  the active enzyme is unknown, but it may be a 2 p 2 . The a subunit is coded  for by the HexA gene, whereas the p subunit is coded for by the HexB gene. In Tay-  Sachs disease, the a subunit is defective, and hexosaminidase A activity is lost.  However, the p subunit can form active tetramers in the absence of the a subunit,  and this activity, named hexosaminidase B, which cleaves the glycolipid globoside,  retains activity in children with Tay-Sachs disease. Thus, children with Tay-Sachs  disease accumulate the ganglioside GM2, but not globoside (Fig. 30.17).   Mutation of the HexB gene, and production of a defective p subunit, leads to  inactivation of both hexosaminidase A and B activity. Such a mutation leads to  Sandhoff disease. Both activities are lost because both activities require a functional  p subunit. The clinical course of this disease is similar to Tay-Sachs but with an  accelerated timetable because of the initial accumulation of both GM2 and globo-  side in the lysosomes.   hexosaminidase A (a 2 (3 2 )  Block in Sandhoff disease   hexosaminidase A or B (p 4 )  Block in Sandhoff disease   Fig. 30.17. Substrate specificities of hexosaminidase A, B, and the function of the activator  protein. Glc = glucose; gal = galactose; NAcGal = N-acetylgalactosamine.   A third type of mutation also can lead to disease symptoms similar to those of  Tay-Sachs disease. Children were identified with Tay-Sachs symptoms, but when  both hexosaminidase A and B activities were measured in a test tube, they were nor-  mal. This disease, ultimately named Sandhoff activator disease, is caused by a  mutation in a protein that is needed to activate hexosaminidase A activity. In the  absence of the activator, hexosaminidase A activity is minimal, and GM2 initially  accumulates in lysosomes. This mutation has no effect on hexosaminidase B  activity.   When a glycolipid cannot be degraded because of an enzymatic mutation, it  accumulates in residual bodies (vacuoles that contain material that lysosomal  enzymes cannot digest). Normal cells contain a small number of residual bodies, but  in diseases of lysosomal enzymes, large numbers of residual bodies accumulate  within the cell, eventually interfering with normal cell function.   In 70% of the cases of Tay-Sachs disease in persons of Ashkenazai Jewish back-  ground, exon 1 1 of the gene for the a chain of hexosaminidase A contains a muta-  tion. The normal gene sequence encodes a protein with the amino acids arg-ile-ser-  tyr-gly-pro-asp in this region, as shown below:   5' - CGTATATCTATCCTATGGCCCTGAC  arg - ile -ser - ile - leu- leu -trp-pro-   A four-base insertion (underlined) occurs in the mutated gene, which alters the  reading frame of the protein, and also introduces a premature stop codon further  down the protein, such that no functional a subunit can be produced.   Gravel RA, Kaback MM, Proia RL, Sandhoff K, et al. The G M2 gangliosidoses. In: Scriver CR, Beaudet  AL, Valle D, Sly WS, et al eds. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed.  New York: McGraw-Hill, 2001:3827-3876.   Kornfeld S, Sly WS. I-cell disease and pseudohurler poly dystrophy: disorders of lysosomal enzyme  phosphorylation and localization. In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds. The Meta-  bolic and Molecular Bases of Inherited Disease, 8th ed. New York: McGraw-Hill, 2001:3469-3482.   Lloyd KO. The chemistry and immunochemistry of blood group A, B, H and Lewis antigens: past, pres-  ent and future. Glycoconjugate J 2000:531-541.   Which of the following best describes a mother with galactosemia caused by a deficiency of galactose 1 -phosphate uridylyl  transferase?   (A) She can convert galactose to UDP-galactose for lactose synthesis during lactation.   (C) She can use galactose as a precursor to glucose production.   (E) She will have lower than normal levels of serum galactose after drinking milk.   2. The immediate carbohydrate precursors for glycolipid and glycoprotein synthesis are which of the following?   3. A newborn is diagnosed with neonatal jaundice. In this patient, the bilirubin produced lacks which of the following carbohy-  drates?   4. The nitrogen donor for the formation of amino sugars is which of the following?   5. Which of the following glycolipids would accumulate in a patient with Sandhoff 's disease?   During fasting, many of the reactions of glycolysis are reversed as the liver pro-  duces glucose to maintain blood glucose levels. This process of glucose produc-  tion is called gluconeogenesis.   Gluconeogenesis , which occurs primarily in the liver, is the pathway for the  synthesis of glucose from compounds other than carbohydrates. In humans, the  major precursors of glucose are lactate, glycerol, and amino acids, particularly  alanine .Except for three key sequences, the reactions of gluconeogenesis are  reversals of the steps of glycolysis (Fig. 31.1). The sequences of gluconeogenesis  that do not use enzymes of glycolysis involve the irreversible, regulated steps of  glycolysis. These three steps are the conversion of (a) pyruvate to phospho-  enolpyruvate, (b) fructose 1 ,6-bisphosphate to fructose 6-phosphate, and (c) glu-  cose 6 -phosphate to glucose.   Some tissues of the body, such as the brain and red blood cells, cannot synthe-  size glucose on their own, yet depend on glucose for energy. On a long-term  basis, most tissues also require glucose for other functions such as the synthesis of  the ribose moiety of nucleotides or the carbohydrate portion of glycoproteins and  glycolipids. Therefore, to survive, humans must have mechanisms for maintaining  blood glucose levels.   After a meal containing carbohydrates, blood glucose levels rise (Fig. 31.2).  Some of the glucose from the diet is stored in the liver as glycogen. After 2 or 3  hours of fasting, this glycogen begins to be degraded by the process of  glycogenolysis, and glucose is released into the blood. As glycogen stores  decrease, adipose triacylglycerols are also degraded, providing fatty acids as an  alternative fuel and glycerol for the synthesis of glucose by gluconeogenesis.  Amino acids are also released from the muscle to serve as gluconeogenic precur-  sors.   During an overnight fast, blood glucose levels are maintained by both  glycogenolysis and gluconeogenesis. However, after approximately 30 hours of  fasting, liver glycogen stores are mostly depleted. Subsequently, gluconeogenesis  is the only source of blood glucose.   Changes in the metabolism of glucose that occur during the switch from the fed  to the fasting state are regulated by the hormones insulin and glucagon. Insulin is  elevated in the fed state, and glucagon is elevated during fasting. Insulin stimu-  lates the transport of glucose into certain cells such as those in muscle and adi-  pose tissue. Insulin also alters the activity of key enzymes that regulate metabo-  lism, stimulating the storage of fuels. Glucagon counters the effects of insulin,  stimulating the release of stored fuels and the conversion of lactate, amino acids,  and glycerol to glucose.    Fig. 31.1. Glycolysis and gluconeogenesis in the liver. The gluconeogenic pathway is almost the reverse of the glycolytic  pathway, except for three reaction sequences. At these three steps, the reactions are catalyzed by different enzymes. The  energy requirements of these reactions differ, and one pathway can be activated while the other is inhibited.   Fig. 31.2. Sources of blood glucose in the fed,  fasting, and starved states. RBC = red blood  cells.   What clinical signs and symptoms  ■ B* help to distinguish a coma caused  by an excess of blood glucose and  ketone bodies due to a deficiency of insulin  (diabetic ketoacidosis [DKA]) from a coma  caused by a sudden lowering of blood glucose  (hypoglycemic coma) induced by the inadver-  tent injection of excessive insulin — the current  problem experienced by Di Abietes?   Blood glucose levels are maintained not only during fasting, but also during  exercise, when muscle cells take up glucose from the blood and oxidize it for  energy. During exercise, the liver supplies glucose to the blood by the processes of  glycogenolysis and gluconeogenesis.   Al Martini, a known alcoholic, was brought to the emergency room by his  Hfl ) landlady, who stated that he had been drinking heavily for the past week.  " During this time his appetite had gradually diminished, and he had not  eaten any food for the past 3 days. He was confused, combative, tremulous, and  sweating profusely. His speech was slurred. His heart rate was rapid (1 10 beats per  minute). As his blood pressure was being determined, he had a grand mal seizure.  His blood glucose, drawn just before the onset of the seizure, was 28 mg/dL or  1.6 mM (reference range for overnight fasting blood glucose = 80-100 mg/dL or  4.4-5.6 mM). His blood ethanol level drawn at the same time was 295 mg/dL  (intoxication level, i.e., "confused" stage = 150-300 mg/dL).   Emma Wheezer presented to the emergency room 3 days after dis-  charge from the hospital following a 10-day admission for severe  refractory bronchial asthma. She required high-dose intravenous dex-  amethasone (an anti-inflammatory synthetic glucocorticoid) for the first 8 days  of her stay. After 2 additional days receiving oral dexamethasone, she was dis-  charged on substantial pharmacologic doses of this steroid and instructed to  return to her physician's office in 5 days. She presented now with marked  polyuria (increased urination), polydipsia (increased thirst), and muscle weak-  ness. Her blood glucose was 275 mg/dL or 15 mM (reference range = 80-100  mg/dL or 4.4-5.6 mM).   Di Abietes could not remember whether she had taken her 6:00 PM insulin  dose, when, in fact, she had done so. Unfortunately, she decided to give her-  self the evening dose (for the second time). When she did not respond to her  alarm clock at 6:00 AM the following morning, her roommate tried unsuccessfully to  awaken her. The roommate called an ambulance, and Di was rushed to the hospital  emergency room in a coma. Her pulse and blood pressure at admission were normal.  Her skin was flushed and slightly moist. Her respirations were slightly slow.   • Ann O'Rexia continues to resist efforts on the part of her psychiatrist and  flP ) family physician to convince her to increase her caloric intake. Her body   " weight varies between 97 and 99 lb, far below the desirable weight for a  woman who is 5 feet 7 inches tall. In spite of her severe diet, her fasting blood glucose  levels range from 55 to 70 mg/dL. She denies having any hypoglycemic symptoms.   • Otto Shape has complied with his calorie-restricted diet and aerobic exer-  1|l ) cise program. He has lost another 7 lb and is closing in on his goal of  weighing 154 lb. He notes increasing energy during the day, and remains  alert during lectures and assimilates the lecture material noticeably better than he  did before starting his weight loss and exercise program. He jogs for 45 minutes  each morning before breakfast.    Diabetes mellitus (DM) should be suspected if a venous plasma glucose level  drawn irrespective of when food was last eaten (a "random" sample of blood  glucose) is "unequivocally elevated" (i.e., >200 mg/dL), particularly in a patient  who manifests the classic signs and symptoms of chronic hyperglycemia (polydipsia,  polyuria, blurred vision, headaches, rapid weight loss, sometimes accompanied by nau-  sea and vomiting). To confirm the diagnosis, the patient should fast overnight (10-16  hours), and the blood glucose measurement should be repeated. Values of less than 110  mg/dL are considered normal. Values greater than 140 mg/dL are indicative of DM. Gly-  cosylated hemoglobin should be measured to determine the extent of hyperglycemia  over the past 4 to 8 weeks. Values of fasting blood glucose between 111 and 140 mg/dL  are designated impaired fasting glucose tolerance (IGT), and further testing should be  performed to determine whether these individuals will eventually develop overt diabetes  mellitus.   Although the oral glucose tolerance test (OGTT) is contraindicated for patients who  clearly have diabetes mellitus, it is used for patients with fasting blood glucose in the IGT  range (between 115 and 140 mg/dL). In the OGTT, a nonpregnant patient who has fasted  overnight drinks 75 g glucose in an aqueous solution. Blood samples are drawn  before the oral glucose load and at 30, 60, 90, and 120 minutes thereafter. If any one of  the 30-, 60-, and 90-minute samples and the 120-minute sample are greater than 200  mg/dL, overt DM is indicated.   The diagnosis of IGT and the more severe form of glucose intolerance (DM) is based  on blood glucose levels because no more specific characteristic for the disorder exists.  The distinction between IGT and DM is clouded by the fact that a patient's blood glucose  level may vary significantly with serial testing over time under the same conditions of  diet and activity.   The renal tubular transport maximum in the average healthy subject is such that glu-  cose will not appear in the urine until the blood glucose level exceeds 180 mg/dL. As a  result, reagent tapes (Tes-Tape or Dextrostix) designed to detect the presence of glucose  in the urine are not sensitive enough to establish a diagnosis of early DM.   Glucose serves as a fuel for most tissues of the body. It is the major fuel for cer-  tain tissues such as the brain and red blood cells. After a meal, food is the source  of blood glucose. The liver oxidizes glucose and stores the excess as glycogen. The  liver also uses the pathway of glycolysis to convert glucose to pyruvate, which  provides carbon for the synthesis of fatty acids. Glycerol 3 -phosphate, produced  from glycolytic intermediates, combines with fatty acids to form triacylglycerols,  which are secreted into the blood in very-low-density lipoproteins (VLDL; further  explained in Chapter 32). During fasting, the liver releases glucose into the blood,  so that glucose-dependent tissues do not suffer from a lack of energy. Two mech-  anisms are involved in this process: glycogenolysis and gluconeogenesis. Hor-  mones, particularly insulin and glucagon, dictate whether glucose flows through  glycolysis or whether the reactions are reversed and glucose is produced via glu-  coneogenesis.   Gluconeogenesis, the process by which glucose is synthesized from noncarbohy-  drate precursors, occurs mainly in the liver under fasting conditions. Under the  more extreme conditions of starvation, the kidney cortex also may produce glucose.  For the most part, the glucose produced by the kidney cortex is used by the kidney  medulla, but some may enter the bloodstream.   Starting with pyruvate, most of the steps of gluconeogenesis are simply reversals  of those of glycolysis (Fig. 31.3). In fact, these pathways differ at only three points.  Enzymes involved in catalyzing these steps are regulated so that either glycolysis or  gluconeogenesis predominates, depending on physiologic conditions.   ® Comatose patients in diabetic  ketoacidosis have the smell of ace-  tone (a derivative of the ketone  body acetoacetate) on their breath. In addi-  tion, DKA patients have deep, relatively  rapid respirations typical of acidotic patients  (Kussmaul respirations). These respirations  result from an acidosis-induced stimulation  of the respiratory center in the brain. More  C0 2 is exhaled in an attempt to reduce the  amount of acid in the body: H + + HC0 3 ~— >  H 2 C0 3 -> H 2 0 + C0 2 (exhaled).   The severe hyperglycemia of DKA also  causes an osmotic diuresis (i.e., glucose  entering the urine carries water with it),  which, in turn, causes a contraction of blood  volume. Volume depletion may be aggra-  vated by vomiting, which is common in  patients with DKA. DKA may cause dehydra-  tion (dry skin), a low blood pressure, and a  rapid heartbeat. These respiratory and  hemodynamic alterations are not seen in  patients with hypoglycemic coma. The  flushed, wet skin of hypoglycemic coma is in  contrast to the dry skin observed in DKA.   Q o Glucocorticoids are naturally  j /~oA j occurring steroid hormones. In  ™ humans, the major glucocorticoid  is Cortisol. Glucocorticoids are produced in  the adrenal cortex in response to various  types of stress (see Chapter 43). One of their  actions is to stimulate the degradation of  muscle protein. Thus, increased amounts of  amino acids become available as substrates  for gluconeogenesis. Emma Wheezer noted  muscle weakness, a result of the muscle-  degrading action of the synthetic glucocorti-  coid dexamethasone, which she was taking  for its anti-inflammatory effects.   Fig. 31.3. Key reactions of gluconeogenesis. The precursors are amino acids (particularly ala-  nine), lactate, and glycerol. Heavy arrows indicate steps that differ from those of glycolysis.   Because ethanol metabolism only  \\ ) gives rise to acetyl-CoA, the car-  bons of ethanol cannot be used for  gluconeogenesis.   Most of the steps of gluconeogenesis use the same enzymes that catalyze the  process of glycolysis. The flow of carbon, of course, is in the reverse direction.  Three reaction sequences of gluconeogenesis differ from the corresponding steps of  glycolysis. They involve the conversion of pyruvate to phosphoenolpyruvate (PEP)  and the reactions that remove phosphate from fructose 1,6-bisphosphate to form  fructose 6-phosphate and from glucose 6-phosphate to form glucose (see Fig. 31.3).  The conversion of pyruvate to PEP is catalyzed during gluconeogenesis by a series  of enzymes instead of the single enzyme used for glycolysis. The reactions that  remove phosphate from fructose 1,6-bisphosphate and from glucose 6-phosphate  each use single enzymes that differ from the corresponding enzymes of glycolysis.  Although phosphate is added during glycolysis by kinases, which use adenosine  triphosphate (ATP), it is removed during gluconeogenesis by phosphatases that  release Pi via hydrolysis reactions.   The three major carbon sources for gluconeogenesis in humans are lactate, glyc-  erol, and amino acids, particularly alanine. Lactate is produced by anaerobic gly-  colysis in tissues such as exercising muscle or red blood cells, as well as by  adipocytes during the fed state. Glycerol is released from adipose stores of   triacylglycerol, and amino acids come mainly from amino acid pools in muscle,  where they may be obtained by degradation of muscle protein. Alanine, the major  gluconeogenic amino acid, is produced in the muscle from other amino acids and  from glucose (see Chapter 38).   The carbon sources for gluconeogenesis form pyruvate, intermediates of the tricar-  boxylic acid (TCA) cycle, or intermediates common both to glycolysis and gluco-  neogenesis.   Pyruvate is produced in the liver from the gluconeogenic precursors lactate and  alanine. Lactate dehydrogenase oxidizes lactate to pyruvate, generating NADH  (Fig. 31.4A), and alanine aminotransferase converts alanine to pyruvate (see  Fig. 31.4B).   Although alanine is the major gluconeogenic amino acid, other amino acids,  such as serine, serve as carbon sources for the synthesis of glucose because they  also form pyruvate, the substrate for the initial step in the process. Some amino  acids form intermediates of the TCA cycle (see Chapter 20), which can enter the  gluconeogenic pathway.   The carbons of glycerol are gluconeogenic because they form dihydroxyacetone  phosphate (DHAP), a glycolytic intermediate (see Fig. 31.4C)   Fatty acids with an odd number of carbon atoms, which are obtained mainly  from vegetables in the diet, produce propionyl CoA from the three carbons at  the co-end of the chain (see Chapter 23). These carbons are relatively minor pre-  cursors of glucose in humans. Propionyl CoA is converted to methylmalonyl  CoA, which is rearranged to form succinyl CoA, a 4-carbon intermediate of the  TCA cycle that can be used for gluconeogenesis. The remaining carbons of an  odd-chain fatty acid form acetyl CoA, from which no net synthesis of glucose  occurs.   p-Oxidation of fatty acids produces acetyl CoA. Because the pyruvate dehydro-  genase reaction is thermodynamically and kinetically irreversible, acetyl CoA does  not form pyruvate for gluconeogenesis. Therefore, if acetyl CoA is to produce glu-  cose, it must enter the TCA cycle and be converted to malate. For every two carbons  of acetyl CoA that are converted to malate, two carbons are released as C0 2 : one in  the reaction catalyzed by isocitrate dehydrogenase and the other in the reaction cat-  alyzed by a-ketoglutarate dehydrogenase. Therefore, there is no net synthesis of  glucose from acetyl CoA.    In a fatty acid with 19 carbons, how  I many carbons (and which ones)  form glucose?   neogenic precursors. Cells have  limited amounts of NAD, which exist either  as NAD + or as NADH. As the levels of NADH  rise, those of NAD + fall, and the ratio of the  concentrations of NADH and NAD +  ([NADH]/[NAD + ]) increases. In the presence  of ethanol, which is very rapidly oxidized in  the liver, the [NADH]/[NAD + ] ratio is much  higher than it is in the normal fasting liver.  High levels of NADH drive the lactate dehy-  drogenase reaction toward lactate. There-  fore, lactate cannot enter the gluconeogenic  pathway, and pyruvate that is generated  from alanine is converted to lactate. Because  glycerol is oxidized by NAD + during its con-  version to DHAP, the conversion of glycerol  to glucose is also inhibited when NADH lev-  els are elevated. Consequently, the major  precursors lactate, alanine, and glycerol are  not used for gluconeogenesis under  conditions in which alcohol metabolism is  high.   In some species, propionate is a major source of carbon for gluconeogenesis.   Ruminants can produce massive amounts of glucose from propionate. In cows,  *r the cellulose in grass is converted to propionate by bacteria in the rumen. This  substrate is then used to generate more than 5 lb glucose each day by the process of glu-  coneogenesis.   Fig. 31.4. Metabolism of gluconeogenic precursors. A. Conversion of lactate to pyruvate. B. Conversion of alanine to pyruvate. In this reaction,  alanine aminotransferase transfers the amino group of alanine to a-ketoglutarate to form glutamate. The coenzyme for this reaction, pyridoxal  phosphate, accepts and donates the amino group. C. Conversion of glycerol to dihydroxyacetone phosphate.   to malate, which enters the cytosol  and is converted to oxaloacetate, which pro-  ceeds through gluconeogenesis to form glu-  cose. When excessive amounts of ethanol  are ingested, elevated NADH levels inhibit  the conversion of malate to oxaloacetate in  the cytosol. Therefore, carbons from amino  acids that form intermediates of the TCA  cycle cannot be converted to glucose as  readily.   Only the three carbons at the co-  A •) end of an odd chain fatty acid that  \ ^^ Jr form propionyl CoA are converted  to glucose. The remaining 16 carbons of a  fatty acid with 19 carbons form acetyl CoA,  which does not form any net glucose.   Gluconeogenesis occurs by a pathway that reverses many, but not all, of the steps  of glycolysis.   In glycolysis, PEP is converted to pyruvate by pyruvate kinase. In gluconeogenesis,  a series of steps are required to accomplish the reversal of this reaction (Fig. 31.5).  Pyruvate is carboxylated by pyruvate carboxylase to form oxaloacetate. This  enzyme, which requires biotin, is the catalyst of an anaplerotic (refilling) reaction  of the TCA cycle (see Chapter 20). In gluconeogenesis, this reaction replenishes the  oxaloacetate that is used for the synthesis of glucose (Fig. 31.6).   The C0 2 that was added to pyruvate to form oxaloacetate is released in the reac-  tion catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), which generates  PEP (Fig. 3 1.7 A). For this reaction, GTP provides a source of energy as well as the  phosphate group of PEP. Pyruvate carboxylase is found in mitochondria. In various  species, PEPCK is located either in the cytosol or in mitochondria, or it is distrib-  uted between these two compartments. In humans, the enzyme is distributed about  equally in each compartment.   Oxaloacetate, generated from pyruvate by pyruvate carboxylase or from amino  acids that form intermediates of the TCA cycle, does not readily cross the mito-  chondrial membrane. It is either decarboxylated to form PEP by the mitochondrial  PEPCK or it is converted to malate or aspartate (see Figs. 31.7B and 31.7C). The  conversion of oxaloacetate to malate requires NADH. PEP, malate, and aspartate  can be transported into the cytosol.   After malate or aspartate traverse the mitochondrial membrane (acting as carri-  ers of oxaloacetate) and enter the cytosol, they are reconverted to oxaloacetate by  reversal of the reactions given above (see Figs. 31.7B and C). The conversion of  malate to oxaloacetate generates NADH. Whether oxaloacetate is transported   OAA exits from the  mitochondrion either  as ©aspartate or (2) malate   Fig. 31.5. Conversion of pyruvate to phosphoenolpyruvate (PEP). Follow the shaded circled numbers on the diagram, starting with the precursors  alanine and lactate. The first step is the conversion of alanine and lactate to pyruvate. Pyruvate then enters the mitochondria and is converted to  OAA (circle 2) by pyruvate carboxylase. Pyruvate dehydrogenase has been inactivated by both the NADH and acetyl-CoA generated from fatty  acid oxidation, which allows oxaloacetate production for gluconeogenesis. The oxaloacetate formed in the mitochondria is converted to either  malate or aspartate to enter the cytoplasm via the malate/aspartate shuttle. Once in the cytoplasm the malate or aspartate is converted back into  oxaloacetate (circle 3), and phosphoenolpyruvate carboxykinase will convert it to PEP (circle 4). The white circled numbers are alternate routes  for exit of carbon from the mitochondrion using the malate/aspartate shuttle. OAA = oxaloacetate; FA = fatty acid; TG = triacylglycerol.   across the mitochondrial membrane as malate or aspartate depends on the need for  reducing equivalents in the cytosol. NADH is required to reduce 1,3-bisphospho-  glycerate to glyceraldehyde 3 -phosphate during gluconeogenesis.   Oxaloacetate, produced from malate or aspartate in the cytosol, is converted to  PEP by the cytosolic PEPCK (see Fig. 3 1.7 A).   The remaining steps of gluconeogenesis occur in the cytosol (Fig. 31.8). Starting  with PEP as a substrate, the steps of glycolysis are reversed to form glyceraldehyde  3 -phosphate. For every two molecules of glyceraldehyde 3 -phosphate that are  formed, one is converted to dihydroxy acetone phosphate (DHAP). These two triose   Fig. 31.7. The generation of PEP from gluconeogenic precursors. A. Conversion of oxaloac-  etate to phosphoenolpyruvate, using PEP carboxy kinase. B. Interconversion of oxaloacetate  and malate. C. Transamination of aspartate to form oxaloacetate. Note that the cytosolic reac-  tion is the reverse of the mitochondrial reaction as shown in Figure 31.5.   phosphates, DHAP and glyceraldehyde 3-phosphate, condense to form fructose 1,6-  bisphosphate by a reversal of the aldolase reaction.   Because glycerol forms DHAP, it enters the gluconeogenic pathway at this level.   The enzyme fructose 1 ,6-bisphosphatase releases inorganic phosphate from fruc-  tose 1 ,6-bisphosphate to form fructose 6-phosphate. This is not a reversal of the  PFK-1 reaction; ATP is not produced when the phosphate is removed from the 1  position of fructose 1 ,6-bisphosphate, because that is a low-energy phosphate bond.  Rather, inorganic phosphate is released in this hydrolysis reaction. In the next reac-  tion of gluconeogenesis, fructose 6-phosphate is converted to glucose 6-phosphate  by the same isomerase used in glycolysis (phosphoglucoisomerase).   Q o Al Martini had not eaten for 3 days,  I ) so he had no dietary source of glu-  ^ cose, and his liver glycogen stores  were essentially depleted. He was solely  dependent on gluconeogenesis to maintain  his blood glucose levels. One of the conse-  quences of ethanol ingestion and the subse-  quent rise in NADH levels is that the major  carbon sources for gluconeogenesis cannot  readily be converted to glucose. After his  alcoholic binges, Mr. Martini became hypo-  glycemic. His blood glucose was 28 mg/dL.   Glucose 6-phosphatase hydrolyzes Pi from glucose 6-phosphate, and free glucose is  released into the blood. As with fructose 1 ,6-bisphosphatase, this is not a reversal  of the glucokinase reaction, because the phosphate bond in glucose 6-phosphate is  a low-energy bond, and ATP is not generated at this step.   Glucose 6-phosphatase is located in the membrane of the endoplasmic reticulum.  It is used not only in gluconeogenesis, but also to produce blood glucose from the  breakdown of liver glycogen.   Although gluconeogenesis occurs during fasting, it is also stimulated during pro-  longed exercise, by a high-protein diet, and under conditions of stress. The factors  that promote the overall flow of carbon from pyruvate to glucose include the avail-  ability of substrate and changes in the activity or amount of certain key enzymes of  glycolysis and gluconeogenesis.   Phosphoenolpyruvate (PEP)  Fig. 31.8. Conversion of phosphoenolpyruvate and glycerol to glucose.   Gluconeogenesis is stimulated by the flow of its major substrates from peripheral  tissues to the liver. Glycerol is released from adipose tissue whenever the levels of  insulin are low and the levels of glucagon or the "stress" hormones, epinephrine and  Cortisol (a glucocorticoid), are elevated in the blood (see Chapter 26). Lactate is  produced by muscle during exercise and by red blood cells. Amino acids are  released from muscle whenever insulin is low or when Cortisol is elevated. Amino  acids are also available for gluconeogenesis when the dietary intake of protein is  high and intake of carbohydrate is low.   Fig. 31.9. Enzymes involved in regulating the substrate cycles of glycolysis and gluconeo-  genesis. Heavy arrows indicate the three substrate cycles. F-2,6-P, fructose 2,6-bisphosphate;  + , activated by; -, inhibited by; circled T, inducible enzyme.   PEPCK is an example of an  Kl ) inducible enzyme. The gene for the   cytosolic form of the enzyme con-  tains regulatory sequences in its  5'-flanking region. cAMP, which is produced  as a result of the binding of hormones such  as glucagon or epinephrine to receptors on  the cell surface, causes one of these  sequences to be activated. Glucocorticoids  affect a different regulatory sequence. cAMP  or glucocorticoids independently cause  increased transcription of the PEPCK gene.  The mRNA produced during transcription  travels to the cytosol combines with ribo-  somes and is translated, producing  increased amounts of the enzyme PEPCK.   These steps correspond to those in glycolysis that are catalyzed by regulatory  enzymes. The enzymes involved in these steps of gluconeogenesis differ from those  that catalyze the reverse reactions in glycolysis. The net flow of carbon, whether  from glucose to pyruvate (glycolysis) or from pyruvate to glucose (gluconeogene-  sis), depends on the relative activity or amount of these glycolytic or gluconeogenic  enzymes (Fig. 31.9 and Table 31.1).   Pyruvate, a key substrate for gluconeogenesis, is derived from lactate and amino  acids, particularly alanine. Pyruvate is not converted to acetyl CoA under conditions  favoring gluconeogenesis because pyruvate dehydrogenase is relatively inactive.  Instead, pyruvate is converted to oxaloacetate by pyruvate carboxylase. Subse-  quently, oxaloacetate is converted to PEP by PEPCK. Because of the activity state  of the enzymes discussed in subsequent sections, PEP reverses the steps of glycol-  ysis, ultimately forming glucose.   Pyruvate dehydrogenase is inactive. Under conditions of fasting, insulin levels  are low, and glucagon levels are elevated. Consequently, fatty acids and glycerol are   Table 31.1. Regulation of Enzymes of Glycolysis and Gluconeogenesis  in Liver   Inhibited by phosphorylation  (glucagon and epinephrine lead to an  increase in cAMP levels, which  activates protein kinase A)   Induced by glucagon, epinephrine,  glucocorticoids  Repressed by insulin  Inhibited by F-2,6-P, AMP  Induced during fasting  Induced during fasting   released from the triacylglycerol stores of adipose tissue. Fatty acids travel to the  liver, where they undergo p -oxidation, producing acetyl CoA, NADH, and ATP. As  a consequence, the concentration of ADP decreases. These changes result in the  phosphorylation of pyruvate dehydrogenase to the inactive form. Therefore, pyru-  vate is not converted to acetyl CoA.   Pyruvate carboxylase is active. Acetyl CoA, which is produced by oxidation of  fatty acids, activates pyruvate carboxylase. Therefore, pyruvate, derived from lac-  tate or alanine, is converted to oxaloacetate.   Phosphoenolpyruvate carboxykinase is induced. Oxaloacetate produces PEP  in a reaction catalyzed by PEPCK. Cytosolic PEPCK is an inducible enzyme, which  means that the quantity of the enzyme in the cell increases because of increased  transcription of its gene and increased translation of its mRNA. The major inducer  is cyclic adenosine monophosphate (cAMP), which is increased by hormones that  activate adenylate cyclase. Adenylate cyclase produces cAMP from ATP. Glucagon  is the hormone that causes cAMP to rise during fasting, whereas epinephrine acts  during exercise or stress. cAMP activates protein kinase A, which phosphorylates a  set of specific transcription factors (CREB) that stimulate transcription of the  PEPCK gene (see Chapter 16 and Fig. 16.18). Increased synthesis of mRNA for  PEPCK results in increased synthesis of the enzyme. Cortisol, the major human glu-  cocorticoid, also induces PEPCK.   Pyruvate kinase is inactive. When glucagon is elevated, pyruvate kinase is  phosphorylated and inactivated by a mechanism involving cAMP and protein kinase  A. Therefore, PEP is not reconverted to pyruvate. Rather, it continues along the  pathway of gluconeogenesis. If PEP were reconverted to pyruvate, these substrates  would simply cycle, causing a net loss of energy with no net generation of useful  products. The inactivation of pyruvate kinase prevents such futile cycling and pro-  motes the net synthesis of glucose.    The mechanism of action of steroid  hormones on glucose homeostasis  differs from that of glucagon or  epinephrine (see Chapters 16 and 26). Glu-  cocorticoids are steroid hormones that stim-  ulate gluconeogenesis, in part because they  induce the synthesis of PEPCK. Emma  Wheezer had elevated levels of blood glu-  cose because she was being treated with  large pharmacologic doses of dexametha-  sone, a potent synthetic glucocorticoid.   The carbons of PEP reverse the steps of glycolysis, forming fructose 1,6-bisphos-  phate. Fructose 1 ,6-bisphosphatase acts on this bisphosphate to release inorganic  phosphate and produce fructose 6-phosphate. A futile substrate cycle is prevented at   Fig. 31.10. Location and function of glucose  6-phosphatase. Glucose 6-phosphate travels on  a transporter (shaded oval) into the endoplas-  mic reticulum (ER), where it is hydrolyzed by  glucose 6-phosphatase (black oval) to glucose  and Pj. These products travel back to the  cytosol on transporters (shaded ovals).   this step because, under conditions that favor gluconeogenesis, the concentrations  of the compounds that activate the glycolytic enzyme PFK-1 are low. These same  compounds, fructose 2,6-bisphosphate (whose levels are regulated by insulin and  glucagon) and AMP, are allosteric inhibitors of fructose 1,6-bisphosphatase. When  the concentrations of these allosteric effectors are low, PFK-1 is less active, fructose  1 ,6-bisphosphatase is more active, and the net flow of carbon is toward fructose 6-  phosphate and, thus, toward glucose. The synthesis of fructose 1,6-bisphosphatase  is also induced during fasting.   Glucose 6-phosphatase catalyzes the conversion of glucose 6-phosphate to glucose,  which is released from the liver cell (Fig. 31.10). The glycolytic enzyme glucoki-  nase, which catalyzes the reverse reaction, is relatively inactive during gluconeoge-  nesis. Glucokinase, which has a high S 0 5 (K m ) for glucose (see Fig. 9.3), is not very  active during fasting because the blood glucose level is lower (approximately 5  mM) than the S 0 5 of the enzyme.   Glucokinase is also an inducible enzyme. The concentration of the enzyme  increases in the fed state, when blood glucose and insulin levels are elevated, and  decreases in the fasting state, when glucose and insulin are low.   During the gluconeogenic reactions, 6 moles of high-energy phosphate bonds are  cleaved. Two moles of pyruvate are required for the synthesis of 1 mole of glucose.  As 2 moles of pyruvate are carboxylated by pyruvate carboxylase, 2 moles of ATP  are hydrolyzed. PEPCK requires 2 moles of GTP (the equivalent of 2 moles of ATP)  to convert 2 moles of oxaloacetate to 2 moles of PEP. An additional 2 moles of ATP  are used when 2 moles of 3-phosphoglycerate are phosphorylated, forming 2 moles  of 1,3-bisphosphoglycerate. Energy in the form of reducing equivalents (NADH) is  also required for the conversion of 1,3-bisphosphoglycerate to glyceraldehyde 3-  phosphate. Under fasting conditions, the energy required for gluconeogenesis is  obtained from (3 -oxidation of fatty acids.   Fig. 31.11. Blood glucose concentrations at  various times after a meal.   The metabolic transitions that occur as a person eats a meal and progresses through  the various stages of fasting have been described in detail in the previous chapters.  This chapter summarizes the concepts presented in these previous chapters.  Because a thorough understanding of these concepts is so critical to medicine, a  summary is not only warranted but essential.   After a high-carbohydrate meal, blood glucose rises from a fasting level of  approximately 80 to 100 mg/dL (~5 mM) to a level of approximately 120 to 140  mg/dL (8 mM) within a period of 30 minutes to 1 hour (Fig. 31.11). The concen-  tration of glucose in the blood then begins to decrease, returning to the fasting range  by approximately 2 hours after the meal (see also Chapter 26).   Blood glucose levels increase as dietary glucose is digested and absorbed. The  values go no higher than approximately 140 mg/dL in a normal, healthy person  because tissues take up glucose from the blood, storing it for subsequent use and  oxidizing it for energy. After the meal is digested and absorbed, blood glucose lev-  els decline because cells continue to metabolize glucose.   If blood glucose levels continued to rise after a meal, the high concentration of  glucose would cause the release of water from tissues as a result of the osmotic  effect of glucose. Tissues would become dehydrated, and their function would be  affected. A hyperosmolar coma could result from dehydration of the brain.   Conversely, if blood glucose levels continued to drop after a meal, tissues that  depend on glucose would suffer from a lack of energy. If blood glucose levels  dropped abruptly, the brain would not be able to produce an adequate amount of  ATP. Light-headedness and dizziness would result, followed by drowsiness and,  eventually, coma. Red blood cells would not be able to produce enough ATP to  maintain the integrity of their membranes. Hemolysis of these cells would decrease  the transport of oxygen to the tissues of the body. Eventually, all tissues that rely on  oxygen for energy production would fail to perform their normal functions. If the  problem were severe enough, death could result.   Devastating consequences of glucose excess or insufficiency are normally  avoided because the body is able to regulate its blood glucose levels. As the con-  centration of blood glucose approaches the normal fasting range of 80 to 100 mg/dL  roughly 2 hours after a meal, the process of glycogenolysis is activated in the liver.  Liver glycogen is the primary source of blood glucose during the first few hours of  fasting. Subsequently, gluconeogenesis begins to play a role as an additional source  of blood glucose. The carbon for gluconeogenesis, a process that occurs in the liver,  is supplied by other tissues. Exercising muscle and red blood cells provide lactate  through glycolysis; muscle also provides amino acids by degradation of protein; and  glycerol is released from adipose tissue as triacylglycerol stores are mobilized.   Even during a prolonged fast, blood glucose levels do not decrease dramatically.  After 5 to 6 weeks of starvation, blood glucose levels decrease to only approxi-  mately 65 mg/dL (Table 31.2).   Source of data: Ruderman NB, Aoki TT, Cahill GF Jr.  Gluconeogenesis and its disorders in man. In: Han-  son RW, Mehlman MA, eds. Gluconeogenesis: Its  Regulation in Mammalian Species. New York, John  Wiley. 1976:517.   The major factors involved in regulating blood glucose levels are the blood glucose  concentration itself and hormones, particularly insulin and glucagon.   As blood glucose levels rise after a meal, the increased glucose concentration  stimulates the p cells of the pancreas to release insulin (Fig. 31.12). Certain amino  acids, particularly arginine and leucine, also stimulate insulin release from the pan-  creas.   Blood levels of glucagon, which is secreted by the a cells of the pancreas, may  increase or decrease, depending on the content of the meal. Glucagon levels decrease  in response to a high-carbohydrate meal, but they increase in response to a high-pro-  tein meal. After a typical mixed meal containing carbohydrate, protein, and fat,  glucagon levels remain relatively constant, whereas insulin levels increase (Fig. 31.13).   When Di Abietes inadvertently injected an excessive amount of insulin, she caused an acute reduction in her blood glucose levels  4 to 5 hours later while she was asleep. Had she been awake, she would have first experienced symptoms caused by a hypo-  glycemia-induced hyperactivity of her sympathetic nervous system (e.g., sweating, tremulousness, palpitations). Eventually, as her  hypoglycemia became more profound, she would have experienced symptoms of "neuroglycopenia" (inadequate glucose supply to the  brain), such as confusion, speech disturbances, emotional instability, possible seizure activity, and, finally, coma. While sleeping, she had  reached this neuroglycopenic stage of hypoglycemia and could not be aroused at 6:00 am.   Ann O' Rexia, whose intake of glucose and of glucose precursors has been severely restricted, has not developed any of these manifes-  tations. Her lack of hypoglycemic symptoms can be explained by the very gradual reduction of her blood glucose levels as a consequence of  near starvation and her ability to maintain blood glucose levels within an acceptable fasting range through hepatic gluconeogenesis. In addi-  tion, lipolysis of adipose triacylglycerols produces fatty acids, which are used as fuel and converted to ketone bodies by the liver. The oxida-  tion of fatty acids and ketone bodies by the brain and muscle reduces the need for blood glucose.   In Di Abiete's case, the excessive dose of insulin inhibited lipolysis and ketone body synthesis, so these alternative fuels were not available  to spare blood glucose. The rapidity with which hypoglycemia was induced could not be compensated for quickly enough by hepatic gluco-  neogenesis, which was inhibited by the insulin, and hypoglycemia ensued.   A stat finger stick revealed that Di's capillary blood glucose level was less than 20 mg/dL. An intravenous infusion of a 50% solution of  glucose was started, and her blood glucose level was determined frequently. When Di regained consciousness, the intravenous solution  was eventually changed to 10% glucose. After 6 hours, her blood glucose levels stayed in the upper normal range, and she was able to tol-  erate oral feedings. She was transferred to the metabolic unit for overnight monitoring. By the next morning, her previous diabetes treat-  ment regimen was reestablished. The reasons that she had developed hypoglycemic coma were explained to Di, and she was discharged  to the care of her family doctor.   After a meal, the liver oxidizes glucose to meet its immediate energy needs. Any  excess glucose is converted to stored fuels. Glycogen is synthesized and stored in  the liver, and glucose is converted to fatty acids and to the glycerol moiety that  reacts with the fatty acids to produce triacylglycerols. These triacylglycerols are  packaged in very-low-density lipoproteins (VLDL) and transported to adipose tis-  sue, where the fatty acids are stored in adipose triacylglycerols.   Regulatory mechanisms control the conversion of glucose to stored fuels. As  the concentration of glucose increases in the hepatic portal vein, the concentration  of glucose in the liver may increase from the fasting level of 80 to 100 mg/dL (~5  mM) to a concentration of 180 to 360 mg/dL (10-20 mM). Consequently, the  velocity of the glucokinase reaction increases because this enzyme has a high S 0 5  (K m ) for glucose (Fig. 31.14). Glucokinase is also induced by a high-carbohydrate  diet; the quantity of the enzyme increases in response to elevated insulin levels.   Insulin promotes the storage of glucose as glycogen by countering the effects of  glucagon-stimulated phosphorylation. The response to insulin activates the phos-  phatases that dephosphorylate glycogen synthase (which leads to glycogen synthase  activation) and glycogen phosphorylase (which leads to inhibition of the enzyme)  (Fig. 3 1.1 5 A). Insulin also promotes the synthesis of the triacylglycerols that are  released from the liver into the blood as VLDL. The regulatory mechanisms for this  process are described in Chapter 33.   Almost every cell in the body oxidizes glucose for energy. Certain critical tissues,  particularly the brain, other nervous tissue, and red blood cells, especially depend  on glucose for their energy supply. The brain requires approximately 150 g glucose  per day. In addition, approximately 40 g/day glucose is required by other glucose-  dependent tissues. Furthermore, all tissues require glucose for the pentose phos-  phate pathway, and many tissues use glucose for synthesis of glycoproteins and  other carbohydrate-containing compounds.    Fig. 31.13. Blood glucose, insulin, and glucagon levels over a 24-hour period in a normal per-  son eating mixed meals. Reprinted with permission from Tasaka Y, Sekine M, Wakatsuki M,  et al. Horm Metab Res 1975;7:206. Copyright © Thieme Medical Publishers, Inc.   Insulin stimulates the transport of glucose into adipose and muscle cells by  promoting the recruitment of glucose transporters to the cell membrane (see  Fig. 31.15C). Other tissues, such as the liver, brain, and red blood cells, have a  different type of glucose transporter that is not as significantly affected by  insulin.   In muscle, glycogen is synthesized after a meal by a mechanism similar to that  in the liver (see Fig. 31.15B). Metabolic differences exist between these tissues (see  Chapter 28), but, in essence, insulin stimulates glycogen synthesis in resting mus-  cle as it does in the liver. A key difference between muscle and liver is that insulin  greatly stimulates the transport of glucose into muscle cells but only slightly stim-  ulates its transport into liver cells.   After a meal has been digested and absorbed, blood glucose levels reach a peak and  then begin to decline. The uptake of dietary glucose by cells, particularly those in  the liver, muscle, and adipose tissue, lowers blood glucose levels. By 2 hours after  a meal, blood glucose levels return to the normal fasting level of less than  140 mg/dL.   During fasting, as blood glucose levels decrease, insulin levels decrease, and  glucagon levels rise. These hormonal changes cause the liver to degrade glycogen  by the process of glycogenolysis and to produce glucose by the process of gluco-  neogenesis so that blood glucose levels are maintained.   Within a few hours after a high-carbohydrate meal, glucagon levels begin to rise.  Glucagon binds to cell surface receptors and activates adenylate cyclase, causing  cAMP levels in liver cells to rise (Fig. 31.16). cAMP activates protein kinase A,  which phosphorylates and inactivates glycogen synthase. Therefore, glycogen  synthesis decreases.   At the same time, protein kinase A stimulates glycogen degradation by a two-  step mechanism. Protein kinase A phosphorylates and activates phosphorylase  kinase. This enzyme, in turn, phosphorylates and activates glycogen phosphory-  lase.    Fig. 31.15. Glucose metabolism in various tis-  sues. A. Effect of insulin on glycogen synthesis  and degradation and on VLDL synthesis in the  liver. B. Glucose metabolism in resting muscle  in the fed state. The transport of glucose into  cells and the synthesis of glycogen are stimu-  lated by insulin. C. Glucose metabolism in adi-  pose tissue in the fed state. FA = fatty acids;  DHAP = dihydroxy acetone phosphate. FA =  fatty acids; TG = triacylglycerols; + = stimu-  lated by insulin; — = inhibited by insulin.   Fig. 31.16. Regulation of glycogenolysis in the liver by glucagon. 1. Glucagon binding to its  receptor leads to the activation of adenylate cyclase, which leads to an increase in cAMP  levels. 2. cAMP binds to the regulatory subunits of protein kinase A, thereby activating the  catalytic subunit. 3. Active protein kinase A phosphorylates and activates phosphorylase  kinase, while simultaneously inactivating glycogen synthase (4). 5. Active phosphorylase  kinase converts glycogen phosphorylase b to glycogen phosphorylase a. 6. Phosphorylase a  degrades glycogen, producing glucose 1 -phosphate, which is converted to glucose 6-phos-  phate, then glucose, for export into the blood.   Q o The pathophysiology leading to an  ( /~oA ) elevation of blood glucose after a  ™ meal differs between patients with  type 1 diabetes mellitus and those with type  2 diabetes mellitus. Di Abietes, who has type  1 disease, cannot secrete insulin adequately  in response to a meal because of a defect in  the p cells of her pancreas.   Ann Sulin, however, has type 2 disease.  In this form of the disorder, the cause of glu-  cose intolerance is more complex, involving  at least a delay in the release of relatively  appropriate amounts of insulin after a meal  combined with a degree of resistance to the  actions of insulin in skeletal muscle and  adipocytes. Excessive hepatic gluconeogen-  esis occurs even though blood glucose lev-  els are elevated.   Glycogen phosphorylase catalyzes the phosphorolysis of glycogen, producing  glucose 1 -phosphate, which is converted to glucose 6-phosphate. Dephosphoryla-  tion of glucose 6-phosphate by glucose 6-phosphatase produces free glucose, which  then enters the blood.   By 4 hours after a meal, the liver is supplying glucose to the blood not only by the  process of glycogenolysis but also by the process of gluconeogenesis. Hormonal  changes cause peripheral tissues to release precursors that provide carbon for glu-  coneogenesis, specifically lactate, amino acids, and glycerol.   Regulatory mechanisms promote the conversion of gluconeogenic precursors to  glucose (Fig. 31.17). These mechanisms prevent the occurrence of potential futile  cycles, which would continuously convert substrates to products while consuming  energy but producing no useful result.   These regulatory mechanisms inactivate the glycolytic enzymes pyruvate kinase,  phosphofructokinase- 1 (PFK-1), and glucokinase during fasting and promote the  flow of carbon to glucose via gluconeogenesis. These mechanisms operate at the  three steps where glycolysis and gluconeogenesis differ:   1. Pyruvate (derived from lactate and alanine) is converted by the gluconeogenic  pathway to phosphoenolpyruvate (PEP). PEP is not reconverted to pyruvate (a  potential futile cycle) because glucagon-stimulated phosphorylation inactivates  pyruvate kinase. Therefore, PEP reverses the steps of glycolysis and forms fruc-  tose 1,6-bisphosphate.   2. Fructose 1,6-bisphosphate is converted to fructose 6-phosphate by a bisphos-  phatase. Because the glycolytic enzyme PFK-1 is relatively inactive mainly as a  result of low fructose 2,6-bisphosphate levels, fructose 6-phosphate is not con-  verted back to fructose 1,6-bisphosphate, and a second potential futile cycle is  avoided. The low fructose 2,6-bisphosphate levels are attributable in part to the  phosphorylation of phosphofructokinase-2 by protein kinase A, which has been  activated in response to glucagon. Fructose 6-phosphate is converted to glucose  6-phosphate.   3. Glucose 6-phosphate is dephosphorylated by glucose 6-phosphatase, forming  free glucose. Because glucokinase has a high S 0 5 (K m ) for glucose, and glucose  concentrations are relatively low in liver cells during fasting, glucose is released  into the blood. Therefore, the third potential futile cycle does not occur.   Enzymes that participate in gluconeogenesis, but not in glycolysis, are active  under fasting conditions. Pyruvate carboxylase is activated by acetyl Co A,  derived from oxidation of fatty acids. Phosphoenolpyruvate carboxykinase, fruc-  tose 1 ,6-bisphosphatase, and glucose 6-phosphatase are induced; that is, the  quantity of the enzymes increases. Fructose 1 ,6-bisphosphatase is also active  because levels of fructose 2,6-bisphosphate, an inhibitor of the enzyme, are low.   The hormonal changes that occur during fasting stimulate the breakdown of adipose  triacylglycerols (see Chapters 3, 33, and 43). Consequently, fatty acids and glycerol  are released into the blood (Fig. 31.18). Glycerol serves as a source of carbon for glu-  coneogenesis. Fatty acids become the major fuel of the body and are oxidized to C0 2  and H 2 0 by various tissues, which enables these tissues to decrease their utilization of  glucose. Fatty acids are also oxidized to acetyl CoA in the liver to provide energy for  gluconeogenesis. In a prolonged fast, acetyl CoA is converted to ketone bodies, which  enter the blood and serve as an additional fuel source for the muscle and the brain.   Fig. 31.17. Regulation of gluconeogenesis in the  liver. GK = glucokinase; G-6-Pase = glucose 6-  phosphatase; PK = pyruvate kinase; OAA =  oxaloacetate; PEPCK = phosphoenolpyruvate  carboxykinase; F-1,6-Pase = fructose 1,6-bis-  phosphatase; F-2,6-P = fructose 2,6-bisphos-  phate; PFK-1 = phosphofructokinase- 1.   During prolonged fasting, a number of changes in fuel utilization occur. These  changes cause tissues to use less glucose than they use during a brief fast and to use  predominantly fuels derived from adipose triacylglycerols (i.e., fatty acids and their  derivatives, the ketone bodies). Therefore, blood glucose levels do not decrease  drastically. In fact, even after 5 to 6 weeks of starvation, blood glucose levels are  still in the range of 65 mg/dL (Fig. 31.19, see Table 31.2).   The major change that occurs in starvation is a dramatic elevation of blood  ketone body levels after 3 to 5 days of fasting (see Fig. 31.19). At these levels, the  brain and other nervous tissues begin to use ketone bodies and, consequently, they  oxidize less glucose, requiring roughly one third as much glucose (approximately  40 g/day) as under normal dietary conditions. As a result of reduced glucose uti-  lization, the rate of gluconeogenesis in the liver decreases, as does the production  of urea (see Fig. 31.19). Because in this stage of starvation amino acids, obtained    Fig. 31.18. Tissue interrelationships during fasting. 1. Blood glucose levels drop, decreasing insulin and raising blood glucagon levels. 2.  Glycogenolysis is induced in the liver to raise blood glucose levels. 3. The brain uses the glucose released by the liver, as do the red blood cells  (4). 5. Adipose tissues are induced to release free fatty acids and glycerol from stored triglycerides. 6. The muscle and liver use fatty acids for  energy. 7. The liver converts fatty acid derived acetyl-CoA to ketone bodies for export, which the muscles (8) and brain can use for energy. 9.  Protein turnover is induced in muscle, and amino acids leave the muscle and travel to the liver for use as gluconeogenic precursors. 10. The high  rate of amino acid metabolism in the liver generates urea, which travels to the kidney for excretion. 11. Red blood cells produce lactate, which  returns to the liver as a substriate for gluconeogenesis. 12. The glycenol released from adipose tissue is used by the liver set gluconeogenesis.  KB = ketone bodies; FA = fatty acids; AA = amino acids; TG = triacylglycerols.   from the degradation of existing proteins, are the major gluconeogenic precursor,  reducing glucose requirements in tissues reduces the rate of protein degradation,  and, hence, the rate of urea formation. Protein from muscle and other tissues is  therefore spared, because there is less need for amino acids for gluconeogenesis.   Body protein, particularly muscle protein, is not primarily a storage form of fuel  in the same sense as glycogen or triacylglycerol; proteins have many functions  beside fuel storage. For example, proteins function as enzymes, as structural pro-  teins, and in muscle contraction. If tissue protein is degraded to too great an extent,  body function can be severely compromised. If starvation continues and no other  problems, such as infections, occur, a starving individual usually dies because of  severe protein loss that causes malfunction of major organs, such as the heart.  Therefore, the increase in ketone body levels that results in the sparing of body  protein allows individuals to survive for extended periods without ingesting food.   Immediately after a meal, dietary carbohydrates serve as the major source of  blood glucose (Fig. 31.20). As blood glucose levels return to the fasting range   Fig. 31.19. Changes in blood fuels during fasting. The units for fatty acids, glucose, and ketone bodies are millimolar (on left) and for urinary  nitrogen and ammonia are grams/day (on right). Modified from Linder MC. Nutritional Biochemistry and Metabolism, 2nd Ed. Stamford, CT:  Appleton & Lange, 1991:103. © 1991 Appleton & Lange.   within 2 hours after a meal, glycogenolysis is stimulated and begins to sup-  ply glucose to the blood. Subsequently, glucose is also produced by gluconeoge-  nesis.   During a 12-hour fast, glycogenolysis is the major source of blood glucose.  Thus, it is the major pathway by which glucose is produced in the basal state  (after a 12-hour fast). However, by approximately 16 hours of fasting,  glycogenolysis and gluconeogenesis contribute equally to the maintenance of  blood glucose.   By 30 hours after a meal, liver glycogen stores are substantially depleted. Sub-  sequently, gluconeogenesis is the primary source of blood glucose.   The mechanisms that cause fats to be used as the major fuel and that allow blood  glucose levels to be maintained during periods of food deprivation result in the  conservation of body protein and, consequently, permit survival during prolonged  fasting for periods often exceeding 1 or more months.   During exercise, mechanisms very similar to those that are used during fasting oper-  ate to maintain blood glucose levels. The liver maintains blood glucose levels  through both glucagon- and epinephrine-induced glycogenolysis and gluconeogen-  esis. The use of fuels by muscle during exercise, including the uptake and use of  blood glucose, is discussed in Chapter 47.   Otto Shape is able to jog for 45 minutes before eating breakfast without devel-  oping symptoms of hypoglycemia in spite of enhanced glucose utilization by  skeletal muscle during exercise. He maintains his blood glucose level in an ade-  quate range through hepatic glycogenolysis and gluconeogenesis.   Fig. 31.20. Sources of blood glucose in fed,  fasting, and starved states. Note that the scale  changes from hours to days. From Ruderman  NB, et al. In: Hanson RW, Mehlman MA, eds.  Gluconeogenesis: Its Regulation in Mammalian  Species. 1976:518. © 1976 John Wiley & Sons.   Remember that muscle glycogen is  Pi) not used to maintain blood glucose   levels; muscle cells lack glucose 6-  phosphatase, so glucose cannot be pro-  duced from glucose 6-phosphate for export.   Epidemiologic studies have corre-  lated the results of the OGTT with  the current and subsequent devel-  opment of the microvascular complications  of DM in the eye (diabetic retinopathy) and  in the kidney (diabetic nephropathy). Their  development is usually limited to those  patients whose fasting blood glucose level  exceeds 140 mg/dL (7.8 mM) or whose 2-  hour postprandial blood glucose level  exceeds 200 mg/dL (11.1 mM).   As a consequence, the success of therapy  with diet alone or with diet plus oral hypo-  glycemic agents is often determined by  measuring blood glucose levels during fast-  ing, before breakfast, and 2 hours after a  meal. For patients taking an intermediate-  acting insulin, blood glucose also may be  measured at other times, such as when  insulin action is expected to peak after its  injection (e.g., around 5:00 pm, when inter-  mediate-acting insulin has been given at  7:00 am the same day).   The chronic excessive ingestion of ethanol concurrent with a recent reduc-  tion in nutrient intake caused Al Martini's blood glucose level to decrease  to 28 mg/dL. This degree of hypoglycemia caused the release of a number  of "counterregulatory" hormones into the blood, including glucagon, growth hor-  mone, Cortisol, and epinephrine (adrenaline).   Some of the patient's signs and symptoms are primarily the result of an increase  in adrenergic nervous system activity after a rapid decrease in blood glucose. The  subsequent increase in epinephrine levels in the blood leads to tremulousness,  excessive sweating, and rapid heart rate. Other manifestations arise when the brain  has insufficient glucose, hence the term "neuroglycopenic symptoms." Mr. Martini  was confused, combative, had slurred speech, and eventually had a grand mal  seizure. If not treated quickly by intravenous glucose administration, Mr. Martini  may have lapsed into a coma. Permanent neurologic deficits and even death may  result if severe hypoglycemia is not corrected in 6 to 10 hours.   The elevation in blood glucose that occurred in Emma Wheezer's case  was primarily a consequence of the large pharmacologic doses of a gluco-  corticoid that she received in an effort to reduce the intrabronchial inflam-  matory reaction characteristic of asthmatic bronchospasm. Although the develop-  ment of hyperglycemia in this case could be classified as a "secondary" form of  diabetes mellitus, most patients treated with glucocorticoids do not develop glucose  intolerance. Ms. Wheezer, therefore, may have a predisposition to the eventual  development of "primary" diabetes mellitus.   In hyperglycemia, increased amounts of glucose enter the urine, causing large  amounts of water to be excreted. This "osmotic diuresis" is responsible for the  increased volume of urine (polyuria) noted by the patient. Because of increased  urinary water loss, the effective circulating blood volume is reduced. Therefore,  less blood reaches volume- sensitive receptors in the central nervous system,  which then trigger the sensation of thirst, causing increased drinking activity  (polydipsia).   A diabetic diet and the tapering of her steroid dose over a period of several weeks  gradually returned Ms. Wheezer's blood glucose level into the normal range.   a Chronically elevated levels of glucose in the blood may contribute to the  development of the microvascular complications of diabetes mellitus, such  — as diabetic retinal damage, kidney damage, and nerve damage, as well as  macrovascular complications such as cerebrovascular, peripheral vascular, and  coronary vascular insufficiency. The precise mechanism by which long-term hyper-  glycemia induces these vascular changes is not fully established.   One postulated mechanism proposes that nonenzymatic glycation (glycosyla-  tion) of proteins in vascular tissue alters the structure and functions of these pro-  teins. A protein exposed to chronically increased levels of glucose will covalently  bind glucose, a process called glycation or glycosylation. This process is not regu-  lated by enzymes (see Chapter 9). These nonenzymatically glycated proteins slowly  form cross-linked protein adducts (often called advanced glycosylation products)  within the micro vasculature and macrovasculature.   By cross-linking vascular matrix proteins and plasma proteins, chronic hyper-  glycemia may cause narrowing of the luminal diameter of the micro vessels in the  retina (causing diabetic retinopathy), the renal glomeruli (causing diabetic  nephropathy), and the microvessels supplying peripheral and autonomic nerve  fibers (causing diabetic neuropathy). The same process has been postulated to  accelerate atherosclerotic change in the macrovasculature, particularly in the brain    (causing strokes), the coronary arteries (causing heart attacks), and the peripheral  arteries (causing peripheral arterial insufficiency and gangrene). The abnormal lipid  metabolism associated with poorly controlled diabetes mellitus also may contribute  to the accelerated atherosclerosis associated with this metabolic disorder (see  Chapters 33 and 34).   Until recently, it was argued that meticulous control of blood glucose levels in  a diabetic patient would not necessarily prevent or even slow these complications  of chronic hyperglycemia. The publication of the Diabetes Control and Compli-  cations Trial, however, suggests that maintaining long-term euglycemia (normal  blood glucose levels) in diabetic patients slows the progress of unregulated gly-  cation of proteins as well as corrects their dyslipidemia. In this way, careful con-  trol may favorably affect the course of the microvascular and macrovascular com-  plications of diabetes mellitus in patients such as Di Abietes and Ann Sulin.   Plants are the ultimate source of the earth's supply of glucose. Plants pro-  duce glucose from atmospheric C0 2 by the process of photosynthesis  (Fig. 3 1.21 A). In contrast to plants, humans cannot synthesize glucose by  the fixation of C0 2 . Although we have a process called gluconeogenesis, the term  may really be a misnomer. Glucose is not generated anew by gluconeogenesis; com-  pounds produced from glucose are simply recycled to glucose. We obtain glucose  from the plants, including bacteria, that we eat and, to some extent, from animals in  our food supply. We use this glucose both as a fuel and as a source of carbon for the  synthesis of fatty acids, amino acids, and other sugars (see Fig. 31.21B). We store  glucose as glycogen, which, along with gluconeogenesis, provides glucose when  needed for energy (see Fig. 31.21C).   Lactate, one of the carbon sources for gluconeogenesis, is actually produced  from glucose by tissues that obtain energy by oxidizing glucose to pyruvate through  glycolysis. The pyruvate is then reduced to lactate, released into the bloodstream,  and reconverted to glucose by the process of gluconeogenesis in the liver. This  process is known as the Cori cycle (Fig. 3 1.2 ID).   Carbons of alanine, another carbon source for gluconeogenesis, may be pro-  duced from glucose. In muscle, glucose is converted via glycolysis to pyruvate and  transaminated to alanine. Alanine from muscle is recycled to glucose in the liver.  This process is known as the glucose-alanine cycle (Fig. 31.21E). Glucose also may  be used to produce nonessential amino acids other than alanine, which are subse-  quently reconverted to glucose in the liver by gluconeogenesis. Even the essential  amino acids that we obtain from dietary proteins are synthesized in plants and bac-  teria using glucose as the major source of carbon. Therefore, all amino acids that  are converted to glucose in humans, including the essential amino acids, were orig-  inally synthesized from glucose.   The production of glucose from glycerol, the third major source of carbon for  gluconeogenesis, is also a recycling process. Glycerol is derived from glucose via  the dihydroxy acetone phosphate intermediate of glycolysis. Fatty acids are then  esterified to the glycerol and stored as triacylglycerol. When these fatty acids are  released from the triacylglycerol, the glycerol moiety can travel to the liver and be  reconverted to glucose (see Fig. 31.21F).   Dimitriadis GD, Raptis SA, Newsholme EA. Integration of some biochemical and physiologic effects of  insulin that may play a role in the control of blood glucose concentration. In LeRoith D, Taylor SI,   Olefsky JM, eds. Diabetes Mellitus: A Fundamental and Clinical Text. 2nd Ed. New York: Lippin-   cott, Williams and Wilkins, 2000:161-176.  Granner D, Pilkis S. The genes of hepatic glucose metabolism. J Biol Chem 1990;265:10173-10176.  Gurney AL, Park EA, Liu J, et al. Metabolic regulation of gene transcription. J Nutr   Pilkis S, Granner D. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.  Annu Rev Physiol 1992;54:885-909.   The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of dia-  betes on the development and progression of long-term complications in insulin-dependent diabetes  mellitus. N Engl J Med 1993;329:977-986.   1 . A common intermediate in the conversion of glycerol and lactate to glucose is which of the following?   2. A patient presented with a bacterial infection that produced an endotoxin that inhibits phosphoenolpyruvate carboxykinase. In   this patient, then, under these conditions, glucose production from which of the following precursors would be inhibited?   (D) Its concentration in the blood increases after a high-carbohydrate meal.   (E) Its concentration increases in the blood when insulin levels increase.   4. Which of the following is most likely to occur in a normal individual after ingesting a high-carbohydrate meal?   5. A patient arrives at the hospital in an ambulance. She is currently in a coma. Before lapsing into the coma, her symptoms  included vomiting, dehydration, low blood pressure, and a rapid heartbeat. She also had relatively rapid respirations, result-  ing in more carbon dioxide exhaled. These symptoms are consistent with which of the following conditions?   Most of the lipids found in the body fall into the categories of fatty  acids and triacylglycerols; glycerophospholipids and sphin-  golipids; eicosanoids; cholesterol, bile salts, and steroid hor-  mones; and fat- soluble vitamins. These lipids have very diverse  chemical structures and functions. However, they are related by a  common property: their relative insolublity in water.  Fatty acids, which are stored as triacylglycerols, serve as fuels, providing the  body with its major source of energy (Fig. VI. 1). Glycerophospholipids and sphin-  golipids, which contain esterified fatty acids, are found in membranes and in blood  lipoproteins at the interfaces between the lipid components of these structures and  the surrounding water. These membrane lipids form hydrophobic barriers between  subcellular compartments and between cellular constituents and the extracellular  milieu. Polyunsaturated fatty acids containing 20 carbons form the eicosanoids,  which regulate many cellular processes (Fig. VI. 2).   Cholesterol adds stability to the phospholipid bilayer of membranes. It serves as  the precursor of the bile salts, detergent-like compounds that function in the process  of lipid digestion and absorption (Fig. VI. 3). Cholesterol also serves as the precur-  sor of the steroid hormones, which have many actions, including the regulation of  metabolism, growth, and reproduction.   The fat- soluble vitamins are lipids that are involved in such varied functions as  vision, growth, and differentiation (vitamin A), blood clotting (vitamin K), preven-  tion of oxidative damage to cells (vitamin E), and calcium metabolism (vitamin D).   Triacylglycerols, the major dietary lipids, are digested in the lumen of the intes-  tine (Fig. VI.4). The initial digestive products, free fatty acids and 2-monoacylglyc-  erol, are reconverted to triacylglycerols in intestinal epithelial cells, packaged in  lipoproteins known as chylomicrons (so they can safely enter the circulation), and  secreted into the lymph. Ultimately, chylomicrons enter the blood, serving as one of  the major blood lipoproteins.   Very low density lipoprotein (VLDL) is produced in the liver, mainly from  dietary carbohydrate. Lipogenesis is an insulin- stimulated process through which  glucose is converted to fatty acids, which are subsequently esterified to glycerol to  form the triacylglycerols that are packaged in VLDL and secreted from the liver.  Thus, chylomicrons primarily transport dietary lipids, and VLDL transports  endogenously synthesized lipids.   The triacylglycerols of chylomicrons and VLDL are digested by lipoprotein  lipase (LPL), an enzyme found attached to capillary endothelial cells (see Fig.  VI.4). The fatty acids that are released are taken up by muscle and many other tis-  sues and oxidized to C0 2 and water to produce energy (see Chapter 23). After a  meal, these fatty acids are taken up by adipose tissue and stored as triacylglycerols.   LPL converts chylomicrons to chylomicron remnants and VLDL to intermediate  density lipoprotein (IDL). These products, which have a relatively low triacylglyc-  erol content, are taken up by the liver by the process of endocytosis and degraded  by lysosomal action. IDL may also be converted to low density lipoprotein (LDL)  by further digestion of triacylglycerol. Endocytosis of LDL occurs in peripheral tis-  sues as well as the liver (Table VI. 1), and is the major means of cholesterol trans-  port and delivery to peripheral tissues.    Fig. VI.4. Overview of triacylglycerol metabolism in the fed state. TG = triacylglyc-  erol; 2-MG = 2-monoacylglycerol; FA = fatty acid; circled TG = triacylglycerols of  VLDL and chylomicrons; LPL = lipoprotein lipase.   The principal function of high density lipoprotein (HDL) is to transport excess  cholesterol obtained from peripheral tissues to the liver and to exchange proteins  and lipids with chylomicrons and VLDL. The protein exchange converts "nascent"  particles to "mature" particles.   During fasting, fatty acids and glycerol are released from adipose triacylglycerol  stores (Fig. VI. 5). The glycerol travels to the liver and is used for gluconeogenesis.  Only the liver contains glycerol kinase, which is required for glycerol metabolism.   • endocytosed by liver or converted to LDL  LDL (low density lipoprotein)   • endocytosed by liver and peripheral tissues  HDL (high density lipoprotein)   • functions in the return of cholesterol from peripheral tissues to the liver   The fatty acids form complexes with albumin in the blood and are taken up by  muscle, kidney, and other tissues, where ATP is generated by their oxidation to  CO 2 and water. Liver also converts some of the carbon to ketone bodies, which are  released into the blood. Ketone bodies are oxidized for energy in muscle, kidney,  and other tissues during fasting, and in the brain during prolonged starvation (see  Chapter 23).   Triacylglycerols are the major fat in the human diet, consisting of three fatty  acids esterified to a glycerol backbone. Limited digestion of these lipids occurs in  the mouth (lingual lipase) and stomach (gastric lipase) because of the low solu-  bility of the substrate. In the intestine, however, the fats are emulsified by bile  salts that are released from the gallbladder. This increases the available surface  area of the lipids for pancreatic lipase and colipase to bind and to digest the  triglycerides. Degradation products are free fatty acids and 2-monoacylglycerol.  When partially digested food enters the intestine, the hormone cholecystokinin is  secreted by the intestine, which signals the gallbladder to contract and release  bile acids, and the pancreas to release digestive enzymes.   In addition to triacylglycerols, phospholipids, cholesterol,, and cholesterol  esters (cholesterol esterified to fatty acids) are present in the foods we eat. Phos-  pholipids are hydrolyzed in the intestinal lumen by phospholipase A2, and choles-  terol esters are hydrolyzed by cholesterol esterase. Both of these enzymes are  secreted from the pancreas.   The products of enzymatic digestion (free fatty acids, glycerol, lysophospho-  lipids, cholesterol) form micelles with bile acids in the intestinal lumen. The  micelles interact with the enterocyte membrane and allow diffusion of the lipid  soluble components across the enterocyte membrane into the cell. The bile acids,  however, do not enter the enterocyte at this time. They remain in the intestine,  travel further down, and are then reabsorbed and sent back to the liver by the  enterohepatic circulation. This allows the bile salts to be used multiple times in  fat digestion.   The intestinal epithelial cells will resynthesize triacyl glycerol from free fatty  acids and 2 -monacy I glycerol and will package them with a protein, apolipopro-  tein B-48, phospholipids, and cholesterol esters into a soluble lipoprotein particle  known as a chylomicron. The chylomicrons are secreted into the lymph and even-  tually end up in the circulation, where they can distribute dietary lipids to all tis-  sues of the body.   Once in circulation, the newly released ("nascent" ) chylomicrons interact with  another lipoprotein particle, HDL (high-density lipoprotein) and acquire two  apoproteins from HDL, apoprotein CH and E. This converts the nascent chylomi-  cron to a "mature" chylomicron. The apoCII on the mature chylomicron activates  the enzyme lipoprotein lipase (LPL), which is located on the inner surface of the  capillary endothelial cells of muscle and adipose tissue. The LPL digests the  triglyceride in the chylomicron, producing free fatty acids and glycerol. The fatty  acids enter the adjacent organs either for energy production (muscle) or fat stor-  age (adipocyte). The glycerol that is released is metabolized in the liver.   As the chylomicron loses triglyceride, its density increases and it becomes a  chylomicron remnant, which is taken up by the liver by receptors that recognize  apolipoprotein E. In the liver, the chylomicron remnant is degraded into its com-  ponent parts for further disposition by the liver.   The lymph system is a network of  vessels that surround interstitial  cavities in the body. Cells secrete  various compounds into the lymph, and the  lymph vessels transport these fluids away  from the interstitial spaces in the body tissues  and into the bloodstream. In the case of the  intestinal lymph system, the lymph enters the  bloodstream through the thoracic duct. These  vessels are designed such that under normal  conditions the contents of the blood cannot  enter the lymphatic system. The lymph fluid  is similar in composition to that of the blood  but lacks the cells found in blood.   Will Sichel had several episodes of mild back and lower extremity pain  over the last year, probably caused by minor sickle cell crises. He then  developed severe right upper abdominal pain radiating to his lower right  chest and his right flank 36 hours before admission to the emergency room. He  states that the pain is not like his usual crisis pain. Intractable vomiting began 12  hours after the onset of these new symptoms. He reports that his urine is the color  of iced tea and his stool now has a light clay color.   On physical examination, his body temperature is slightly elevated, and his heart  rate is rapid. The whites of his eyes (the sclerae) are obviously jaundiced (a yellow  discoloration caused by the accumulation of bilirubin pigment). He is exquisitely  tender to pressure over his right upper abdomen.   The emergency room physician suspects that Michael is not in sickle cell cri-  sis but instead has either acute cholecystitis (gallbladder inflammation) or a gall-  stone lodged in his common bile duct, causing cholestasis (the inability of the  bile from the liver to reach his small intestine). His hemoglobin level was low at  7.6 mg/dL (reference range = 12-16) but unchanged from his baseline 3 months  earlier. His serum total bilirubin level was 3.2 mg/dL (reference range =  0.2-1.0), and his direct (conjugated) bilirubin level was 0.9 mg/dL (reference  range = 0 -0.2).   Intravenous fluids were started, he was not allowed to take anything by mouth,  a nasogastric tube was passed and placed on constant suction, and symptomatic  therapy was started for pain and nausea. When his condition had stabilized,  Michael was sent for an ultrasonographic (ultrasound) study of his upper  abdomen.   Al Martini has continued to abuse alcohol and to eat poorly. After a par-  ticularly heavy intake of vodka, a steady severe pain began in his upper  mid-abdomen. This pain spread to the left upper quadrant and eventually  radiated to his mid-back. He began vomiting nonbloody material and was brought  to the hospital emergency room with fever, a rapid heart beat, and a mild reduction  in blood pressure. On physical examination, he was dehydrated and tender to pres-  sure over the upper abdomen. His vomitus and stool were both negative for occult  blood.   Blood samples were sent to the laboratory for a variety of hematologic and  chemical tests, including a measurement of serum amylase and lipase, digestive  enzymes normally secreted from the exocrine pancreas through the pancreatic ducts  into the lumen of the small intestine.   Currently, 38% of the calories  \m ) (kcal) in the typical American diet  *r come from fat. The content of fat  in the diet increased from the early 1900s  until the 1960s, and then decreased as we  became aware of the unhealthy effects of a  high-fat diet. According to current recom-  mendations, fat should provide no more  than 30% of the total calories of a healthy  diet.   Triacylglycerols are the major fat in the human diet because they are the major stor-  age lipid in the plants and animals that constitute our food supply. Triacylglycerols  contain a glycerol backbone to which three fatty acids are esterified (Fig. 32.1). The  main route for digestion of triacylglycerols involves hydrolysis to fatty acids and 2-  monoacylglycerols in the lumen of the intestine. However, the route depends to some  extent on the chain length of the fatty acids. Lingual and gastric lipases are produced  by cells at the back of the tongue and in the stomach, respectively. These lipases  preferentially hydrolyze short- and medium-chain fatty acids (containing 12 or fewer  carbon atoms) from dietary triacylglycerols. Therefore, they are most active in   CH 3 - (CH 2 ) 7 - CH = CH - (CH 2 ) 7 - C - O - CH   J CH 2 - O 7 C - (CH 2 ) 16 - CH 3   Fig. 32.1. Structure of a triacylglycerol. The glycerol moiety is highlighted, and its carbons  are numbered.   infants and young children, who drink relatively large quantities of cow's milk,  which contains triacylglycerols with a high percentage of short- and medium-chain  fatty acids.   Dietary fat leaves the stomach and enters the small intestine, where it is emulsified  (suspended in small particles in the aqueous environment) by bile salts (Fig. 32.2).  The bile salts are amphipathic compounds (containing both hydrophobic and  hydrophilic components), synthesized in the liver (see Chapter 34 for the pathway)  and secreted via the gallbladder into the intestinal lumen. The contraction of the  gallbladder and secretion of pancreatic enzymes are stimulated by the gut hormone  cholecystokinin, which is secreted by the intestinal cells when stomach contents  enter the intestine. Bile salts act as detergents, binding to the globules of dietary fat  as they are broken up by the peristaltic action of the intestinal muscle. This emulsi-  fied fat, which has an increased surface area as compared with unemulsified fat, is  attacked by digestive enzymes from the pancreas (Fig. 32.3).   The major enzyme that digests dietary triacylglycerols is a lipase produced in the  pancreas. Pancreatic lipase is secreted along with another protein, colipase, along  with bicarbonate, which neutralizes the acid that enters the intestine with partially  digested food from the stomach. Bicarbonate raises the pH of the contents of the  intestinal lumen into a range (pH ~ 6) that is optimal for the action of all of the  digestive enzymes of the intestine. Bicarbonate secretion from the pancreas is stim-  ulated by the hormone secretin, which is released from the intestine when acid  enters the duodenum.   In patients such as Will Sichel who have severe and recurrent episodes of  increased red blood cell destruction (hemolytic anemia), greater than normal  amounts of the red cell pigment heme must be processed by the liver and  spleen. In these organs, heme (derived from hemoglobin) is degraded to bilirubin, which  is excreted by the liver in the bile.   If large quantities of bilirubin are presented to the liver as a consequence of acute  hemolysis, the capacity of the liver to conjugate it, that is, convert it to the water-soluble  bilirubin diglucuronide, can be overwhelmed. As a result, a greater percentage of the  bilirubin entering the hepatic biliary ducts in patients with hemolysis is in the less water-  soluble forms. In the gallbladder, these relatively insoluble particles tend to precipitate  as gallstones rich in calcium bilirubinate. In some patients, one or more stones may  leave the gallbladder through the cystic duct and enter the common bile duct. Most pass  harmlessly into the small intestine and are later excreted in the stool. Larger stones,  however, may become entrapped in the lumen of the common bile duct, where they  cause varying degrees of obstruction to bile flow (cholestasis) with associated ductal  spasm, producing pain. If adequate amounts of bile salts do not enter the intestinal  lumen, dietary fats cannot readily be emulsified and digested.    The mammary gland produces  milk, which is the major source of  nutrients for the breastfed human  infant. The fatty acid composition of human  milk varies, depending on the diet of the  mother. However, long-chain fatty acids pre-  dominate, particularly palmitic, oleic, and  linoleic acids. Although the amount of fat  contained in human milk and cow's milk is  similar, cow's milk contains more short- and  medium-chain fatty acids and does not con-  tain the long-chain, polyunsaturated fatty  acids found in human milk that are impor-  tant in brain development.   Although the concentrations of pancre-  atic lipase and bile salts are low in the intes-  tinal lumen of the newborn infant, the fat of  human milk is still readily absorbed. This is  true because lingual and gastric lipases pro-  duced by the infant partially compensate for  the lower levels of pancreatic lipase. The  human mammary gland also produces  lipases that enter the milk. One of these  lipases, which requires lower levels of bile  salts than pancreatic lipase, is not inacti-  vated by stomach acid and functions in the  intestine for a number of hours.   Fig. 32.2. Structure of a bile salt. The bile  salts are derived from cholesterol and retain  the cholesterol ring structure. They differ from  cholesterol in that the rings in bile salts contain  more hydroxyl groups and a polar side chain  and lack a 5-6 double bond.   Al Martini's serum levels of pancre-  atic amylase (which digests dietary  starch) and pancreatic lipase were  elevated, a finding consistent with a diagno-  sis of acute and possibly chronic pancreatitis.  The elevated levels of these enzymes in the  blood are the result of their escape from the  inflamed exocrine cells of the pancreas into  the surrounding pancreatic veins. The cause  of this inflammatory pancreatic process in  this case was related to the toxic effect  of acute and chronic excessive alcohol  ingestion.   Fig. 32.4. Action of pancreatic lipase. Fatty  acids (FA) are cleaved from positions 1 and 3  of the triacylglycerol, and a monoacylglycerol  with a fatty acid at position 2 is produced.   Bile salts inhibit pancreatic lipase  \ \ ) activity by coating the substrate and   not allowing the enzyme access to  the substrate. The colipase relieves the bile  salt inhibition, and allows the triglyceride to  enter the active site of the lipase.   The exocrine pancreas secretes  \\ ) phospholipase A2 in an inactive   zymogen form, prophospholipase  A2. The enzyme is activated in the intestinal  lumen by proteolytic cleavage by trypsin.  Pancreatic lipase, however, is secreted in its  active form, and only needs to bind colipase  and substrate to be active.   When he was finally able to toler-  ate a full diet, Al Martini's stools  became bulky, glistening, yellow-  brown, and foul smelling. They floated on  the surface of the toilet water. What caused  this problem?    Fig. 32.3. Digestion of triacylglycerols in the intestinal lumen. TG = triacylglycerol; bs  bile salts; FA = fatty acid; 2-MG = 2-monoacylglycerol.   The colipase binds to the dietary fat and to the lipase, thereby increasing lipase  activity. Pancreatic lipase hydrolyzes fatty acids of all chain lengths from positions  1 and 3 of the glycerol moiety of the triacylglycerol, producing free fatty acids and  2-monoacylglycerol, i.e., glycerol with a fatty acid esterified at position 2  (Fig. 32.4). The pancreas also produces esterases that remove fatty acids from com-  pounds (such as cholesterol esters) and phospholipase A2 that digests phospholipids  to a free fatty acid and a lysophospholipid (Fig. 32.5).   The fatty acids and 2-monoacylglycerols produced by digestion are packaged into  micelles, tiny microdroplets emulsified by bile salts (see Fig. 32.3). Other dietary  lipids, such as cholesterol, lysophospholipids, and fat-soluble vitamins, are also  packaged in these micelles. The micelles travel through a layer of water (the  unstirred water layer) to the microvilli on the surface of the intestinal epithelial  cells, where the fatty acids, 2-monoacylglycerols, and other dietary lipids are  absorbed, but the bile salts are left behind in the lumen of the gut.   The bile salts are extensively resorbed when they reach the ileum. Greater than  95% of the bile salts are recirculated, traveling through the enterohepatic circulation   R 2 -C -O-CH O  I II  H ? C-0-P-0-X  I   y^^v Al Martini's stool changes are char-  •] acteristic of steatorrhea (fat-laden  ^^^y stools caused by malabsorption of  dietary fats), in this case caused by a lack of  pancreatic secretions, particularly pancreatic  lipase, which normally digests dietary fat.   Steatorrhea also may be caused by insuf-  ficient production or secretion of bile salts.  Therefore, Michael Sichel might also develop  this condition.   Fig. 32.5. Action of pancreatic esterases (A) and phospholipase A2 (B).   to the liver, which secretes them into the bile for storage in the gallbladder and ejec-  tion into the intestinal lumen during another digestive cycle (Fig. 32.6).   Short- and medium-chain fatty acids (C4 to CI 2) do not require bile salts for their  absorption. They are absorbed directly into intestinal epithelial cells. Because they  do not need to be packaged to increase their solubility, they enter the portal blood  (rather than the lymph) and are transported to the liver bound to serum albumin.    For bile salt micelles to form, the  concentration of bile salts in the  contents of the intestinal lumen  must reach 5-15 ixmol/mL. This  critical concentration of bile salts is therefore  required for optimal lipid absorption.   Within the intestinal epithelial cells, the fatty acids and 2-monoacylglycerols are  condensed by enzymatic reactions in the smooth endoplasmic reticulum to form tri-  acylglycerols. The fatty acids are activated to fatty acyl Co A by the same process    Fig. 32.6. Recycling of bile salts. Bile salts are synthesized in the liver, stored in the gall-  bladder, secreted into the small intestine, resorbed in the ileum, and returned to the liver via  the enterohepatic circulation. Five percent or less of luminal bile acids are excreted in the  stool under normal circumstances.   Fig. 32.7. Resynthesis of triacylglycerols in intestinal epithelial cells. Fatty acids (FA), produced by digestion, are activated in intestinal epithe-  lial cells and then esterified to the 2-monoacylglycerol produced by digestion. The triacylglycerols are packaged in chylomicrons and secreted  into the lymph.   Because the fat-soluble vitamins (A, D, E,  and K) are absorbed from micelles along  with the long-chain fatty acids and 2-monoa-  cylglycerols, prolonged obstruction of the  duct that carries exocrine secretions from  the pancreas and the gallbladder into the  intestine (via the common duct) could lead  to a deficiency of these metabolically impor-  tant substances. If the obstruction of Michael  Sichel's common duct continues, he will  eventually suffer from a fat-soluble vitamin  deficiency. (Graph from Devlin T. Textbook of  Biochemistry, 3rd Ed. 1992:1084. Copyright  © John Wiley & Sons, Inc.)   used for activation of fatty acids before p-oxidation (see Chapter 23). A fatty acyl  CoA then reacts with a 2-monoacylglycerol to form a diacylglycerol, which reacts  with another fatty acyl CoA to form a triacylglycerol (Fig. 32.7). The reactions for  triacylglycerol synthesis in intestinal cells differ from those in liver and adipose cells  in that 2-monoacylglycerol is an intermediate in triacylglycerol in intestinal cells,  whereas phosphatidic acid is the necessary intermediate in other tissues.   Triacylglycerols are transported in lipoprotein particles because they are insolu-  ble in water. If triacylglycerols directly entered the blood, they would coalesce,  impeding blood flow. Intestinal cells package triacylglycerols together with proteins  and phospholipids in chylomicrons, which are lipoprotein particles that do not read-  ily coalesce in aqueous solutions (Figs. 32.8 and 32.9). Chylomicrons also contain  cholesterol and fat- soluble vitamins. The protein constituents of the lipoproteins are  known as apoproteins.   The major apoprotein associated with chylomicrons as they leave the intestinal  cells is B-48 (Fig. 32.10). The B-48 apoprotein is structurally and genetically   Fig. 32.8. Example of the structure of a blood lipoprotein. VLDL is depicted. Lipoproteins  contain phospholipids and proteins on the surface, with their hydrophilic regions interacting  with water. Hydrophobic molecules are in the interior of the lipoprotein. The hydroxyl group  of cholesterol is near the surface. In cholesterol esters, the hydroxyl group is esterified to a  fatty acid. Cholesterol esters are found in the interior of lipoproteins and are synthesized by  reaction of cholesterol with an activated fatty acid (see Chapter 33).    Fig. 32.10. Formation and secretion of chylomicrons. The triacylglycerol is produced in the  smooth endoplasmic reticulum (SER) of intestinal epithelial cells from the digestive prod-  ucts, fatty acids, and 2-monoacylglycerols. The protein is synthesized in the rough endoplas-  mic reticulum (RER). The major apoprotein in chylomicrons is B-48. Assembly of the  lipoproteins occurs in both the ER and the Golgi complex.   Fig. 32.9. Composition of a typical chylomi-  cron. Although the composition varies to some  extent, the major component is triacylglycerol  (TG). C = cholesterol; CE = cholesterol ester;  PL = phospholipid.   related to the B-100 apoprotein synthesized in the liver that serves as a major pro-  tein of another lipid carrier, very-low-density lipoprotein (VLDL). These two  apoproteins are encoded by the same gene. In the intestine, the primary transcript  of this gene undergoes RNA editing (Fig. 32. 1 1 and see Chapter 15). A stop codon  is generated that causes a protein to be produced in the intestine that is 48% of the  size of the protein produced in the liver; hence the designations B-48 and B-100.   Fig. 32.11. B-apoprotein gene. The gene, located on chromosome 2, is transcribed and trans-  lated in liver to produce apoB-100, which is 4,536 amino acids in length (one of the longest  single-polypeptide chains). In intestinal cells, RNA editing converts a cytosine (C) to an  adenine (A), producing a stop codon. Consequently, the B-apoprotein of intestinal cells  (apoB-48) contains only 2,152 amino acids. ApoB-48 is 48% of the size of apoB-100.   Olestra is an artificial fat substitute  designed to allow individuals to  obtain the taste and food consis-  tency of fat, without the calories from fat.  The structure of Olestra is shown below and  consists of a sucrose molecule to which  fatty acids are esterified to the hydroxyl  groups.   The fatty acids attached to sucrose are  resistant to hydrolysis by pancreatic lipase,  so Olestra passes through the intestine  intact and is eliminated in the feces. As a  result, no useful calories can be obtained  through the metabolism of Olestra.   Because of their high triacylglyc-  erol content, chylomicrons are the  least dense of the blood lipopro-  teins. When blood is collected from patients  with certain types of hyperlipoproteinemias  (high concentrations of lipoproteins in the  blood) in which chylomicron levels are ele-  vated, and the blood is allowed to stand in  the refrigerator overnight, the chylomicrons  float to the top of the liquid and coalesce,  forming a creamy layer.   ^> One manner in which individuals  rj J can lose weight is to inhibit the  activity of pancreatic lipase. This  would result in reduced fat digestion and  absorption and a reduced caloric yield from  the diet. The drug Orlistat is a chemically  synthesized derivative of lipstatin, a natural  lipase inhibitor found in certain bacteria. The  drug works in the intestinal lumen and forms  a covalent bond with the active site serine  residue of both gastric and pancreatic lipase,  thereby inhibiting their activities. Nondi-  gested triglycerides are not absorbed by the  intestine and are eliminated in the feces.  Under normal use of the drug, approxi-  mately 30% of dietary fat absorption is inhib-  ited. Because excessive nondigested fat in  the intestines can lead to gastrointestinal  distress related to excessive intestinal gas  formation, individuals taking this drug need  to follow a reduced daily intake of fat in their  diet, which should be evenly distributed  amongst the meals of the day.    Fig. 32.12. Transfer of proteins from HDL to  chylomicrons. Newly synthesized chylomi-  crons (nascent chylomicrons) mature as they  receive apoproteins CII and E from HDL.  HDL functions in the transfer of these apopro-  teins and also in transfer of cholesterol from  peripheral tissues to the liver (see Table 1 in  the introduction to this section).    The protein component of the lipoproteins is synthesized on the rough endo-  plasmic reticulum. Lipids, which are synthesized in the smooth endoplasmic retic-  ulum, are complexed with the proteins to form the chylomicrons (see Fig. 32.10).   By the process of exocytosis, chylomicrons are secreted by the intestinal epithelial  cells into the chyle of the lymphatic system and enter the blood through the thoracic  duct. Chylomicrons begin to enter the blood within 1 to 2 hours after the start of a  meal; as the meal is digested and absorbed, they continue to enter the blood for many  hours. Initially, the particles are called nascent (newborn) chylomicrons. As they  accept proteins from HDL within the lymph and the blood, they become "mature"  chylomicrons.   HDL transfers proteins to the nascent chylomicrons, particularly apoprotein E  (apoE) and apoprotein CII (apoCII) (Fig. 32.12). ApoE is recognized by membrane  receptors, particularly those on the surface of liver cells, allowing ApoE-bearing  lipoproteins to enter these cells by endocytosis for subsequent digestion by lyso-  somes. ApoCII acts as an activator of LPL, the enzyme on capillary endothelial  cells, primarily within muscle and adipose tissue, that digests the triacylglycerols of  the chylomicrons and VLDL in the blood.   The triacylglycerols of the chylomicrons are digested by LPL attached to the pro-  teoglycans in the basement membranes of endothelial cells that line the capillary  walls (Fig. 32.13). LPL is produced by adipose cells, muscle cells (particularly car-  diac muscle), and cells of the lactating mammary gland. The isozyme synthesized in  adipose cells has a higher K m than the isozyme synthesized in muscle cells. There-  fore, adipose LPL is more active after a meal, when chylomicrons levels are elevated  in the blood. Insulin stimulates the synthesis and secretion of adipose LPL, such that  after a meal, when triglyceride levels increase in circulation, LPL has been upregu-  lated (through insulin release) to facilitate the hydrolysis of fatty acids from the  triglyceride.   The fatty acids released from triacylglycerols by LPL are not very soluble in  water. They become soluble in blood by forming complexes with the protein albu-  min. The major fate of the fatty acids is storage as triacylglycerol in adipose tissue.  However, these fatty acids also may be oxidized for energy in muscle and other tis-  sues (see Fig. 32. 13). The LPL in the capillaries of muscle cells has a lower K m than  adipose LPL. Thus, muscle cells can obtain fatty acids from blood lipoproteins  whenever they are needed for energy, even if the concentration of the lipoproteins  is low.   The glycerol released from chylomicron triacylglycerols by LPL may be used for  triacylglycerol synthesis in the liver in the fed state.   The portion of a chylomicron that remains in the blood after LPL action is  known as a chylomicron remnant. This remnant binds to receptors on hepatocytes  (the major cells of the liver), which recognize apoprotein E, and is taken up by the  process of endocytosis. Lysosomes fuse with the endocytic vesicles, and the   ^Heparin is a complex polysaccharide that is a component of proteoglycans (see  | Chapter 49). Isolated heparin is frequently used as an anticoagulant, because it  binds to antithrombin III (ATM I), and the activated ATIII then binds factors nec-  essary for clotting and inhibits them from working. As LPL is bound to the capillary  endothelium through binding to proteoglycans, heparin also can bind to LPL and dis-  lodge it from the capillary wall. This leads to loss of LPL activity and an increase of  triglyceride content in the blood.   Fig. 32.13. Fate of chylomicrons. Chylomicrons are synthesized in intestinal epithelial cells, secreted into the lymph, pass into the blood, and  become mature chylomicrons (see Fig. 32.11). On capillary walls in adipose tissue and muscle, lipoprotein lipase (LPL) activated by ApoC n  digests the triacylglycerols (TG) of chylomicrons to fatty acids and glycerol. Fatty acids (FA) are oxidized in muscle or stored in adipose cells  as triacylglycerols. The remnants of the chylomicrons are taken up by the liver by receptor-mediated endocytosis. Lysosomal enzymes within the  hepatocyte digest the remnants, releasing the products into the cytosol.   chylomicron remnants are degraded by lysosomal enzymes. The products of lyso-  somal digestion (e.g., fatty acids, amino acids, glycerol, cholesterol, phosphate) can  be reused by the cell.   The upper abdominal ultrasound study showed a large gallstone lodged in  Will Sichel's common duct with dilation of this duct proximal to the stone.  Michael was scheduled for endoscopic retrograde cholangiopancreatogra-  phy (ERCP). (An ERCP involves cannulation of the common bile duct — and, if nec-  essary, the pancreatic duct — through a tube placed through the mouth and stomach  and into the upper small intestine.) With this technique, a stone can be snared in the  common duct and removed to relieve an obstruction.   If common duct obstruction is severe enough, bilirubin flows back into the  venous blood draining from the liver. As a consequence, serum bilirubin levels, par-  ticularly the indirect (unconjugated) fraction, increase. Tissues such as the sclerae  of the eye take up this pigment, which causes them to become yellow (jaundiced).  Will Sichel's condition was severe enough to cause jaundice by this mechanism.   Alcohol excess may produce proteinaceous plugs in the small pancreatic  ducts, causing back pressure injury and autodigestion of the pancreatic  acini drained by these obstructed channels. This process causes one form  of acute pancreatitis. Al Martini had an episode of acute alcohol-induced pancre-  atitis superimposed on a more chronic alcohol-related inflammatory process in the  pancreas — in other words, a chronic pancreatitis. As a result of decreased secretion  of pancreatic lipase through the pancreatic ducts and into the lumen of the small  intestine, dietary fat was not absorbed at a normal rate, and steatorrhea (fat-rich   stools) occurred. If abstinence from alcohol does not allow adequate recovery of  the enzymatic secretory function of the pancreas, Mr. Martini will have to take a  commercial preparation of pancreatic enzymes with meals that contain even mini-  mal amounts of fat.   The assembly of chylomicrons within the endoplasmic reticulum of the  enterocyte requires the activity of microsomal triglyceride transfer protein  (MTP). The protein is a dimer of two nonidentical subunits. The smaller  subunit (57 kDa) is protein disulfide isomerase (PDI, see Chapter 7, section IX. A),  whereas the larger subunit (97 kDa) contains the triglyceride transfer activity. MTP  accelerates the transport of triglycerides, cholesterol esters, and phospholipids  across membranes of subcellular organelles. The role of PDI in this complex is not  known; the disulfide isomerase activity of this subunit is not needed for triglyceride  transport to occur. The lack of triglyceride transfer activity leads to the disease  abetalipoproteinemia. This disease affects both chylomicron assembly in the  intestine and VLDL assembly in the liver. Both particles require a B apoprotein for  their assembly (ApoB-48 for chylomicrons, ApoB-100 for VLDL), and MTP binds  to the B apoproteins. For both chylomicron and VLDL assembly, a small  ApoB-containing particle is first produced within the lumen of the ER. The appro-  priate apoB is made on the rough endoplasmic reticulum (RER) and is inserted into  the ER lumen during its synthesis (see Chapter 15, section IX). As the protein is  being translated, lipid (a small amount of triglyceride) begins to associate with the  protein, and the lipid association is catalyzed by MTP. This leads to the generation  of small ApoB-containing particles; these particles are not formed in patients with  abetalipoproteinemia. Thus, it appears as though MTP activity is necessary to trans-  fer triacylglycerol formed within the ER to the ApoB protein. The second stage of  particle assembly is the fusion of the initial ApoB particle with triacylglycerol  droplets within the ER. MTP also may be required for the transfer of triacylglycerol  from the cytoplasm to the lumen of the ER to form this lipid droplet. These steps  are depicted in Fig. 32.14.   The symptoms of abetalipoproteinemia include lipid malabsorption (and its  accompanying symptoms, such as steatorrhea and vomiting), which can result in  caloric deficiencies and weight loss. Because lipid-soluble vitamin distribution  occurs through chylomicron circulation, signs and symptoms of deficiencies in the  lipid-soluble vitamins may be seen in these patients.   Fig. 32.14. A model of microsomal triglyceride transfer protein (MTP) action. MTP is  required to transfer lipid to apoB-48 as it is synthesized, and to transfer lipid from the cyto-  plasm to the ER lumen.   Berriot-Varoqueaux N, Aggerbeck LP, Samson-Bouma ME, Wetterau JR. The role of the microsomal  triglyceride transfer protein in abetalipoproteinemia. Annu Rev Nutr 2000;20:663-697.   Havel RJ, Kane JP. Introduction: structure and metabolism of plasm lipoproteins. In: Scriver CR,  Beaudet AL, Valle D, Sly WS, et al., eds. The Metabolic and Molecular Bases of Inherited Disease,  vol II, 8th Ed. New York: McGraw-Hill, 2001:2705-2716.   Knopp RH. Drug treatment of lipid disorders. N Engl J Med 1999;341:498-51 1.   Linder MC, ed. Nutrition and metabolism of fats. In: Nutritional Biochemistry and Metabolism with  Clinical Applications. New York: Elsevier, 1991:51-85.   Zlotkin SH. Neonatal nutrition. In: Linder MC, ed. Nutritional Biochemistry and Metabolism with Clin-  ical Applications. New York: Elsevier, 1991:357-360.   1 . The most abundant component of chylomicrons is which of the following?   2. The conversion of nascent chylomicrons to mature chylomicrons requires which of the following?   3. The apoproteins B-48 and B-100 are similar with respect to which of the following?   (B) They are derived by alternative spicing of the same hnRNA.   4. Bile salts must reach a particular concentration within the intestinal lumen before they are effective agents for lipid digestion.  This is because of which of the following?   (A) The bile salt concentration must be equal to the triglyceride concentration.   (B) The bile salt solubility in the lumen is a critical factor.   (C) The ability of bile salts to bind lipase is concentration dependant.   (D) The bile salts cannot be reabsorbed in the ileum until they reach a certain concentration.   (E) The bile salts do not activate lipase until they reach a particular concentration.   5. Type III hyperlipidemia is caused by a deficiency of apoprotein E. Analysis of the serum of patients with this disorder would  exhibit which of the following?   Fatty acids are synthesized mainly in the liver in humans, with dietary glucose serv-  ing as the major source of carbon. Glucose is converted through glycolysis to pyru-  vate, which enters the mitochondrion and forms both acetyl Co A and oxaloacetate  (Fig. 33.1). These two compounds condense, forming citrate. Citrate is transported  to the cytosol, where it is cleaved to form acetyl Co A, the source of carbon for the  reactions that occur on the fatty acid synthase complex. The key regulatory enzyme  for the process, acetyl Co A carboxylase, produces malonyl Co A from acetyl Co A.   The growing fatty acid chain, attached to the fatty acid synthase complex in the  cytosol, is elongated by the sequential addition of 2 -carbon units provided by mal-  onyl Co A. NADPH, produced by the pentose phosphate pathway and the malic  enzyme, provides reducing equivalents. When the growing fatty acid chain is 16  carbons in length, it is released as palmitate. After activation to a Co A derivative,  palmitate can be elongated and desaturated to produce a series of fatty acids.    Fig. 33.1. Lipogenesis, the synthesis of triacylglycerols from glucose. In humans, the syn-  thesis of fatty acids from glucose occurs mainly in the liver. Fatty acids (FA) are converted to  triacylglycerols (TG), packaged in VLDL, and secreted into the blood. OAA = oxaloacetate.   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS    Fig. 33.2. Fate of VLDL triacylglycerol (TG). The TG of VLDL, produced in the liver, is  digested by lipoprotein lipase (LPL) present on the lining cells of the capillaries in adipose  and skeletal muscle tissue. Fatty acids are released and either oxidized or stored in tissues as  TG. Glycerol is used by the liver and other tissues that contain glycerol kinase. FA = fatty  acid (or fatty acyl group).   Fatty acids, produced in cells or obtained from the diet, are used by various  tissues for the synthesis of triacylglycerols (the major storage form of fuel) and  the glycerophospholipids and sphingolipids (the major components of cell mem-  branes).   In the liver, triacylglycerols are produced from fatty acyl Co A and glycerol 3-  phosphate. Phosphatidic acid serves as an intermediate in this pathway. The tria-  cylglycerols are not stored in the liver but rather packaged with apoproteins and  other lipids in very-low-density lipoprotein (VLDL) and secreted into the blood  (see Fig. 33.1).   In the capillaries of various tissues (particularly adipose tissue, muscle, and  the lactating mammary gland), lipoprotein lipase (LPL) digests the triacylglyc-  erols of VLDL, forming fatty acids and glycerol (Fig. 33.2). The glycerol travels to  the liver and other tissues where it is used. Some of the fatty acids are oxidized by  muscle and other tissues. After a meal, however, most of the fatty acids are con-  verted to triacylglycerols in adipose cells, where they are stored. These fatty acids  are released during fasting and serve as the predominant fuel for the body.   Glycerophospholipids are also synthesized from fatty acyl Co A, which forms  esters with glycerol 3-phosphate, producing phosphatidic acid. Various head groups  are added to carbon 3 of the glycerol 3-phosphate moiety of phosphatidic acid, gen-  erating amphipathic compounds such as phosphatidylcholine, phosphatidylinositol,  and cardiolipin (Fig. 33.3). In the formation of plasmalogens and platelet-activat-  ing factor (PAF), a long-chain fatty alcohol forms an ether with carbon 1, replac-  ing the fatty acyl ester (Fig. 33.4). Cleavage of phospholipids is catalyzed by phos-  pholipases found in cell membranes, lysosomes, and pancreatic juice.   Sphingolipids, which are prevalent in membranes and the myelin sheath of the  central nervous system, are built on serine rather than glycerol. In the synthesis of  sphingolipids, serine and palmityl Co A condense , forming a compound that is  related to sphingosine. Reduction of this compound, followed by addition of a  second fatty acid in amide linkage, produces cer amide. Carbohydrate groups  attach to ceramide, forming glycolipids such as the cerebrosides, globosides, and  gangliosides (Fig. 33.5). The addition of phosphocholine to ceramide produces  sphingomyelin. These sphingolipids are degraded by lysosomal enzymes.   Fig. 33.3. General structure of a glycerophos-  pholipid. The fatty acids are joined by ester  bonds to the glycerol moiety. Various combi-  nations of fatty acids may be present. The fatty  acid at carbon 2 of the glycerol is usually  unsaturated. The head group is the group  attached to the phosphate on position 3 of the  glycerol moiety. The most common head  group is choline, but ethanolamine, serine,  inositol, or phosphatidylglycerol also may be  present. The phosphate group is negatively  charged, and the head group may carry a posi-  tive charge (choline and ethanolamine), or  both a positive and a negative charge (serine).  The inositol may be phosphorylated and, thus,  negatively charged.   Fig. 33.4. General structure of a plasmalogen.  Carbon 1 of glycerol is joined to a long-chain  fatty alcohol by an ether linkage. The fatty  alcohol group has a double bond between car-  bons 1 and 2. The head group is usually  ethanolamine or choline.   Fig. 33.5. General structures of the sphin-  golipids. The "backbone" is sphingosine rather  than glycerol. Ceramide is sphingosine with a  fatty acid joined to its amino group by an  amide linkage. Sphingomyelin contains phos-  phocholine, whereas glycolipids contain car-  bohydrate groups.   0 o The dietician did a careful analysis   of Percy Veere's diet, which was  ^ indeed low in fat, adequate in pro-  tein, but excessive in carbohydrates, espe-  cially in refined sugars. Percy's total caloric  intake averaged about 430 kilocalories (kcal)  a day in excess of his isocaloric require-  ments. This excess carbohydrate was being  converted to fats, accounting for Percy's  weight gain. A new diet with a total caloric  content that would prevent further gain in  weight was prescribed.   Percy Veere's mental depression slowly responded to antidepressant med-  ication, to the therapy sessions with his psychiatrist, and to frequent visits  from an old high school sweetheart whose husband had died several years  earlier. While hospitalized for malnutrition, Mr. Veere's appetite returned. By the  time of discharge, he had gained back 8 of the 22 lb he had lost and weighed 133 lb.   During the next few months, Mr. Veere developed a craving for "sweet foods"  such as the candy he bought and shared with his new friend. After 6 months of this  high-carbohydrate courtship, Percy had gained another 22 lb and now weighed 155  lb, just 8 lb more than he weighed when his depression began. He became con-  cerned about the possibility that he would soon be overweight and consulted his die-  titian, explaining that he had faithfully followed his low-fat diet but had "gone over-  board" with carbohydrates. He asked whether it was possible to become fat without  eating fat.   Cora Nari's hypertension and heart failure have been well controlled on  medication, and she has lost 10 lb since she had her recent heart attack. Her  fasting serum lipid profile on discharge from the hospital indicated signif-  icantly elevated serum low-density lipoprotein (LDL) cholesterol level of 175  mg/dL (recommended level for a patient with known coronary artery disease =100  mg/dL or less), a serum triacylglycerol level of 280 mg/dL (reference range =  60-150), and a serum high-density lipoprotein (HDL) cholesterol level of 34 mg/dL  (reference range > 50 for healthy women). While still in the hospital, she was asked  to obtain the most recent serum lipid profiles of her older brother and her younger  sister, both of whom were experiencing chest pain. Her brother's profile showed  normal triacylglycerols, moderately elevated LDL cholesterol, and significantly  suppressed HDL cholesterol levels. Her sister's profile showed only hypertriglyc-  eridemia (high blood triacylglycerols).    Colleen Lakker was born 6 weeks prematurely. She appeared normal until  about 30 minutes after delivery, when her respirations became rapid at 64  breaths/minute with audible respiratory grunting. The spaces between her  ribs (intercostal spaces) retracted inward with each inspiration, and her lips and fin-  gers became cyanotic from a lack of oxygen in her arterial blood. An arterial blood  sample indicated a low partial pressure of oxygen (p0 2 ) and a slightly elevated partial  pressure of carbon dioxide (pC0 2 ). The arterial pH was somewhat suppressed, in part  from an accumulation of lactic acid secondary to the hypoxemia (a low level of oxy-  gen in her blood). A chest x-ray showed a fine reticular granularity of the lung tissue,  especially in the left lower lobe area. From these clinical data, a diagnosis of  respiratory distress syndrome (RDS), also known as hyaline membrane disease, was  made.   Colleen was immediately transferred to the neonatal intensive care unit, where,  with intensive respiration therapy, she slowly improved.   Fatty acids are synthesized whenever an excess of calories is ingested. The major  source of carbon for the synthesis of fatty acids is dietary carbohydrate. An excess  of dietary protein also can result in an increase in fatty acid synthesis. In this case,  the carbon source is amino acids that can be converted to acetyl CoA or tricarboxylic   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   acid (TCA) cycle intermediates (see Chapter 39). Fatty acid synthesis occurs mainly  in the liver in humans, although the process also occurs in adipose tissue.   When an excess of dietary carbohydrate is consumed, glucose is converted to  acetyl CoA, which provides the 2-carbon units that condense in a series of reactions  on the fatty acid synthase complex, producing palmitate (see Fig. 33.1). Palmitate  is then converted to other fatty acids. The fatty acid synthase complex is located in  the cytosol, and, therefore, it uses cytosolic acetyl CoA.   The pathway for the synthesis of cytosolic acetyl CoA from glucose begins with gly-  colysis, which converts glucose to pyruvate in the cytosol (Fig. 33.6). Pyruvate enters  mitochondria, where it is converted to acetyl CoA by pyruvate dehydrogenase and to  oxaloacetate by pyruvate carboxylase. The pathway pyruvate follows is dictated by  the acetyl CoA levels in the mitochondria. When acetyl CoA levels are high, pyru-  vate dehydrogenase is inhibited, and pyruvate carboxylase activity is stimulated. As  oxaloacetate levels increase because of the activity of pyruvate carboxylase, oxaloac-  etate condenses with acetyl CoA to form citrate. This condensation reduces the  acetyl CoA levels, which leads to the activation of pyruvate dehydrogenase and inhi-  bition of pyruvate carboxylase. Through such reciprocal regulation, citrate can be  continuously synthesized and transported across the inner mitochondrial membrane.  In the cytosol, citrate is cleaved by citrate lyase to re-form acetyl CoA and oxaloac-  etate. This circuitous route is required because pyruvate dehydrogenase, the enzyme  that converts pyruvate to acetyl CoA, is found only in mitochondria and because  acetyl CoA cannot directly cross the mitochondrial membrane.   The NADPH required for fatty acid synthesis is generated by the pentose phos-  phate pathway (see Chapter 29) and from recycling of the oxaloacetate produced by  citrate lyase (Fig. 33.7). Oxaloacetate is converted back to pyruvate in two steps: the  reduction of oxaloacetate to malate by NAD + -dependent malate dehydrogenase and  the oxidative decarboxylation of malate to pyruvate by an NADP + -dependent  malate dehydrogenase (malic enzyme) (Fig. 33.8). The pyruvate formed by malic  enzyme is reconverted to citrate. The NADPH that is generated by malic enzyme,  along with the NADPH generated by glucose 6-phosphate and gluconate 6-phos-  phate dehydrogenases in the pentose phosphate pathway, is used for the reduction  reactions that occur on the fatty acid synthase complex (Fig. 33.9).   The generation of cytosolic acetyl CoA from pyruvate is stimulated by elevation  of the insulin/glucagon ratio after a carbohydrate meal. Insulin activates pyruvate  dehydrogenase by stimulating the phosphatase that dephosphorylates the enzyme to   Fig. 33.6. Conversion of glucose to cytosolic  acetyl CoA. OAA = oxaloacetate.   Fig. 33.7. Fate of citrate in the cytosol. Citrate lyase is also called citrate cleavage enzyme.  OAA = oxaloacetate; circled t = inducible enzyme.   Fig. 33.8. Reaction catalyzed by malic  enzyme. This enzyme is also called the decar-  boxylating or NADP-dependent malate dehy-  drogenase.   Fig. 33.10. Reaction catalyzed by acetyl CoA  carboxylase. C0 2 is covalently attached to  biotin, which is linked by an amide bond to the  e-amino group of a lysine residue of the  enzyme. Hydrolysis of ATP is required for the  attachment of C0 2 to biotin.   of the adenylate kinase reaction.  The [AMP] to [ATP] ratio is proportional to  the square of the [ADP] to [ATP] ratio, so a  fivefold change in ADP levels corresponds to  a 25-fold change in AMP levels.   Fig. 33.9. Sources of NADPH for fatty acid synthesis. NADPH is produced by the pentose  phosphate pathway and by malic enzyme. OAA = oxaloacetate.   an active form (see Chapter 20). The synthesis of malic enzyme, glucose 6-phosphate  dehydrogenase, and citrate lyase is induced by the high insulin/glucagon ratio. The  ability of citrate to accumulate, and leave the mitochondrial matrix for the synthe-  sis of fatty acids, is attributable to the allosteric inhibition of isocitrate dehydroge-  nase by high energy levels within the matrix under these conditions. The concerted  regulation of glycolysis and fatty acid synthesis is described in Chapter 36.   Cytosolic acetyl CoA is converted to malonyl CoA, which serves as the immediate  donor of the 2-carbon units that are added to the growing fatty acid chain on the  fatty acid synthase complex. To synthesize malonyl CoA, acetyl CoA carboxylase  adds a carboxyl group to acetyl CoA in a reaction requiring biotin and adenosine  triphosphate (ATP) (Fig. 33.10).   Acetyl CoA carboxylase is the rate-limiting enzyme of fatty acid synthesis. Its  activity is regulated by phosphorylation, allosteric modification, and induction/  repression of its synthesis (Fig. 33.11). Citrate allosterically activates acetyl CoA  carboxylase by causing the individual enzyme molecules (each composed of 4 sub-  units) to polymerize. Palmityl CoA, produced from palmitate (the endproduct of  fatty acid synthase activity), inhibits acetyl CoA carboxylase. Phosphorylation by  an AMP-dependent protein kinase inhibits the enzyme in the fasting state when  energy levels are low. The enzyme is activated by dephosphorylation in the fed state  when energy and insulin levels are high. A high insulin/glucagon ratio also results  in induction of the synthesis of both acetyl CoA carboxylase and the next enzyme  in the pathway, fatty acid synthase.   As an overview, fatty acid synthase sequentially adds 2-carbon units from malonyl  CoA to the growing fatty acyl chain to form palmitate. After the addition of each  2-carbon unit, the growing chain undergoes two reduction reactions that require  NADPH.   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   Fig. 33.11. Regulation of acetyl CoA carboxylase. This enzyme is regulated allosterically,  both positively and negatively, by phosphorylation (circled P) and dephosphorylation, and by  diet-induced induction (circled t). It is active in the dephosphorylated state when citrate  causes it to polymerize. Dephosphorylation is catalyzed by an insulin- stimulated phos-  phatase. Low energy levels, via activation of an AMP-dependent protein kinase, cause the  enzyme to be phosphorylated and inactivated. The ultimate product of fatty acid synthesis,  palmitate, is converted to its CoA derivative palmityl CoA, which inhibits the enzyme. A  high-calorie diet increases the rate of transcription of the gene for acetyl CoA carboxylase,  whereas a low-calorie diet reduces transcription of this gene.   Fatty acid synthase is a large enzyme composed of two identical dimers, which  each have seven catalytic activities and an acyl carrier protein (ACP) segment in a  continuous polypeptide chain. The ACP segment contains a phosphopantetheine  residue that is derived from the cleavage of coenzyme A (Fig. 33.12). The two  dimers associate in a head-to-tail arrangement, so that the phosphopantetheinyl  sulfhydryl group on one subunit and a cysteinyl sulfhydryl group on another sub-  unit are closely aligned.   In the initial step of fatty acid synthesis, an acetyl moiety is transferred from  acetyl CoA to the ACP phosphopantetheinyl sulfhydryl group of one subunit,  and then to the cysteinyl sulfhydryl group of the other subunit. The malonyl  moiety from malonyl CoA then attaches to the ACP phosphopantetheinyl  sulfhydryl group of the first subunit. The acetyl and malonyl moieties condense,  with the release of the malonyl carboxyl group as C0 2 . A 4-carbon a-keto acyl  chain is now attached to the ACP phosphopantetheinyl sulfhydryl group (Fig.  33.13).   A series of three reactions reduces the 4-carbon keto group to an alcohol,  removes water to form a double bond, and reduces the double bond (Fig. 33.14).  NADPH provides the reducing equivalents for these reactions. The net result is that  the original acetyl group is elongated by two carbons.   The 4-carbon fatty acyl chain is then transferred to the cysteinyl sulfhydryl group  and subsequently condenses with a malonyl group. This sequence of reactions is  repeated until the chain is 16 carbons in length. At this point, hydrolysis occurs, and  palmitate is released (Fig. 33.15).   Palmitate is elongated and desaturated to produce a series of fatty acids. In the  liver, palmitate and other newly synthesized fatty acids are converted to triacyl-  glycerols that are packaged into VLDL for secretion.   Fig. 33.12. Phosphopantetheinyl residue of  the fatty acid synthase complex. The portion  derived from the vitamin, pantothenic acid, is  indicated. Phosphopantetheine is covalently  linked to a serine residue of the acyl carrier  protein (ACP) segment of the enzyme. The  sulfhydryl group reacts with malonyl CoA to  form a thioester.   Fig. 33.14. Reduction of a p-ketoacyl group  on the fatty acid synthase complex. NADPH is  the reducing agent.   Fig. 33.13. Addition of a 2-carbon unit to an acetyl group on fatty acid synthase. The mal-  onyl group attaches to the phosphopantetheinyl residue (P) of the ACP of the fatty acid syn-  thase. The acetyl group, which is attached to a cysteinyl sulfhydryl group, condenses with the  malonyl group. C0 2 is released, and a 3-ketoacyl group is formed. The carbon that eventu-  ally forms the co-methyl group of palmitate is labeled co.   In the liver, the oxidation of newly synthesized fatty acids back to acetyl CoA via  the mitochondrial p-oxidation pathway is prevented by malonyl CoA.  Carnitine :palmitoy transferase I, the enzyme involved in the transport of long-chain  fatty acids into mitochondria (see Chapter 23), is inhibited by malonyl CoA (Fig.  33.16). Malonyl CoA levels are elevated when acetyl CoA carboxylase is activated,  and, thus, fatty acid oxidation is inhibited while fatty acid synthesis is proceeding.  This inhibition prevents the occurrence of a futile cycle.   After synthesis on the fatty acid synthase complex, palmitate is activated, forming  palmityl CoA. Palmityl CoA and other activated long-chain fatty acids can be   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS    Fig. 33.15. Synthesis of palmitate on the fatty acid synthase complex. Initially, acetyl CoA adds to the synthase. It provides the co-methyl group  of palmitate. Malonyl CoA provides the 2-carbon units that are added to the growing fatty acyl chain. The addition and reduction steps are  repeated until palmitate is produced. 1. Transfer of the malonyl group to the phosphopantetheinyl residue. 2. Condensation of the malonyl and  fatty acyl groups. 3. Reduction of the p-ketoacyl group. 4. Dehydration. 5. Reduction of the double bond. P = a phosphopantetheinyl group  attached to the fatty acid synthase complex; Cys-SH = a cysteinyl residue.   elongated, two carbons at a time, by a series of reactions that occur in the endo-  plasmic reticulum (Fig. 33.17). Malonyl CoA serves as the donor of the 2-carbon  units, and NADPH provides the reducing equivalents. The series of elongation reac-  tions resemble those of fatty acid synthesis except that the fatty acyl chain is  attached to coenzyme A rather than to the phosphopantetheinyl residue of an ACP.  The major elongation reaction that occurs in the body involves the conversion of  palmityl CoA (CI 6) to stearyl CoA (CI 8). Very-long-chain fatty acids (C22 to C24)  are also produced, particularly in the brain.   Desaturation of fatty acids involves a process that requires molecular oxygen (0 2 ),  NADH, and cytochrome b 5 . The reaction, which occurs in the endoplasmic reticu-  lum, results in the oxidation of both the fatty acid and NADH (Fig. 33.18). The most  common desaturation reactions involve the placement of a double bond between  carbons 9 and 10 in the conversion of palmitic acid to palmitoleic acid (16:1, A 9 )  and the conversion of stearic acid to oleic acid (18:1, A 9 ). Other positions that can  be desaturated in humans include carbons 4, 5, and 6.   ®The methyl group of acetyl CoA  becomes the co-carbon (the termi-  nal methyl group) of palmitate.  Each new 2-carbon unit is added to the car-  boxyl end of the growing fatty acyl chain  (see Fig. 33.13).   Fig. 33.17. Elongation of long-chain fatty  acids in the endoplasmic reticulum.   Fig. 33.16. Inhibition of carnitine ipalmitoyltransferase (CPTI, also called carnitine :acyl-  transf erase I) by malonyl CoA. During fatty acid synthesis, malonyl CoA levels are high.  This compound inhibits CPTI, which is involved in the transport of long-chain fatty acids  into mitochondria for p-oxidation. This mechanism prevents newly synthesized fatty acids  from undergoing immediate oxidation.   Polyunsaturated fatty acids with double bonds three carbons from the methyl end  (o)3 fatty acids) and six carbons from the methyl end (oo6 fatty acids) are required for  the synthesis of eicosanoids (see Chapter 35). Because humans cannot synthesize these  fatty acids de novo (i.e., from glucose via palmitate), they must be present in the diet  or the diet must contain other fatty acids that can be converted to these fatty acids. We  obtain w6 and 0)3 polyunsaturated fatty acids mainly from dietary plant oils that con-  tain the 0)6 fatty acid linoleic acid (18:2, A 912 ) and the 0)3 fatty acid a-linolenic acid  (18:3, A 9 ' 12 ' 15 ). In the body, linoleic acid can be converted by elongation and desatura-  tion reactions to arachidonic acid (20:4, A 5 ' 8 ' 11 ' 14 ), which is used for the synthesis of the  major class of human prostaglandins and other eicosanoids (Fig. 33.19). Elongation  and desaturation of a-linolenic acid produces eicosapentaenoic acid (EPA; 20:5,  A 5,8,111417 ), which is the precursor of a different class of eicosanoids (see Chapter 35).   Plants are able to introduce double bonds into fatty acids in the region between  | I ) C10 and the co-end and therefore can synthesize co3 and co6 polyunsaturated  fatty acids. Fish oils also contain co3 and co6 fatty acids, particularly eicosapen-  taenoic acid (EPA; oo3, 20:5, A5, 8, 11, 14, 17) and docosahexaenoic acid (DHA;  co3,22:6, A4,7,10,13,16,19). The fish obtain these fatty acids by eating phytoplankton  (plants that float in water).   Arachidonic acid is listed in some textbooks as an essential fatty acid. Although it is  an w6 fatty acid, it is not essential in the diet if linoleic acid is present because arachi-  donic acid can be synthesized from dietary linoleic acid (see Fig. 33.19).   The essential fatty acid linoleic acid is required in the diet for at least three rea-  \% ) sons: (a) It serves as a precursor of arachidonic acid from which eicosanoids  are produced, (b) It covalently binds another fatty acid attached to cerebrosides  in the skin, forming an unusual lipid (acylglucosylceramide) that helps to make the skin  impermeable to water. This function of linoleic acid may help to explain the red, scaly  dermatitis and other skin problems associated with a dietary deficiency of essential fatty  acids, (c) It is the precursor of C22:6co3, an important neuronal fatty acid.   The other essential fatty acid, a-linolenic acid (18:3, A9, 12, 15), also forms eicosanoids.   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   CH3 (CH 2 ) n "~ J CH2 CH2 J~ (CH 2 ) m   CH 3 - (CH 2 ) n CH = CH r- (CH 2 ) m - C   Fig. 33.18. Desaturation of fatty acids. The process occurs in the endoplasmic reticulum and uses molecular oxygen. Both the fatty acid and NADH  are oxidized. Human desaturases cannot introduce double bonds between carbon 9 and the methyl end. Therefore, m is equal to or less than 7.    Arachidonyl CoA (A 5 ' 8 ' 11 ' 14 -eicosatetraenoyl CoA)   Fig. 33.19. Conversion of linoleic acid to arachidonic acid. Dietary linoleic acid (as linoleoyl CoA) is desaturated at carbon 6, elongated by 2  carbons, and then desaturated at carbon 5 to produce arachidonyl CoA.   In liver and adipose tissue, triacylglycerols are produced by a pathway containing a  phosphatidic acid intermediate (Fig. 33.20). Phosphatidic acid is also the precursor  of the glycerolipids found in cell membranes and the blood lipoproteins.   The sources of glycerol 3-phosphate, which provides the glycerol moiety for tria-  cylglycerol synthesis, differ in liver and adipose tissue. In liver, glycerol 3-phosphate   muscle cells. The significance of  this finding is under investigation, but it may  indicate that muscle has a greater capacity for  fatty acid synthesis than previously believed.   is produced from the phosphorylation of glycerol by glycerol kinase or from the  reduction of dihydroxy acetone phosphate derived from glycolysis. Adipose tissue  lacks glycerol kinase and can produce glycerol 3 -phosphate only from glucose via  dihydroxyacetone phosphate. Thus, adipose tissue can store fatty acids only when  glycolysis is activated, i.e., in the fed state.   In both adipose tissue and liver, triacylglycerols are produced by a pathway in  which glycerol 3 -phosphate reacts with fatty acyl Co A to form phosphatidic acid.  Dephosphorylation of phosphatidic acid produces diacylglycerol. Another fatty acyl  CoA reacts with the diacylglycerol to form a triacylglycerol (see Fig. 33.20).   Fig. 33.20. Synthesis of triacylglycerol in liver and adipose tissue. Glycerol 3 -phosphate is  produced from glucose in both tissues. It is also produced from glycerol in liver, but not in  adipose tissue, which lacks glycerol kinase. The steps from glycerol 3-phosphate are the  same in the two tissues. FA = fatty acyl group.   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   Adipose tissue also undergoes glyceroneogenesis, the process of synthesizing  \ \ ) glycerol from gluconeogenic precursors in the blood, such as alanine, aspar-   tate, and malate. Glyceroneogenesis occurs primarily in the fasting state and is  dependent on the induction of cytoplasmic PEPCK in the adipocyte. The re-synthesis of  triglycerides by adipose tissue during fasting modulates the release of fatty acids in the  circulation. Mice that have been engineered to not express PEPCK in adipose tissue dis-  play reduced levels of triglyceride in their adipocytes; mice that overproduce adipocyte  PEPCK were obese. Thus, although activation of hormone-sensitive lipase during fasting  results in the release of fatty acids from adipocytes, the release is carefully modulated  through glyceroneogenesis and re-synthesis of triglycerides.   The triacylglycerol, which is produced in the smooth endoplasmic reticulum of  the liver, is packaged with cholesterol, phospholipids, and proteins (synthesized in  the rough endoplasmic reticulum) to form VLDL (Fig. 33.21). The microsomal  triglyceride transfer protein (MTP), which is required for chylomicron assembly, is  also required for VLDL assembly. The major protein of VLDL is apoB-100. There  is one long apoB-100 molecule wound through the surface of each VLDL particle.  ApoB-100 is encoded by the same gene as the apoB-48 of chylomicrons, but is a  longer protein (see Fig. 32.11). In intestinal cells, RNA editing produces a smaller  mRNA and, thus, a shorter protein, apoB-48.   VLDL is processed in the Golgi complex and secreted into the blood by the liver  (Figs. 33.22 and 33.23). The fatty acid residues of the triacylglycerols ultimately are  stored in the triacylglycerols of adipose cells. Note that, in comparison to chylomi-  crons (see Chapter 32), VLDL particles are more dense, as they contain a lower per-  centage of triglyceride than do the chylomicrons. Similar to chylomicrons, VLDL  particles are first synthesized in a nascent form, and on entering the circulation they  acquire apoproteins CII and E from HDL particles to become mature VLDL  particles.   Abetalipoproteinemia, which is due  \\ to a lack of MTP (microsomal  *S triglyceride transfer protein; see  Chapter 32) activity, results in an inability to  assemble both chylomicrons in the intestine  and VLDL particles in the liver.    The fact that a number of different abnormal lipoprotein profiles were found in  Cora Nari and her siblings, and that each had evidence of coronary artery dis-  ease, suggests that Cora has familial combined hyperlipidemia (FCH). This  diagnostic impression is further supported by the finding that Cora's profile of lipid  abnormalities appeared to change somewhat from one determination to the next, a  characteristic of FCH. This hereditary disorder of lipid metabolism is believed to be quite  common, with an estimated prevalence of about 1 per 100 population.   The mechanisms for FCH are incompletely understood but may involve a genetically  determined increase in the production of apoprotein B-100. As a result, packaging of  VLDL is increased, and blood VLDL levels may be elevated. Depending on the efficiency  of lipolysis of VLDL by LPL, VLDL levels may be normal and LDL levels may be elevated,  or both VLDL and LDL levels may be high. In addition, the phenotypic expression of FCH  in any given family member may be determined by the degree of associated obesity, the  diet, the use of specific drugs, or other factors that change over time.   Fig. 33.21. Composition of a typical VLDL  particle. The major component is triacylglyc-  erol (TG). C = cholesterol; CE = cholesterol  ester; PL = phospholipid.   Fig. 33.23. Synthesis, processing, and secre-  tion of VLDL. Proteins synthesized on the  rough endoplasmic reticulum (RER) are pack-  aged with triacylglycerols in the ER and Golgi  complex to form VLDL. VLDL are transported  to the cell membrane in secretory vesicles and  secreted by endocytosis. Blue dots represent  VLDL particles. An enlarged VLDL particle is  depicted at the bottom of the figure.    Fig. 33.22. Synthesis of VLDL from glucose in the liver. G-6-P = glucose 6-phosphate;  F-6-P = fructose 6-phosphate; F-1,6-BP = fructose 1 ,6-bisphosphate; FA = fatty acyl  group; TG = triacylglycerol.   Lipoprotein lipase (LPL), which is attached to the basement membrane proteoglycans  of capillary endothelial cells, cleaves the triacylglycerols in both VLDL and chylomi-  crons, forming fatty acids and glycerol. The C-II apoprotein, which these lipoproteins  obtain from HDL, activates LPL. The low K m of the muscle LPL isozyme permits  muscle to use the fatty acids of chylomicrons and VLDL as a source of fuel even when  the blood concentration of these lipoproteins is very low. The isozyme in adipose tis-  sue has a high K m and is most active after a meal, when blood levels of chylomicrons  and VLDL are elevated. The fate of the VLDL particle after triglyceride has been  removed by LPL is the generation of an IDL particle (intermediate-density lipopro-  tein), which can further lose triglyceride to become an LDL particle (low-density  lipoprotein). The fate of the IDL and LDL particles is discussed in Chapter 34.   ^ Fatty acids for VLDL synthesis in the liver may be obtained from the blood or they  may be synthesized from glucose. In a healthy individual, the major source of the  fatty acids of VLDL triacylglycerol is excess dietary glucose. In individuals with  diabetes mellitus, fatty acids mobilized from adipose triacylglycerols in excess of the oxida-  tive capacity of tissues are a major source of the fatty acids re-esterified in liver to VLDL tri-  acylglycerol. These individuals frequently have elevated levels of blood triacylglycerols.   In alcoholism, NADH levels in the liver are elevated (see Chapter 25). High lev-  els of NADH inhibit the oxidation of fatty acids. Therefore, fatty acids, mobilized  from adipose tissue, are re-esterified to glycerol in the liver, forming triacyl-  glycerols, which are packaged into VLDL and secreted into the blood. Elevated VLDL is  frequently associated with chronic alcoholism. As alcohol-induced liver disease pro-  gresses, the ability to secrete the triacylglycerols is diminished, resulting in a fatty liver.    CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   After a meal, the triacylglycerol stores of adipose tissue increase (Fig. 33.24).  Adipose cells synthesize LPL and secrete it into the capillaries of adipose tissue  when the insulin/glucagon ratio is elevated. This enzyme digests the triacylglyc-  erols of both chylomicrons and VLDL. The fatty acids enter adipose cells and are  activated, forming fatty acyl Co A, which reacts with glycerol 3 -phosphate to form  triacylglycerol by the same pathway used in the liver (see Fig. 33.20). Because  adipose tissue lacks glycerol kinase and cannot use the glycerol produced by LPL,  the glycerol travels through the blood to the liver, which uses it for the synthesis  of triacylglycerol. In adipose cells, glycerol 3-phosphate is derived from glucose.   In addition to stimulating the synthesis and release of LPL, insulin stimulates  glucose metabolism in adipose cells. Insulin leads to the activation of the gly-  colytic enzyme phosphofructokinase- 1 by an activation of PFK-2, which increases  fructose 2,6-bisphosphate levels. Insulin also stimulates the dephosphorylation of  pyruvate dehydrogenase, so that the pyruvate produced by glycolysis can be oxi-  dized in the TCA cycle. Furthermore, insulin stimulates the conversion of glucose  to fatty acids in adipose cells, although the liver is the major site of fatty acid syn-  thesis in humans.   During fasting, the decrease of insulin and the increase of glucagon cause cAMP  levels to rise in adipose cells, stimulating lipolysis (Fig. 33.25). Protein kinase A  phosphorylates hormone- sensitive lipase to produce a more active form of the  enzyme. Hormone- sensitive lipase, also known as adipose triacylglycerol lipase,  cleaves a fatty acid from a triacylglycerol. Subsequently, other lipases complete the  process of lipolysis, and fatty acids and glycerol are released into the blood. Simul-  taneously, to regulate the amount of fatty acids released into circulation, triglyceride  synthesis occurs along with glyceroneogenesis.    In some cases of hyperlipidemia,  1 LPL is defective. If a blood lipid  profile is performed on patients  with an LPL deficiency, which lipids would  be elevated?   Because the fatty acids of adipose  triacylglycerols come both from  chylomicrons and VLDL, we pro-  duce our major fat stores both from dietary  fat (which produces chylomicrons) and  dietary sugar (which produces VLDL). An  excess of dietary protein also can be used to  produce the fatty acids for VLDL synthesis.   The dietician carefully explained to Percy  Veere that we can become fat from eating  excess fat, excess sugar, or excess protein.    Fig. 33.24. Conversion of the fatty acid (FA) from the triacylglycerols (TG) of chylomicrons  and VLDL to the TG stored in adipose cells. Note that insulin stimulates both the transport  of glucose into adipose cells and the secretion of LPL from the cells. Glucose provides the  glycerol 3-phosphate for TG synthesis. Insulin also stimulates the synthesis and secretion of  lipoprotein lipase (LPL). Apoprotein C-II activates LPL.    Individuals with a defective LPL  have high blood triacylglycerol lev-  els. Their levels of chylomicrons   and VLDL (which contain large amounts of  triacylglycerols) are elevated because they  are not digested at the normal rate by LPL.   LPL can be dissociated from capillary  walls by treatment with heparin (a gly-  cosaminoglycan). Measurements can be  made on blood after heparin treatment to  determine whether LPL levels are abnormal.   Fig. 33.25. Mobilization of adipose triacylglycerol (TG). In the fasted state, when insulin  levels are low and glucagon is elevated, intracellular cAMP increases and activates protein  kinase A, which phosphorylates hormone- sensitive lipase (HSL). Phosphorylated HSL is  active and initiates the breakdown of adipose TG. Recall, however, that re-esterification of  fatty acids does occur, along with glyceroneogenesis, in the fasted state. HSL is also called  triacylglycerol lipase. FA = fatty acid.   The fatty acids, which travel in the blood complexed with albumin, enter cells of  muscle and other tissues, where they are oxidized to C0 2 and water to produce  energy. During prolonged fasting, acetyl Co A produced by p -oxidation of fatty  acids in the liver is converted to ketone bodies, which are released into the blood.  The glycerol derived from lipolysis in adipose cells is used by the liver during fast-  ing as a source of carbon for gluconeogenesis.   Fatty acids, obtained from the diet or synthesized from glucose, are the precursors of  glycerophospholipids and of sphingolipids (Fig. 33.26). These lipids are major com-  ponents of cellular membranes. Glycerophospholipids are also components of blood  lipoproteins, bile, and lung surfactant. They are the source of the polyunsaturated fatty  acids, particularly arachidonic acid, that serve as precursors of the eicosanoids (e.g.,  prostaglandins, thromboxanes, leukotrienes; see Chapter 35). Ether glycerophospho-  lipids differ from other glycerophospholipids in that the alkyl or alkenyl chain (an alkyl  chain with a double bond) is joined to carbon 1 of the glycerol moiety by an ether rather  than an ester bond. Examples of ether lipids are the plasmalogens and platelet activat-  ing factor. Sphingolipids are particularly important in forming the myelin sheath sur-  rounding nerves in the central nervous system, and in signal transduction.   In glycerolipids and ether glycerolipids, glycerol serves as the backbone to  which fatty acids and other substituents are attached. Sphingosine, derived from ser-  ine, provides the backbone for sphingolipids.   The initial steps in the synthesis of glycerophospholipids are similar to those of tri-  acylglycerol synthesis. Glycerol 3 -phosphate reacts with fatty acyl Co A to form   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   Fig. 33.26. Types of glycerolipids and sphingolipids. Glycerolipids contain glycerol, and sphingolipids contain sphingosine. The category of  phospholipids overlaps both glycerolipids and sphingolipids. The head groups include choline, ethanolamine, serine, inositol, glycerol, and phos-  phatidylglycerol. The carbohydrates are monosaccharides (which may be sulfated), oligosaccharides, and oligosaccharides with branches of N-  acetylneuraminic acid. P = phosphate.   phosphatidic acid. Two different mechanisms are then used to add a head group to  the molecule (Fig. 33.27). A head group is a chemical group, such as choline or ser-  ine, attached to carbon 3 of a glycerol moiety that contains hydrophobic groups,  usually fatty acids, at positions 1 and 2. Head groups are hydrophilic, either charged  or polar.   In the first mechanism, phosphatidic acid is cleaved by a phosphatase to form  diacylglycerol (DAG). DAG then reacts with an activated head group. In the syn-  thesis of phosphatidylcholine, the head group choline is activated by combining  with CTP to form CDP-choline (Fig. 33.28). Phosphocholine is then transferred to  carbon 3 of DAG, and CMP is released. Phosphatidylethanolamine is produced by  a similar reaction involving CDP-ethanolamine.   Various types of interconversions occur among these phospholipids (see Fig. 33.28).  Phosphatidylserine is produced by a reaction in which the ethanolamine moiety of   Fig. 33.27. Strategies for addition of the head group to form glycerophospholipids. In both  cases, CTP is used to drive the reaction.   3 CH 2 -0-P-0-CH 2 - CH 2 NH 3 j   Fig. 33.28. Synthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The multiple pathways reflect the importance  of phospholipids in membrane structure. For example, phosphatidylcholine (PC) can be synthesized from dietary choline when it is available. If  choline is not available, PC can be made from dietary carbohydrate, although the amount synthesized is inadequate to prevent choline deficiency.  SAM is S-adenosylmethionine, a methyl group donor for many biochemical reactions (see Chapter 40).   Phosphatidylcholine (lecithin) is  \\ J not required in the diet because it   can be synthesized in the body.  The components of phosphatidylcholine  (including choline) all can be produced, as  shown in Figure 33.28. A pathway for de  novo choline synthesis from glucose exists,  but the rate of synthesis is inadequate to  provide for the necessary amounts of  choline. Thus, choline has been classified as  an essential nutrient, with an Al (adequate  intake) of 425 mg/day in females and 550  mg/day in males.   Because choline is widely distributed in  the food supply, primarily in phosphatidyl-  choline (lecithin), deficiencies have not been  observed in humans on a normal diet. Defi-  ciencies may occur, however, in patients on  total parental nutrition (TPN), i.e., supported  solely by intravenous feeding. The fatty liv-  ers that have been observed in these  patients probably result from a decreased  ability to synthesize phospholipids for VLDL  formation.   phosphatidylethanolamine is exchanged for serine. Phosphatidylserine can be con-  verted back to phosphatidylethanolamine by a decarboxylation reaction. Phos-  phatidylethanolamine can be methylated to form phosphatidylcholine (see Chapter 40).   In the second mechanism for the synthesis of glycerolipids, phosphatidic acid  reacts with CTP to form CDP-diacylglycerol (Fig. 33.29). This compound can react  with phosphatidylglycerol (which itself is formed from the condensation of CDP-dia-  cylglycerol and glycerol 3 -phosphate) to produce cardiolipin or with inositol to pro-  duce phosphatidylinositol. Cardiolipin is a component of the inner mitochondrial  membrane. Phosphatidylinositol can be phosphorylated to form phosphatidylinositol  4,5-bisphosphate (PIP 2 ), which is a component of cell membranes. In response to sig-  nals such as the binding of hormones to membrane receptors, PIP 2 can be cleaved to  form the second messengers diacylglycerol and inositol triphosphate (see Chapter 11).   The ether glycerolipids are synthesized from the glycolytic intermediate dihydroxy-  acetone phosphate (DHAP). A fatty acyl Co A reacts with carbon 1 of DHAP, form-  ing an ester (Fig. 33.30). This fatty acyl group is exchanged for a fatty alcohol, pro-  duced by reduction of a fatty acid. Thus, the ether linkage is formed. Then the keto  group on carbon 2 of the DHAP moiety is reduced and esterified to a fatty acid. Addi-  tion of the head group proceeds by a series of reactions analogous to those for syn-  thesis of phosphatidylcholine. Formation of a double bond between carbons 1 and 2  of the alkyl group produces a plasmalogen. Ethanolamine plasmalogen is found in   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   O CH 2 -0-C-R 1  II I  Ro-C-O-C-H O  I II  CH 2 -0-P    The respiratory distress syndrome (RDS) of a premature infant such as Colleen  Lakker is, in part, related to a deficiency in the synthesis of a substance known as  lung surfactant. The major constituents of surfactant are dipalmitoylphosphatidyl-  choline, phosphatidylglycerol, apoproteins (surfactant proteins: Sp-A,B,C), and cholesterol.   O H 2 C - O - C - (CH 2 )i4 " CH 3  II I   CH 3 -(CH 2 ) 14 -C-0-CH O CH 3  I II + l 3   H 2 C -O - P - O - CH 2 - CH 2 - N - CH 3   These components of lung surfactant normally contribute to a reduction in the sur-  face tension within the air spaces (alveoli) of the lung, preventing their collapse. The pre-  mature infant has not yet begun to produce adequate amounts of lung surfactant.   Without lung surfactant,  sac collapses. Ten times  the normal pressure is  needed for re-inflation.   Lung surfactant reduces  the surface tension of  water (fluid) lining the  surface of the aveolar  sac, preventing collapse.   Less pressure is needed  to re-inflate sac when  surfactant is present.   brane lipids damaged by oxidative  free radical reactions. Arachidonic acid,  which is a polyunsaturated fatty acid, can be  peroxidatively cleaved in free radical reac-  tions to malondialdehyde and other prod-  ucts. Phospholipase A 2 recognizes the dis-  tortion of membrane structure caused by the  partially degraded fatty acid and removes it.  Acyltransferases then add back a new  arachidonic acid molecule.   myelin and choline plasmalogen in heart muscle. Platelet-activating factor (PAF) is  similar to choline plasmalogen except that an acetyl group replaces the fatty acyl  group at carbon 2 of the glycerol moiety, and the alkyl group on carbon 1 is satu-  rated. PAF is released from phagocytic blood cells in response to various stimuli. It  causes platelet aggregation, edema, and hypotension, and it is involved in the aller-  gic response. Plasmalogen synthesis occurs within peroxisomes, and, in individuals  with Zellweger's syndrome (a defect in peroxisome biogenesis), plasmalogen syn-  thesis is compromised. If severe enough, this syndrome leads to death at an early age.   Phospholipases located in cell membranes or in lysosomes degrade glycerophospho-  lipids. Phospholipase Al removes the fatty acyl group on carbon 1 of the glycerol  moiety, and phospholipase A2 removes the fatty acid on carbon 2 (Fig. 33.31). The  C2 fatty acid in cell membrane phospholipids is usually an unsaturated fatty acid,   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   which is frequently arachidonic acid. It is removed in response to signals for the syn-  thesis of eicosanoids. The bond joining carbon 3 of the glycerol moiety to phosphate  is cleaved by phospholipase C. Hormonal stimuli activate phospholipase C, which  hydrolyzes PIP 2 to produce the second messengers DAG and inositol triphosphate  (IP 3 ). The bond between the phosphate and the head group is cleaved by phospholi-  pase D, producing phosphatidic acid and the free alcohol of the head group.   Sphingolipids serve in intercellular communication and as the antigenic determi-  nants of the ABO blood group. Some are used as receptors by viruses and bacterial  toxins, although it is unlikely that this was the purpose for which they originally  evolved. Before the functions of the sphingolipids were elucidated, these com-  pounds appeared to be inscrutable riddles. They were, therefore, named for the  Sphinx of Thebes, who killed passersby that could not solve her riddle.   The synthesis of sphingolipids begins with the formation of ceramide (Fig.  33.32). Serine and palmityl Co A condense to form a product that is reduced. A  very-long-chain fatty acid (usually containing 22 carbons) forms an amide with the  amino group, a double bond is generated, and ceramide is formed.   Ceramide reacts with phosphatidylcholine to form sphingomyelin, a component  of the myelin sheath (Fig. 33.33). Ceramide also reacts with UDP-sugars to form  cerebrosides (which contain a single monosaccharide, usually galactose or glucose).  Galactocerebroside may react with 3'-phosphoadenosine 5' -phospho sulfate (PAPS,  an active sulfate donor; Figure 33.34) to form sulfatides, the major sulfolipids of the  brain.   Additional sugars may be added to ceramide to form globosides, and ganglio-  sides are produced by the addition of TV-acetylneuraminic acid (NANA) as branches  from the oligosaccharide chains (see Fig. 33.33 and Chapter 30).   Sphingolipids are degraded by lysosomal enzymes (see Chapter 30). Deficien-  cies of these enzymes result in a group of lysosomal storage diseases known as the  sphingolipidoses.   ^ If Percy Veere had continued to eat a hypercaloric diet rich in carbohydrates,  J he would have become obese. In an effort to define obesity, it has been  agreed internationally that the ratio of the patient's body weight in kilograms  and their height in meters squared (W/H 2 ) is the most useful and reproducible meas-  ure. This ratio is referred to as the body mass index or BMI. Normal men and women  fall into the range of 20 to 25. Percy's current value is 21.3 and rising.   CH 3 -(CH 2 ) 14 -C~SCoA  Palmitoyl CoA  I PLP   Fig. 33.32. Synthesis of ceramide. The  changes that occur in each reaction are high-  lighted. PLP = pyridoxal phosphate.   | Ceramide | - O - P - OCH 2 - CH 2 - N - CH 3   Fig. 33.33. Synthesis of sphingolipids from ceramide. Phosphocholine or sugars add to the  hydroxymethyl group of ceramide (in blue) to form sphingomyelins, cerebrosides, sulfatides,  globosides, and gangliosides. Gal = galactose; Glc = gucose; GalNAc = ^-acetylgalac-  tosamine; NANA = Af-acetylneuraminic acid.   Fig. 33.34. The synthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an active sul-  fate donor. PAPS donates sulfate groups to cerebrosides to form sulfatides and is also  involved in glycosaminoglycan biosynthesis (see Chapter 49). Ad = adenosine.   Approximately 36 million people in the United States have a BMI greater than  27.8 (for men) or 27.3 (for women). At this level of obesity, which is quite close to  a 20% weight increase above the "ideal" or desirable weight, an attempt at weight  loss should be strongly advised. The idea that obesity is a benign condition unless  accompanied by other risk factors for cardiovascular disease is disputed by several  long-term, properly controlled prospective studies. These studies show that obesity   CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   is an independent risk factor not only for heart attacks and strokes, but for the devel-  opment of insulin resistance, type 2 diabetes mellitus, hypertension, and gallblad-  der disease.   Percy did not want to become overweight and decided to follow his new diet  faithfully.   v Because Cora Nari's lipid profile indicated an elevation in both serum  ) triacylglycerols and LDL cholesterol, she was classified as having a com-  bined hyperlipidemia. The dissimilarities in the lipid profiles of Cora and  her two siblings, both of whom were experiencing anginal chest pain, is charac-  teristic of the multigenic syndrome referred to as familial combined hyperlipi-  demia (FCH).   Approximately 1% of the North American population has FCH. It is the most  common cause of coronary artery disease in the United States. In contrast to  patients with familial hypercholesterolemia (FH), patients with FCH do not have  fatty deposits within the skin or tendons (xanthomas) (see Chapter 34). In FCH,  coronary artery disease usually appears by the fifth decade of life.   Treatment of FCH includes restriction of dietary fat. Patients who do not respond  adequately to dietary therapy are treated with antilipidemic drugs. Selection of the  appropriate antilipidemic drugs depends on the specific phenotypic expression of  the patient's multigenic disease as manifest by their particular serum lipid profile.  In Cora's case, a decrease in both serum triacylglycerols and LDL cholesterol must  be achieved. If possible, her serum HDL cholesterol level should also be raised to a  level above 40 mg/dL.   To accomplish these therapeutic goals, her physician initially prescribed fast-  release nicotinic acid (niacin), because this agent has the potential to lower serum  triacylglycerol levels and cause a reciprocal rise in serum HDL cholesterol levels,  as well as to lower serum total and LDL cholesterol levels. The mechanisms sug-  gested for niacin's triacylglycerol-lowering action include enhancement of the  action of LPL, inhibition of lipolysis in adipose tissue, and a decrease in esterifi-  cation of triacylglycerols in the liver (see Table 34.5). The mechanism by which  niacin lowers the serum total and LDL cholesterol levels is related to the decrease  in hepatic production of VLDL. When the level of VLDL in the circulation  decreases, the production of its daughter particles, IDL and LDL, also decreases.  Cora found niacin's side effects of flushing and itching to be intolerable, and the  drug was discontinued.   Pravastatin was given instead. Pravastatin inhibits cholesterol synthesis by  inhibiting hydroxymethylglutaryl CoA (HMG-CoA) reductase, the rate-limiting  enzyme in the pathway (see Chapter 34). After 3 months of therapy, pravastatin  decreased Cora's LDL cholesterol from a pretreatment level of 175 to 122 mg/dL  (still higher than the recommended treatment goal of 100 mg/dL or less in a patient  with established coronary artery disease). Her fasting serum triacylglycerol con-  centration was decreased from a pretreatment level of 280 to 178 mg/dL (a treat-  ment goal for serum triacylglycerol when the pretreatment level is less than 500  mg/dL has not been established).   ^ Colleen Lakker suffered from respiratory distress syndrome (RDS),  ) which is a major cause of death in the newborn. RDS is preventable if pre-  maturity can be avoided by appropriate management of high-risk preg-  nancy and labor. Before delivery, the obstetrician must attempt to predict and pos-  sibly treat pulmonary prematurity in utero. For example, estimation of fetal head  circumference by ultrasonography, monitoring for fetal arterial oxygen saturation,  and determination of the ratio of the concentrations of phosphatidylcholine  (lecithin) and that of sphingomyelin in the amniotic fluid may help to identify pre-  mature infants who are predisposed to RDS (Fig. 33.35).   Fig. 33.35. Comparison of phosphatidyl-  choline and sphingomyelin in amniotic fluid.  Phosphatidylcholine is the major lipid in lung  surfactant. The concentration of phosphatidyl-  choline relative to sphingomyelin rises at 35  weeks of gestation, indicating pulmonary  maturity.   The administration of synthetic corticosteroids 48 to 72 hours before delivery of  a fetus of less than 33 weeks of gestation in women who have toxemia of pregnancy,  diabetes mellitus, or chronic renal disease may reduce the incidence or mortality of  RDS by stimulating fetal synthesis of lung surfactant.   The administration of one dose of surfactant into the trachea of the premature  infant immediately after birth may transiently improve respiratory function but does  not improve overall mortality. In Colleen's case, intensive therapy allowed her to  survive this acute respiratory complication of prematurity.   of fat an individual can store depends on the number of fat cells in the body   and the amount of triacylglycerol each cell can accommodate. In obese  individuals, both the number of fat cells and the size of the cells (i.e., the total stor-  age capacity) is greater than in individuals with no history of obesity. To fill these  stores, however, an individual must eat more than required to support the basal  metabolic rate and physical activity.   Fat cells begin to proliferate early in life, starting in the third trimester of gesta-  tion. Proliferation essentially ceases before puberty, and thereafter fat cells change  mainly in size. However, some increase in the number of fat cells can occur in adult-  hood if preadipocytes are induced to proliferate by growth factors and changes in  the nutritional state. Weight reduction results in a decrease in the size of fat cells  rather than a decrease in number. After weight loss, the amount of LPL, an enzyme  involved in the transfer of fatty acids from blood triacylglycerols to the triacylglyc-  erol stores of adipocytes, increases. In addition, the amount of mRNA for LPL also  increases. All of these factors suggest that individuals who become obese, particu-  larly those who do so early in life, will have difficulty losing weight and maintain-  ing a lower body adipose mass.   Signals that initiate or inhibit feeding are extremely complex and include psy-  chological and hormonal factors as well as neurotransmitter activity. These signals  are integrated and relayed through the hypothalamus. Destruction of specific  regions of the hypothalamus can lead to overeating and obesity or to anorexia and  weight loss. Overeating and obesity are associated with damage to the ventromedial  or the paraventricular nucleus, whereas weight loss and anorexia are related to dam-  age to more lateral hypothalamic regions. Compounds that act as satiety signals  have been identified in brain tissue and include leptin and glucagon-like peptide- 1  (GLP-1). Appetite suppressors developed from compounds such as these may be  used in the future for the treatment of obesity.   Recently it has become apparent that the adipocyte, in addition to storing triacyl-  glycerol, secretes hormones that regulate both glucose and fat metabolism. The hor-  mones leptin, resistin (resists insulin action), and adiponectin (also known as Acrp30)  are all secreted from adipocytes under different conditions. The role of these hormones  has been best understood in mouse models; unfortunately, extrapolation to the human  condition has been difficult. In mice, leptin is released from adipocytes as triglyceride  levels increase and signals the hypothalamus to reduce eating and to increase physical  activity. Mice lacking the ability to secrete leptin (the ob mouse), or respond to leptin  (the db mouse) are obese. Injecting leptin into ob mice allows them to lose weight.   The adipocytes in mice have been shown to release a hormone known as resistin.  This hormone may contribute to insulin resistance in these animals. The mechanism  by which resistin causes an insensitivity of cells to the actions of insulin is unknown.  It is of great interest, however, that the class of drugs known as thiazolidinediones,  which are given to individuals with type 2 diabetes, suppress resistin transcription,  reduce resistin levels, and increase sensitivity to insulin in these patients. Addition-    CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS   ally, thiazolidinediones may upregulate adipose PEPCK, resulting in a reduced fatty  acid output from the adipocyte because of increased glyceroneogenesis.   In humans, adiponectin is secreted from adipocytes in inverse proportion to their  adipose mass, lean individuals secreting more adiponectin than obese individuals.  This is the exact opposite of leptin secretion. The effects of adiponectin, and how it  interacts with resistin and leptin, are active areas of current research.   Further complicating the issue of glucose and lipid homeostasis is the effect of  nuclear receptors known as peroxisome proliferator activated receptors (PPAR).  These nuclear receptors (see Chapter 10) exist in three forms; a, (3/8, and 7. PPAR7  is found in highest levels in adipocytes, and activation of the receptor leads to gene  transcription, which is necessary for adipocyte differentiation and regulation of  lipid metabolism. The thiazolidinediones activate PPAR7, which leads to a decrease  in circulating resistin levels. Understanding more about the physiologic regulators  of PPAR7 is also an active area of research. The role of PPAR in liver is discussed  in Chapter 46.   Although an increase in food intake beyond the daily requirements results in an  increase in body weight and in fat stores, there is a large variation among individu-  als in the amount of weight gained for a given number of excess calories consumed.  Both genetic and environmental factors influence the development of obesity. Stud-  ies of identical twins who were purposely overfed showed that the amount of weight  gained was more similar within sets than between sets. Other studies of identical  and fraternal twins, in which the members of a set were reared apart, support the  conclusion that heredity plays a major role in determining body weight.   Beale EG, Hammer RE, Antoine B, Forest C. Glyceroneogenesis comes of age. FASEB J.  2002;16:1695-1696.   Berg AH, Combs TP, Scherer PE. ACRP30/adiponectin: an adipokine regulating glucose and lipid  metabolism. Trends in Endocrinology and Metabolism 2002;13:84-89.   Bouchard C, Tremblay A, Despres J-P, et al. The response to long-term overfeeding in identical twins.  N Engl J Med 1990;322:1477-1482.   Girard J, Perderbeau D, Foufelle F, Prip-Buus C, Ferre P. Regulation of lipogenic enzyme gene expres-  sion by nutrients and hormones. FASEB J 1994;8:36-42.   Kern PA, Ong JM, Bahman S, Carty J. The effects of weight loss on the activity and expression of adi-  pose-tissue lipoprotein lipase in very obese humans. N Engl J Med 1990;322:1053-1059.   Picard F, Auwerx, J. PPAR^and glucose homeostasis. Annu Rev Nutr 2002;22:167-197.   Steppen CM et al. The hormone resistin links obesity to diabetes. Nature 2001;409:307-312.   Stunkard A, Harris J, Pedersen N, McClearn G. The body-mass index of twins who have been reared  apart. N Engl J Med 1990;322:1483-1487.   1. Which of the following is involved in the synthesis of triacylgycerols in adipose tissue?   2. A molecule of palmitic acid, attached to carbon 1 of the glycerol moiety of a triacylglycerol, is ingested and digested. It passes  into the blood, is stored in a fat cell, and ultimately is oxidized to carbon dioxide and water in a muscle cell. Choose the molec-  ular complex in the blood in which the palmitate residue is carried from the lumen of the gut to the surface of the gut epithe-  lial cell.   3. A patient with hyperlipoproteinemia would be most likely to benefit from a low-carbohydrate diet if the lipoproteins that are  elevated in blood are which of the following?   (A) It is converted to ceramide by reacting with a UDP-sugar.   5. Newly synthesized fatty acids are not immediately degraded because of which of the following?   (A) Tissues that synthesize fatty acids do not contain the enzymes that degrade fatty acids.   (C) In the presence of insulin, the key fatty acid degrading enzyme is not induced.   (D) Newly synthesized fatty acids cannot be converted to their Co A derivatives.   (E) Transport of fatty acids into mitochondria is inhibited under conditions in which fatty acids are being synthesized.   Cholesterol is one of the most highly recognized molecules in human biology, in  part because of a direct relationship between its concentrations in blood and tis-  sues and the development of atherosclerotic vascular disease. Cholesterol, which  is transported in the blood in lipoproteins because of its absolute insolubility in  water, serves as a stabilizing component of cell membranes and as a precursor of  the bile salts and steroid hormones. Precursors of cholesterol are converted to  ubiquinone, dolichol, and, in the skin, to cholecalciferol, the active form of vita-  min D. As a major component of blood lipoproteins, cholesterol can appear in  its free, unesterified form in the outer shell of these macromolecules and as cho-  lesterol esters in the lipoprotein core.   Cholesterol is obtained from the diet or synthesized by a pathway that occurs  in most cells of the body, but to a greater extent in cells of the liver and intestine.  The precursor for cholesterol synthesis is acetyl Co A, which can be produced  from glucose, fatty acids, or amino acids. Two molecules of acetyl CoAform ace-  toacetyl Co A, which condenses with another molecule of acetyl CoA to form  hydroxymethylglutaryl CoA (HMG-CoA). Reduction of HMG-CoA produces  mevalonate. This reaction, catalyzed by HMG-CoA reductase, is the major rate-  limiting step of cholesterol synthesis. Mevalonate produces isoprene units that  condense, eventually forming squalene. Cyclization of squalene produces the  steroid ring system, and a number of subsequent reactions generate cholesterol.  The adrenal cortex and the gonads also synthesize cholesterol in significant  amounts and use it as a precursor for steroid hormone synthesis.   Cholesterol is packaged in chylomicrons in the intestine and in very-low-den-  sity lipoprotein (VLDL) in the liver. It is transported in the blood in these lipopro-  tein particles, which also transport triacy I glycerols. As the triacylglycerols of the  blood lipoproteins are digested by lipoprotein lipase, chylomicrons are converted  to chylomicron remnants, and VLDL is converted to intermediate-density  lipoprotein (IDL) and subsequently to low-density lipoprotein (LDL). These prod-  ucts return to the liver, where they bind to receptors in cell membranes and are  taken up by endocytosis and digested by lysosomal enzymes. LDL is also endocy-  tosed by nonhepatic (peripheral) tissues. Cholesterol and other products of lyso-  somal digestion are released into the cellular pools. The liver uses this recycled  cholesterol, and the cholesterol that is synthesized from acetyl CoA, to produce  VLDL and to synthesize bile salts.   Intracellular cholesterol obtained from blood lipoproteins decreases the synthesis  of cholesterol within cells, stimulates the storage of cholesterol as cholesterol esters,  and decreases the synthesis of LDL receptors. LDL receptors are found on the sur-  face of the cells and bind various classes of lipoproteins prior to endocytosis.   Although high-density lipoprotein (HDL) contains triacylglycerols and choles-  terol, its function is very different from that of the chylomicrons and VLDL, which  transport triacylglycerols. HDL exchanges proteins and lipids with the other  lipoproteins in the blood. HDL transfers apolipoprotein E (apoE) and apoC u to  chylomicrons and VLDL. After digestion of the VLDL triacylglycerols, apoE and  apoC n are transferred back to HDL. In addition, HDL obtains cholesterol from   other lipoproteins and from cell membranes and converts it to cholesterol esters  by the lecithin '.cholesterol acyltransf erase (LCAT) reaction. Then HDL either  directly transports cholesterol and cholesterol esters to the liver or transfers cho-  lesterol esters to other lipoproteins via the cholesterol ester transfer protein  (CETP). Ultimately, lipoprotein particles carry the cholesterol and cholesterol  esters to the liver, where endocytosis and lysosomal digestion occur Thus,  u reverse cholesterol transport" (i.e., the return of cholesterol to the liver) is a  major function of HDL.   Elevated levels of cholesterol in the blood are associated with the formation of  atherosclerotic plaques that can occlude blood vessels, causing heart attacks and  strokes. Although high levels ofLDL cholesterol are especially atherogenic, high  levels of HDL cholesterol are protective because HDL particles are involved in  the process of removing cholesterol from tissues, such as the lining cells of ves-  sels, and returning it to the liver.   Bile salts, which are produced in the liver from cholesterol obtained from the  blood lipoproteins or synthesized from acetyl Co A, are secreted into the bile.  They are stored in the gallbladder and released into the intestine during a meal.  The bile salts emulsify dietary triacylglycerols, thus aiding in digestion. The  digestive products are absorbed by intestinal epithelial cells from bile salt  micelles, tiny microdroplets that contain bile salts at their water interface. After  the contents of the micelles are absorbed, most of the bile salts travel to the  ileum, where they are resorbed and recycled by the liver. Less than 5% of the  bile salts that enter the lumen of the small intestine are eventually excreted in  the feces.   Although the fecal excretion of bile salts is relatively low, it is a major means  by which the body disposes of the steroid nucleus of cholesterol. Because the ring  structure of cholesterol cannot be degraded in the body, it is excreted mainly in  the bile as free cholesterol and bile salts.   The steroid hormones, derived from cholesterol, include the adrenal cortical  hormones (e.g., Cortisol, aldosterone, and the adrenal sex steroids dehy-  droepiandrosterone [DHEA] and androstenedione) and the gonadal hormones  (e.g., the ovarian and testicular sex steroids, such as testosterone and estrogen).   At his current office visit, Ivan Applebod's case was reviewed by his  physician. Mr. Applebod has several of the major risk factors for coronary  heart disease (CHD). These include a sedentary lifestyle, marked obesity,  hypertension, hyperlipidemia, and early non-insulin-dependent diabetes mellitus  (NIDDM). Unfortunately, he has not followed his doctor's advice with regard to a  diabetic diet designed to affect a significant loss of weight, nor has he followed an  aerobic exercise program. As a consequence, his weight has gone from 270 to 281  lb. After a 14-hour fast, his serum glucose is now 214 mg/dL (normal, <110), and  his serum total cholesterol level is 314 mg/dL (desired level is 200 or less). His  serum triacylglycerol level is 295 mg/dL (desired level is 150 or less), and his serum  HDL cholesterol is 24 mg/dL (desired level is >40 for a male). His calculated serum  LDL cholesterol level is 231 mg/dL (desired level for a person with two or more  risk factors for CHD is 130 mg/dL or less, unless one of the risk factors is diabetes  mellitus, in which case, the LDL cholesterol level should be < 100 mg/dL).   • Ann Jeina was carefully followed by her physician after she survived her  flP ) heart attack. Before discharge from the hospital, after a 14-hour fast, her   " serum triacylglycerol level was 158 mg/dL (slightly above the upper range  of normal), and her HDL cholesterol level was low at 32 mg/dL (normal for women  is >50). Her serum total cholesterol level was elevated at 420 mg/dL (reference  range, <200 for a female with known CHD). From these values, her LDL choles-  terol level was calculated to be 356 mg/dL (desirable level for a person with estab-  lished heart disease is <100).   Both of Ms. Jeina's younger brothers had "very high" serum cholesterol levels,  and both had suffered heart attacks in their mid-forties. With this information, a ten-  tative diagnosis of familial hypercholesterolemia, type IIA was made, and the  patient was started on a step I diet as recommended by the National Cholesterol  Education Program (NCEP) Adult Treatment Panel III. This panel recommends that  decisions with regard to when dietary and drug therapy are initiated based on the  serum LDL cholesterol level, as depicted in Table 34. 1 .   Because a Step I diet (Table 34.2) usually lowers serum total and LDL choles-  terol levels by no more than 15%, it is likely that Ms. Jeina's diet will eventually  have to be further restricted in cholesterol and fat and that one or more lipid-lower-  ing drugs will have to be added to her treatment plan.   • Vera Leizd is a 34-year-old woman in whom pubertal changes began at  flP ) age 12, leading to the development of normal secondary sexual character-   " istics and the onset of menses at age 13. Her menstrual periods occurred  on a monthly basis over the next 7 years, but the flow was scant. At age 20, she  noted a gradual increase in the intermenstrual interval from her normal of 28 days  to 32 to 38 days. The volume of her menstrual flow also gradually diminished. After  7 months, her menstrual periods ceased. She complained of increasing oiliness of  her skin, the appearance of acnelike lesions on her face and upper back, and the  appearance of short dark terminal hairs on the mustache and sideburn areas of her  face. The amount of extremity hair also increased, and she noticed a disturbing loss  of hair from her scalp.   Until recently, the concentration of  LDL cholesterol could only be  directly determined by sophisticated  laboratory techniques not available for routine  clinical use. As a consequence, the LDL choles-  terol concentration in the blood was derived  indirectly by using the Friedewald formula: the  sum of the HDL cholesterol level and the tria-  cylglycerol (TG) level divided by 5 (which  gives an estimate of the VLDL cholesterol level)  subtracted from the total cholesterol level.   This equation yields inaccurate LDL choles-  terol levels 15 to 20% of the time and fails  completely when serum triacylglycerol levels  exceed 400 mg/dL.   A recently developed test called "LDL  direct" isolates LDL cholesterol by using a  special immunoseparation reagent. Not only is  this direct assay for LDL cholesterol more  accurate than the indirect Friedewald calcula-  tion, it also is not affected by mildly to moder-  ately elevated serum triacylglycerol levels and  can be used for a patient who has not fasted. It  does not require the expense of determining  serum total cholesterol, HDL cholesterol, and  triacylglycerol levels.   Cholesterol absorption by intestinal cells is a key regulatory point in human sterol  metabolism because it ultimately determines what percentage of the 1,000 mg of  biliary cholesterol produced by the liver each day and what percentage of the   Table 34.1. ATP III: LDL-C Goals and Cut Points for Therapy in Different Risk  Categories   Source: Executive summary of the third report of the National Cholesterol Education Programs (NCEP)  Expert panel on detection, evaluation, and treatment of high blood cholesterol in Adults (Adult Treatment  Panel III). Final Report, Circulation 2002;106:3145-3457.   Based on: NICER Second Report of the Adult Treatment Panel, JAMA, 1993;269(23):301 5-3023.  a The Step II diet is applied if 3 months on the Step I diet has failed to reduce blood cholesterol to the  desired level (see Table 34.1).   b Except for the values given in mg/day, all the values are percentage of total calories eaten daily.    Fig. 34.1. The basic ring structure of sterols; the  perhy drocy clopentanophenanthrene nucleus .  Each ring is labeled either A, B, C, or D.   300 mg of dietary cholesterol entering the gut per day is eventually absorbed into  the blood. In normal subjects, approximately 55% of this intestinal pool enters the  blood through the enterocyte each day. The details of cholesterol absorption from  dietary sources was outlined in Chapter 32.   Although the absorption of cholesterol from the intestinal lumen is a diffusion-  controlled process, there is also a mechanism to remove unwanted or excessive  cholesterol and plant sterols from the enterocyte. The transport of sterols out of the  enterocyte, and into the lumen, is related to the products of genes that code for the  adenosine triphosphate (ATP)-binding cassette (ABC) protein family, ABC1,  ABCG5, and ABCG8. These proteins couple ATP hydrolysis to the transport of  unwanted or excessive cholesterol and plant sterols (phytosterols) from the entero-  cyte back into the gut lumen. Cholesterol cannot be metabolized to C0 2 and water  and is, therefore, principally eliminated from the body in the feces as unreabsorbed  sterols and bile acids. ABC protein expression increases the amount of sterols pres-  ent in the gut lumen, with the potential to increase elimination of the sterols into  the feces. Patients with a condition known as phytosterolemia (a rare autosomal  recessive disease, also known as sitosterolemia) have a defect in the function of  either ABCG5 or ABCG8 in the enterocytes, thereby leading to the accumulation  of cholesterol and phytosterols within these cells. These eventually reach the  bloodstream, markedly elevating the level of cholesterol and phytosterol in the  blood. This accounts for the increased cardiovascular morbidity in individuals with  this disorder. From these experiments of nature, it is clear that agents that either  amplify the expression of the ABC proteins within enterocytes, or block choles-  terol absorption from the lumen, have therapeutic potential in the treatment of  patients with hypercholesterolemia. Ezetimibe, now available for clinical use, is a  compound that is structurally different from the sterols. Its primary action in low-  ering serum cholesterol levels is to block cholesterol absorption through a specific  but as yet poorly characterized cholesterol absorption mechanism in the brush bor-  der of enterocytes. It also may induce ABC protein expression, but this action is  relatively unimportant in reducing net cholesterol absorption. The reduction of  cholesterol absorption from the intestinal lumen has been shown to reduce blood  levels of LDL cholesterol.   Cholesterol is an alicyclic compound whose basic structure includes the perhy-  drocyclopentanophenanthrene nucleus containing four fused rings (Figure 34.1).  In its "free" form, the cholesterol molecule contains 27 carbon atoms, a simple  hydroxyl group at C3, a double bond between C5 and C6, an eight-membered  hydrocarbon chain attached to carbon 17 in the D ring, a methyl group (carbon  19) attached to carbon 10, and a second methyl group (carbon 18) attached to car-  bon 13 (Figure 34.2).   Approximately one third of plasma cholesterol exists in the free (or unesterified)  form. The remaining two thirds exists as cholesterol esters in which a long-chain  fatty acid (usually linoleic acid) is attached by ester linkage to the hydroxyl group  at C-3 of the A ring. The proportions of free and esterified cholesterol in the blood  can be measured using methods such as high-performance liquid chromatography  (HPLC).   The structure of cholesterol suggests that its synthesis involves multimolecular  interactions; yet all of the 27 carbons are derived from one precursor, acetyl Co A.  Acetyl Co A can be obtained from several sources, including the beta oxidation of  fatty acids, the oxidation of ketogenic amino acids, such as leucine and lysine, and  the pyruvate dehydrogenase reaction. Carbons 1, 2, 5, 7, 9, 13, 15, 18, 19, 20, 22,  24, 26, and 27 of cholesterol are derived from the methyl group of acetyl CoA and  the remaining 12 carbons of cholesterol from the carboxylate atom of acetyl CoA.   The synthesis of cholesterol requires significant reducing power, which is sup-  plied in the form of NADPH. The latter is provided by glucose-6-phosphate dehy-  drogenase and 6-phosphogluconate dehydrogenase of the hexose monophosphate  shunt pathway (see Chapter 29). Cholesterol synthesis occurs in the cytosol, requir-  ing hydrolysis of high-energy thioester bonds of acetyl CoA and phosphoanhydride  bonds of ATP. Its synthesis occurs in four stages.   The first stage of cholesterol synthesis leads to the production of the intermediate  mevalonate (Fig. 34.3). The synthesis of mevalonate is the committed, rate-limiting  step in cholesterol formation. In this cytoplasmic pathway, two molecules of acetyl  CoA condense, forming acetoacetyl CoA, which then condenses with a third mole-  cule of acetyl CoA to yield the 6-carbon compound p-hydroxy- (3-methylglutaryl-  CoA (HMG-CoA). The HMG-CoA synthase in this reaction is present in the  cytosol and is distinct from the mitochondrial HMG-CoA synthase that catalyses  HMG-CoA synthesis involved in ketone body production. The committed step and  major point of regulation of cholesterol synthesis in stage 1 involves reduction of  HMG-CoA to mevalonate, a reaction catalyzed by HMG-CoA reductase, an enzyme  embedded in the membrane of the endoplasmic reticulum. HMG-CoA reductase  contains eight membrane-spanning domains, and the amino terminal domain, which  faces the cytoplasm, contains the enzymatic activity. The reducing equivalents for  this reaction are donated by two molecules of NADPH. The regulation of the activ-  ity of HMG-CoA reductase is controlled in multiple ways.   CH 3 - C - OH glutaryl CoA  ^ H (HMG-CoA)   Fig. 34.3. The conversion of three molecules  of acetyl-CoA to mevalonic acid.    Ann Jeina's serum total and LDL  cholesterol levels improved only  modestly after 3 months on a Step I  diet. Three additional months on a more severe  low-fat diet (Step II diet) brought little further  improvement. The next therapeutic step would  be to initiate lipid-lowering drug therapy (see  Table 34.5).   The rate of synthesis of HMG-CoA reductase messenger RNA (mRNA) is con-  trolled by one of the family of sterol regulatory element binding proteins  (SREBPs)(Fig. 34.4A). These transcription factors belong to the helix-loop-helix-   Fig. 34.4. Regulation of HMG-CoA reductase activity. See text for details. A. Transcriptional  control. B. Regulation by proteolysis. C. Regulation by phosphorylation.   leucine zipper (bHIH-Zip) family of transcription factors that directly activate the  expression of more than 30 genes dedicated to the synthesis and uptake of choles-  terol, fatty acids, triacylglycerols, and phospholipids as well as the production of the  NADPH cof actors required to synthesize these molecules.   SREBPl-a specifically enhances transcription of genes required for HMG-CoA  reductase expression by binding to the sterol regulatory element (SRE) upstream of  the reductase gene. When bound, the rate of transcription is increased. SREBPs,  after synthesis, are integral ER proteins, and the active component of the protein is  released by two proteases, SCAP (SREBP cleavage-activating protein) and S2P  (site 2 protease). Once released, the active amino terminal component travels to the  nucleus to bind to SREs. The soluble SREBPs are rapidly turned over and need to  be continuously produced to effectively stimulate reductase mRNA transcription.  When cytoplasmic sterol levels rise, the sterols bind to SCAP and inactivate it,  thereby leading to a decrease in transcription of the reductase gene, and less reduc-  tase protein being produced.   Rising levels of cholesterol and bile salts in cells that synthesize these molecules  also may cause a change in the oligomerization state of the membrane domain of  HMG-CoA reductase, rendering the enzyme more susceptible to proteolysis (see  Fig. 34. 4B). This, in turn, decreases its activity. The membrane domains of  HMG-CoA reductase contain sterol-sensing regions, which are similar to those  in SCAR   In addition to the inductive and repressive influences cited above, the activity of  the reductase is also regulated by phosphorylation and dephosphorylation (see  Fig. 34. 4C). Elevated glucagon levels increase phosphorylation of the enzyme,  thereby inactivating it, whereas hyperinsulinemia increases the activity of the  reductase by activating phosphatases, which dephosphorylate the reductase.  Increased levels of intracellular sterols also may increase phosphorylation of  HMG-CoA reductase, thereby reducing its activity as well (feedback suppres-  sion). Thyroid hormone also increases enzyme activity, whereas glucocorticoids  decrease its activity. The enzyme that phosphorylates HMG-CoA reductase is the  adenosine monophosphate (AMP) -activated protein kinase, which itself is regu-  lated by phosphorylation by the AMP-activated protein kinase kinase. Thus, cho-  lesterol synthesis decreases when ATP levels are low and increases when ATP lev-  els are high. This will become very clear once the further reactions of the  biosynthetic pathway of cholesterol are discussed.   In the second stage of cholesterol synthesis, three phosphate groups are trans-  ferred from three molecules of ATP to mevalonate (Fig. 34.5). The purpose of  these phosphate transfers is to activate both carbon 5 and the hydroxyl group on  carbon 3 for further reactions in which these groups will leave the molecule. The  phosphate group attached to the C-3 hydroxyl group of mevalonate in the 3-  phospho-5-pyrophosphomevalonate intermediate is removed along with the car-  boxyl group on C-l. This produces a double bond in the 5 -carbon product, A 3 -  isopentenyl pyrophosphate, the first of two activated isoprenes necessary for the  synthesis of cholesterol. The second activated isoprene is formed when A 3 -  isopentenyl pyrophosphate is isomerized to dimethylallyl pyrophosphate (see  Fig. 34.5).   C. Stage 3: Condensation of Six Activated 5-Carbon  Isoprenes to Form the 30-Carbon Squalene   The next stage in the biosynthesis of cholesterol involves the head-to-tail conden-  sation of isopentenylpyrophosphate and dimethylallyl pyrophosphate. In this reac-  tion, one pyrophosphate group is displaced, and a 10-carbon chain, known as ger-  anyl pyrophosphate, is generated (Fig. 34.6). (The "head" refers to the end to which  pyrophosphate is joined.) Geranyl pyrophosphate then undergoes another head-to-  tail condensation with isopentenyl pyrophosphate, resulting in the formation of the  15-carbon intermediate, farnesyl pyrophosphate. After this, two molecules of farne-  syl pyrophosphate undergo a head-to-head fusion, and both pyrophosphate groups  are removed to form squalene, a compound first isolated from the liver of sharks  (genus Squalus). Squalene contains 30 carbons (24 in the main chain and 6 in the  methyl group branches; see Fig. 34.6).   Farnesyl and geranyl groups can  \m ) form covalent bonds with proteins,  particularly the G proteins and cer-  tain protooncogene products involved in signal  transduction. These hydrophobic groups  anchor the proteins in the cell membrane.   -O - C - CH 2 - C - CH 2 - CH 2 OH  OH  Mevalonate  -ATP   "0 - C - CH 2 - C - CH 2 - CH 2 - O - P -O"  I I   "0 - C - CH 2 - C - CH 2 - CH 2 - O - P -O - P - 0"  I I I   "0 - C - CH 2 - C - CH 2 - CH 2 - O - P -O - P - 0"  h . I I   CH 2 = C - CH 2 - CH 2 - O - P -O - P - O"  II   Fig. 34.5. The formation of activated isoprene units (A 3 -isopentenyl pyrophosphate and  dimethylallyl pyrophosphate) from mevalonic acid. Note the large ATP requirement for these  steps.   D. Stage 4: Conversion of Squalene to the Four-Ring  Steroid Nucleus   The enzyme squalene monooxygenase adds a single oxygen atom from 0 2 to the  end of the squalene molecule, forming an epoxide. NADPH then reduces the other  oxygen atom of 0 2 to H 2 0. The unsaturated carbons of the squalene 2, 3- epoxide  are aligned in a way that allows conversion of the linear squalene epoxide into a  cyclic structure. The cyclization leads to the formation of lanosterol, a sterol with  the four-ring structure characteristic of the steroid nucleus. A series of complex   Fig. 34.6. The formation of squalene from six isoprene units. The activation of the isoprene  units drives their condensation to form geranyl pyrophosphate, farnesyl pyrophosphate, and  squalene.   reactions, containing many steps and elucidated in the late 1950s, leads to the for-  mation of cholesterol (Fig. 34.7).   Almost all mammalian cells are capable of producing cholesterol. Most of the  biosynthesis of cholesterol, however, occurs within liver cells, although the gut, the  adrenal cortex, and the gonads (as well as the placenta in pregnant women) also pro-  duce significant quantities of the sterol. Although a fraction of hepatic cholesterol  is used for the synthesis of hepatic membranes, the bulk of synthesized cholesterol  is secreted from the hepatocyte as one of three moieties: cholesterol esters, biliary  cholesterol, or bile acids. Cholesterol ester production in the liver is catalyzed by  acyl-CoA-cholesterol acyl transferase (AC AT). AC AT catalyzes the transfer of a  fatty acid from coenzyme A to the hydroxyl group on carbon 3 of cholesterol  (Fig. 34.8). Cholesterol esters are more hydrophobic than is free cholesterol. The  liver packages some of the esterified cholesterol into the hollow core of lipopro-  teins, primarily VLDL. VLDL is secreted from the hepatocyte into the blood and  transports the cholesterol esters (and triacylglycerols, phospholipids, apoproteins,  etc.) to the tissues that require greater amounts of cholesterol than they can synthe-  size de novo. These tissues then use the cholesterol for the synthesis of membranes,   Fig. 34.7. The conversion of squalene to cho-  lesterol. Squalene is shown in a different con-  formation than that of Fig. 34.6 to better indi-  cate how the cyclization reaction occurs.   Fig. 34.8. The ACAT reaction, producing cho-  lesterol esters. ACAT = acyl-CoA:cholesterol  acyl transferase.   for the formation of steroid hormones, and for the biosynthesis of vitamin D. The  residual cholesterol esters not used in these ways are stored in the liver for later use.  The hepatic cholesterol pool also serves as a source of cholesterol for the synthesis  of the relatively hydrophilic bile acids and their salts. These derivatives of choles-  terol are highly effective detergents because they contain both polar and nonpolar  regions. They are introduced in the biliary ducts of the liver. They are stored and  concentrated in the gallbladder and later discharged into the gut in response to the  ingestion of food. They aid in the digestion of intraluminal lipids by forming  micelles with them, which increases the surface area of lipids exposed to the diges-  tive action of intraluminal lipases. Free cholesterol also enters the gut lumen via the  biliary tract (approximately 1,000 mg daily, which mixes with 300 mg dietary cho-  lesterol to form an intestinal pool, roughly 55% of which is resorbed by the entero-  cytes and enters the bloodstream daily). On a low-cholesterol diet, the liver will syn-  thesize approximately 800 mg cholesterol per day to replace bile salts and  cholesterol lost from the enterohepatic circulation into the feces. Conversely, a  greater intake of dietary cholesterol suppresses the rate of hepatic cholesterol syn-  thesis (feedback repression).   Bile salts are synthesized in the liver from cholesterol by reactions that hydroxy-  late the steroid nucleus and cleave the side chain. In the first reaction, an a-  hydroxyl group is added to carbon 7 (on the a side of the B ring). The activity of  the 7 a-hydroxylase that catalyzes this rate-limiting step is decreased by bile salts  (Fig. 34.9).   In subsequent steps, the double bond in the B ring is reduced, and an additional  hydroxylation may occur. Two different sets of compounds are produced. One set  has a-hydroxyl groups at positions 3,7, and 12, and produces the cholic acid series   Fig. 34.9. The reaction catalyzed by 7a-hydroxylase. An a-hydroxyl group is formed at posi-  tion 7 of cholesterol. This reaction, which is inhibited by bile salts, is the rate-limiting step  in bile salt synthesis.   Fig. 34.10. Synthesis of bile salts. Two sets of bile salts are generated; one with a-hydroxyl  groups at positions 3 and 7 (the chenocholate series), and the other with a-hydroxyls at posi-  tions 3, 7 and 12 (the cholate series).   of bile salts. The other set has a-hydroxyl groups only at positions 3 and 7 and pro-  duces the chenocholic acid series (Fig. 34.10). Three carbons are removed from the  side chain by an oxidation reaction. The remaining 5 -carbon fragment attached to  the ring structure contains a carboxyl group (see Fig. 34.10).   The pK of the bile acids is approximately 6. Therefore, in the contents of the  intestinal lumen, which normally have a pH of 6, approximately 50% of the mole-  cules are present in the protonated form, and 50% are ionized, which forms bile  salts. (The terms bile acids and bile salts are often used interchangeably, but bile  salts actually refer to the ionized form of the molecule.)   The carboxyl group at the end of the side chain of the bile salts is activated by a  reaction that requires ATP and coenzyme A (Co A). The Co A derivatives can react  with either glycine or taurine (which is derived from cysteine), forming amides that  are known as the conjugated bile salts. In glycocholic acid and glycochenocholic  acid, the bile acids are conjugated with glycine. These compounds have a pK of  approximately 4, so compared to their unconjugated forms, a higher percentage of  the molecules is present in the ionized form at the pH of the intestine. The taurine  conjugates, taurocholic and taurochenocholic acid, have a pK of approximately 2.  Therefore, compared with the glycoconjugates, an even greater percentage of the  molecules of these conjugates are ionized in the lumen of the gut (Fig. 34.11).   The bile salts are produced in the liver and secreted into the bile (Fig. 34.12), They  are stored in the gallbladder and released into the intestine during a meal, where  they serve as detergents that aid in the digestion of dietary lipids (see Chapter 32).   Intestinal bacteria deconjugate and dehydroxylate the bile salts, removing the  glycine and taurine residues and the hydroxyl group at position 7. The bile salts that  lack a hydroxyl group at position 7 are called secondary bile salts. The deconjugated  and dehydroxylated bile salts are less soluble and, therefore, less readily resorbed  from the intestinal lumen than the bile salts that have not been subjected to bacterial   Fig. 34.11. Conjugation of bile salts. Conjugation lowers the pK of the bile salts, making them better detergents; i.e., they are more ionized in  the contents of the intestinal lumen (pH ~ 6) than are the unconjugated bile salts (pK ~ 6). The reactions are the same for the chenocholic acid  series of bile salts.   Bile salts  reabsorbed  (12-32 g/day) and  returned to liver for  recycling  > 95% efficiency   action (Fig. 34.13). Lithocholic acid, a secondary bile salt that has a hydroxyl group  only at position 3, is the least soluble bile salt. Its major fate is excretion.   Greater than 95% of the bile salts are resorbed in the ileum and return to the liver  via the enterohepatic circulation (via the portal vein; see Fig. 34.12). The second-  ary bile salts may be reconjugated in the liver, but they are not rehydroxylated. The  bile salts are recycled by the liver, which secretes them into the bile. This entero-  hepatic recirculation of bile salts is extremely efficient. Less than 5% of the bile  salts entering the gut are excreted in the feces each day. Because the steroid nucleus  cannot be degraded in the body, the excretion of bile salts serves as a major route  for removal of the steroid nucleus and, thus, of cholesterol from the body.   Because they are hydrophobic and essentially insoluble in the water of the blood,  cholesterol and cholesterol esters, like triacylglycerols and phospholipids, must be  transported through the bloodstream packaged as lipoproteins. These macromole-  cules are water-soluble. Each lipoprotein particle is composed of a core of hydropho-  bic lipids such as cholesterol esters and triacylglycerols surrounded by a shell of  polar lipids (the phospholipids), which allows a hydration shell to form around the  lipoprotein (see Fig. 32.9). This occurs when the positive charge of the nitrogen atom  of the phospholipid (phosphatidylcholine, phosphatidylethanolamine, or phos-  phatidylserine) forms an ionic bond with the negatively charged hydroxyl ion of the  environment. In addition, the shell contains a variety of apoproteins that also increase  the water solubility of the lipoprotein. Free cholesterol molecules are dispersed  throughout the lipoprotein shell to stabilize it in a way that allows it to maintain its  spherical shape. The major carriers of lipids are chylomicrons (see Chapter 32),  VLDL, and HDL. Metabolism of VLDL will lead to IDL and LDL. Metabolism of  chylomicrons leads to chylomicron remnant formation.   Through this carrier mechanism, lipids leave their tissue of origin, enter the  bloodstream, and are transported to the tissues, where their components will be  either used in synthetic or oxidative process or stored for later use. The apoproteins  ("apo" describes the protein within the shell of the particle in its lipid-free form) not  only add to the hydrophilicity and structural stability of the particle but have other  functions as well: (1) they activate certain enzymes required for normal lipoprotein  metabolism and (2) they act as ligands on the surface of the lipoprotein that target  specific receptors on peripheral tissues that require lipoprotein delivery for their  innate cellular function.   Ten principal apoproteins have been characterized. Their tissue source, molecu-  lar mass, distribution within lipoproteins, and metabolic functions are shown in  Table 34.3.   The lipoproteins themselves are distributed among eight major classes. Some of  their characteristics are shown in Table 34.4. Each class of lipoprotein has a specific  function determined by its apolipoprotein content, its tissue of origin, and the pro-  portion of the macromolecule made up of triacylglycerols, cholesterol esters, free  cholesterol, and phospholipids (see Tables 34.3 and 34.4).   Chylomicrons are the largest of the lipoproteins and the least dense because of their  rich triacylglycerol content. They are synthesized from dietary lipids (the "exoge-  nous" lipoprotein pathway) within the epithelial cells of the small intestine and then  secreted into the lymphatic vessels draining the gut (see Fig. 32.13). They enter the  bloodstream via the left subclavian vein. The major apoproteins of chylomicrons are  apoB-48, apoC n , and apoE (see Table 34.3). The apoC n activates lipoprotein lipase   Fig. 34.13. Structures of the primary and sec-  ondary bile salts. Primary bile salts form con-  jugates with taurine or glycine in the liver.  After secretion into the intestine, they may be  deconjugated and dehydroxylated by the bac-  terial flora, forming secondary bile salts. Note  that dehydroxylation occurs at position 7,  forming the deoxy family of bile salts.   (LPL), an enzyme that projects into the lumen of capillaries in adipose tissue, cardiac  muscle, skeletal muscle, and the acinar cells of mammary tissue. This activation  allows LPL to hydrolyze the chylomicrons, leading to the release of free fatty acids  derived from core triacylglycerides of the lipoprotein into these target cells. The mus-  cle cells then oxidize the fatty acids as fuel while the adipocytes and mammary cells  store them as triacylglycerols (fat) or, in the case of the lactating breast, use them for  milk formation. The partially hydrolyzed chylomicrons remaining in the bloodstream  (the chylomicron remnants), now partly depleted of their core triacylglycerols, retain  their apoE and apoB48 proteins. Receptors in the plasma membranes of the liver cells  bind to apoE on the surface of these remnants, allowing them to be taken up by the  liver through a process of receptor-mediated endocytosis (see below).   If dietary intake of fatty acids exceeds the immediate fuel requirements of the liver,  the excess fatty acids are converted to triacylglycerols, which, along with free and  esterified cholesterol, phospholipids, and a variety of apoproteins (see Table 34.3),   Abbreviations: TG, Triacylglycerols; Choi, the sum of free and esterified cholesterol; PL, phospholipid; VLDL = very-low-density lipoproteins;  IDL, intermediate-density lipoproteins; LDL, low-density lipoproteins; HDL, high-density lipoproteins.   including apoB-100, apoC n , and apoE, are packaged to form VLDL. These particles  are then secreted from the liver (the "endogenous" pathway of lipoprotein metabo-  lism) into the bloodstream (Fig. 34.14). The density, particle size, and lipid content  of VLDL particles are given in Table 34.3. These particles are then transported from  the hepatic veins to capillaries in skeletal and cardiac muscle and adipose tissue, as  well as lactating mammary tissues, where lipoprotein lipase is activated by apoC n in  the VLDL particles. The activated enzyme facilitates the hydrolysis of the triacyl-  glycerol in VLDL, causing the release of fatty acids and glycerol from a portion of  core triacylglycerols. These fatty acids are oxidized as fuel by muscle cells, used in  the resynthesis of triacylglycerols in fat cells, and used for milk production in the lac-  tating breast. The residual particles remaining in the bloodstream are called VLDL  remnants. Approximately 50% of these remnants are taken up from the blood by  liver cells through the binding of VLDL apoE to the hepatocyte plasma membrane  apoE receptor followed by endocytic internalization of the VLDL remnant.   Approximately half of the VLDL remnants are not taken up by the liver but, instead,  have additional core triacylglycerols removed to form IDL, a specialized class of  VLDL remnants. With the removal of additional triacylglycerols from IDL through  the action of hepatic triglyceride lipase within hepatic sinusoids, LDL is generated  from IDL. As seen in Table 34.4, the LDL particles are rich in cholesterol and cho-  lesterol esters. Approximately 60% of the LDL is transported back to the liver,  where its apoB-100 binds to specific apoB-100 receptors in the liver cell plasma  membranes, allowing particles to be endocytosed into the hepatocyte. The remain-  ing 40% of LDL particles are carried to extrahepatic tissues such as adrenocortical    Fig. 34.14. Fate of VLDL. VLDL triacylglycerol (TG) is degraded by LPL, forming IDL. IDL can either be endocytosed by the liver through a  receptor-mediated process or further digested, mainly by hepatic triacylglycerol lipase (HTGL), to form LDL. LDL may be endocytosed by  receptor-mediated processes in the liver or in peripheral cells. LDL also may be oxidized and taken up by "scavenger" receptors on macrophages.  The scavenger pathway plays a role in atherosclerosis. FA = fatty acids; Pi = inorganic phosphate.   and gonadal cells that also contain apoB-100 receptors, allowing them to internal-  ize the LDL particles and use their cholesterol for the synthesis of steroid hormones.  Some of the cholesterol of the internalized LDL is used for membrane synthesis and  vitamin D synthesis as well. If an excess of LDL particles is present in the blood,  this specific receptor-mediated uptake of LDL by hepatic and nonhepatic tissue  becomes saturated. The "excess" LDL particles are now more readily available for  nonspecific uptake of LDL by macrophages (scavenger cells) present near the  endothelial cells of arteries. This exposure of vascular endothelial cells to high lev-  els of LDL is believed to induce an inflammatory response by these cells, a process  suggested to initiate the complex cascade of atherosclerosis discussed below.   The fourth class of lipoproteins is HDL, which plays several roles in whole body  lipid metabolism.   HDL particles can be created by a number of mechanisms. The first is synthesis of  nascent HDL by the liver and intestine as a relatively small molecule whose shell,  like that of other lipoproteins, contains phospholipids, free cholesterol, and a vari-  ety of apoproteins, predominant among which are apoAl, apoAII, apoQ, and  apoC n (see Table 34.3). Very low levels of triacylglycerols or cholesterol esters are  found in the hollow core of this early, or nascent, version of HDL.   A second method for HDL generation is the budding of apoproteins from chy-  lomicrons and VLDL particles as they are digested by lipoprotein lipase. The  apoproteins (particularly AI) and shells can then accumulate more lipid, as  described below.   A third method for HDL generation is free apoprotein AI, which may be shed  from other circulating lipoproteins. AI will acquire cholesterol and phospholipids  from other lipoproteins and cell membranes, to form a nascent-like HDL particle  within the circulation.   In the process of maturation, the nascent HDL particles accumulate phospholipids  and cholesterol from cells lining the blood vessels. As the central hollow core of  nascent HDL progressively fills with cholesterol esters, HDL takes on a more glob-  ular shape to eventually form the mature HDL particle. The transfer of lipids to nas-  cent HDL does not require enzymatic activity.   A major benefit of HDL particles derives from their ability to remove cholesterol  from cholesterol-laden cells and to return the cholesterol to the liver, a process  known as reverse cholesterol transport. This is particularly beneficial in vascular tis-  sue; by reducing cellular cholesterol levels in the subintimal space, the likelihood  that foam cells (lipid-laden macrophages that engulf oxidized LDL-cholesterol and  represent an early stage in the development of atherosclerotic plaque) will form  within the blood vessel wall is reduced.   Reverse cholesterol transport requires a directional movement of cholesterol from  the cell to the lipoprotein particle. Cells contain the protein ABC1 (ATP-binding cas-  sette protein 1) which uses ATP hydrolysis to move cholesterol from the inner leaflet  of the membrane to the outer leaflet. Once the cholesterol has reached the outer mem-  brane leaflet, the HDL particle can accept it, but if the cholesterol is not modified  within the HDL particle, the cholesterol can leave the particle by the same route that  it entered. To trap the cholesterol within the HDL core, the HDL particle acquires the   enzyme LCAT from the circulation (LCAT is synthesized and secreted by the liver).  LCAT catalyzes the transfer of a fatty acid from the 2-position of lecithin (phos-  phatidylcholine) in the phospholipid shell of the particle to the 3-hydroxyl group of  cholesterol, forming a cholesterol ester (Fig. 34.15). The cholesterol ester migrates to  the core of the HDL particle and is no longer free to return to the cell.   Elevated levels of lipoprotein-associated cholesterol in the blood, particularly  that associated with LDL but also that in the more triacylglycerol-rich lipoproteins,  are associated with the formation of cholesterol-rich atheromatous plaque in the  vessel wall, eventually leading to diffuse atherosclerotic vascular disease resulting  in acute cardiovascular events, such as a myocardial infarction, a stroke, or symp-  tomatic peripheral vascular insufficiency. High levels of HDL in the blood, there-  fore, are believed to be vasculoprotective, because these high levels increase the rate  of reverse cholesterol transport "away" from the blood vessels and "toward" the  liver ("out of harm's way").   Mature HDL particles can bind to specific receptors on hepatocytes (such as the  apoE receptor), but the primary means of clearance of HDL from the blood is  through its uptake by the scavenger receptor SR-B1. This receptor is present on  many cell types. It does not carry out endocytosis per se, but once the HDL particle   II ©  HC-O-P-0 - CH 2 CH 2 N(CH 3 ) 3   II ©  HC-O-P-0 - CH 2 CH 2 N(CH 3 ) 3   Fig. 34.15. The reaction catalyzed by LCAT. Rl = saturated fatty acid. R2 = unsaturated  fatty acid.   Two genetically determined disor-  ders, familial HDL deficiency and  Tangier disease, result from muta-  tions in the ATP-binding cassette 1 (ABC 1)  protein. Cholesterol-depleted HDL cannot  transport free cholesterol from cells that lack  the ability to express this protein. As a conse-  quence, HDL is rapidly degraded. These disor-  ders have established a role for ABC 1 protein  in the regulation of HDL levels in the blood.   Because Ann Jeina continued to  experience intermittent chest pain, in  spite of good control of her hyper-  tension and a 20-lb weight loss, her physician  decided that a 2-drug regimen to lower her  blood LDL cholesterol level must be added to  the dietary measures already in place. Conse-  quently, treatment with cholestyramine, a resin  that binds some of the bile salts in the intes-  tinal lumen, and the HMG-CoA reductase  inhibitor pravastatin was initiated.    Fig. 34.16. Functions and fate of HDL. Nascent HDL is synthesized in liver and intestinal cells. It exchanges proteins with chylomicrons and  VLDL. HDL picks up cholesterol (C) from cell membranes. This cholesterol is converted to cholesterol ester (CE) by the LCAT reaction. HDL  transfers CE to VLDL in exchange for triacylglycerol (TG). The cholesterol ester transfer protein (CETP) mediates this exchange. PL =  phospholipids.   Fig. 34.17. Function of cholesterol ester trans-  fer protein (CETP). CETP transfers cholesterol  esters (CE) from HDL to VLDL in exchange  for triacylglycerol (TG).   is bound to the receptor, its cholesterol and cholesterol esters are transferred into the  cells. When depleted of cholesterol and its esters, the HDL particle dissociates from  the SR-B 1 receptor and re-enters the circulation. SR-B 1 receptors can be upregu-  lated in certain cell types that require cholesterol for biosynthetic purposes, such as  the cells that produce the steroid hormones. The SR-B1 receptors are not downreg-  ulated when cholesterol levels are high.   In addition to its ability to pick up cholesterol from cell membranes, HDL also  exchanges apoproteins and lipids with other lipoproteins in the blood. For exam-  ple, HDL transfers apolipoprotein E (apoE) and apolipoprotein CII (apoC n ) to chy-  lomicrons and to VLDL. The apoC n stimulates the degradation of the triacylglyc-  erols of chylomicrons and VLDL by activating LPL (Fig. 34.16). After digestion  of the chylomicrons and the VLDL triacylglycerols, apoE and apoCII are trans-  ferred back to HDL. When HDL obtains free cholesterol from cell membranes, the  free cholesterol is esterified at the third carbon of the A ring via the LCAT reaction  (see Fig. 34.14). From this point, HDL either transports the free cholesterol and  cholesterol esters directly to the liver, as described above, or by CETP to circulat-  ing triacylglycerol-rich lipoproteins such as VLDL and VLDL remnants (see Fig.  34.16). In exchange, triacylglycerols from the latter lipoproteins are transferred to  HDL (Fig. 34.17). The greater the concentration of triacylglycerol-rich lipopro-  teins in the blood, the greater the rate of these exchanges. Thus, the CETP  exchange pathway may explain the observation that whenever triacylglycerol-rich  lipoproteins are present in the blood in high concentrations, the amount of choles-  terol reaching the liver via cholesterol-enriched VLDL and VLDL remnants  increases, and a proportional reduction in the total amount of cholesterol and cho-  lesterol esters that are directly transferred to the liver via HDL occurs. Mature   HDL particles are designated as HDL 3 ; after the CETP reaction and loss of cho-  lesterol and gain of triacylglycerol, the particles become larger and are designated  as HDL 2 particles (see Table 34.4).   As stated earlier, each lipoprotein particle contains specific apoproteins on its  surface that act as ligands for specific plasma membrane receptors on target tis-  sues such as the liver, the adrenal cortex, the gonads, and other cells that require  one or more of the components of the lipoproteins. With the exception of the  scavenger receptor SR-B1, the interaction of ligand and receptor initiates the  process of endocytosis depicted for LDL in Figure 34.18. The receptors for LDL,  for example, are found in specific areas of the plasma membrane of the target  cell for circulating lipoproteins. These are known as coated pits, and they con-  tain a unique protein called clathrin. The plasma membrane in the vicinity of the  receptor-LDL complex invaginates and fuses to form an endocytic vesicle.  These vesicles then fuse with lysosomes, acidic subcellular vesicles that contain  a number of degradative enzymes. The cholesterol esters of LDL are hydrolyzed  to form free cholesterol, which is rapidly reesterified through the action of  AC AT. This rapid reesterification is necessary to avoid the damaging effect of  high levels of free cholesterol on cellular membranes. The newly esterified cho-  lesterol contains primarily oleate or palmitoleate (monounsaturated fatty acids),  unlike those of the cholesterol esters in LDL, which are rich in linoleate, a  polyunsaturated fatty acid.   As is true for the synthesis and activity of HMG CoA reductase, the synthesis of  the LDL receptor itself is subject to feedback inhibition by increasing levels of cho-  lesterol within the cell. One probable mechanism for this feedback regulation  involves one or more of the SREBP described earlier. These proteins or the cofactors  that are required for the full expression of genes that code for the LDL receptor are  also capable of sensing the concentration of sterols within the cell. When sterol lev-  els are high, the process that leads to the binding of the SREBP to the sterol regula-  tory element of these genes is suppressed (see Fig. 34.4). The rate of synthesis from  mRNA for the LDL receptor is diminished under these circumstances. This, in turn,  appropriately reduces the amount of cholesterol that can enter these cholesterol-rich  cells by receptor-mediated endocytosis (downregulation of receptor synthesis).  When the intracellular levels of cholesterol decrease, these processes are reversed,  and cells act to increase their cholesterol levels. Both synthesis of cholesterol from  acetyl CoA and synthesis of LDL receptors are stimulated. An increased number of  receptors (upregulation of receptor synthesis) results in an increased uptake of LDL  cholesterol from the blood, with a subsequent reduction of LDL-cholesterol levels.  At the same time, the cellular cholesterol pool is replenished.   The best-characterized lipoprotein receptor, the LDL receptor, specifically recog-  nizes apoB-100 and apo E. Therefore, this receptor binds VLDL, IDL, and chy-  lomicron remnants in addition to LDL. The binding reaction is characterized by its  saturability and occurs with high affinity and a narrow range of specificity. Other  receptors, such as the LDL receptor-related proteins (LRP) and the macrophage  scavenger receptor (notably types SR-A1 and SR-A2, which are located primarily  near the endothelial surface of vascular endothelial cells), have broad specificity  and bind many other ligands in addition to the blood lipoproteins.   The LDL receptor has a mosaic structure encoded by a gene that was assembled  by a process known as exon shuffling. It is composed of six different regions  (Fig. 34.19). The first region, at the amino terminus, contains the LDL-binding  region, a cysteine-rich sequence of 40 residues. Acidic side chains in this region  bind ionic calcium. When these side chains are protonated, calcium is released  from its binding sites. This release leads to conformational changes that allow  the LDL to disconnect from its receptor docking site. Disulfide bonds, formed  from the cysteine residues, have a stabilizing influence on the structural integrity  of this portion of the receptor.   The second region of the receptor contains domains that are homologous with  epidermal growth factor (EGF) as well as a complex consisting of six repeats that  resemble the blades of the transducin beta subunit forming a propeller-like moiety.   The third region of the LDL receptor contains a chain of N-linked oligosaccha-  rides, whereas the fourth region contains a domain that is rich in serine and threo-  nine and contains O-linked sugars. This region may have a role in physically  extending the receptor away from the membrane so that the LDL-binding region is  accessible to the LDL molecule.   The fifth region contains 22 hydrophobic residues constituting the membrane-  spanning unit of the receptor, whereas the sixth region extends into the cytosol,  where it regulates the interaction between the C-terminal domain of the LDL recep-  tor and the clathrin-containing coated pit where the process of receptor-mediated  endocytosis is initiated.   The number of LDL receptors, the binding of LDL to its receptors, and the  postreceptor binding process can be diminished for a variety of reasons, all of which   Fig. 34.19. Structure of the LDL receptor. The protein has six major regions.   may lead to an accumulation of LDL cholesterol in the blood and premature ather-  osclerosis. These abnormalities can result from mutations in one (heterozygous —  seen in approximately 1 in 500 people) or both (homozygous — seen in about 1 in 1  million people) alleles for the LDL receptor (familial hypercholesterolemia). Het-  erozygotes produce approximately half of the normal complement of LDL recep-  tors, whereas the homozygotes produce almost no LDL receptor protein (receptor  negative familial hypercholesterolemia). The latter have serum total cholesterol lev-  els in the range of 500 to 800 mg/dL. In a subset of patients with familial hyperc-  holesterolemia, the LDL receptor is normally synthesized and transported to the cell  surface, but an amino acid substitution or deletion leads to changes in the protein's  structure such that LDL-binding is impaired. As a result, cholesterol does not enter  the target cell from the bloodstream and, therefore, cannot feedback negatively on  cholesterol biosynthesis in the cell. As a result, the serum cholesterol level rises. A  third form of familial hypercholesterolemia involves a genetic defect in the trans-  port or migration mechanism that normally delivers the LDL receptor from its point  of synthesis within the cell to its proper location in that cell's plasma membrane.  Genetic mutations can lead to yet another form of familial hypercholesterolemia in  which a structural change occurs in the carboxy terminus of the LDL receptor.   Q <z Ann Jeina's blood lipid levels (in  yv\) mg/dL) were:  ™ Triacylglycerol  Total cholesterol  HDL cholesterol  LDL cholesterol   She was diagnosed as having familial  hypercholesterolemia (FH) type IIA, which is  caused by genetic defects in the gene that  encodes the LDL receptor (see Biochemical  Comments). As a result of the receptor defect,  LDL cannot readily be taken up by cells, and  its concentration in the blood is elevated.   LDL particles contain a high percentage,  by weight, of cholesterol and cholesterol  esters, more than other blood lipoproteins.  However, LDL triacylglycerol levels are low  because LDL is produced by digestion of the  triacylglycerols of VLDL and IDL. Therefore,  individuals with a type IIA hyperlipoproteine-  mia have very high blood cholesterol levels,  but their levels of triacylglycerols may be in or  near the normal range (see Table 34.4).   Triacylglycerol 295  Total cholesterol 314  HDL cholesterol 24  LDL cholesterol 23 1  The elevated serum levels of LDL choles-  terol found in patients such as Ivan Applebod  who have type 2 diabetes mellitus is multifac-  torial. One of the mechanisms responsible for  this increase involves the presence of chroni-  cally elevated levels of glucose in the blood of  poorly controlled diabetics. This prolonged  hyperglycemia increases the rate of nonenzy-  matic attachment of glucose to various pro-  teins in the body, a process referred to as gly-  cation or glycosylation of proteins.   Glycation may adversely affect the struc-  ture or the function of the protein involved. For  example, glycation of the LDL receptor and of  proteins in the LDL particle may interfere with  the normal "fit" of LDL particles with their  specific receptors. As a result, less circulating  LDL is internalized into cells by receptor-  mediated endocytosis, and the serum LDL  cholesterol level rises.   Although such patients are able to place structurally normal LDL receptors in the  plasma membrane of the cell, they are unable to internalize the LDL-LDL receptor  complex because they cannot properly translocate the complex into the clathrin-  containing coated pits. The spectrum of mutations of the LDL receptor gene is  shown in Figure 34.20.   LRP is structurally related to the LDL receptor but recognizes a broader spectrum  of ligands. In addition to lipoproteins, it binds the blood proteins a 2 -macroglobulin  (a protein that inhibits blood proteases) and tissue plasminogen activator (TPA) and  its inhibitors. The LRP receptor recognizes the apoE of lipoproteins and binds rem-  nants produced by the digestion of the triacylglycerols of chylomicrons and VLDL  by LPL. Thus, one of its functions is believed to be the clearance of these remnants  from the blood. The LRP receptor is abundant in the cell membranes of the liver,  brain, and placenta. In contrast to the LDL receptor, synthesis of the LRP receptor  is not significantly affected by an increase in the intracellular concentration of cho-  lesterol. However, insulin causes the number of these receptors on the cell surface  to increase, consistent with the need for removal of chylomicron remnants that oth-  erwise would accumulate after eating a meal.   Some cells, particularly the phagocytic macrophages, have nonspecific receptors  known as "scavenger" receptors that bind various types of molecules, including  oxidatively modified LDL particles. There are a number of different types of scav-  enger receptors. SR-B1 is used primarily for HDL binding, whereas the scavenger  receptors expressed on macrophages are SR-A1 and SR-A2. Modification of LDL  frequently involves oxidative damage, particularly of polyunsaturated fatty acyl  groups (see Chapter 24). In contrast to the LDL receptors, the scavenger receptors   Fig. 34.20. Location of 353 point mutations and small deletions/insertions (<25 bp) in the LDL receptor gene in individuals with familial hyper-  cholesterolemia (FH). Exons are shown as vertical boxes and introns as the lines connecting them. The figure was obtained from Goldstein JL  Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, et al., eds. The Metabolic and Molecular  Bases of Inherited Disease. 8 th Ed., vol III. New York: McGraw-Hill, 2001:2863-2913.   are not subject to downregulation. The continued presence of scavenger receptors in  the cell membrane allows the cells to take up oxidatively modified LDL long after  intracellular cholesterol levels are elevated. When the macrophages become  engorged with lipid, they are called foam cells. An accumulation of these foam cells  in the subendothelial space of blood vessels form the earliest gross evidence of a  developing atherosclerotic plaque known as a fatty streak.   The processes that cause oxidation of LDL involve superoxide radicals, nitric  oxide, hydrogen peroxide, and other oxidants (see Chapter 24). Antioxidants, such  as vitamin E, ascorbic acid (vitamin C), and carotenoids, may be involved in pro-  tecting LDL from oxidation.   The normal artery is composed of three distinct layers (Fig. 34.21). That which is  closest to the lumen of the vessel, the intima, is lined by a monolayer of endothelial  cells that are bathed by the circulating blood. Just beneath these specialized cells  lies the subintimal extracellular matrix, in which some vascular smooth muscle cells  are embedded (the subintimal space). The middle layer, known as the tunica media,  is separated from the intima by the internal elastic lamina. The tunica media con-  tains lamellae of smooth muscle cells surrounded by an elastin- and collagen-rich  matrix. The external elastic lamina forms the border between the tunica media and  the outermost layer, the adventitia. This layer contains nerve fibers and mast cells.  It is the origin of the vasa vasorum, which supply blood to the outer two thirds of  the tunica media.   The initial step in the development of an atherosclerotic lesion within the wall  of an artery is the formation of a fatty streak. The latter represents an accumula-  tion of lipid-ladened macrophages or foam cells in the subintimal space. These  fatty streaks are visible as a yellow-white linear streak that bulges slightly into the  lumen of the vessel. These streaks are initiated when one or more known "vascu-  lar risk factors for atherosclerosis," all of which have the potential to injure the  vascular endothelial cells, reach a critical threshold at the site of future lesions.  Examples of such risk factors include elevated intra- arterial pressure (arterial  hypertension), elevated circulating levels of various lipids such as LDL, chylomi-  cron remnants, and VLDL remnants, or low levels of circulating HDL, cigarette  smoking, chronic elevations in blood glucose levels, high circulating levels of the  vasoconstricting octapeptide angiotensin II, and others. The resulting insult to   In addition to dietary therapy, aimed  at reducing her blood cholesterol  levels, Ann Jeina was treated with  pravastatin, an HMG-CoA reductase inhibitor.  The HMG-CoA reductase inhibitors decrease  the rate of synthesis of cholesterol in cells. As  cellular cholesterol levels decrease, the synthe-  sis of LDL receptors increases. As the number  of receptors rises on the cell surface, the  uptake of LDL is increased. Consequently, the  blood level of LDL cholesterol decreases.   HDL is considered to be the "good  cholesterol," because it accepts free  cholesterol from peripheral tissues,  such as cells in the walls of blood vessels. This  cholesterol is converted to cholesterol ester,  part of which is transferred to VLDL by CETP,  and returned to the liver by IDL and LDL. The  remainder of the cholesterol is transferred  directly as part of the HDL molecule to the  liver. The liver reutilizes the cholesterol in the  synthesis of VLDL, converts it to bile salts, or  excretes it directly into the bile. HDL therefore  tends to lower blood cholesterol levels. Lower  blood cholesterol levels correlate with a lower  rates of death of atherosclerosis.   In patients such as Ann Jeina and  Ivan Applebod, who have elevated  levels of VLDL or LDL, HDL levels  are often low. These patients are predisposed  to atherosclerosis and suffer from a high inci-  dence of heart attacks and strokes.   Exercise and estrogen administration both  increase HDL levels. This is one of the reasons  exercise is often recommended to aid in the  prevention or treatment of heart disease, and  estrogen replacement therapy (ERT) is often  prescribed for postmenopausal women. Before  menopause, the incidence of heart attacks is  relatively low in women, but it rises after  menopause and increases to the level found in  men by the age of 65 or 70 years. Moderate  consumption of ethanol (alcohol) has also been  correlated with increased HDL levels. Recent  studies suggest that the beneficial amount of  ethanol may be quite low, about two small  glasses of wine a day, and that beneficial effects  ascribed to ethanol may result from other com-  ponents of wine and alcoholic beverages. In  spite of the evidence that postmenopausal  estrogen replacement therapy decreases circu-  lating levels of LDL and increases HDL levels,  recent data suggest that ERT may actually  increase the rate of atherosclerotic vascular dis-  ease in these women. As a result, the accepted  indications for ERT are now limited to intoler-  able "hot flashes" or vaginal dryness.    endothelial cells may trigger these cells to secrete adhesion molecules that bind  to circulating monocytes and markedly slow their rate of movement past the  endothelium. When sufficiently slowed, these monocytic cells accumulate and  have access to the physical spaces that exist between endothelial cells. This accu-  mulation of monocytic cells resembles the classical inflammatory response to  injury. These changes have led to the suggestion that atherosclerosis is, in fact, an  inflammatory disorder and, therefore, is one that might be prevented or attenuated  through the use of anti-inflammatory agents such as acetylsalicylic acid (e.g.,  aspirin) and HMG Co A reductase inhibitors (statins), which have been shown to  suppress the inflammatory cascade as well as to inhibit the action of HMG CoA  reductase.   The monocytic cells are transformed into macrophages that migrate through the  spaces between endothelial cells. They enter the subintimal space under the influ-  ence of chemoattractant cytokines (e.g., chemokine macrophage chemoattractant  protein I) secreted by vascular cells in response to exposure to oxidatively modified  fatty acids within the lipoproteins.   The macrophages can replicate and exhibit augmented expression of receptors  that recognize oxidatively modified lipoproteins. Unlike the classic LDL receptors  on liver and many nonhepatic cells, these macrophage-bound receptors are high-  capacity, low- specificity receptors (scavenger receptors). They bind to and internal-  ize oxidatively modified fatty acids within LDLs to become subintimal foam cells  as described previously. As these foam cells accumulate, they deform the overlying  endothelium, causing microscopic separations between endothelial cells, exposing  these foam cells and underlying extracellular matrix to the blood. These exposed  areas serve as sites for platelet adhesion and aggregation. Activated platelets secrete  cytokines that perpetuate this process and increase the potential for thrombus (clot)  formation locally. As the evolving plaque matures, a fibrous cap forms over its  expanding "roof," which now bulges into the vascular lumen, thereby partially  occluding it. Vascular smooth muscle cells now migrate from the tunica media to  the subintimal space and secrete additional plaque matrix material. The smooth  muscle cells also secrete metalloproteinases that thin the fibrous cap near its  "elbow" at the periphery of the plaque. This thinning progresses until the fibrous  cap ruptures, allowing the plaque contents to physically contact the procoagulant  elements present within the circulation. This leads to acute thrombus formation. If  this thrombus completely occludes the remaining lumen of the vessel, an infarction  of tissues distal to the occlusion (i.e., an acute myocardial infarction) may occur  (Fig. 34.22). Most plaques that rupture also contain focal areas of calcification,  which appears to result from the induction of the same cluster of genes as those that  promote the formation of bone. The inducers for this process include oxidized  sterols as well as transforming growth factor beta (TGF-(3) derived from certain  vascular cells.   Finally, high intraluminal shear forces develop in these thinning or eroded areas  of the plaque's fibrous cap, inducing macrophages to secrete additional metallopro-  teinases that further degrade the arterial-fibrous cap matrix. This contributes further  to plaque rupture and thrombus formation (see Fig. 34.22). The consequence is a  macrovascular ischemic event such as an acute myocardial infarction (AMI) or an  acute cerebrovascular accident (CVA).   Lipoprotein(a) is essentially an LDL particle that is covalently bound to apopro-  tein^). It is called "lipoprotein little a" to avoid confusion with the apoprotein A  found in HDL. The structure of apoprotein(a) is very similar to that of plasmino-  gen, a precursor of the protease plasmin that degrades fibrin, a major component of blood  clots. Lipoprotein(a), however, cannot be converted to active plasmin. There are reports that  high concentrations of lipoprotein(a) correlate with an increased risk of coronary artery dis-  ease, even in patients in whom the lipid profile is otherwise normal.   Fig. 34.22. Evolution of an atherosclerotic plaque. Plaque capsule eroded near the "elbow"  of plaque creating an early plaque fissure (A), which may heal as plaque increases in size (B)  or may grow as thrombus expands, having an intraluminal portion and an intraintimal por-  tion (C). If the fissure is not properly sealed, the thrombus may grow and completely occlude  the vessel lumen (D), causing an acute infarction of tissues downstream of the vessel occlu-  sion.   Cholesterol is the precursor of all five classes of steroid hormones: glucocorticoids,  mineralcorticoids, androgens, estrogens, and progestins. These hormones are syn-  thesized in the adrenal cortex, ovaries, testes, and ovarian corpus luteum. Steroid  hormones are transported through the blood from their sites of synthesis to their tar-  get organs, where, because of their hydrophobicity, they cross the cell membrane  and bind to specific receptors in either the cytoplasm or nucleus. The bound recep-  tors then bind to DNA to regulate gene transcription (see Chapter 16, section  III.C.2, and Fig. 16.13). Because of their hydrophobicity, steroid hormones must be  complexed with a serum protein. Serum albumin can act as a nonspecific carrier for  the steroid hormones, but there are specific carriers as well. The cholesterol used for  steroid hormone synthesis is either synthesized in the tissues from acetyl Co A,  extracted from intracellular cholesterol ester pools, or taken up by the cell in the  form of cholesterol-containing lipoproteins (either internalized by the LDL-recep-  tor, or absorbed by the SR-B1 receptor). In general, glucocorticoids and progestins  contain 21 carbons, androgens contain 19 carbons, and estrogens contain 18 car-  bons. The specific complement of enzymes present in the cells of an organ deter-  mines which hormones the organ can synthesize.   The oxidative reactions that lead to the synthesis and secretion of glucocorticoids  such as Cortisol are stimulated by adrenal corticotrophic hormone (ACTH). The role  of Cortisol as a stress-released hormone is discussed in Chapter 43.   Mineralocorticoids such as aldosterone are also synthesized in the adrenal cor-  tex and are secreted in response to angiotensin II or III, rising potassium levels in  the blood, or hyponatremia (low levels of sodium ions in the blood). Aldosterone  stimulates sodium reuptake in the kidney, sweat glands, salivary glands, and other   Vera Leizd consulted her gynecolo-  gist, who confirmed that her prob-  lems were probably the result of an  excess production of androgens (virilization)  and ordered blood and urine studies to deter-  mine whether Vera's adrenal cortices or her  ovaries were causing her virilizing syndrome.   Cytochrome P450 C11 , another  \m ) enzyme located in the mitochondrial  membrane, catalyzes p-hydroxyla-  tion at Cll. Hydroxylations at C17 and C21  are catalyzed by two enzymes located in the  membranes of the endoplasmic reticulum  (P450 C17 for 17a-hydroxylation and P450 C21  for 21-hydroxylation).    tissues, with a resultant increase in extracellular fluid volume and eventually in  blood pressure. The angiotensins are produced in response to a reduction in extra-  cellular fluid volume, which may occur as a result of such things as excessive  sweating, persistent vomiting without sufficient rehydration, or bleeding without  adequate replacement of blood.   Androgens such as testosterone are synthesized in the Leydig cells of the testes  and to a lesser extent in the ovary and are secreted in response to luteinizing hor-  mone (LH). In males, testosterone is commonly converted to dihydrotestosterone, a  higher- affinity form of the hormone, within specific target tissues. This active form  of the hormone stimulates the production of sperm proteins in Sertoli cells and the  development of secondary sex characteristics.   Estrogens such as 17-p-estradiol are synthesized in the ovarian follicle and the  corpus luteum, from which their secretion is stimulated by follicle-stimulating hor-  mone (FSH). In the female, 17 p-estradiol feeds back negatively on the synthesis  and secretion of the pituitary gonadotropins, such as FSH. Estrogen and proges-  terone prepare the uterine endometrium for implantation of the fertilized ovum, and  among other actions promotes differentiation of the mammary gland.   Progestogens such as progesterone are synthesized in the corpus luteum, and  their secretion is stimulated by LH. As mentioned, in concert with estradiol, prog-  esterone prepares the uterine endometrium for implantation of the fertilized ovum  and acts as a differentiation factor in mammary gland development.   The biosynthesis of glucocorticoids and mineralocorticoids (in the adrenal cor-  tex), and that of sex steroids (in the adrenal cortex and gonads), requires four  cytochrome P450 enzymes (see Chapter 24). These monooxygenases are involved  in the transfer of electrons from NADPH through electron transfer protein interme-  diates to molecular oxygen, which then oxidizes a variety of the ring carbons of  cholesterol.   Cholesterol is converted to progesterone in the first two steps of synthesis of all  steroid hormones. Cytochrome P450 scc side-chain cleavage enzyme (previously  referred to as cholesterol desmolase) is located in the mitochondrial inner mem-  brane and removes six carbons from the side chain of cholesterol, forming preg-  nenolone, which has 21 carbons (Fig. 34.23). The next step, the conversion of preg-  nenolone to progesterone, is catalyzed by 3 p -hydroxy steroid dehydrogenase, an  enzyme that is not a member of the cytochrome P450 family. Other steroid hor-  mones are produced from progesterone by reactions that involve members of the  P450 family. As the synthesis of the steroid hormones is discussed, notice how cer-  tain enzymes are used in more than one pathway. Defects in such enzymes will lead  to multiple abnormalities in steroid synthesis, which, in turn, results in a variety of  abnormal phenotypes.   The adrenocortical biosynthetic pathway that leads to Cortisol synthesis occurs in  the middle layer of the adrenal cortex known as the zona fasciculata. Free choles-  terol is transported by an intracellular carrier protein to the inner mitochondrial  membrane of cells (Fig. 34.24), where the side chain is cleaved to form preg-  nenolone. Pregnenolone returns to the cytosol, where it forms progesterone.   In the membranes of the endoplasmic reticulum, the enzyme P450 C17 catalyzes  the hydroxy lation of C 17 of progesterone or pregnenolone and can also catalyze the  cleavage of the 2-carbon side chain of these compounds at C17 (a C17-C20 lyase  activity). These two separate functions of the same enzyme allow further steroid  synthesis to proceed along two separate pathways: the 17 -hydroxy lated steroids that  retain their side chains are precursors of Cortisol (C21), whereas those from which  the side chain was cleaved (CI 9 steroids) are precursors of androgens (male sex  hormones) and estrogens (female sex hormones).   Fig. 34.23. Synthesis of the steroid hormones. The rings of the precursor, cholesterol, are lettered. Dihydrotestosterone is produced from testos-  terone by reduction of the carbon-carbon double bond in ring A. Structural changes between the precursor and final hormone are noted in blue.  DHEA = dehydroepiandrosterone. The dashed lines indicate alternative pathways to the major pathways indicated. The starred enzymes are those  that may be defective in the condition congenital adrenal hyperplasia.    Fig. 34.24. Cellular route for Cortisol synthesis. Cholesterol is synthesized from acetyl CoA  or derived from low-density lipoprotein (LDL), which is endocytosed and digested by lyso-  somal enzymes. Cholesterol is stored in cells of the adrenal cortex as cholesterol esters.  ACTH signals the cell to convert cholesterol to Cortisol. 1 = cholesterol desmolase (involved  in side chain cleavage); 2 = 3 p -hydroxy steroid dehydrogenase; 3 = 17a-hydroxylase; 4 =  21 -hydroxylase; 5 = 1 1 (^-hydroxylase.   In the pathway of Cortisol synthesis, the 17-hydroxylation of progesterone yields  17-a-hydroxyprogesterone, which, along with progesterone, is transported to the  smooth endoplasmic reticulum. There the membrane-bound P450 C2 i (21-a-hydrox-  ylase) enzyme catalyzes the hydroxylation of C21 of 17-a-hydroxyprogesterone to  form 11-deoxycortisol (and of progesterone to form deoxycorticosterone [DOC], a  precursor of the mineralocorticoid, aldosterone; see Fig. 34.23).   The final step in Cortisol synthesis requires transport of 1 1-deoxycortisol back to  the inner membrane of the mitochondria, where P450 C11 (11 -|3 -hydroxylase)  receives electrons from electron transport protein intermediates (adrenodoxin,  which when oxidized is reduced by adrenodoxin reductase). The enzyme then trans-  fers these reducing equivalents by way of oxygen to 11-deoxycortisol for hydroxy-  lation at CI 1 to form Cortisol. The rate of biosynthesis of Cortisol and other adrenal  steroids is dependent on stimulation of the adrenal cortical cells by adrenocorti-  cotropic hormone (ACTH).   The synthesis of the potent mineralocorticoid aldosterone in the zona glomeru-  losa of the adrenal cortex also begins with the conversion of cholesterol to prog-  esterone (see Figs. 34.23 and 34.24). Progesterone is then hydroxylated at C21,   a reaction catalyzed by P450 C21 , to yield DOC. The P450 C11 enzyme system then  catalyzes the reactions that convert DOC to corticosterone. The terminal steps in  aldosterone synthesis, catalyzed by the P450 aldosterone system, involve the oxi-  dation of corticosterone to 18 -hydroxy corticosterone, which is oxidized to  aldosterone.   The primary stimulus for aldosterone production is the octapeptide angiotensin II,  although hyperkalemia (greater than normal levels of potassium in the blood) or  hyponatremia (less than normal levels of sodium in the blood) may directly stimu-  late aldosterone synthesis as well. ACTH has a permissive action in aldosterone pro-  duction. It allows cells to respond optimally to their primary stimulus, angiotensin II.   Adrenal androgen biosynthesis proceeds from cleavage of the 2-carbon side chain of  17-hydroxypregnenolone at C17 to form the 19-carbon adrenal androgen dehy-  droepiandrosterone (DHEA) and its sulfate derivative (DHEAS) in the zona reticu-  losum of the adrenal cortex (see Fig. 34.23). These compounds, which are weak  androgens, represent a significant percentage of the total steroid production by the  normal adrenal cortex, and are the major androgens synthesized in the adrenal gland.   Androstenedione, another weak adrenal androgen, is produced when the 2-car-  bon side chain is cleaved from 17a-hydroxyprogesterone by the C17-C20 lyase  activity of P450 C17 . This androgen is converted to testosterone primarily in extra-  adrenal tissues. Although the adrenal cortex makes very little estrogen, the weak  adrenal androgens may be converted to estrogens in the peripheral tissues, particu-  larly in adipose tissue (Fig. 34.25).   Luteinizing hormone (LH) from the anterior pituitary stimulates the synthesis of  testosterone and other androgens by the Leydig cells of the human testicle. In  many ways, the pathways leading to androgen synthesis in the testicle are similar  to those described for the adrenal cortex. In the human testicle, the predominant  pathway leading to testosterone synthesis is through pregnenolone to 17-a-  hydroxypregnenolone to DHEA (the A 5 pathway), and then from DHEA to  androstenedione, and from androstenedione to testosterone (see Fig. 34.23). As  for all steroids, the rate-limiting step in testosterone production is the conversion  of cholesterol to pregnenolone. LH controls the rate of side-chain cleavage from  cholesterol at carbon 21 to form pregnenolone, and thus regulates the rate of   Hyperplasia or tumors of the adrenal  cortex that produce excess aldos-  terone result in a condition known as  primary aldosteronism, which is characterized  by enhanced sodium and water retention, result-  ing in hypertension.   Although aldosterone is the major  mineralocorticoid in humans, exces-  sive production of a weaker miner-  alocorticoid, DOC, which occurs in patients  with a deficiency of the 11 -hydroxylase (the  P450 C11 enzyme), may lead to clinical signs  and symptoms of mineralocorticoid excess  even though aldosterone secretion is sup-  pressed in these patients.   Androstenedione can be purchased  \\ ) at health food stores under the name   Andros. It is touted to improve ath-  letic performance through its ability to be con-  verted to testosterone. Its use has been banned  by most major sports, although in 1998 it was  a legal supplement in baseball. During that  year, the drug received a lot of publicity, as the  supplement had been used by a player who  broke the major league home run record.    Congenital adrenal hyperplasia (CAH) is a group of diseases caused by a geneti-  cally determined deficiency in a variety of enzymes required for Cortisol synthe-  sis. The most common deficiency is that of 21 -a hydroxylase, the activity of  which is necessary to convert progesterone to 11 -deoxycorticosterone and 17-a hydroxy  progesterone to 11-deoxycortisol. Thus, this deficiency reduces both aldosterone and Corti-  sol production, without affecting androgen production. If the enzyme deficiency is severe,  the precursors for aldosterone and Cortisol production are shunted to androgen synthesis,  producing an overabundance of androgens, which leads to prenatal masculinization in  females and postnatal virilization of males. Another enzyme deficiency in this group of dis-  eases is that of ll-p hydroxylase, which results in the accumulation of 11 -deoxycorticos-  terone. An excess of this mineralocorticoid leads to hypertension (through binding of 11-  deoxy corticosterone to the aldosterone receptor). In this form of CAH, 11-deoxycortisol  also accumulates, but its biologic activity is minimal, and no specific clinical signs and  symptoms result. The androgen pathway is unaffected, and the increased ACTH levels may  increase the levels of adrenal androgens in the blood. A third possible enzyme deficiency is  that of 17-a hydroxylase. A defect in 17-a hydroxylase leads to aldosterone excess and  hypertension; however, because adrenal androgen synthesis requires this enzyme, no viril-  ization occurs in these patients.   Fig. 34.25. Adrenal androgens. These weak  androgens are converted to testosterone or  estrogens in other tissues.   imately 50% of the testosterone in the  blood in a normal woman is produced equally in  the ovaries and in the adrenal cortices. The  remaining half is derived from ovarian and  adrenal androstenedione, which, after secretion  into the blood, is converted to testosterone in  adipose tissue, muscle, liver, and skin. The adre-  nal cortex, however, is the major source of the  relatively weak androgen dehydroepiandros-  terone (DHEA). The serum concentration of its  stable metabolite, DHEAS, is used as a measure  of adrenal androgen production in hyperandro-  genic patients with diffuse excessive growth of  secondary sexual hair, e.g., facial hair as well as  that in the axillae, the suprapubic area, the chest,  and the upper extremities.   The results of the blood tests on  ( §1 •) Vera Leizd showed that her level of   testosterone was normal but that her  serum dehydroepiandrosterone sulfate  (DHEAS) level was significantly elevated.  Which tissue was the most likely source of the  androgens that caused Vera's hirsutism (a male  pattern of secondary sexual hair growth)?   Ergosterol is the provitamin of vita-  I ■ min D 2 , which differs from 7-dehy-  drocholesterol and vitamin D 3 ,  respectively, only by having a double bond  between C22 and C23 and a methyl group at  C24. Vitamin D 2 is the constituent in many  commercial vitamin preparations and in irradi-  ated milk and bread. The antirachitic potencies  of D 2 and D 3 in humans are equal, but both  must be converted to 25-(OH)-cholecalciferol  and eventually to the active form calcitriol  (l,25-(OH) 2 D 3 ) for biologic activity.   Rickets is a disorder of young chil-  dren caused by a deficiency of vita-  min D. Low levels of calcium and  phosphorus in the blood are associated with  skeletal deformities in these patients.   testosterone synthesis. In its target cells, the double bond in ring A of testosterone  is reduced through the action of 5 -a reductase, forming the active hormone dihy-  drotestosterone (DHT).   Ovarian production of estrogens, progestins (compounds related to progesterone),  and androgens requires the activity of the cytochrome P450 family of oxidative  enzymes used for the synthesis of other steroid hormones. Ovarian estrogens are  CI 8 steroids with a phenolic hydroxyl group at C3 and either a hydroxyl group  (estradiol) or a ketone group (estrone) at CI 7. Although the major steroid-produc-  ing compartments of the ovary (the granulosa cell, the theca cell, the stromal cell,  and the cells of the corpus luteum) have all of the enzyme systems required for the  synthesis of multiple steroids, the granulosa cells secrete primarily estrogens, the  thecal and stromal cells secrete primarily androgens, and the cells of the corpus  luteum secrete primarily progesterone.   The ovarian granulosa cell, in response to stimulation by follicle- stimulating  hormone (FSH) from the anterior pituitary gland and through the catalytic activity  of P450 aromatase, converts testosterone to estradiol, the predominant and most  potent of the ovarian estrogens (see Fig. 34.23). Similarly, androstenedione is con-  verted to estrone in the ovary, although the major site of estrone production from  androstenedione occurs in extraovarian tissues, principally skeletal muscle and adi-  pose tissue.   Vitamin D is unique in that it can be either obtained from the diet (as vitamin D 2 or  D 3 ) or synthesized from a cholesterol precursor, a process that requires reactions in  the skin, liver, and intestine. The calciferols, including several forms of vitamin D,  are a family of steroids that affect calcium homeostasis (Fig. 34.26). Cholecalciferol  (vitamin D 3 ) requires ultraviolet light for its production from 7-dehydrocholesterol  present in cutaneous tissues (skin) in animals and from ergosterol in plants. This irra-  diation cleaves the carbon-carbon bond at C9-C10 to open the B ring to form chole-  calciferol, an inactive precursor of l,25-(OH) 2 -cholecalciferol (calcitriol). Calcitriol  is the most potent biologically active form of vitamin D (see Fig. 34.26).   The formation of calcitriol from cholecalciferol begins in the liver and ends in  the kidney, where the pathway is regulated. In this activation process, carbon 25 of  vitamin D 2 or D 3 is hydroxylated in the microsomes of the liver to form 25-hydrox-  ycholecalciferol (calcidiol). Calcidiol circulates to the kidney bound to vitamin  D-binding globulin (transcalciferin). In the proximal convoluted tubule of the kid-  ney, a mixed function oxidase, which requires molecular 0 2 and NADPH as cofac-  tors, hydroxy lates carbon 1 on the A ring to form calcitriol. This step is tightly reg-  ulated and is the rate-limiting step in the production of the active hormone.   l,25-(OH) 2 D 3 (calcitriol) is approximately 100 times more potent than 25-  (OH)D 3 in its actions, yet 25-(OH)D 3 is present in the blood in a concentration that  may be 100 times greater, which suggests that it may play some role in calcium and  phosphorus homeostasis.   The biologically active forms of vitamin D are sterol hormones and, like other  steroids, diffuse passively through the plasma membrane. In the intestine, bone, and  kidney, the sterol then moves into the nucleus and binds to specific vitamin D 3  receptors. This complex activates genes that encode proteins mediating the action  of active vitamin D 3 . In the intestinal mucosal cell, for example, transcription of  genes encoding calcium-transporting proteins is activated. These proteins are capa-  ble of carrying Ca 2+ (and phosphorus) absorbed from the gut lumen across the cell,  making it available for eventual passage into the circulation.   ^ Ann Jeina is typical of patients with essentially normal serum triacyl-  ) glycerol levels and elevated serum total cholesterol levels that are repeat-  edly in the upper 1% of the general population (e.g., 325-500 mg/dL).  When similar lipid abnormalities are present in other family members in a pattern  of autosomal dominant inheritance and no secondary causes for these lipid alter-  ations (e.g., hypothyroidism) are present, the entity referred to as "familial hyper-  cholesterolemia (FH), type IIA" is the most likely cause of this hereditary dys-  lipidemia.   FH is a genetic disorder caused by an abnormality in one or more alleles respon-  sible for the formation or the functional integrity of high-affinity LDL receptors on  the plasma membrane of cells that normally initiate the internalization of circulat-  ing LDL and other blood lipoproteins. Heterozygotes for FH (1 in 500 of the pop-  ulation) have roughly one half of the normal complement or functional capacity of  such receptors, whereas homozygotes (1 in 1 million of the population) have essen-  tially no functional LDL receptors. The rare patient with the homozygous form of  FH has a more extreme elevation of serum total and LDL cholesterol than does the  heterozygote and, as a result, has a more profound predisposition to premature coro-  nary artery disease.   Chronic hypercholesterolemia not only may cause the deposition of lipid within  vascular tissues leading to atherosclerosis but also may cause the deposition of lipid  within the skin and eye. When this occurs in the medial aspect of the upper and  lower eyelids, it is referred to as xanthelasma. Similar deposits known as xanthomas  may occur in the iris of the eye (arcus lipidalis) as well as the tendons of the hands  ("knucklepads") and Achilles tendons.   Although therapy aimed at inserting competent LDL receptor genes into the cells  of patients with homozygous FH is undergoing clinical trials, the current approach  in the heterozygote is to attempt to increase the rate of synthesis of LDL receptors  in cells pharmacologically.   Ann Jeina was treated with cholestyramine, a resin that binds some of the bile  salts in the intestine, causing these resin-bound salts to be carried into the feces  rather than recycled to the liver. The liver must now synthesize more bile salts,  which lowers the intrahepatic free cholesterol pool. As a result, hepatic LDL recep-  tor synthesis is induced, and more circulating LDL is taken up by the liver.   HMG-CoA reductase inhibitors, such as pravastatin, also stimulate the synthesis  of additional LDL receptors but do so by inhibiting HMG-CoA reductase, the rate-  limiting enzyme for cholesterol synthesis. The subsequent decline in the intracellu-  lar free cholesterol pool also stimulates the synthesis of additional LDL receptors.  These additional receptors reduce circulating LDL-cholesterol levels by increasing  receptor-mediated endocytosis of LDL particles.   A combination of strict dietary and dual pharmacologic therapy, aimed at  decreasing the cholesterol levels of the body, is usually quite effective in cor-   Ann Jeina was treated with a statin (pravastatin) and cholestyramine, a bile acid  sequestrant. With the introduction of the cholesterol absorption blocker ezetimibe, the  use of cholestyramine with its high level of gastrointestinal side effects may decline.  Ezetimibe reduces the percentage of absorption of free cholesterol present in the lumen of the  gut and hence the amount of cholesterol available to the enterocyte to package into chylomi-  crons. This, in turn, reduces the amount of cholesterol returning to the liver in chylomicron rem-  nants. The net result is a reduction in the cholesterol pool in hepatocytes. The latter induces the  synthesis of an increased number of LDL receptors by the liver cells. As a consequence, the  capacity of the liver to increase hepatic uptake of LDL from the circulation leads to a decrease  in serum LDL levels.   H-C-CH 2 -CH 2 -CH 2 -C-OH  H 3 C | CH,   Fig. 34.26. Synthesis of active vitamin D. (1,  25-di (OH) 2 D 3 ) is produced from 7-dehydroc-  holesterol, a precursor of cholesterol. In the  skin, ultraviolet (UV) light produces cholecal-  ciferol, which is hydroxylated at the 25-posi-  tion in the liver and the 1 -position in the kid-  ney to form the active hormone.   Vera Leizd's hirsutism was most  likely the result of a problem in her  adrenal cortex that caused excessive  production of DHEA.   recting the lipid abnormality and, hopefully, the associated risk of atherosclerotic  cardiovascular disease in patients with heterozygous familial hypercholes-  terolemia.   ^ Low-density lipoprotein cholesterol is the primary target of cholesterol-  lowering therapy because both epidemiologic and experimental evidence  strongly suggest a benefit of lowering serum LDL cholesterol in the pre-  vention of atherosclerotic cardiovascular disease. Similar evidence for raising sub-  normal levels of serum HDL cholesterol is less conclusive but adequate to support  such efforts, particularly in high-risk patients, such as Ivan Applebod, who have  multiple cardiovascular risk factors. The first-line therapy in this attempt is non-  pharmacologic and includes such measures as increasing aerobic exercise, weight  loss in overweight patients, avoidance of excessive alcohol intake, reducing the  intake of refined sugars, and cessation of smoking. If these efforts fail, drug therapy  to raise serum HDL cholesterol levels must be considered.   So far, Mr. Applebod has failed in his attempts to diet and exercise. His LDL  cholesterol level is 231 mg/dL. According to Table 34.1, he is a candidate for more  stringent dietary therapy and for drug treatment. He could be given an HMG CoA  reductase inhibitor such as pravastatin and, perhaps, a bile salt-binding resin such  as cholestyramine. Other lipid-lowering drugs such as the fibric acid derivatives and  ezetimibe, which also decrease triacylglycerol levels and potentially increase HDL  levels, should be considered (Table 34.5).   ^ Vera Leizd was born with a normal female genotype and phenotype, had  ) normal female sexual development, spontaneous onset of puberty, and reg-  ular, although somewhat scanty, menses until the age of 20. At that point,  she developed secondary amenorrhea (cessation of menses) and evidence of male  hormone excess with early virilization (masculinization).   The differential diagnosis included an ovarian versus an adrenocortical  source of the excess androgenic steroids. A screening test to determine whether    Activates LPL;  reduces hepatic  production of VLDL;  reduces catabolism  of HDL    Antagonizes PPAR-a  causing an increase  in LPL activity, a  decrease in  apoprotein C-lll  production, and an  increase in  apoprotein A-l   Abbreviations: LPL, lipoprotein lipase; LDL, low-density lipoprotein; HDL, high-density lipoprotein; triacylglycerols, triglycerides; PPAR, peroxisome proliferators-acti-  vated receptor (the Table is adapted from Circulation 2002; 106:3145-3457).   the adrenal cortex or the ovary is the source of excess male hormone involves  the measurement of the concentration of dehydroepiandrosterone sulfate  (DHEAS) in the patient's plasma, because the adrenal cortex makes most of the  DHEA, and the ovary makes little or none. Vera's plasma DHEAS level was  moderately elevated, identifying her adrenal cortices as the likely source of her  virilizing syndrome.   If the excess production of androgens is not the result of an adrenal tumor, but  the result of a defect in the pathway for Cortisol production, the simple treatment  is to administer glucocorticoids by mouth. The rationale for such treatment can be  better understood by reviewing Fig. 34.23. If Vera Leizd has a genetically deter-  mined partial deficiency in the P450 C11 enzyme system needed to convert 11-  deoxycortisol to Cortisol, her blood Cortisol levels would fall. By virtue of the nor-  mal positive feedback mechanism, a subnormal level of Cortisol in the blood would  induce the anterior pituitary to make more ACTH. The latter would not only stim-  ulate the Cortisol pathway to increase Cortisol synthesis to normal but, in the  process, would also induce increased production of adrenal androgens such as  DHEA and DHEAS. The increased levels of the adrenal androgens (although rel-  atively weak androgens) would cause varying degrees of virilization, depending on  the severity of the enzyme deficiency. The administration of a glucocorticoid by  mouth would suppress the high level of secretion of ACTH from the anterior pitu-  itary gland that occurs in response to the reduced levels of Cortisol secreted from  the adrenal cortex. Treatment with prednisone (a synthetic glucocorticoid), there-  fore, will prevent the ACTH-induced overproduction of adrenal androgens. How-  ever, when ACTH secretion returns to normal, endogenous Cortisol synthesis falls  below normal. The administered prednisone brings the net glucocorticoid activity  in the blood back to physiologic levels. Vera's adrenal androgen levels in the blood  returned to normal after several weeks of therapy with prednisone (a synthetic glu-  cocorticoid). As a result, her menses eventually resumed, and her virilizing fea-  tures slowly resolved.   Because Vera's symptoms began in adult life, her genetically determined adrenal  hyperplasia is referred to as a "nonclassic" or "atypical" form of the disorder. A more  severe enzyme deficiency leads to the "classic" disease, which is associated with  excessive fetal adrenal androgen production in utero and, therefore, manifests itself at  birth, often with ambiguous external genitalia and virilizing features in the female  neonate.   Defects in the LDL receptor gene are responsible for the elevated blood  levels of LDL, and thus of cholesterol, in FH. Over 300 mutations have  been found in the LDL receptor gene, affecting all stages in the production  and functioning of the receptor.   The LDL receptor gene, which contains 18 exons and is 45 kilobases (kb) in  length, is located on the short arm of chromosome 19. The exons share sequences  for the C9 component of complement (a blood protein involved in the immune  response), and the TV-linked oligosaccharide domain is homologous to the genes for  the precursor of EGF and also for three proteases of the blood clotting system, Fac-  tors IX and X and protein C (see Chapter 45). The LDL receptor gene encodes a  glycoprotein that contains 839 amino acids.   Heterozygotes for FH have one normal and one mutant allele. Their cells pro-  duce approximately half the normal amount of receptor and take up LDL at about  half the normal rate. Homozygotes have two mutant alleles, which may either be  identical or differ. They produce very little functional receptor.   The genetic mutations are mainly deletions, but insertions or duplications also  occur, as well as missense and nonsense point mutations (see Fig. 34.20). Four  classes of mutations have been identified. The first class involves "null" alleles that  either direct the synthesis of no protein at all or a protein that cannot be precipitated  by antibodies to the LDL receptor. In the second class, the alleles encode proteins, but  they cannot be transported to the cell surface. The third class of mutant alleles encodes  proteins that reach the cell surface but cannot bind LDL normally. Finally, the fourth  class encodes proteins that reach the surface and bind LDL but fail to cluster and inter-  nalize the LDL particles. The result of each of these mutations is that blood levels of  LDL are elevated because cells cannot take up these particles at a normal rate.   Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly  WS, Valle D, et al., eds. The Metabolic and Molecular Bases of Inherited Disease. 8 th Ed., vol III.  New York: McGraw-Hill, 2001:2863-2913.   Jeon H, Meng W, Takagi J, Eck MG, Springer TA, Blacklow SC. Implications for familial hypercholes-  terolemia from the structure of the LDL receptor YWTD-EGF domain pair. Nature Struct Biol  2001;8:499-504.   Nimpf J, Schneider WJ. From cholesterol transport to signal transduction: Low density lipoprotein  receptor, very low density lipoprotein receptor, and apolipoprotein E receptor-2. Biochim Biophys  Acta 2000;1529:287-298.   Yokoyama S.. Release of cellular cholesterol molecular mechanism for cholesterol homeostasis in cells  and in the body. Biochim Biophys Acta 2000;1529:231-244.   1 . Which of the following steps in the biosynthesis of cholesterol is the committed rate-limiting step?   (A) The condensation of acetoacetyl-CoA with a molecule of acetyl-CoA to yield p-hydroxy-(3 methylglutaryl-CoA  (HMG-CoA)   2. Considering the final steps in cholesterol biosynthesis, when squalene is eventually converted to lanosterol, which of the fol-  lowing statements is correct?   (A) All of the sterols have three fused rings (the steroid nucleus) and are alcohols with a hydroxyl group at C-3.   (B) The action of squalene monooxygenase oxidizes carbon 14 of the squalene chain, forming an epoxide.   (C) Squalene monooxygenase is considered a mixed function oxidase because it catalyzes a reaction in which only one of  the oxygen atoms of 0 2 is incorporated into the organic substrate.   (D) Squalene monooxygenase uses reduced flavin nucleotides (e.g., FAD(2H)) as the cosubstrate in the reaction.   (E) Squalene is joined at carbons 1 and 30 to form the fused ring structure of sterols.   3. Of the major risk factors for the development of atherosclerotic cardiovascular disease (ASCVD) such as sedentary lifestyle,  obesity, cigarette smoking, diabetes mellitus, hypertension, and hyperlipidemia, which one, if present, is the only risk factor  in a given patient without a history of having had a myocardial infarction that requires that the therapeutic goal for the serum  LDL cholesterol level be < lOOmg/dL?   4. Which one of the following apoproteins acts as a cof actor activator of the enzyme lipoprotein lipase (LPL)?   5. Which one of the following sequences places the lipoproteins in the order of most dense to least dense?   The eicosanoids, which include the prostaglandins (PG), thromboxanes (TX),  and leukotrienes (LT), are among the most potent regulators of cellular function  in nature and are produced by almost every cell in the body. They act mainly as  "local" hormones, affecting the cells that produce them or neighboring cells of a  different type.   Eicosanoids participate in many processes in the body, particularly the inflam-  matory response that occurs after infection or injury. The inflammatory response  is the sum of the body's efforts to destroy invading organisms and to repair dam-  age. It includes the control of bleeding through the formation of blood clots. In the  process of protecting the body from a variety of insults, the inflammatory response  can produce symptoms such as pain, swelling, and fever. An exaggerated or inap-  propriate expression of the normal inflammatory response may occur in individuals  who have allergic or hypersensitivity reactions.   In addition to participating in the inflammatory response, eicosanoids also regu-  late smooth muscle contraction (particularly in the intestine and uterus). They  increase water and sodium excretion by the kidney and are involved in regulating  blood pressure . They frequently serve as modulators; some eicosanoids stimulate and  others inhibit the same process. For example, some serve as constrictors and others  as dilators of blood vessels. They are also involved in regulating bronchoconstriction  and bronchodilation.   Eicosanoids are derived from polyunsaturated fatty acids containing 20 carbon  atoms, which are found in cell membranes esterified to membrane phospholipids.  Arachidonic acid, derived from the diet or synthesized from linoleate, is the com-  pound from which most of the eicosanoids are produced in the body. Compounds  that serve as signals for eicosanoid production bind to cell membrane receptors  and activate phospholipases that cleave the polyunsaturated fatty acids from cell  membrane phospholipids (Fig. 35.1).   Arachidonic acid is enzymatically metabolized by three major pathways. The  two pathways that have been most thoroughly studied are the cyclo oxygenase  pathway (which produces prostaglandins and thromboxanes) and the  lipoxygenase pathway (which produces leukotrienes). The cytochrome P450  pathway generates eicosanoids with less well-defined physiologic functions.  Isoprostanes are a relatively new class of eicosanoids derived from  none nzy mat ic free radical-catalyzed reactions. The isoprostanes are similar  to prostaglandins in structure and may play a role in inflammatory responses  and free radical-mediated tissue injury. In brain tissue, arachidonic acid can  be coupled to ethanolamine to generate anandamide. This compound can  bind and activate cannabinoid receptors with actions similar to those of A 9 -  tetrahydrocannabinol (THC).   Many eicosanoids have very short half-lives, in the range of a few minutes or  less. They are rapidly inactivated and excreted.   Fig. 35.1. Overview of eicosanoid metabolism. Eicosanoids are produced from fatty acids  released from membrane phospholipids. In humans, arachidonic acid is the major precursor  of the eicosanoids, which include the prostaglandins, leukotrienes, and thromboxanes. The  circled minus sign = inhibits; cyt = cytochrome.   Since her admission to the hospital for an acute myocardial infarction, Ann  Jeina has been taking the bile salt sequestrant cholestyramine and the  HMG-CoA reductase inhibitor pravastatin to lower her blood cholesterol  levels (see Chapter 34). She also takes 160 mg acetylsalicylic acid (ASA; aspirin)  each day. At her most recent visit to her cardiologist, she asked whether she should   "Eicosa" is the Greek word for the  ) number 20. Eicosanoids are syn-  thesized from polyunsaturated  fatty acids with 20 carbon atoms.   The composition of the diet affects  \\ ) the fatty acid content of membrane  phospholipids. Individuals with a  high content of saturated fatty acids in their  diets have a high content of saturated fatty  acids in their membrane lipids. Likewise,  individuals with a high content of polyunsat-  urated fatty acids in their diets have a high  content of polyunsaturated fatty acids in  their membrane lipids.   0 © Dietary deficiencies of essential  ( /~vA J fatty acids are rare. However, some  ™ cases have been reported in  patients receiving total parenteral nutrition  (TPN). Although the most obvious symptom  is a red scaly dermatitis, deficiencies of  essential fatty acids also result in a  decreased availability of precursors for  eicosanoid synthesis.   continue to take aspirin because she no longer has any chest pain. She was told that  the use of aspirin in her case was not to alleviate pain but to reduce the risk of a sec-  ond heart attack and that she should continue to take this drug for the remainder of  her life unless a complication, such as gastrointestinal bleeding, occurred as a result  of its use.   Emma Wheezer has done well with regard to her respiratory function  since her earlier hospitalization for an acute asthmatic attack. She has been  maintained on two puffs of triamcinolone acetonide, a potent inhaled cor-  ticosteroid, three times per day, and has not required systemic steroids for months.  The glucose intolerance precipitated by high intravenous and oral doses of the syn-  thetic glucocorticoid dexamethasone during her earlier hospitalization resolved  after this drug was discontinued. She has come to her doctor now because she is  concerned that the low-grade fever and cough she has developed over the last 36  hours may trigger another acute asthma attack.   The most abundant and therefore the most common precursor of the eicosanoids is  arachidonic acid (eicosatetraenoic acid, o)6,20:4,A5,8,ll,14), a polyunsaturated  fatty acid with 20 carbons and 4 double bonds (see Fig. 35.1). It is esterified to  phospholipids located in the lipid bilayer that constitutes the plasma membrane of  the cell. Because arachidonic acid cannot be synthesized de novo in the body, the  diet must contain arachidonic acid or other fatty acids from which arachidonic acid  can be produced. The major dietary precursor for arachidonic acid synthesis is the  essential fatty acid linoleate, which is present in plant oils (see Chapter 33).   The arachidonic acid present in membrane phospholipids is released from the  lipid bilayer as a consequence of the activation of membrane-bound phospholipase  A2 or C (see Fig. 33.31 and Fig. 35.2). This activation occurs when a variety of  stimuli (agonists), such as histamine and the cytokines, interact with a specific  plasma membrane receptor on the target cell surface. Phospholipase A2 is specific  for the sn-2 position of phosphoacylglycerols, the site of attachment of arachidonic  acid to the glycerol moiety. Phospholipase C hydrolyzes phosphorylated inositol   Fig. 35.2. Release of arachidonic acid from membrane lipids. The binding of a stimulus to  its receptor activates pathway 1 or 2.   from the inositol glycerophospholipids, generating a diacylglycerol containing  arachidonic acid. This arachidonic acid is subsequently released by the action of  other lipases.   After arachidonic acid is released into the cytosol, it is converted to eicosanoids by  a variety of enzymes with activities that vary among tissues. This variation explains  why some cells, such as those in the vascular endothelium, synthesize prostaglandins  E 2 and I 2 (PGE 2 and PGI 2 ) whereas cells, such as platelets, synthesize primarily  thromboxane A 2 (TXA 2 ) and 12-hydroxyeicosatetraenoic acid (12-HETE).   Three major pathways for the metabolism of arachidonic acid occur in various  tissues (Fig. 35.3). The first of these, the cyclooxygenase pathway, leads to the syn-  thesis of prostaglandins and thromboxanes. The second, the lipoxygenase pathway,  yields the leukotrienes, HETEs, and lipoxins. The third pathway, catalyzed by the  cytochrome P450 system, is responsible for the synthesis of the epoxides, HETEs,  and diHETEs.   Prostaglandins are fatty acids containing 20 carbon atoms, including an internal 5-  carbon ring. In addition to this ring, each of the biologically active prostaglandins  has a hydroxyl group at carbon 15, a double bond between carbons 13 and 14, and  various substituents on the ring (Fig. 35.4)   The nomenclature for the prostaglandins (PGs) involves the assignment of a cap-  ital letter (PGE), an Arabic numeral subscript (PGE^, and, for the PGF family, a  Greek letter subscript (e.g., PGF 2a ). The capital letter, in this case "F," refers to the  ring substituents shown in figure 35.5.   The subscript that follows the capital letter (PGFi) refers to the PG series 1, 2,  or 3, determined by the number of unsaturated bonds present in the linear portion  of the hydrocarbon chain (Fig. 35.6). It does not include double bonds in the inter-  nal ring. Prostaglandins of the 1 -series have one double bond (between carbons 13  and 14). The 2-series has two double bonds (between carbons 13 and 14, and 5 and  6), and the 3-series has three double bonds (between carbons 13 and 14, 5 and 6,  and 17 and 18). The double bonds between carbons 13 and 14 are trans; the others  are cis.   The Greek letter subscript, found only in the F series, refers to the position of the  hydroxyl group at carbon 9. This hydroxyl group primarily exists in the a position,  where it lies below the plane of the ring, as does the hydroxyl group at carbon 1 1  (see Figs. 35.4 and 35.5).    Inflammation involving the  mucosal and smooth muscle layers  of the respiratory tract plays a  major role in the development of acute asth-  matic bronchospasm in patients such as  Emma Wheezer. Dexamethasone and other  potent glucocorticoids are capable of pre-  venting or suppressing this inflammation.   In part, the glucocorticoids act by inhibit-  ing the recruitment of leukocytes and mono-  cytes-macrophages into affected areas.  They also limit the ability of these cells to  elaborate a variety of chemotactic factors  and other substances, such as certain  eicosanoids, which enhance the inflamma-  tory process. Glucocorticoids, for example,  suppress the transcription and translation of  the inducible form of the cyclooxygenase  enzyme, COX-2. Glucocorticoids also induce  the synthesis of a protein or family of pro-  teins (lipocortins or macrocortins) that  inhibit the activity of phospholipase A 2 . As a  result, the synthesis of prostaglandins and  leukotrienes is decreased, and the inflamma-  tory response in bronchial tissues is reduced  (see Figs. 35.1 and 35.2).    Fig. 35.4. Structural features common to the  biologically active prostaglandins. These com-  pounds have 20 carbons, with a carboxyl group  at carbon 1. Carbons 8 through 12 form a five-  membered ring with substituents (usually a  hydroxyl or keto group) at carbons 9 (X) and  11 (Y). Carbon 15 contains a hydroxyl group,  and a trans double bond connects carbons 13  and 14. Double bonds also may be present  between carbons 5 and 6 and between carbons  17 and 18 (see Fig. 35.6).   Fig. 35.5. Ring substituents of the prosta-  glandins (PG). The letter after PG denotes the  configuration of the ring and its substituents.  R 4 , R 7 , and R 8 represent the non-ring portions  of the molecule. R 4 contains four carbons  (including the carboxyl group). R 7 and R 8 con-  tain seven and eight carbons, respectively, and  correspond to the 1-, 2-, or 3-series shown in  Figure 35.6. Note that the prostacyclins (PGI)  contain two rings.   C 20 :A 5 ' 8 ' 11 ' 14 '  (precursor)  co6   Fig. 35.6. Prostaglandins of the 1-, 2-, and 3-series and their precursors. The numeral (as a  subscript in the name of the compound) refers to the number of double bonds in the non-ring  portion of the prostaglandin. Trans double bonds are at position 13, and cis double bonds at  positions 5 and 17. The hydroxyl group at carbon 15 is required for biologic activity.   The thromboxanes, derived from arachidonic acid via the cyclooxygenase pathway,  closely resemble the prostaglandins in structure except that they contain a 6-mem-  The thromboxanes were named for bered ring that includes an oxygen atom (Fig. 35.7). The most common thrombox-  their action in producing blood ane, TXA 2 , contains an additional oxygen atom attached both to carbon 9 and car-  clots (thrombi). bon 11 of the ring.   Only the biosynthesis of those prostaglandins derived from arachidonic acid (e.g.,  the 2-series, such as PGE 2 , PGI 2 , TXA 2 ) are described, because those derived from  eicosatrienoic acid (the 1 -series) or from eicosapentaenoic acid (the 3-series) are  present in very small amounts in humans on a normal diet (see Fig. 35.6).   The biochemical reactions that lead to the synthesis of prostaglandins and throm-  boxanes are illustrated in figure 35.8. The initial step, which is catalyzed by a  cyclooxygenase, forms the five-membered ring and adds four atoms of oxygen (two  between carbons 9 and 11, and two at carbon 15) to form the unstable endoperox-  ide, PGG 2 . The hydroperoxy group at carbon 15 is quickly reduced to a hydroxyl  group by a peroxidase to form another endoperoxide, PGH 2 .   The next step is tissue specific (see Fig. 35.8). Depending on the type of cell  involved, PGH 2 may be reduced to PGE 2 or PGD 2 by specific isomerases (PGE syn-  thase and PGD synthase). PGE 2 may be further reduced by PGE 9-ketoreductase to  form PGF 2a PGF 2a also may be formed directly from PGH 2 by the action of an  endoperoxide reductase. Some of the major functions of the prostaglandins are  listed in Table 35.1.   PGH 2 may be converted to the thromboxane TXA 2 , a reaction catalyzed by TXA  synthase (see Fig. 35.8). This enzyme is present in high concentration in platelets. In  the vascular endothelium, however, PGH 2 is converted to the prostaglandin PGI 2   Fig. 35.7. The thromboxane ring. In contrast  to the prostaglandins, which have a five-mem-  bered carbon ring, thromboxanes have a six-  membered ring (shown in blue) containing an  oxygen atom. Substituents are attached to the  ring at carbons 9 and 1 1 . In the case of TXA 2  (shown above), an oxygen atom connects car-  bons 9 and 1 1 .   Fig. 35.8. Formation of prostaglandins (including the prostacyclin PGI 2 ) and thromboxane TXA 2 from arachidonic acid. The conversion of  arachidonic acid to PGH 2 is catalyzed by a membrane-bound enzyme, prostaglandin endoperoxide synthase, which has cyclooxygenase and per-  oxidase activities. The reducing agent is glutathione (GSH), which is oxidized to GSSG.   The predominant eicosanoid in platelets is TXA 2 , a potent vasoconstrictor and a stimulator of platelet aggregation. The latter  action initiates thrombus formation at sites of vascular injury as well as in the vicinity of a ruptured atherosclerotic plaque in the  lumen of vessels such as the coronary arteries. Such thrombi may cause sudden total occlusion of the vascular lumen, causing  acute ischemic damage to tissues distal to the block (i.e., acute myocardial infarction).   Aspirin, by covalently acetylating the active site of cyclooxygenase, blocks the production of TXA 2 from its major precursor, arachidonic  acid. By causing this mild hemostatic defect, low-dose aspirin has been shown to be effective in prevention of acute myocardial infarction  (see Clinical Comments). For Ivan Applebod (who has symptoms of coronary heart disease), aspirin is used to prevent a first heart attack  (primary prevention). For Ann Jeina and Cora Nari (who already have had heart attacks), aspirin is used to prevent a second heart attack  (secondary prevention).    Q o Diets that include cold water fish  [ ooA ) (e.g., salmon, mackerel, brook  ^ trout, herring), with a high content  of polyunsaturated fatty acids, eicosapen-  taenoic acid (EPA), and docosahexaenoic  acid (DHA) (see Chapter 33), result in a high  content of these fatty acids in membrane  phospholipids. It has been suggested that  such diets are effective in preventing heart  disease, in part because they lead to forma-  tion of more TXA 3 relative to TXA 2 . TXA 3 is  less effective in stimulating platelet aggrega-  tion than its counterpart in the 2-series, TXA 2 .   a IL = interleukin, a cytokine that augments the activ-  ity of many cells in the immune system.   (prostacyclin) by PGI synthase (see Fig. 35.8). TXA 2 and PGI 2 have important antag-  onistic biologic effects on vasomotor and smooth muscle tone and on platelet aggre-  gation. Some of the known functions of the thromboxanes are listed in Table 35.2.   In the 1990s, the cyclooxygenase enzyme was found to exist as two distinct iso-  forms, designated COX-1 and COX-2. COX-1 is regarded as a constitutive form of  the enzyme, widely expressed in almost all tissues, and involved in the production  of prostaglandins and thromboxanes for "normal" physiologic functions. COX-2 is  an inducible form of the enzyme regulated by a variety of cytokines and growth fac-  tors. COX-2 mRNA and protein levels are usually low in most healthy tissue, but  are expressed at high levels in inflamed tissue.   Because of the importance of prostaglandins in mediating the inflammatory  response, drugs that block prostaglandin production should provide relief from  pain. The cyclooxygenase enzyme is inhibited by all nonsteroidal anti-inflamma-  tory drugs (NSAIDs) such as aspirin (acetylsalicylic acid). Aspirin transfers an  acetyl group to the enzyme, irreversibly inactivating it (Fig. 35.9). Other NSAIDs  (e.g., acetaminophen, ibuprofen) act as reversible inhibitors of cyclooxygenase.  Acetaminophen is the major ingredient in Tylenol, and ibuprofen is the major  ingredient in other NSAIDs such as Motrin, Nuprin, and Advil (see Fig. 35.9).  Although having some relative selectivity for inhibiting either COX-1 or COX-2,   NSAIDs block the activity of both isoforms. These findings have provided the  impetus for the development of selective COX-2 inhibitors, which are proposed  to act as potent anti-inflammatory agents by inhibiting COX-2 activity, without  the gastrointestinal and anti-platelet side effects commonly associated with  NSAID use. These NSAID adverse effects are thought to be a result of COX-1  inhibition. Examples of these newer selective COX-2 inhibitors are celecoxib  (Celebrex) and rofecoxib (Vioxx).   Prostaglandins and thromboxanes are rapidly inactivated. Their half-lives (t 1/2 )  range from seconds to minutes. The prostaglandins are inactivated by oxidation of  the 15 -hydroxy group, critical for their activity, to a ketone. The double bond at  carbon 13 is reduced. Subsequently, both (3- and oo-oxidation of the non-ring por-  tions occur, producing dicarboxylic acids that are excreted in the urine. Active  TXA 2 is rapidly metabolized to TXB 2 by cleavage of the oxygen bridge between  carbons 9 and 11 to form two hydroxyl groups. TXB 2 has no biologic activity.   In addition to serving as a substrate for the cyclooxygenase pathway, arachidonic  acid also acts as a substrate for the lipoxygenase pathway. The lipoxygenase  enzymes catalyze the incorporation of an oxygen molecule onto a carbon of one of  several double bonds of arachidonic acid, forming a hydroperoxy (-OOH) group at  these positions. With this oxygenation, the double bond isomerizes to a position one  carbon removed from the hydroperoxy group and is transformed from the cis to the  trans configuration (Fig. 35.10). The unstable hydroperoxy group is then converted  to the more stable hydroxy group.   Fig. 35.10. Action of lipoxygenases in the formation of HPETEs and HETEs. Lipoxygenases add hydroperoxy groups at position 5, 12, or 15  with rearrangement of the double bond. HPETEs are unstable and are rapidly reduced to form HETEs or converted to leukotrienes and lipoxins  (see Figs. 35.11 and 35.12).   Leukotrienes were so named  \ \ ) because they are synthesized in  *r leukocytes and contain the typical  triene structure, i.e., three double bonds in  series (in this case, at positions 7, 9, and 11)  (see Fig. 35.11).   Fig. 35.12. Formation of the lipoxins. These  compounds contain three hydroxy 1 groups.   Lipoxygenases may act at carbons 5, 12, or 15. The type of lipoxygenase varies  from tissue to tissue. For example, polymorphonuclear leukocytes contain primarily  5-lipoxygenase, platelets are rich in 12-lipoxygenase, and eosinophilic leukocytes  contain primarily 15-lipoxygenase.   As shown in figure 35.11, the synthesis of the leukotrienes begins with the forma-  tion of hydroperoxyeicosatetraenoic acids (HPETEs). This product is either reduced  to the corresponding hydroxy metabolites, HETEs (see Fig. 35.10), or it is metabo-  lized to form leukotrienes or lipoxins (see Figs. 35.11 and 35.12). The major  leukotrienes are produced by 5-lipoxygenase.   In leukocytes and mast cells, 5-HPETE is converted to an epoxide, leukotriene  A 4 (LTA 4 ). The subscript number 4 refers to the presence of four double bonds in  the leukotriene. Three of the double bonds (7, 9, 11) are conjugated, that is, they  form a triene.   Other functional leukotrienes are formed from LTA 4 by one of two pathways. In  the first, LTA 4 is converted to LTB 4 , a 5,12-dihydroxy derivative. The second meta-  bolic pathway involves the addition of reduced glutathione to carbon 6 to form  LTC 4 , a reaction catalyzed by glutathione S-transf erase. Glutamate is removed  from the glutathione moiety of LTC 4 through the action of 7-glutamyl transpepti-  dase to form LTD 4 . A dipeptidase then cleaves the glycine residue from LTD 4 to  form LTE 4 (see Fig. 35.11). The major functions of some of the leukotrienes are  listed in Table 35.3.    Fig. 35.11. Formation of leukotrienes and their glutathione (GSH) derivatives. HPETE = hydroper-  oxyeicosatetraenoic acids; LT = leukotriene (thus, LTA 4 is leukotriene A 4 ).   The lipoxins are formed through the action of 15 -lipoxygenase followed by the  action of 5 -lipoxygenase on arachidonic acid. A series of reductions of the resultant  hydroperoxy groups leads to the formation of trihydroxy derivatives of arachidonic  acid known as the lipoxins (see Fig. 35.12). Lipoxins induce chemotaxis and stim-  ulate superoxide anion production in leukocytes.   C. Cytochrome P450 Pathway: Synthesis and Actions  of Epoxides, HETEs, and diHETES   A third mechanism for the oxygenation of arachidonic acid involves the cytochrome  P450 pathway. The activity of the monooxygenases in this microsomal system  yields epoxides, certain forms of HETEs (e.g., oo-hydroxy derivatives), and diol  forms (diHETEs) (Fig. 35.13). The biologic activities of these compounds include  actions in ocular, vascular, endocrine, and renal systems. Some of these actions are  attributed to inhibition of Na + , K + -ATPase. The physiologic role of these com-  pounds remains to be fully characterized.   Isoprostanes are derived from arachidonic acid by lipid peroxidation, initiated by  free radicals. There is no enzymatic mechanism for their production. Arachidonic  acid, while still a component of a phospholipid, undergoes free radical damage, and  then phospholipase A2 removes the altered eicosanoid from the phospholipid and  releases it into circulation (Fig. 35.14). The level of isoprostanes in the urine can be  used as a measure of the oxidative stress of a patient and is a useful biologic marker  for patients undergoing oxidative stress caused by a variety of disorders. Surpris-  ingly, these altered arachidonic acid molecules also have biologic activity when  measured on cultured cells; it is not known whether intracellular levels reach high  enough concentrations to elicit these biologic effects. The best studied isoprostane  is similar to PGF 2a , and this molecule has similar effects on cultured cells as does  PGF 2a (see Table 35.1).   Endocannabinoids are endogenous ligands for the cannabinoid receptor, with effects  primarily in the nervous system. Anandamide was the first such ligand isolated and  identified. Anandamide is synthesized in neurons from phosphatidylethanolamine, as  outlined in figure 35.15. The biosynthetic pathway is unique in transferring an arachi-  donic acid group from the 2-position to the free amino group on ethanolamine, and  then using a unique phospholipase D to cleave the modified ethanolamine from the  phospholipid. The synthesis of anandamide is regulated, in part, by agonists that cause  calcium influx into nerve cells. Once anandamide is released, it acts as a retrograde  messenger, binding to receptors on the presynaptic membrane that alter ion fluxes  such that neurotransmitter release from the presynaptic neuron can be increased and  an analgesic effect obtained. Anandamide is degraded by the enzyme fatty acid amide  hydrolase, which splits anandamide to arachidonic acid and ethanolamine. The hydro-  lase enzyme is the target of drug research, because inhibiting the action of this enzyme  will prolong the analgesic effects induced by anandamide.   The eicosanoids have a wide variety of physiologic effects, which are generally  initiated through an interaction of the eicosanoid with a specific receptor on the  plasma membrane of a target cell (Table 35.4). This eicosanoid-receptor binding  either activates the adenylate cyclase-cAMP-protein kinase A system (PGE, PGD,   Fig. 35.13. Examples of compounds produced  from arachidonic acid by the cytochrome P450  pathway.   Recall that oxidative stress results  from reactive oxygen or nitrogen  species that are not fully neutral-  ized by anti-oxidants (see Chapter 24). The  increase in reactive oxygen and nitrogen  species results in protein, lipid, and nucleic  acid modifications. Many neurodegenerative  diseases, such as Alzheimer disease, Parkin-  son disease, and Lou Gehrig disease, have in  common increased oxidative stress.   HC-0-P-OCH 2 CH 2 -N -C^ / ^ / = v =0    Fig. 35.14. Generation of an isoprostane. Rad-  ical damage to a phospholipid on the arachi-  donic acid residue at position 2 generates an  isoprostane, which is then removed from the  damaged phospholipid by phospholipase A2.  The example of an isoprostane shown in this  figure is just one of many that can be produced.   and PGI series) or causes an increase in the level of calcium in the cytosol of tar-  get cells (PGF 2a , TXA 2 , the endoperoxides, and the leukotrienes).   In some systems, the eicosanoids appear to modulate the degree of activation of  adenylate cyclase in response to other stimuli. In these instances, the eicosanoid  may bind to a regulatory subunit of the GTP-binding proteins (G proteins) within  the plasma membrane of the target cell (see Chapter 1 1). If the eicosanoid binds to  the stimulatory subunit, the effect of the stimulus is amplified. Conversely, if the  eicosanoid binds to the inhibitory subunit, the cellular response to the stimulus is  reduced. Through these influences on the activation of adenylate cyclase,  eicosanoids contribute to the regulation of cell function.   Some of the biologic effects of certain eicosanoids occur as a result of a  paracrine or autocrine action. One paracrine action is the contraction of vascular  smooth muscle cells caused by TXA 2 released from circulating platelets (vasocon-  striction). An autocrine action of eicosanoids is exemplified by platelet aggregation  induced by TXA 2 produced by the platelets themselves.   The eicosanoids influence the cellular function of almost every tissue of the  body. Certain organ systems are affected to a greater degree than others.   In the presence of aspirin, cyclooxygenase is irreversibly inactivated by  acetylation. New cyclooxygenase molecules are not produced in platelets,  because these cells have no nuclei and, therefore, cannot synthesize new  mRNA. Thus, the inhibition of cyclooxygenase by aspirin persists for the lifespan of  the platelet (7-10 days). When aspirin is taken daily at doses between 81 and 325 mg,  new platelets are affected as they are generated. Higher doses do not improve efficacy  but do increase side effects, such as gastrointestinal bleeding and easy bruisability.   Patients with established or suspected atherosclerotic coronary disease, such as  Ann Jeina, Cora Nari, and Ivan Applebod, benefit from the action of low-dose  aspirin (approximately 162 mg/day), which produces a mild defect in hemostasis.  This action of aspirin helps to prevent thrombus formation in the area of an athero-  sclerotic plaque at critical sites in the vascular tree.   Corticosteroids reduce inflammation, in part, through their inhibitory effect  on phospholipase A2. In addition, suppression of COX-2 induction is now  thought to be a primary anti-inflammatory mechanism of action for gluco-  corticoids. Despite the unquestionable value of glucocorticoid therapy in a variety of  diseases associated with acute inflammation of tissues, the suppression of the inflam-  matory response with pharmacologic doses of corticosteroids is potentially haz-  ardous. The sudden appearance of temporary glucose intolerance when Emma  Wheezer was treated with large doses of dexamethasone, a gluconeogenic steroid  (glucocorticoid), is just one of the many potential adverse effects of this class of  drugs when given systemically in pharmacologic doses over an extended period. The  inhaled steroids, conversely, have far fewer systemic side effects because their  absorption across the bronchial mucosa into the circulation is very limited. This  property allows them to be used over prolonged periods in the treatment of asthma.  The fact that inhalation allows direct delivery of the agent to the primary site of  inflammation adds to their effectiveness in the treatment of these patients.   Inflammation is the response of the body to infection or injury, directed at  destroying the infectious agents and repairing the damaged areas. It  involves an increase of the blood supply to the affected region by means of    Although our knowledge of the  spectrum of biologic actions of the  endogenous eicosanoids is incom-  plete, several actions are well-enough estab-  lished to allow their application in a variety  of clinical situations or diseases. For exam-  ple, drugs that are analogs of PGE^ and PGE 2  suppress gastric ulceration, in part by  inhibiting secretion of hydrochloric acid in  the mucosal cells of the stomach. Analogs of  PGEt are used in the treatment of sexual  impotence. Men with certain forms of sexual  impotence can self-inject this agent into the  corpus cavernosum of the penis to induce  immediate but temporary penile tumes-  cence. The erection lasts for 1 to 3 hours.  The stimulatory action of PGE 2 and PGF 2a on  uterine muscle contraction has led to the use  of these prostaglandins to induce labor and  to control postpartum bleeding. PGE-, is also  used as palliative therapy in neonates with  congenital heart defects to maintain patency  of the ductus arteriosus until surgery can be  performed. Analogs of PGI 2 have been  shown to be effective in the treatment of pri-  mary pulmonary hypertension.   Although uncomfortable, the pain,  swelling, and fever that are part of  the inflammatory response serve  as an important warning sign that the host is  threatened and that some specific counter-  actions must be taken against the offending  agent or process. Although the use of anti-  inflammatory drugs may bring welcome  symptomatic relief, their use may, in part,  diminish the effectiveness of the host's  response to the inciting agent.   vasodilation. The capillaries become more permeable so that fluid, large molecules,  and white blood cells can cross, leaving the blood and entering the tissue. White  blood cells (particularly neutrophils and monocytes) move by chemotaxis to the  injured site. Redness (rubor), heat (calor), swelling (tumor), and pain (dolor) are  associated with the inflammatory process. Redness and heat are caused by the  increased blood flow. Swelling is the result of the increased movement of fluid and  white blood cells into the area of inflammation. Pain is caused by the release of  chemical compounds and the compression of nerves in the vicinity of the inflam-  matory process.   The chemical mediators of inflammation usually are produced by activation of  complement (a family of blood proteins that are cleaved to form active fragments)  or of the blood clotting cascade (see Chapter 45). These processes cause the release  of histamine from mast cells, and the production of kinins by cleavage of kinino-  gens. Among their other effects, both histamine and kinins increase vascular per-  meability. They stimulate the synthesis of eicosanoids that act on the motility and  metabolism of white blood cells and cause the aggregation of platelets to arrest  bleeding. Some of the prostaglandins act on thermoregulatory centers of the brain,  producing fever. Cytokines are also released that stimulate the proliferation of cells  involved in the immune response.   Brash AR. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J Biol Chem  1999;274:23679-23682.   Dimarzo V, Bisogno T, and DePetrocellis L. Anandamide: some like it hot. Trends Pharm Sci  2001;22:346-349.   Halushka PV. Prostaglandins and related compounds. In: Goldman L and Bennett JC, eds. Cecil Text-  book of Medicine. Philadelphia: WB Saunders, 2000:1189-1194.   Marnett LJ, Rowlinson SW, Goodwin DC, Kalgutkar AS, Lanzo CA. Arachidonic acid oxygenation  by COX-1 and COX-2: Mechanisms of catalysis and inhibition. J Biol Chem 1999;274:  22903-22906.   Morrow JD, Roberts LJ II. Lipid-derived autacoids. In: Hardman JG, Limbird LE, eds. Goodman  and Gilman's The Pharmacological Basis of Therapeutics. New York: McGraw-Hill, 2001:  669-680.   1. In humans, prostaglandins are primarily derived from which of the following?   3. Which of the following drugs leads to the covalent modification, and inactivation, of both the COX-1 and COX-2 enzymes?   4. Thromboxane A2, which is found in high levels in platelets, aids in wound repair through induction of which of the follow-  ing activities?   5. Certain prostaglandins, when binding to their receptor, induce an increase in intracellular calcium levels. The signal that leads  to the elevation of intracellular calcium is initiated by which of the following enzymes?   The purpose of this chapter is to summarize and integrate the major pathways for  the utilization of carbohydrates and fats as fuels. We will concentrate on review-  ing the regulatory mechanisms that determine the flux of metabolites in the fed  and fasting states, integrating the pathways that were described separately under  carbohydrate and lipid metabolism. The next section of the book covers the mech-  anisms by which the pathways of nitrogen metabolism are coordinated with fat  and carbohydrate metabolism.   For the species to survive, it is necessary for us to store excess food when we  eat and to use these stores when we are fasting. Regulatory mechanisms direct  compounds through the pathways of metabolism involved in the storage and uti-  lization of fuels. These mechanisms are controlled by hormones, by the concen-  tration of available fuels, and by the energy needs of the body.   Changes in hormone levels, in the concentration of fuels, and in energy  requirements affect the activity of key enzymes in the major pathways of metabo-  lism. Intracellular enzymes are generally regulated by activation and inhibition,  by phosphorylation and dephosphorylation (or other covalent modifications), by  induction and repression of synthesis, and by degradation. Activation and inhibi-  tion of enzymes cause immediate changes in metabolism. Phosphorylation and  dephosphorylation of enzymes affect metabolism slightly less rapidly. Induction  and repression of enzyme synthesis are much slower processes, usually affecting  metabolic flux over a period of hours. Degradation of enzymes decreases the  amount available to catalyze reactions.   The pathways of metabolism have multiple control points and multiple regula-  tors at each control point. The function of these complex mechanisms is to pro-  duce a graded response to a stimulus and to provide sensitivity to multiple stimuli  so that an exact balance is maintained between flux through a given step ( or  series of steps) and the need or use for the product. Pyruvate dehydrogenase is an  example of an enzyme that has multiple regulatory mechanisms. Regardless of  insulin levels, the enzyme cannot become fully activated in the presence of  products and absence of substrates.   The major hormones that regulate the pathways of fuel metabolism are insulin  and glucagon. In liver, all effects of glucagon are reversed by insulin, and some of  the pathways that insulin activates are inhibited by glucagon. Thus, the pathways  of carbohydrate and lipid metabolism are generally regulated by changes in the  insulin/ glucagon ratio.   Although glycogen is a critical storage form of fuel because blood glucose lev-  els must be carefully maintained, adipose triacylglycerols are quantitatively the  major fuel store in the human. After a meal, both dietary glucose and fat are  stored in adipose tissue as triacy I glycerol. This fuel is released during fasting,  when it provides the main source of energy for the tissues of the body. The length  of time we can survive without food depends mainly on the size of our bodies fat  stores.   Within 2 months of her surgery to remove a benign insulin- secreting (3 cell  tumor of the pancreas, Bea Selmass was again jogging lightly. She had lost  the 8 lb that she had gained in the 6 weeks before her surgery. Because her  hypoglycemic episodes were no longer occurring, she had no need to eat frequent  carbohydrate snacks to prevent the adrenergic and neuroglycopenic symptoms that  she had experienced when her tumor was secreting excessive amounts of insulin.   A few months after her last hospitalization, Di Abietes was once again  brought to the hospital emergency room in diabetic ketoacidosis (DKA).  Blood samples for glucose and electrolytes were drawn repeatedly during  the first 24 hours. The hospital laboratory reported that the serum in each of these  specimens appeared opalescent rather than having its normal clear or transparent  appearance. This opalescence results from light scattering caused by the presence  of elevated levels of triacylglycerol-rich lipoproteins in the blood   When Ann Sulin initially presented with type 2 diabetes mellitus at age  39, she was approximately 30 lb above her ideal weight. Her high serum  glucose levels were accompanied by abnormalities in her 14-hour fasting  lipid profile. Her serum total cholesterol, low-density lipoprotein (LDL) choles-  terol, and triacylglycerol levels were elevated, and her serum high-density lipopro-  tein (HDL) cholesterol level was below the normal range.   I. REGULATION OF CARBOHYDRATE AND LIPID  METABOLISM IN THE FED STATE   A. Mechanisms That Affect Glycogen and Triacylglyc-  erol Synthesis in Liver   After a meal, the liver synthesizes glycogen and triacylglycerol. The level of glyco-  gen stored in the liver can increase from approximately 80 g after an overnight fast  to a limit of approximately 200-300 g. Although the liver synthesizes triacylglycerol,  it does not store this fuel but rather packages it in very-low-density lipoprotein  (VLDL) and secretes it into the blood. The fatty acids of the VLDL triacylglycerols  secreted from the liver are stored as adipose triacylglycerols. Adipose tissue has an  almost infinite capacity to store fat, limited mainly by the ability of the heart to pump  blood through the capillaries of the expanding adipose mass. Although we store fat  throughout our bodies, it tends to accumulate in places where it does not interfere too  much with our mobility: in the abdomen, hips, thighs, and buttocks.   Both the synthesis of liver glycogen and the conversion by the liver of dietary  glucose to triacylglycerol (lipogenesis) are regulated by mechanisms involving key  enzymes in these pathways.   After a meal, glucose can be converted to glycogen or to triacylglycerol in the liver.  For both processes, glucose is first converted to glucose 6-phosphate by glucoki-  nase, a liver enzyme that has a high K m for glucose (Fig. 36.1). Because of the  enzyme's low affinity for glucose, this enzyme is most active in the fed state, when  the concentration of glucose is particularly high because the hepatic portal vein   Fig. 36.1. Regulation of glucokinase, PFK-1,  and pyruvate kinase in the liver.   Fig. 36.2. Regulation of glycogen synthase.  This enzyme is phosphorylated by a series of  kinases, which are initiated by the cAMP-  dependent protein kinase, under fasting condi-  tions. It is dephosphorylated and active after a  meal, and glycogen is stored. Circled P =  phosphate; a circled + sign = activated by; a  circled — sign = inhibited by.   Fig. 36.3. Regulation of pyruvate dehydroge-  nase (PDH). A kinase associated with the PDH  complex phosphorylates and inactivates the  enzyme.   carries digestive products directly from the intestine to the liver. Synthesis of  glucokinase is also induced by insulin (which is elevated after a meal) and  repressed by glucagon (which is elevated during fasting). In keeping with the  liver's function in maintaining blood glucose levels, this system is set up such that  the liver can only metabolize glucose when sugar levels are high, and not when  sugar levels are low.   In the conversion of glucose 6-phosphate to glycogen, the key regulatory enzyme is  glycogen synthase. This enzyme is activated by the dephosphorylation that occurs  when insulin is elevated and glucagon is decreased (Fig. 36.2) and by the increased  level of glucose.   For lipogenesis, glucose 6-phosphate is converted through glycolysis to pyruvate.  Key enzymes that regulate this pathway in the liver are phosphofructokinase-1  (PFK-1) and pyruvate kinase. PFK-1 is allosterically activated in the fed state by  fructose 2,6-bisphosphate and adenosine monophosphate (AMP) (see Fig. 36.1).  Phosphofructokinase-2, the enzyme that produces the activator fructose 2,6-  bisphosphate, is dephosphorylated and active after a meal (see Chapter 22). Pyru-  vate kinase is also activated by dephosphorylation, which is stimulated by the  increase of the insulin/glucagon ratio in the fed state (see Fig. 36.1).   The conversion of pyruvate to fatty acids requires a source of acetyl CoA in the  cytosol. Pyruvate can only be converted to acetyl CoA in mitochondria, so it enters  mitochondria and forms acetyl CoA through the pyruvate dehydrogenase (PDH)  reaction. This enzyme is dephosphorylated and most active when its supply of sub-  strates and adenosine diphosphate (ADP) is high, its products are used, and insulin  is present (Fig. 36.3).   Pyruvate is also converted to oxaloacetate. The enzyme that catalyzes this reac-  tion, pyruvate carboxylase, is activated by acetyl CoA. Because acetyl CoA cannot  directly cross the mitochondrial membrane to form fatty acids in the cytosol, it con-  denses with oxaloacetate, producing citrate. The citrate that is not required for tri-  carboxylic acid (TCA) cycle activity crosses the membrane and enters the cytosol.   As fatty acids are produced under conditions of high energy, the high  NADH/NAD + ratio in the mitochondria inhibits isocitrate dehydrogenase, which  leads to citrate accumulation within the mitochondrial matrix. As the citrate accu-  mulates, it is transported out into the cytosol to donate carbons for fatty acid  synthesis.   In the cytosol, citrate is cleaved by citrate lyase, an inducible enzyme, to form  oxaloacetate and acetyl CoA (Fig. 36.4). The acetyl CoA is used for fatty acid  biosynthesis and for cholesterol synthesis, pathways that are activated by insulin.  Oxaloacetate is recycled to pyruvate via cytosolic malate dehydrogenase and malic  enzyme, which is inducible. Malic enzyme generates NADPH for the reactions of  the fatty acid synthase complex. NADPH is also produced by the two enzymes of  the pentose phosphate pathway (see Chapter 29), glucose 6-phosphate dehydroge-  nase and 6-phosphogluconate dehydrogenase. Glucose 6-phosphate dehydrogenase  is also induced by insulin.    Fig. 36.4. Regulation of citrate lyase, malic enzyme, glucose 6-phosphate dehydrogenase,  and fatty acid synthase. Citrate lyase, which provides acetyl CoA for fatty acid biosynthesis,  the enzymes that provide NADPH (malic enzyme, glucose 6-phosphate dehydrogenase), as  well as fatty acid synthase, are inducible (circled t).   Acetyl CoA is converted to malonyl CoA, which provides the 2-carbon units for  elongation of the growing fatty acyl chain on the fatty acid synthase complex.  Acetyl CoA carboxylase, the enzyme that catalyzes the conversion of acetyl CoA  to malonyl CoA, is controlled by three of the major mechanisms that regulate  enzyme activity (Fig. 36.5). It is activated by citrate, which causes the enzyme to   Fig. 36.5. Regulation of acetyl CoA carboxylase (AcC). AcC is regulated by activation and  inhibition, by phosphorylation (mediated by the AMP-activated protein kinase) and dephos-  phorylation (via an insulin- stimulated phosphatase), and by induction and repression. It is  active in the fed state.   Fig. 36.6. Inhibition of transport of fatty acids  (FA) into mitochondria by malonyl CoA. In the  fed state, malonyl CoA (the substrate for fatty  acid synthesis produced by acetyl CoA car-  boxylase) is elevated. It inhibits CPTI, prevent-  ing the transport of long-chain fatty acids into  mitochondria. Therefore, substrate is not avail-  able for p-oxidation and ketone body synthesis.   0 o Di Abietes has insulin-dependent  ( /~oA ) diabetes mellitus (type 1), a dis-  ™ ease associated with a severe defi-  ciency or absence of insulin production by  the p cells of the pancreas. One of the effects  of insulin is to stimulate production of LPL.  Because of low insulin levels, Di Abietes  tends to have low levels of this enzyme.  Hydrolysis of the triacylglycerols in chylomi-  crons and in VLDL is decreased, and hyper-  triglyceridemia results.   polymerize, and inhibited by long-chain fatty acyl CoA. A phosphatase stimulated by  insulin activates the enzyme by dephosphorylation. The third means by which this  enzyme is regulated is induction: the quantity of the enzyme increases in the fed state.   Malonyl CoA, the product of the acetyl CoA carboxylase reaction, provides the  carbons for the synthesis of palmitate on the fatty acid synthase complex. Malonyl  CoA also inhibits carnitine :palmitoyltransferase I (CPTI, also known as carni-  tine :acyltransf erase I), the enzyme that prepares long-chain fatty acyl CoA for  transport into mitochondria (Fig. 36.6). In the fed state, when acetyl CoA carboxy-  lase is active and malonyl CoA levels are elevated, newly synthesized fatty acids are  converted to triacylglycerols for storage, rather than being transported into  mitochondria for oxidation and ketone body formation.   In a well-fed individual, the quantity of the fatty acid synthase complex is increased  (see Fig. 36.4). The genes that produce this enzyme complex are induced by  increases in the insulin/glucagon ratio. The amount of the complex increases slowly  after a few days of a high-carbohydrate diet.   Glucose 6-phosphate dehydrogenase, which generates NADPH in the pentose  phosphate pathway, and malic enzyme, which produces NADPH, are also induced  by the increase of insulin.   The palmitate produced by the synthase complex is converted to palmityl CoA  and elongated and desaturated to form other fatty acyl CoA molecules, which are  converted to triacylglycerols. These triacylglycerols are packaged and secreted into  the blood as VLDL.   The lipoprotein triacylglycerols in chylomicrons and VLDL are hydrolyzed to fatty  acids and glycerol by lipoprotein lipase (LPL), an enzyme attached to endothelial  cells of capillaries in muscle and adipose tissue. The enzyme found in muscle, par-  ticularly heart muscle, has a low K m for these blood lipoproteins. Therefore, it acts  even when these lipoproteins are present at very low concentrations in the blood.  The fatty acids enter muscle cells and are oxidized for energy. The enzyme found  in adipose tissue has a higher K m and is most active after a meal when blood  lipoprotein levels are elevated.   Insulin stimulates adipose cells to synthesize and secrete LPL, which hydrolyzes the  chylomicron and VLDL triacylglycerols. The C n apoprotein, donated to chylomi-  crons and VLDL by HDL, activates LPL (Fig. 36.7).   Fatty acids released from chylomicrons and VLDL by LPL are stored as  triacylglycerols in adipose cells. The glycerol released by LPL is not used by adi-  pose cells because they lack glycerol kinase. Glycerol can be used by liver cells,  however, because these cells do contain glycerol kinase. In the fed state, liver  cells convert glycerol to the glycerol moiety of the triacylglycerols of VLDL,  which is secreted from the liver to distribute the newly synthesized triglycerides  to the tissues.   Insulin causes the number of glucose transporters in adipose cell membranes to  increase. Glucose enters these cells, and is oxidized, producing energy and provid-  ing the glycerol 3 -phosphate moiety for triacylglycerol synthesis (via the  dihydroxy acetone phosphate intermediate of glycolysis).    Fig. 36.7. Regulation of the storage of triacylglycerols (TG) in adipose tissue. Insulin stim-  ulates the secretion of LPL from adipose cells and the transport of glucose into these cells.  ApoC n activates LPL. FA = fatty acids.   A. Mechanisms in Liver That Serve to Maintain Blood  Glucose Levels   During fasting, the insulin/glucagon ratio decreases. Liver glycogen is degraded to pro-  duce blood glucose because enzymes of glycogen degradation are activated by cAMP-  directed phosphorylation (Fig. 36.8). Glucagon stimulates adenylate cyclase to produce  cAMP, which activates protein kinase A. Protein kinase A phosphorylates phosphory-  lase kinase, which then phosphorylates and activates glycogen phosphorylase. Protein  kinase A also phosphorylates but, in this case, inactivates glycogen synthase.   Gluconeogenesis is stimulated because the synthesis of phosphoenolpyruvate  carboxykinase, fructose 1 ,6-bisphosphatase, and glucose 6-phosphatase is induced  and because there is an increased availability of precursors. Fructose 1,6-bisphos-  phatase is also activated because the levels of its inhibitor, fructose 2,6-bisphos-  phate, are low (Fig. 36.9). During fasting, the activity of the corresponding enzymes  of glycolysis is decreased.   During fasting, as blood insulin levels fall and glucagon levels rise, the level of  cAMP rises in adipose cells. Consequently, protein kinase A is activated and causes  phosphorylation of hormone- sensitive lipase (HSL). The phosphorylated form of  this enzyme is active and cleaves fatty acids from triacylglycerols (Fig. 36.10).  Other hormones (e.g., epinephrine, adrenocorticotropic hormone [ACTH], growth  hormone) also activate this enzyme (see Chapter 43). Glyceroneogenesis and resyn-  thesis of triglyceride by the adipocyte regulates the rate of release of fatty acids  during fasting.   As fatty acids are released from adipose tissue during fasting, they travel in the  blood complexed with albumin. These fatty acids are oxidized by various tissues,  particularly muscle. In the liver, fatty acids are transported into mitochondria   Ann Sulin has type 2 diabetes mel-  litus. She produces insulin, but her  adipose tissue is resistant to its  actions. Therefore, her adipose tissue does  not produce as much LPL as a normal per-  son, which is one of the reasons why VLDL  and chylomicrons remain elevated in her  blood.   Twenty to thirty percent of patients  with an insulinoma gain weight as  part of their syndrome. Bea Selmass  gained 8 lb in the 6 weeks before her surgery.  Although she was primed by her high insulin  levels both to store and to use fuel more effi-  ciently, she would not have gained weight if  she had not consumed more calories than she  required for her daily energy expenditure dur-  ing her illness (see Chapter 1). Bea consumed  extra carbohydrate calories, mostly as hard  candies and table sugar, to avoid the symp-  toms of hypoglycemia.   Induction of enzyme synthesis  requires activation of transcription  factors. One of the factors that is  activated is CREB, which stands for cAMP  response element binding protein. CREB is  phosphorylated and activated by the cAMP-  dependent protein kinase, which itself is  activated on glucagon or epinephrine stimu-  lation. A second set of transcription factors  activated are the C/EPBs (CCAAT/ enhancer  binding proteins), although the regulation of  their activity is still under investigation.   Di Abietes suffers from hyper-  glycemia because her insulin levels  tend to be low and her glucagon  levels tend to be high. Her muscle and adi-  pose cells do not take glucose up at a normal  rate, and she produces glucose by  glycogenolysis and gluconeogenesis. As a  result, her blood glucose levels are elevated.   Ann Sulin is in a similar metabolic state.  However, in this case, she produces insulin,  but her tissues are resistant to its actions.   y^-^v Because the adipose tissue of indi-  a\ # ) viduals with type 2 diabetes melli-  tus is relatively resistant to  insulin's inhibition of HSL and its stimulation  of LPL, Ann Sulin's serum triacylglycerol  level would be elevated for the same rea-  sons as those that caused the hypertriglyc-  eridemia in Di Abietes.   q q Insulin normally inhibits lipolysis  ( /~vA J by decreasing the lipolytic activity  ™ of HSL in the adipocyte. Individuals  such as Di Abietes, who have a deficiency of  insulin, have an increase in lipolysis and a  subsequent increase in the concentration of  free fatty acids in the blood. The liver, in  turn, uses some of these fatty acids to syn-  thesize triacylglycerols, which then are used  in the hepatic production of VLDL. VLDL is  not stored in the liver but is secreted into the  blood, raising its serum concentration. Di  also has low levels of LPL because of  decreased insulin levels. Her hypertriglyc-  eridemia is the result, therefore, of both  overproduction of VLDL by the liver and  decreased breakdown of VLDL triacylglyc-  erol for storage in adipose cells.   The serum begins to appear cloudy when  the triacylglycerol level reaches 200 mg/dL.  As the triacylglycerol level increases still fur-  ther, the degree of serum opalescence  increases proportionately.   Fig. 36.8. Regulation of the enzymes of glycogen degradation in the liver. 1. Glucagon (or  epinephrine) binds to its cell membrane receptor, initially activating a G protein, which acti-  vates adenylate cyclase. 2. As cAMP levels rise, inhibitory subunits are removed from pro-  tein kinase A, which now phosphorylates phosphorylase kinase (step 3). 4. The cAMP-  dependent protein kinase also phosphorylates glycogen synthase, inactiving the enzyme.  5. Phosphorylated phosphorylase kinase phosphorylates glycogen phosphorylase. 6. Phos-  phorylated glycogen phosphorylase catalyzes the phosphorolysis of glycogen, producing glu-  cose 1 -phosphate. These events occur during fasting and produce glucose to maintain a rela-  tively constant level of blood glucose.   because acetyl CoA carboxylase is inactive, malonyl CoA levels are low, and CPTI  (carnitine :acyltransf erase I) is active (see Fig. 36.6). Acetyl CoA, produced by p-  oxidation, is converted to ketone bodies. Ketone bodies are used as an energy source  by many tissues (Table 36.1) to spare the use of glucose and the necessity of degrad-  ing muscle protein to provide the precursors for gluconeogenesis. The high levels  of acetyl CoA in the liver (derived from fat oxidation) inhibit pyruvate dehydroge-  nase (which prevents pyruvate from being converted to acetyl CoA) and activate  pyruvate carboxylase, which produces oxaloacetate for gluconeogenesis. The  oxaloacetate does not condense with acetyl CoA to form citrate for two reasons.  The first is that under these conditions (a high rate of fat oxidation in the liver mito-  chondria), energy levels in the mitochondrial matrix are high; that is, there are high  levels of NADH and ATP present. The high NADH level inhibits isocitrate dehy-  drogenase. As a result, citrate accumulates and inhibits citrate synthase from pro-  ducing more citrate. The second reason that citrate synthesis is depressed is that the   Fig. 36.9. Regulation of gluconeogenesis and glycolysis during fasting. The gluconeogenic  enzymes phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose  6-phosphatase are induced. Fructose 1,6-bisphosphatase is also active because, during fast-  ing, the level of its inhibitor, fructose 2,6-bisphosphate, is low. The corresponding enzymes  of glycolysis are not very active during fasting. The rate of glucokinase is low because it has  a high K m for glucose and the glucose concentration is low. Phosphofructokinase-1 is not  very active because the concentration of its activator fructose 2,6-bisphosphate is low. Pyru-  vate kinase is inactivated by cAMP-mediated phosphorylation.   Chapter 47), through the actions of  the AMP-activated protein kinase. Thus, if  energy levels are very low, and AMP levels  increase, glucose can still be transported  from the circulation into the muscle to pro-  vide energy. This most often occurs during  exercise.    Fig. 36.10. Regulation of hormone- sensitive lipase (HSL) in adipose tissue. During fasting,  the glucagon/insulin ratio rises, causing cAMP levels to be elevated. Protein kinase A is acti-  vated and phosphorylates HSL, activating this enzyme. HSL-P initiates the mobilization of  adipose triacylglycerol by removing a fatty acid (FA). Other lipases then act, producing fatty  acids and glycerol. Insulin stimulates the phosphatase that inactivates HSL in the fed state.   high NADH/NAD + ratio also diverts oxaloacetate into malate, such that the malate  can exit the mitochondria (via the malate/aspartate shuttle) for use in  gluconeogenesis.   D. Regulation of the Use of Glucose and Fatty Acids  by Muscle   During exercise, the fuel that is used initially by muscle cells is muscle glycogen. As  exercise continues and the blood supply to the tissue increases, glucose is taken up  from the blood and oxidized. Liver glycogenolysis and gluconeogenesis replenish the  blood glucose supply. However, as insulin levels drop, the concentration of GLUT4  transporters in the membrane is reduced, thereby reducing glucose entry from the cir-  culation into the muscle.   Thus, as fatty acids become available because of increased lipolysis of adipose  triacylglycerols, the exercising muscle begins to oxidize fatty acids, p -Oxidation  produces NADH and acetyl CoA, which slow the flow of carbon from glucose  through the reaction catalyzed by pyruvate dehydrogenase (see Fig. 36.3). Thus, the  oxidation of fatty acids provides a major portion of the increased demand for ATP  generation and spares blood glucose.   The switch between catabolic and anabolic pathways is often regulated by the lev-  els of AMP and fructose 2,6-bisphosphate in cells, particularly the liver. It is logi-  cal for AMP to be a critical regulator. As a cell uses ATP in energy-requiring path-  ways, the levels of AMP accumulate more rapidly than that of ADP because of the  adenylate kinase reaction (2 ADP — > ATP and AMP). The rise in AMP levels then  signals that more energy is required (usually through allosteric binding sites on  enzymes and the activation of the AMP-activated protein kinase), and the cell will  switch to the activation of catabolic pathways. As AMP levels drop, and ATP levels  rise, the anabolic pathways are now activated to store the excess energy.   The levels of fructose 2,6-bisphosphate are also critical in regulating glycolysis  versus gluconeogenesis in the liver. Under conditions of high blood glucose, and  insulin release, fructose 2,6 bisphosphate levels will be high because PFK-2 will be  in its activated state. The fructose 2,6 bisphosphate activates PFK-1, and inhibits  fructose 2,6 bisphosphatase, thereby allowing glycolysis to proceed. When blood  glucose levels are low, and glucagon is released, PFK-2 is phosphorylated by the  cAMP-dependent protein kinase and is inhibited, thereby lowering fructose  2,6-bisphosphate levels and inhibiting glycolysis, while favoring gluconeogenesis.   Bea Selmass's younger sister was very concerned that Bea's pancreatic  tumor might be genetically determined or potentially malignant, so she  accompanied Bea to her second postoperative visit to the endocrinologist.  The doctor explained that insulinomas may be familial in up to 20% of cases and  that in 10% of patients with insulinomas, additional secretory neoplasms may occur  in the anterior pituitary or the parathyroid glands (a genetically determined syn-  drome known as multiple endocrine neoplasia, type I or, simply, MEN I). Bea's  tumor showed no evidence of malignancy, and the histologic slides, although not  always definitive, showed a benign-appearing process. The doctor was careful to  explain, however, that close observation for recurrent hypoglycemia and for the  signs and symptoms suggestive of other facets of MEN I would be necessary for the  remainder of Bea's and her immediate family's life.   2 Diabetes mellitus is a well-accepted risk factor for the development of  ) coronary artery disease; the risk is three to four times higher in the dia-  betic than in the nondiabetic population. Although chronically elevated  serum levels of chylomicrons and VLDL may contribute to this atherogenic pre-  disposition, the premature vascular disease seen in Di Abietes and other patients  with type 1 diabetes mellitus, as well as Ann Sulin and other patients with type  2 diabetes mellitus, is also related to other abnormalities in lipid metabolism.  Among these are the increase in glycation (nonenzymatic attachment of glucose  molecules to proteins) of LDL apoproteins as well as glycation of the proteins of  the LDL receptor, which occurs when serum glucose levels are chronically ele-  vated. These glycations interfere with the normal interaction or "fit" of the cir-  culating LDL particles with their specific receptors on cell membranes. As a con-  sequence, the rate of uptake of circulating LDL by the normal target cells is  diminished. The LDL particles, therefore, remain in the circulation and eventu-  ally bind nonspecifically to "scavenger" receptors located on macrophages adja-  cent to the endothelial surfaces of blood vessels, one of the early steps in the  process of atherogenesis.   All of the material in this chapter was presented previously. However,  because this information is so critical for understanding biochemistry in a  way that will allow it to be used in interpreting clinical situations, it was  summarized in this chapter. In addition, the information previously presented under  carbohydrate metabolism was integrated with lipid metabolism. We have, for the most  part, left out the role of allosteric modifiers and other regulatory mechanisms that  finely coordinate these processes to an exquisite level. Because such details may be  important for specific clinical situations, we hope this summary will serve as a frame-  work to which the details can be fitted as students advance in their clinical studies.   Because Di Abietes produces very  little insulin, she is prone to develop-  ing ketoacidosis. When insulin levels  are low, HSL of adipose tissue is very active,  resulting in increased lipolysis. The fatty acids  that are released travel to the liver, where they  are converted to the triacylglycerols of VLDL.  They also undergo p-oxidation and conversion  to ketone bodies. If Di does not take exoge-  nous insulin or if her insulin levels decrease  abruptly for some physiologic reason, she may  develop a ketoacidosis (DKA). In fact, she has  had repeated bouts of DKA.   For reasons that are not as well under-  stood, individuals with type 2 diabetes melli-  tus, such as Ann Sulin, do not tend to  develop ketoacidosis. One possible explana-  tion is that the insulin resistance is tissue-  specific; the insulin sensitivity of adipocytes  may be greater than that of muscle and liver.  Such a tissue-specific sensitivity would lead  to less fatty acids being released from  adipocytes in type 2 diabetes than in type 1  diabetes, although in both cases the release  of fatty acids would be greater than that of  an individual without the disease.   Fig. 36.11. Regulation of carbohydrate and lipid metabolism in the liver. Solid blue arrows indicate the flow of metabolites in the fed state. Solid black  arrows indicate the flow during fasting. G = glucose; GK = glucokinase; F = fructose; PFK = phosphofructokinase-1; PEP = phosphoenolpyru-  vate; PK = pyruvate kinase; OAA = oxaloacetate; aKG = a-ketoglutarate; GS = glycogen synthase; Phos = glycogen phosphorylase; PhK = phos-  phorylase kinase; AcC = acetyl CoA carboxylase; FA = fatty acid or fatty acyl group; TG = triacylglycerol; circled P = phosphate group.   Figure 36.11 is a comprehensive figure, and Tables 36.2 and 36.3 provide a  list of the major regulatory enzymes of carbohydrate and lipid metabolism in the  liver, an order to the events that occur, and the mechanisms by which they are  controlled. This figure and tables should help students to integrate this mass of  material.   Now that many of the details of the pathways have been presented, it would  be worthwhile to re-read the first three chapters of this book. A student who  understands biochemistry within the context of fuel metabolism is in a very good  position to solve clinical problems that involve metabolic derangements.   When blood sugar increases:  Insulin is released, which leads  to the dephosphorylation of:   When blood sugar decreases:  Glucagon is released, which  leads to the phosphorylation  of:   Table 36.3. Regulation of Liver Enzymes Involved in Glycogen, Blood Glucose,  and Triacylglycerol Synthesis and Degradation   LIVER ENZYMES REGULATED BY PHOSPHORYLATION/DEPHOSPHORYLATION  Enzyme Active Form State in Which Active   Glycogen synthase  Phosphorylase kinase  Glycogen phosphorylase  Phosphofructokinase-2/F-2,  6-bisphosphatase (acts as  a kinase, increasing  fructose- 2,6-bisP levels)  Phosphofructokinase-2/F-2,  6-bisphosphatase (acts as  a phosphatase, decreasing  fructose- 2,6-bisP levels)  Pyruvate kinase  Pyruvate dehydrogenase  Acetyl CoA carboxylase   Iritani N. Nutritional and hormonal regulation of lipogenic -enzyme gene expression in rat liver. Eur J  Biochem 1992:205:433-442.   Jump DB, Clarke SD. Regulation of gene expression by dietary fat. Annu Rev Nutr 1999; 19:63-90.   Klip A, Tsakiridis T, Marette A, Ortiz PA. Regulation of expression of glucose transporters by glucose:  a review of studies in vivo and in cell cultures. FASEB J 1994;8:43-53.   Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and glycoly-  sis. Annu Rev Physiol 1992;54:885-909.   Roesler WJ. What is a cAMP response unit? Mol Cell Endocrinol 2000;162:1-7.   Sugden MC, Holness MJ. Interactive regulation of the pyruvate dehydrogenase complex and the carni-  tine palmitoyltransferase system. FASEB J 1994;8:54-61.   Vaulont S, Kahn A. Transcriptional control of metabolic regulation genes by carbohydrates. FASEB J  1994:8:28-35.   1. A 20-year-old woman with diabetes mellitus was admitted to the hospital in a semiconscious state with fever, nausea, and  vomiting. Her breath smelled of acetone. A urine sample was strongly positive for ketone bodies. Which of the following  statements is correct concerning this woman?   (A) A blood glucose test would probably show that her blood glucose level was well below 80 mg/dL.   (C) She should be given a glucose infusion to regain consciousness.   (D) Glucagon should be administered to stimulate glycogenolysis and gluconeogenesis in the liver.   (E) The acetone was produced by decarboxylation of the ketone body p-hydroxybutyrate.   2. A woman was told by her physician to go on a low-fat diet. She decided to continue to consume the same number of calories  by increasing her carbohydrate intake while decreasing her fat intake. Which of the following blood lipoprotein levels would  be decreased as a consequence of her diet?   3. Assume that an individual has been eating excess calories daily such that they will gain weight. Under which of the follow-  ing conditions would the person gain weight most rapidly?   (C) If all the excess calories were split 50%-50% between carbohydrate and triacylgycerol   (D) If all the excess calories were split 25%-75% between carbohydrate and triacylgycerol   (E) It makes no difference what form the excess calories are in   4. A chronic alcoholic has been admitted to the hospital because of a severe hypoglycemic episode brought about by excessive  alcohol consumption for the past 5 days. A blood lipid analysis indicates much higher than expected VLDL levels. The ele-  vated VLDL is attributable to which of the following underlying causes?   5. Certain patients with abetalipoproteinemia frequently have difficulties in maintaining blood volume; their blood has trouble  clotting. This symptom is attributable to which of the following?   Dietary proteins are the primary source of the nitrogen that is metabo-  lized by the body (Fig. 1). Amino acids, produced by digestion of  dietary proteins, are absorbed through intestinal epithelial cells and  enter the blood. Various cells take up these amino acids, which enter  the cellular pools. They are used for the synthesis of proteins and other  nitrogen-containing compounds or they are oxidized for energy.  Protein synthesis, the translation of mRNA on ribosomes (see Chapter 15), is a  dynamic process. Within the body, proteins are constantly being synthesized and  degraded, partially draining and then refilling the cellular amino acid pools.   Compounds derived from amino acids include cellular proteins, hormones, neu-  rotransmitters, creatine phosphate, the heme of hemoglobin and the cytochromes,  the skin pigment melanin, and the purine and pyrimidine bases of nucleotides and  nucleic acids. In fact, all of the nitrogen-containing compounds of the body are syn-  thesized from amino acids. Many of these pathways are outlined in the following  chapters of the book.   In addition to serving as the precursors for the nitrogen-containing compounds of  the body and as the building blocks for protein synthesis, amino acids are also a source  of energy. Amino acids are directly oxidized or they are converted to glucose and then  oxidized or stored as glycogen. They also may be converted to fatty acids and stored  as adipose triacylglycerols. Glycogen and triacylglycerols are oxidized during periods  of fasting. The liver is the major site of amino acid oxidation. However, most tissues  can oxidize the branched-chain amino acids (leucine, isoleucine, and valine).   Before the carbon skeletons of amino acids are oxidized, the nitrogen must be  removed. Amino acid nitrogen forms ammonia, which is toxic to the body. In the  liver, ammonia and the amino groups from amino acids are converted to urea, which  is nontoxic, water-soluble, and readily excreted in the urine. The process by which  urea is produced is known as the urea cycle. The liver is the organ responsible for  producing urea. Branched-chain amino acids can be oxidized in many tissues, but  the nitrogen must always travel to the liver for disposal.   Although urea is the major nitrogenous excretory product of the body, nitrogen  is also excreted in other compounds (Table 1). Uric acid is the degradation product   Fig. 1. Summary of amino acid metabolism.  Dietary proteins are digested to amino acids in  the stomach and intestine, which are absorbed  by the intestinal epithelium, transferred to the  circulation, and taken up by cells. Amino acids  are used to synthesize proteins and other nitro-  gen-containing compounds. The carbon skele-  tons of amino acids are also oxidized for  energy, and the nitrogen is converted to urea  and other nitrogenous excretory products.   Table 1. Major Nitrogenous Urinary Excretory Products  Amount excreted in urine/day a   a The amounts are expressed in units generally reported by clinical laboratories. Note that the amounts for  creatinine and uric acid are for the whole compound, whereas those for urea and ammonia are for the  nitrogen content.   b Under normal circumstances, approximately 90% of the nitrogen excreted in the urine is in the form of  urea. The exact amounts of each component vary, however, depending on dietary protein intake and phys-  iologic state. For instance, NH 4 + excretion increases during an acidosis because the kidney secretes  ammonia to bind protons in the urine.   The healthy human adult is in nitro-  \\ gen balance; in other words, the  amount of nitrogen excreted each  day (mainly in the urine) equals the amount  consumed (mainly as dietary protein). Nega-  tive nitrogen balance occurs when the amount  of nitrogen excreted is greater than the amount  consumed, and positive nitrogen balance  occurs when the amount excreted is less than  that consumed (see Chapter 1).   a These amino acids are called "nonessential" or "dispensable," terms that refer to dietary requirements.  Of course, within the body, they are necessary. We cannot survive without them.  b Although the carbons of cysteine can be derived from glucose, its sulfur is obtained from the essential  amino acid methionine.   of the purine bases, creatinine is produced from creatine phosphate, and ammonia is  released from glutamine, particularly by the kidney, where it helps to buffer the urine  by reacting with protons to form ammonium ions (NH 4 + ). These compounds are  excreted mainly in the urine, but substantial amounts are also lost in the feces and  through the skin. Small amounts of nitrogen-containing metabolites are formed from  the degradation of neurotransmitters, hormones, and other specialized amino acid  products and excreted in the urine. Some of these degradation products, such as  bilirubin (formed from the degradation of heme), are excreted mainly in the feces.   Eleven of the 20 amino acids used to form proteins are synthesized in the body if  an adequate amount is not present in the diet (Table 2). Ten of these amino acids can  be produced from glucose; the 1 1th, tyrosine, is synthesized from the essential amino  acid phenylalanine. It should be noted that cysteine, one of the 10 amino acids pro-  duced from glucose, obtains its sulfur atom from the essential amino acid methionine.   (NAD,NADP)  Serotonin  Melatonin  Norepinephrine  Epinephrine  Histamine  Melanin  Pyrimidines  Purines ---.^  -Creatine-P *\  Thyroxine }  -Sphingosine /   Fig. 2. Overview of nitrogen metabolism. The metabolism of nitrogen-containing compounds is shown on  the right and that of glucose and fatty acids is shown on the left. This figure shows a hypothetical, com-  posite cell. No single cell type has all of these pathways. Many of the pathways shown are described in the  next few chapters. aKG = a-ketoglutarate; OAA = oxaloacetate; G-6-P = glucose 6-phosphate; G-l-P =  glucose 1 -phosphate.   Nine amino acids are essential in the human. "Essential" means that the carbon  skeleton cannot be synthesized and, therefore, these amino acids are required in the  diet (Table 3). The essential amino acids are also called the indispensable amino  acids. Arginine is essential during periods of growth; in adults it is no longer con-  sidered essential.   After nitrogen is removed from amino acids, the carbon skeletons are oxidized  (Fig. 2). Most of the carbons are converted to pyruvate, intermediates of the tricar-  boxylic acid (TCA) cycle, or to acetyl CoA. In the liver, particularly during fasting,  these carbons may be converted to glucose or to ketone bodies and released into the  blood. Other tissues then oxidize the glucose and ketone bodies. Ultimately, the car-  bons of the amino acids are converted to C0 2 and H 2 0.   Proteolytic enzymes (also called proteases) break down dietary proteins into their  constituent amino acids in the stomach and the intestine. Many of these digestive  proteases are synthesized as larger, inactive forms known as zymogens. After  zymogens are secreted into the digestive tract, they are cleaved to produce the  active proteases.   In the stomach, pepsin begins the digestion of proteins by hydrolyzing them to  smaller polypeptides. The contents of the stomach pass into the small intestine,  where enzymes produced by the exocrine pancreas act. The pancreatic proteases  (trypsin, chymotrypsin, elastase, and the carboxypeptidases) cleave the polypep-  tides into oligopeptides and amino acids.   Further cleavage of the oligopeptides to amino acids is accomplished by  enzymes produced by the intestinal epithelial cells. These enzymes include  aminopeptidases located on the brush border and other peptidases located within  the cells. Ultimately, the amino acids produced by protein digestion are absorbed  through the intestinal epithelial cells and enter the blood.   A large number of overlapping transport systems exist for amino acids in cells.  Some systems contain facilitative transporters, whereas others express sodium-  linked tranporters, which allow the active transport of amino acids into cells.  Defects in amino acid transport can lead to disease.   Proteins are also continually synthesized and degraded (turnover) in cells. A  wide variety of proteases exist in cells to carry out this activity. Lysosomal pro-  teases (cathepsins) degrade proteins that enter lysosomes. Cytoplasmic proteins  targeted for turnover are covalently linked to the small protein ubiquitin, which  then interacts with a large protein complex, the proteasome, to degrade the pro-  tein in an adenosine triphosphate (ATP) -dependent process. The amino acids  released from proteins during turnover can then be used for the synthesis of new  proteins or for energy generation.   Sissy Fibrosa, a young child with cystic fibrosis, has had repeated bouts  of bronchitis caused by Pseudomonas aeruginosa. With each of these  infections, her response to aerosolized antibiotics has been good. However,  her malabsorption of food continues, resulting in foul- smelling, glistening, bulky  stools. Her growth records show a slow decline. She is now in the 24th percentile for  height and the 20th percentile for weight. She is often listless and irritable, and she  tires easily. When her pediatrician discovered that her levels of the serum proteins   albumin, transferrin, and thyroid hormone binding prealbumin (transthyretin) were  low to low-normal (indicating protein malnutrition), Sissy was given enteric-coated  microspheres of pancreatic enzymes. Almost immediately, the character of Sissy's  stools became more normal and she began gaining weight. In the next 6 months, her  growth curves showed improvement, and she seemed brighter, more active, and less  irritable.   Fig. 37.1. Digestion of proteins. The prote-  olytic enzymes, pepsin, trypsin, chymotrypsin,  elastase, and the carboxypeptidases, are pro-  duced as zymogens (the [pro] and [ogen]  accompanying the enzyme name) that are acti-  vated by cleavage after they enter the gastroin-  testinal lumen (see Fig. 37.2).   F° r the first few months after a painful episode of renal colic, during which  H|I ( he passed a kidney stone (see Chapter 6), Cal Kulis had faithfully main-   " tained a high daily fluid intake and had taken the medication required to  increase the pH of his urine. Because he has cystinuria, these measures were nec-  essary to increase the solubility of the large amounts of cystine present in his urine  and, thereby, to prevent further formation of kidney stones (calculi). With time,  however, he became increasingly complacent about his preventive program. After  failing to take his medication for a month, he experienced another severe episode of  renal colic with grossly bloody urine. Fortunately, he passed the stone sponta-  neously, after which he vowed to faithfully comply with therapy.   His mother heard that some dietary amino acids were not absorbed in patients  with cystinuria and asked whether any dietary changes would reduce Cal's chances  of developing additional renal stones.   The digestion of proteins begins in the stomach and is completed in the intestine  (Fig. 37.1). The enzymes that digest proteins are produced as inactive precursors  (zymogens) that are larger than the active enzymes. The inactive zymogens are  secreted from the cells in which they are synthesized and enter the lumen of the  digestive tract, where they are cleaved to smaller forms that have proteolytic activ-  ity (Fig. 37.2). These active enzymes have different specificities; no single enzyme  can completely digest a protein. However, by acting in concert, they can digest  dietary proteins to amino acids and small peptides, which are cleaved by peptidases  associated with intestinal epithelial cells   q q Kwashiorkor, a common problem of  ( /~0A ) children in Third World countries, is  ™ caused by a deficiency of protein in  a diet that is adequate in calories. Children  with kwashiorkor suffer from muscle wasting  and a decreased concentration of plasma pro-  teins, particularly albumin. The result is an  increase in interstitial fluid that causes edema  and a distended abdomen that make the chil-  dren appear "plump" (see Chapter 44). The  muscle wasting is caused by the lack of  essential amino acids in the diet; existing pro-  teins must be broken down to produce these  amino acids for new protein synthesis.   These problems may be compounded by  a decreased ability to produce digestive  enzymes and new intestinal epithelial cells  because of a decreased availability of amino  acids for the synthesis of new proteins.   Fig. 37.2. Activation of the gastric and pancreatic zymogens. Pepsinogen catalyzes its own  cleavage as the pH of the stomach drops. Trypsinogen is cleaved by enteropeptidase in the  intestine to form the active protease trypsin. Trypsin then plays a key role by catalyzing the  cleavage and activation of the other pancreatic zymogens.   Pepsinogen is secreted by the chief cells of the stomach. The gastric parietal cells  secrete HC1. The acid in the stomach lumen alters the conformation of pepsinogen  so that it can cleave itself, producing the active protease pepsin. Thus, the activation  of pepsinogen is autocatalytic.   Dietary proteins are denatured by the acid in the stomach. This serves to inacti-  vate the proteins and partially unfolds them such that they are better substrates for  proteases. However, at the low pH of the stomach, pepsin is not denatured and acts  as an endopeptidase, cleaving peptide bonds at various points within the protein  chain. Although pepsin has a fairly broad specificity, it tends to cleave peptide  bonds in which the carboxyl group is provided by an aromatic or acidic amino acid  (Fig. 37.3). Smaller peptides and some free amino acids are produced.   As the gastric contents empty into the intestine, they encounter the secretions from  the exocrine pancreas. One of these secretions is bicarbonate, which, in addition to  neutralizing the stomach acid, raises the pH such that the pancreatic proteases,  which are also present in pancreatic secretions, can be active. As secreted, these  pancreatic proteases are in the inactive proenzyme form (zymogens). Because the  active forms of these enzymes can digest each other, it is important for their zymo-  gen forms all to be activated within a short span of time. This feat is accomplished  by the cleavage of trypsinogen to the active enzyme trypsin, which then cleaves the  other pancreatic zymogens, producing their active forms (see Fig. 37.2).   The zymogen trypsinogen is cleaved to form trypsin by enteropeptidase (a protease,  formerly called enterokinase) secreted by the brush-border cells of the small intestine.  Trypsin catalyzes the cleavages that convert chymotrypsinogen to the active enzyme  chymotrypsin, proelastase to elastase, and the procarboxypeptidases to the car-  boxypeptidases. Thus, trypsin plays a central role in digestion because it both cleaves  dietary proteins and activates other digestive proteases produced by the pancreas.   Trypsin, chymotrypsin, and elastase are serine proteases (see Chapter 9) that act  as endopeptidases. Trypsin is the most specific of these enzymes, cleaving peptide  bonds in which the carboxyl (carbonyl) group is provided by lysine or arginine (see  Fig. 37.3). Chymotrypsin is less specific but favors residues that contain hydropho-  bic or acidic amino acids. Elastase cleaves not only elastin (for which it was named)  but also other proteins at bonds in which the carboxyl group is contributed by amino  acid residues with small side chains (alanine, glycine, or serine). The actions of  these pancreatic endopeptidases continue the digestion of dietary proteins begun by  pepsin in the stomach.   Elastase is also found in neutrophils, white blood cells that have the job of  engulfing and destroying invading bacteria. Neutrophils frequently act in the  lung, and elastase is sometimes released into the lung as the neutrophils work.  In normal individuals, the released elastase is blocked from destroying lung cells by the  action of circulating a-1-antitrypsin, a protease inhibitor synthesized and secreted by the  liver. Certain individuals have a genetic mutation that leads to the production of an inac-  tive a-1-antitrypsin protein (a-1-antitrypsin deficiency). The lack of this enzyme activity  leads to the development of emphysema caused by proteolytic destruction of lung cells,  which results in a reduction in the expansion/contraction capability of the lungs.   Fig. 37.3. Action of the digestive proteases.  Pepsin, trypsin, chymotrypsin, and elastase are  endopeptidases; they hydrolyze peptide bonds  within chains. The others are exopeptidases;  aminopeptidases remove the amino acid at the  N-terminus and the carboxypeptidases remove  the amino acid at the C terminus. For each pro-  teolytic enzyme, the amino acid residues  involved in the peptide bond that is cleaved are  listed beside the R group to the right of the  enzyme name.   Recall that the exocrine pancreas,  in addition to secreting proteolytic  zymogens, also secretes amylase  for starch digestion and lipase and co-lipase  for dietary triacylglycerol digestion.   Patients with cystic fibrosis, such as Sissy Fibrosa, have a genetically determined  defect in the function of the chloride channels. In the pancreatic secretory ducts,  which carry pancreatic enzymes into the lumen of the small intestines, this  defect causes inspissation (drying and thickening) of pancreatic exocrine secretions, even-  tually leading to obstruction of these ducts. One result of this problem is the inability of  pancreatic enzymes to enter the intestinal lumen to digest dietary proteins.   The pancreas synthesizes and  stores the zymogens in secretory  granules. The pancreas also syn-  thesizes a secretory trypsin inhibitor. The  need for the inhibitor is to block any trypsin  activity that may occur from accidental  trypsinogen activation. If the inhibitor were  not present, trypsinogen activation would  lead to the activation of all of the zymogens  in the pancreas, which would lead to the  digestion of intracellular pancreatic proteins.  Such episodes can lead to pancreatitis.   The smaller peptides formed by the action of trypsin, chymotrypsin, and elastase  are attacked by exopeptidases, which are proteases that cleave one amino acid at a  time from the end of the chain. Procarboxypeptidases, zymogens produced by the  pancreas, are converted by trypsin to the active carboxypeptidases. These exopepti-  dases remove amino acids from the carboxyl ends of peptide chains.  Carboxypeptidase A preferentially releases hydrophobic amino acids, whereas car-  boxypeptidase B releases basic amino acids (arginine and lysine).   Exopeptidases produced by intestinal epithelial cells act within the brush border and  also within the cell. Aminopeptidases, located on the brush border, cleave one  amino acid at a time from the amino end of peptides. Intracellular peptidases act on  small peptides that are absorbed by the cells.   The concerted action of the proteolytic enzymes produced by cells of the stomach,  pancreas, and intestine cleaves dietary proteins to amino acids. The digestive enzymes  digest themselves as well as dietary protein. They also digest the intestinal cells that are  regularly sloughed off into the lumen. These cells are replaced by cells that mature from  precursor cells in the duodenal crypts. The amount of protein that is digested and  absorbed each day from digestive juices and cells released into the intestinal lumen may  be equal to, or greater than, the amount of protein consumed in the diet (50-100 g).   Why do patients with cystinuria  and Hartnup disease have a hyper-  aminoaciduria without an associ-  ated hyperaminoacidemia?   0 o Hartnup disease is another geneti-  j /vA ) cally determined and relatively rare  ™ autosomal recessive disorder. It is  caused by a defect in the transport of neutral  amino acids across both intestinal and renal  epithelial cells. The signs and symptoms are,  in part, caused by a deficiency of essential  amino acids (see Clinical Comments). Cystin-  uria and Hartnup disease involve defects in  two different transport proteins. In each  case, the defect is present both in intestinal  cells, causing malabsorption of the amino  acids from the digestive products in the  intestinal lumen and in kidney tubular cells,  causing a decreased resorption of these  amino acids from the glomerular filtrate.   ^Pf^ Trace amounts of polypeptides pass  /^oA J into the blood. They may be trans-  ™ ported through intestinal epithelial  cells, probably by pinocytosis, or they may slip  between the cells that line the gut wall. This  process is particularly troublesome for prema-  ture infants, because it can lead to allergies  caused by proteins in their food.   Amino acids are absorbed from the intestinal lumen through secondary active Na + -  dependent transport systems and through facilitated diffusion.   Amino acids are absorbed from the lumen of the small intestine principally by  semispecific Na + -dependent transport proteins in the luminal membrane of the intes-  tinal cell brush border, similar to that already seen for carbohydrate transport (Fig 37.4).  The cotransport of Na + and the amino acid from the outside of the apical membrane to  the inside of the cell is driven by the low intracellular Na + concentration. Low intracel-  lular Na + results from the pumping of Na + out of the cell by a Na + ,K + -ATPase on the  serosal membrane. Thus, the primary transport mechanism is the creation of a sodium  gradient; the secondary transport process is the coupling of amino acids to the influx of  sodium. This mechanism allows the cells to concentrate amino acids from the intestinal  lumen. The amino acids are then transported out of the cell into the interstitial fluid prin-  cipally by facilitated transporters in the serosal membrane (see Fig. 37.4).   At least six different Na + -dependent amino acid carriers are located in the apical  brush border membrane of the epithelial cells. These carriers have an overlapping  specificity for different amino acids. One carrier preferentially transports neutral  amino acids, another transports proline and hydroxyproline, a third preferentially  transports acidic amino acids, and a fourth transports basic amino acids (lysine, argi-  nine, the urea cycle intermediate ornithine) and cystine (two cysteine residues linked  by a disulfide bond). In addition to these Na + -dependent carriers, some amino acids  are transported across the luminal membrane by facilitated transport carriers. Most  amino acids are transported by more than one transport system.   As with glucose transport, the Na + -dependent carriers of the apical membrane of  the intestinal epithelial cells are also present in the renal epithelium. However, differ-  ent isozymes are present in the cell membranes of other tissues. Conversely, the  facilitated transport carriers in the serosal membrane of the intestinal epithelia are sim-  ilar to those found in other cell types in the body. During starvation, the intestinal  epithelia, like these other cells, take up amino acids from the blood to use as an energy  source. Thus, amino acid transport across the serosal membrane is bidirectional.   Table 37.1. A Partial Listing of Amino Acid Transport Systems 3   ® Patients with cystinuria and Hart-  nup disease have defective trans-  port proteins in both the intestine  and the kidney. These patients do not absorb  the affected amino acids at a normal rate  from the digestive products in the intestinal  lumen. They also do not readily resorb these  amino acids from the glomerular filtrate into  the blood. Therefore, they do not have a  hyperaminoacidemia (a high concentration  in the blood). Normally, only a few percent  of the amino acids that enter the glomerular  filtrate are excreted in the urine; most are  resorbed. In these diseases, much larger  amounts of the affected amino acids are  excreted in the urine, resulting in a hyper-  aminoaciduria.   Amino acids that enter the blood are transported across cell membranes of the var-  ious tissues principally by Na + -dependent cotransporters and, to a lesser extent, by  facilitated transporters (Table 37.1). In this respect, amino acid transport differs  from glucose transport, which is Na + -dependent transport in the intestinal and renal  epithelium but facilitated transport in other cell types. The Na + dependence of  amino acid transport in liver, muscle, and other tissues allows these cells to con-  centrate amino acids from the blood. These transport proteins have a different  genetic basis, amino acid composition, and somewhat different specificity than  those in the luminal membrane of intestinal epithelia. They also differ somewhat  between tissues. For instance, the N system for glutamine uptake is present in the  liver but either not present in other tissues or present as an isoform with different  properties. There is also some overlap in specificity of the transport proteins, with  most amino acids being transported by more than one carrier.   III. PROTEIN TURNOVER AND REPLENISHMENT OF THE  INTRACELLULAR AMINO ACID POOL   The amino acid pool within cells is generated both from dietary amino acids and  from the degradation of existing proteins within the cell. All proteins within cells  have a half-life (t 1/2 ), a time at which 50% of the protein that was synthesized at a  particular time will have been degraded. Some proteins are inherently short-lived,  with half-lives of 5 to 20 minutes. Other proteins are present for extended periods,  with half -lives of many hours, or even days. Thus, proteins are continuously being    Cal Kulis and other patients with cystinuria have a genetically determined  defect in the transport of cystine and the basic amino acids, lysine, arginine,  and ornithine, across the brush-border membranes of cells in both their small  intestine and renal tubules. However, they do not appear to have any symptoms of  amino acid deficiency, in part because the amino acids cysteine (which is oxidized in  blood and urine to form the disulfide cystine) and arginine can be synthesized in the  body (i.e., they are "nonessential" amino acids). Ornithine (an amino acid that is not  found in proteins but serves as an intermediate of the urea cycle) can also be synthe-  sized. The most serious problem for these patients is the insolubility of cystine, which  can form kidney stones that may lodge in the ureter, causing bleeding and severe pain.   Fig. 37.4. Transepithelial amino acid transport.  Na + -dependent carriers transport both Na + and  an amino acid into the intestinal epithelial cell  from the intestinal lumen. Na + is pumped out  on the serosal side (across the basolateral mem-  brane) in exchange for K + by the Na + ,K + -  ATPase. On the serosal side, the amino acid is  carried by a facilitated transporter down its con-  centration gradient into the blood. This process  is an example of secondary active transport.   Protein turnover is quite extensive,  rj J For example, red blood cells have a  lifespan of 120 days. Every day 3 X  10 11 (300,000 million) red blood  cells die and are phagocytosed. The hemo-  globin in these cells is degraded to amino  acids by lysosomal proteases, and these  amino acids are reutilized. Approximately  6 lb hemoglobin is recycled in this way every  year. As the aged cells are dying, newly gen-  erated reticulocytes are synthesizing hemo-  globin in preparation for their conversion  into new red blood cells, which replace the  dying cells.   Adults cannot increase the amount  of muscle or other body proteins  by eating an excess amount of pro-  tein. If dietary protein is consumed in excess  of our needs, it is converted to glycogen and  triacylglycerols, which are then stored.   synthesized and degraded in the body, using a variety of enzyme systems to do so  (Table 37.2). Examples of proteins that undergo extensive synthesis and degradation  are hemoglobin, muscle proteins, digestive enzymes, and the proteins of cells  sloughed off from the gastrointestinal tract. Hemoglobin is produced in reticulo-  cytes and reconverted to amino acids by the phagocytic cells that remove mature red  blood cells from the circulation on a daily basis. Muscle protein is degraded during  periods of fasting, and the amino acids are used for gluconeogenesis. After inges-  tion of protein in the diet, muscle protein is resynthesized.   A large amount of protein is recycled daily in the form of digestive enzymes, which  are themselves degraded by digestive proteases. In addition, approximately one fourth  of the cells lining the walls of the gastrointestinal tract are lost each day and replaced  by newly synthesized cells. As cells leave the gastrointestinal wall, their proteins and  other components are digested by enzymes in the lumen of the gut, and the products  are absorbed. Only approximately 6% (roughly 10 g) of the protein that enters the  digestive tract (including dietary proteins, digestive enzymes, and the proteins in  sloughed-off cells) is excreted in the feces each day. The remainder is recycled.   Proteins are also recycled within cells. The differences in amino acid composi-  tion of the various proteins of the body, the vast range in turnover times (t 1/2 ), and  the recycling of amino acids are all important factors that help to determine the  requirements for specific amino acids and total protein in the diet. The synthesis of  many enzymes is induced in response to physiologic demand (such as fasting or  feeding). These enzymes are continuously being degraded. Intracellular proteins are  also damaged by oxidation and other modifications that limit their function. Mech-  anisms for intracellular degradation of unnecessary or damaged proteins involve  lysosomes and the ubiquitin/proteasome system.   Lysosomes participate in the process of autophagy, in which intracellular compo-  nents are surrounded by membranes that fuse with lysosomes, and endocytosis (see  Chapter 10). Autophagy is a complex regulated process in which cytoplasm is  sequestered into vesicles and delivered to the lysosomes. Within the lysosomes, the  cathepsin family of proteases degrades the ingested proteins to individual amino  acids. The recycled amino acids can then leave the lysosome and rejoin the intra-  cellular amino acid pool. Although the details of how autophagy is induced are still  not known, starvation of a cell is a trigger to induce this process. This will allow old  proteins to be recycled and the newly released amino acids used for new protein  synthesis, to enable the cell to survive starvation conditions.   Model extracellular matrix  components; regulated by  TIMPs (tissue inhibitors of  matrix metalloproteinases)   Ubiquitin is a small protein (76 amino acids) that is highly conserved. Its amino  acid sequence in yeast and humans differs by only three residues. Ubiquitin targets  intracellular proteins for degradation by covalently binding to the e-amino group  of lysine residues. This is accomplished by a three-enzyme system that adds ubiq-  uitin to proteins targeted for degradation. Oftentimes, the target protein is polyu-  biquitinylated, in which additional ubiquitin molecules are added to previous ubiq-  uitin molecules, forming a long ubiquitin tail on the target protein. After  polyubiquitinylation is complete, the targeted protein is released from the three-  enzyme complex.   A protease complex, known as the proteasome, then degrades the targeted pro-  tein, releasing intact ubiquitin that can again mark other proteins for degradation  (Figure 37.5). The basic proteasome is a cylindrical 20S protein complex with mul-  tiple internal proteolytic sites. ATP hydrolysis is used both to unfold the tagged pro-  tein and to push the protein into the core of the cylinder. The complex is regulated  by cap protein complexes, which bind the ubiquinylated protein (a step that requires  ATP) and deliver them to the complex. After the target protein is degraded, the ubiq-  uitin is released intact and recycled. The resultant amino acids join the intracellular  pool of free amino acids. Different cap complexes alter the specificity of the pro-  teasome. For example, the PA700 cap is required for ubiquinylated proteins,  whereas the PA28 cap targets only short peptides to the complex.   ^ Sissy Fibrosa's growth and weight curves were both subnormal until her  ) pediatrician added pancreatic enzyme supplements to her treatment plan.  These supplements digest dietary protein, releasing essential and other  amino acids from the dietary protein that are then absorbed by the endothelial cells  of Sissy's small intestine, through which they are transported into the blood. A dis-  cernable improvement in Sissy's body weight and growth curves was noted within  months of the start of this therapy.   Another protein modification, which  \ \ ) occurs through a three-enzyme  complex similar to that required for  ubiquitin addition, is SUMOylation. SUMO  stands for small ubiqutin-like modifier, and  when proteins are tagged with SUMO their  activites are altered (either positively or nega-  tively, depending on the protein). SUMOyla-  tion presents yet another means of fine-tuning  existing regulatory systems.   Many proteins that contain regions  \\ ) rich in the amino acids proline (P),   glutamate (E), serine (S), and threo-  nine (T) have short half-lives. These regions  are known as PEST sequences, based on the  one-letter abbreviations used for these  amino acids. Most of the amino acids that  contain PEST sequences are hydrolyzed by  the ubiquitin-proteasome system.   Fig 37.5. The proteasome and cap proteins. The cap proteins (PA700 and PA28) regulate the  activity of this proteolytic complex by recruiting to the complex the substrates for proteoly-  sis. The ATP requirement is to unfold and denature the proteins targeted for destruction,  although the PA28 protease complex does not require ATP.   Apart from the proportions of essential amino acids present in various foods, the  quality of a dietary protein is also determined by the rate at which it is digested and,  in a more general way, by its capacity to contribute to the growth of the infant. In  this regard, the proteins in foods of animal origin are more digestible than are those  derived from plants. For example, the digestibility of proteins in eggs is approxi-  mately 97%; that for meats, poultry, and fish is 85 to 100%, and that from wheat,  soybeans, and other legumes ranges from 75 to 90%.   The official daily dietary "protein requirement" accepted by the U.S. and  Canadian governments is 0.8 g of protein per kilogram of desirable body weight  for adults (approximately 56 g for an adult male and 44 g for an adult female).  On an average weight basis, the requirement per kilogram is much greater for  infants and children. This fact underscores the importance of improving Sissy  Fibrosa's protein digestion to optimize her potential for normal growth and  development.   • In patients with cystinuria, such as Cal Kulis, the inability to normally  H|I ) absorb cystine and basic amino acids from the gut and the increased loss   " of these amino acids in the urine would be expected to cause a defi-  ciency of these compounds in the blood. However, because three of these amino  acids can be synthesized in the body (i.e., they are nonessential amino acids),  their concentrations in the plasma remain normal, and clinical manifestations of  a deficiency state do not develop. It is not clear why symptoms related to a lysine  deficiency have not been observed.   In the disorder that was first observed in the Hartnup family and bears their  name, the intestinal and renal transport defect involves the neutral amino acids  (monoamine, monocarboxylic acids), including a number of the essential amino  acids (isoleucine, leucine, phenylalanine, threonine, tryptophan, and valine) as well  as certain nonessential amino acids (alanine, serine, and tyrosine). A reduction in  the availability of these essential amino acids would be expected to cause a variety  of clinical disorders. Yet children with the Hartnup disorder identified by routine  newborn urine screening almost always remain clinically normal.   However, some patients with the Hartnup biochemical phenotype eventually  develop pellagra-like manifestations, which usually include a photosensitivity rash,  ataxia, and neuropsychiatric symptoms. Pellagra results from a dietary deficiency of  the vitamin niacin or the essential amino acid tryptophan, which are both precursors  for the nicotinamide moiety of NAD and NADP. In asymptomatic patients, the  transport abnormality may be incomplete and so subtle as to allow no phenotypic  expression of Hartnup disease. These patients also may be capable of absorbing  some small peptides that contain the neutral amino acids.   The only rational treatment of patients having pellagra-like symptoms is the  administration of niacin (nicotinic acid) in oral doses up to 300 mg/day.  Although the rash, ataxia, and neuropsychiatric manifestations of niacin defi-  ciency may disappear, the hyperaminoaciduria and intestinal transport defect do  not respond to this therapy. In addition to niacin, a high-protein diet may benefit  some patients.   The 7-glutamyl cycle is necessary for the synthesis of glutathione, a com-  pound that protects cells from oxidative damage (see Chapter 24). When  originally discovered, the cycle was thought to be important in amino acid  transport, but its involvement in such transport is now thought to be limited to sal-  vage of cysteine. The enzymes of the cycle are present in many tissues, although  certain tissues lack one or more of the enzymes of the cycle.   Fig. 37.6. 7-Glutamyl cycle. In cells of the intestine and kidney, amino acids can be trans-  ported across the cell membrane by reacting with glutathione (7-glutamyl-cysteinyl-glycine)  to form a 7-glutamyl amino acid. The amino acid is released into the cell, and glutathione is  resynthesized. However, the major role of this cycle is glutathione synthesis, and many  tissues lack the transpeptidase and 5-oxo-prolinase activities.   The entire cycle is presented in Figure 37.6. In this case, the extracellular amino  acid reacts with glutathione (7-glutamyl-cysteinyl-glycine) in a reaction catalyzed  by a transpeptidase present in the cell membrane. A 7-glutamyl amino acid is  formed, which travels across the cell membrane and releases the amino acid into  the cell. The other products of these two reactions are reconverted to glutathione.   The reactions converting glutamate to glutathione in the 7-glutamyl cycle are the  same reactions required for the synthesis of glutathione. The enzymes for  glutathione synthesis, but not the transpeptidase, are found in most tissues. The oxo-  prolinase is also missing from many tissues, such that the major role of this pathway  is one of glutathione synthesis from glutamate, cysteine, and glycine. The transpep-  tidase is the only protease in the cell that can break the 7-glutamyl linkage in  glutathione. Glutathione is also involved in reducing compounds such as hydrogen  peroxide (see Chapter 21). It also protects cells, in particular erythrocytes, from  oxidative damage, through formation of oxidized glutathione, two glutathione  residues connected by a disulfide bond.   Argiles JF, Lopez-Soriano FJ. Intestinal amino acid transport: an overview. Int J Biochem  1990;22:931-939.   Christensen HN. Role of amino acid transport and countertransport in nutrition and metabolism. Phys-  iol Rev 1990;70:43-77.   Biol Chem 1999;274:22123-22126.  Larrson A, Anderson ME. Glutathione synthetase deficiency and other disorders of the 7-glutamyl cycle.   In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds. The Metabolic and Molecular Bases of   Inherited Disease, vol II, 8th Ed. New York: McGraw-Hill, 2001:2205-2216.  Palacin M, Estevez R, Bertran J, Zorzano A. Molecular biology of mammalian plasma membrane amino   1 . An individual with a deficiency in the conversion of trypsinogen to trypsin would be expected to experience a more detrimen-   tal effect on protein digestion than an individual who was defective in any of the other digestive proteases. This is due to which  of the following?   (A) Trypsin has a greater and wider range of substrates to act on.   (B) Trypsin activates pepsinogen, so digestion can begin in the stomach.   (C) Trypsin activates the other zymogens that are secreted by the pancreas.   (D) Trypsin activates enteropeptidase, which is needed to activate the other pancreatic zymogens.   (E) Trypsin inhibits intestinal motility, so the substrates can be hydrolyzed for longer periods.   2. An individual has been shown to have a deficiency in an intestinal epithelial cell amino acid transport system for leucine. However,   the individual shows no symptoms of amino acid deficiency. This could be due to which of the following?   (A) The body synthesizes leucine to compensate for the transport defect.   (B) The kidney reabsorbs leucine and sends it to other tissues.   (A) Consuming a calorie-deficient diet that is also deficient in protein   (C) Consuming a calorie-adequate diet that is deficient in fatty acids   4. Which of the following enzymes is activated through an autocatalytic process?   5. Children with kwashiorkor usually have a fatty liver. This is due to which of the following?   (E) The lack of substrates for protein synthesis in the liver   (F) The lack of substrates for glycogen synthesis in the liver   Fig 38.1. Fate of amino acid carbons and  nitrogen. Amino acid carbon can be used either  for energy storage (glycogen, fatty acids) or  for energy. Amino acid nitrogen is used for  urea synthesis. One nitrogen of urea comes  from NH 4 + , the other from aspartate.   In comparison with carbohydrate and lipid metabolism, the metabolism of amino  acids is complex. We must be concerned not only with the fate of the carbon atoms  of the amino acids but also with the fate of the nitrogen. During their metabolism,  amino acids travel in the blood from one tissue to another. Ultimately, most of the  nitrogen is converted to urea in the liver and the carbons are oxidized to C0 2 and  H 2 0 by a number of tissues (Fig. 38.1).   After a meal that contains protein, amino acids released by digestion ( see  Chapter 37) pass from the gut through the hepatic portal vein to the liver (see Fig.  38. 2A). In a normal diet containing 60 to 100 g protein, most of the amino acids  are used for the synthesis of proteins in the liver and in other tissues. Excess  amino acids may be converted to glucose or triacy I glycerol.   During fasting, muscle protein is cleaved to amino acids. Some of the amino  acids are partially oxidized to produce energy (see Fig. 38.2B). Portions of these  amino acids are converted to alanine and glutamine, which, along with other  amino acids, are released into the blood. Glutamine is oxidized by various tissues,  including the lymphocytes, gut, and kidney, which convert some of the carbons  and nitrogen to alanine. Alanine and other amino acids travel to the liver, where  the carbons are converted to glucose and ketone bodies and the nitrogen is con-  verted to urea, which is excreted by the kidneys. Glucose, produced by gluconeo-  genesis, is subsequently oxidized to C0 2 and H 2 0 by many tissues, and ketone  bodies are oxidized by tissues such as muscle and kidney.   Several enzymes are important in the process of interconverting amino acids and in  removing nitrogen so that the carbon skeletons can be oxidized. These include dehy-  dratases, transaminases, glutamate dehydrogenase, glutaminase, and deaminases.   The conversion of amino acid nitrogen to urea occurs mainly in the liver. Urea  is formed in the urea cycle from NH 4 + , bicarbonate, and the nitrogen of aspartate  (see Fig. 38.1 ). Initially, NH 4 + , bicarbonate, and ATP react to produce carbamoyl  phosphate, which reacts with ornithine to form citrulline. Aspartate then reacts  with citrulline to form arginino succinate , which releases fumarate , forming argi-  nine. Finally, arginase cleaves arginine to release urea and regenerate ornithine.  The cycle is regulated in a feed-forward manner, such that when amino acid  degradation is occurring, the rate of the cycle is increased.   Percy Veere and his high school friend decided to take a Caribbean cruise,  during which they sampled the cuisine of many of the islands on their itin-  erary. One month after their return to the United States, Percy complained of    Fig 38.2. Roles of various tissues in amino acid metabolism. A. In the fed state, amino acids released by digestion of dietary proteins travel  through the hepatic portal vein to the liver, where they are used for the synthesis of proteins, particularly the blood proteins, such as serum albu-  min. Excess amino acids are converted to glucose or to triacylglycerols. The latter are then packaged and secreted in VLDL. The glucose pro-  duced from amino acids in the fed state is stored as glycogen or released into the blood if blood glucose levels are low. Amino acids that pass  through the liver are converted to proteins in cells of other tissues. B. During fasting, amino acids are released from muscle protein. Some enter  the blood directly. Others are partially oxidized and the nitrogen stored in the form of alanine and glutamine, which enter the blood. In the kid-  ney, glutamine releases ammonia into the urine and is converted to alanine and serine. In the cells of the gut, glutamine is converted to alanine.  Alanine (the major gluconeogenic amino acid) and other amino acids enter the liver, where their nitrogen is converted to urea, which is excreted  in the urine, and their carbons to glucose and ketone bodies, which are oxidized by various tissues for energy.   severe malaise, loss of appetite, nausea, vomiting, and arthralgias (joint pains). He  had a low-grade fever and noted a persistent and increasing pain in the area of his  liver. His friend noted a yellow discoloration of the whites of Percy's eyes and skin.  Percy's urine turned the color of iced tea, and his stool became a light-clay color.  His doctor found his liver to be enlarged and tender. Liver function tests were  ordered.   Serologic testing for viral hepatitis type B, C, and D were nonreactive, but fecal  studies showed "shedding" of hepatitis virus type A. Tests for antibodies to antigens  of the hepatitis A virus (anti-HAV) in the serum were positive for the immunoglob-  ulin M type.   A diagnosis of acute viral hepatitis type A was made, probably contracted from  virus -contaminated food Percy had eaten while on his cruise. His physician  explained that there was no specific treatment for type A viral hepatitis but recom-  mended symptomatic and supportive care and prevention of transmission to others  by the fecal-oral route. Percy took acetaminophen 3 to 4 times a day for fever and  arthralgias throughout his illness.   Transamination is the major process for removing nitrogen from amino acids. In  most instances, the nitrogen is transferred as an amino group from the original  amino acid to a-ketoglutarate, forming glutamate, whereas the original amino acid  is converted to its corresponding a-keto acid (Fig. 38.3). For example, the amino  acid aspartate can be transaminated to form its corresponding a-keto acid, oxaloac-  etate. In the process, the amino group is transferred to a-ketoglutarate, which is con-  verted to its corresponding amino acid, glutamate.   All amino acids except lysine and threonine undergo transamination reactions.  The enzymes catalyzing these reactions are known as transaminases or aminotrans-  ferases. For most of these reactions, a-ketoglutarate and glutamate serve as one of  the a-keto acid-amino acid pairs. Pyridoxal phosphate is the cof actor, and the  mechanism of the reaction is indicated in Figure 38.4.   Overall, in a transamination reaction, an amino group from one amino acid  becomes the amino group of a second amino acid. Because these reactions are read-  ily reversible, they can be used to remove nitrogen from amino acids or to transfer  nitrogen to a-keto acids to form amino acids. Thus, they are involved both in amino  acid degradation and in amino acid synthesis.   Cells in the body and bacteria in the gut release the nitrogen of certain amino acids  as ammonia or ammonium ion (NH 4 + ) (Fig. 38.5). Because these two forms of  nitrogen can be interconverted, the terms are sometimes used interchangeably.  Ammonium ion releases a proton to form ammonia by a reaction with a pK of 9.3  (Fig. 38.6). Therefore, at physiologic pH, the equilibrium favors NH 4 + by a factor  of approximately 100/1 (see Chapter 4, the Henderson-Hasselbalch equation).  However, it is important to note that NH 3 is also present in the body, because this is  the form that can cross cell membranes. For example, NH 3 passes into the urine  from kidney tubule cells and decreases the acidity of the urine by binding protons,  forming NH 4 + . Once the NH 4 + is formed, the compound can no longer freely dif-  fuse across membranes.   Glutamate can be oxidatively deaminated by a reaction catalyzed by glutamate  dehydrogenase that produces ammonium ion and a-ketoglutarate (Fig. 38.7).  Either NAD + or NADP + can serve as the cof actor. This reaction, which occurs in  the mitochondria of most cells, is readily reversible; it can incorporate ammonia   Fig. 38.3. Transamination. The amino group  from one amino acid is transferred to another.  Pairs of amino acids and their corresponding  a-keto acids are involved in these reactions, a-  ketoglutarate and glutamate are usually one of  the pairs. The reactions, which are readily  reversible, use pyridoxal phosphate (PLP) as a  cofactor. The enzymes are called transaminases  or aminotransferases. A. A generalized reac-  tion. B. The aspartate transaminase reaction.   Whereas pyridoxal phosphate is  used primarily for reactions involv-  ing amino acids, it is also required  for the glycogen phosphorylase reaction, in  which it acts as a general acid/base catalyst.   Fig. 38.4. Function of pyridoxal phosphate (PLP) in transamination reactions. The order in which the reactions occur is 1 to 4. Pyridoxal phos-  phate (enzyme-bound) reacts with amino acid l9 forming a Schiff base (a carbon-nitrogen double bond). After a shift of the double bond, a-keto  acid! is released through hydrolysis of the Schiff base, and pyridoxamine phosphate is produced. Pyridoxamine phosphate then forms a Schiff  base with a-keto acid 2 . After the double bond shifts, amino acid 2 is released through hydrolysis of the Schiff base and enzyme-bound pyridoxal  phosphate is regenerated. The net result is that the amino group from amino acidj is transferred to amino acid 2 .   Glutamate dehydrogenase is one  of three mammalian enzymes that  can "fix" ammonia into organic  molecules. The other two are glutamine syn-  thetase and carbamoyl phosphate syn-  thetase I.   into glutamate or release ammonia from glutamate. Glutamate can collect nitrogen  from other amino acids as a consequence of transmination reactions and then  release ammonia through the glutamate dehydrogenase reaction. This process pro-  vides one source of the ammonia that enters the urea cycle.   In addition to glutamate, a number of amino acids release their nitrogen as  NH 4 + (see Fig. 38.5). Histidine may be directly deaminated to form NH 4 + and  urocanate. The deaminations of serine and threonine are dehydration reactions  that require pyridoxal phosphate and are catalyzed by serine dehydratase. Serine  forms pyruvate, and threonine forms a-ketobutyrate. In both cases, NH 4 + is  released.   Glutamine and asparagine contain R group amides that may be released as NH 4 +  by deamidation. Asparagine is deamidated by asparaginase, yielding aspartate and  NH 4 + . Glutaminase acts on glutamine, forming glutamate and NH 4 + . The glutaminase    Fig. 38.5. Summary of the sources of NH 4 + for the urea cycle. All of the reactions are irre-  versible except glutamate dehydrogenase (GDH). Only the dehydratase reactions, which pro-  duce NH 4 + from serine and threonine, require pyridoxal phosphate as a cofactor. The reac-  tions that are not shown occurring in the muscle or the gut can all occur in the liver, where  the NH 4 + generated can be converted to urea. The purine nucleotide cycle of the brain and  muscle is further described in Chapter 41.   reaction is particularly important in the kidney, where the ammonium ion produced  is excreted directly into the urine, where it forms salts with metabolic acids, facili-  tating their removal in the urine.   In muscle and brain, but not in liver, the purine nucleotide cycle allows NH 4 + to be  released from amino acids (see Fig. 38.5). Nitrogen is collected by glutamate from  other amino acids by means of transamination reactions. Glutamate then transfers its  amino group to oxaloacetate to form aspartate, which supplies nitrogen to the purine  nucleotide cycle (see Chapter 41). The reactions of the cycle release fumarate and  NH 4 + . The ammonium ion formed can leave the muscle in the form of glutamine.   Fig. 38.7. Reaction catalyzed by glutamate dehydrogenase. This reaction is readily reversible  and can use either NAD + or NADP + as a cofactor. The oxygen on a-ketoglutarate is derived  from H 2 0.   Percy Veere's laboratory studies  showed that his serum alanine  transaminase (ALT) level was 294  units/L (reference range = 5-30),  and his serum aspartate transaminase (AST)  level was 268 units/L (reference range =  10-30). His serum alkaline phosphatase level  was 284 units/L (reference range for an adult  male = 40-125), and his serum total bilirubin  was 9.6 mg/dL (reference range = 0.2-1.0).  Bilirubin is a degradation product of heme,  as described in Chapter 44.   Cellular enzymes such as AST, ALT, and  alkaline phosphatase leak into the blood  through the membranes of hepatic cells that  have been damaged as a result of the inflam-  matory process. In acute viral hepatitis, the  serum ALT level is often elevated to a  greater extent than the serum AST level.  Alkaline phosphatase, which is present on  membranes between liver cells and the bile  duct, is also elevated in the blood in acute  viral hepatitis.   The rise in serum total bilirubin occurs as  a result of the inability of the infected liver to  conjugate bilirubin and of a partial or com-  plete occlusion of the hepatic biliary  drainage ducts caused by inflammatory  swelling within the liver. In fulminant hepatic  failure, the serum bilirubin level may exceed  20 mg/dL, a poor prognostic sign.   Fig. 38.6. Formation of ammonia from ammo-  nium ion. Because the pK is 9.3, at physiologic  pH, the concentration of NH 4 + is almost 100  times that of NH 3 .   Vitamin B 6 deficiency symptoms  include dermatitis, a microcytic,  hypochromic anemia, weakness,  irritability, and, in some cases, convulsions.  Xanthurenic acid (a degradation product of  tryptophan) and other compounds appear in  the urine because of an inability to com-  pletely metabolize amino acids. A decreased  ability to synthesize heme from glycine may  cause the microcytic anemia (see Chapter  44), and decreased decarboxylation of  amino acids to form neurotransmitters (see  Chapter 48) may explain the convulsions.   In summary, NH 4 + that enters the urea cycle is produced in the body by deami-  nation or deamidation of amino acids (see Fig. 38.5). A significant amount of NH 4 +  is also produced by bacteria that live in the lumen of the intestinal tract. This ammo-  nium ion enters the hepatic portal vein and travels to the liver.   Fig. 38.8. Role of glutamate in amino acid syn-  thesis. Glutamate transfers nitrogen by means  of transamination reactions to a-keto acids to  form amino acids. This nitrogen is either  obtained by glutamate from transamination of  other amino acids or from NH 4 + by means of  the glutamate dehydrogenase (GDH) reaction.  PLP = pyridoxal phosphate.   Pyridoxal phosphate is derived  r| ) from pyridoxine (vitamin B 6 ). Pyri-  *S doxal phosphate is the cofactor not  only for transamination reactions but also  for decarboxylations and a number of other  reactions involving amino acids.    C. Role of Glutamate in the Metabolism of Amino Acid  Nitrogen   Glutamate plays a pivotal role in the metabolism of amino acids. It is involved in  both synthesis and degradation.   Glutamate provides nitrogen for amino acid synthesis (Fig. 38.8). In this process,  glutamate obtains its nitrogen either from other amino acids by transamination reac-  tions or from NH 4 + by the glutamate dehydrogenase reaction. Transamination reac-  tions then serve to transfer amino groups from glutamate to a-keto acids to produce  their corresponding amino acids.   When amino acids are degraded and urea is formed, glutamate collects nitrogen  from other amino acids by transamination reactions. Some of this nitrogen is  released as ammonia by the glutamate dehydrogenase reaction, but much larger  amounts of ammonia are produced from the other sources shown in Figure 38.5.  NH 4 + is one of the two forms in which nitrogen enters the urea cycle (Fig. 38.9).   The second form of nitrogen for urea synthesis is provided by aspartate (see Fig.  38.9). Glutamate can be the source of the nitrogen. Glutamate transfers its amino  group to oxaloacetate, and aspartate and a-ketoglutarate are formed.   D. Role of Alanine and Glutamine in Transporting  Amino Acid Nitrogen to the Liver   Protein turnover and amino acid degradation occur in all tissues; however, the urea  cycle enzymes are primarily active in the liver (the intestine expresses low levels of  activity of these enzymes; see Chapter 42). Thus, a mechanism needs to be in place  to transport amino acid nitrogen to the liver. Alanine and glutamine are the major  carriers of nitrogen in the blood. Alanine is primarily exported by the muscle.  Because the muscle is metabolizing glucose through glycolysis, pyruvate is avail-  able in the muscle. The pyruvate is transaminated by glutamate to form alanine,  which travels to the liver (Fig. 38.10). The glutamate is formed by transamination  of an amino acid that is being degraded. On arriving at the liver, alanine is transam-  inated to pyruvate, and the nitrogen will be used for urea synthesis. The pyruvate  formed is used for gluconeogenesis and the glucose exported to the muscle for use  as energy. This cycle of moving carbons and nitrogen between the muscle and liver  is known as the glucose/alanine cycle.   Compounds that contain "glut" in their name have five carbons in a straight  KJ ) chain. At each end of the chain, the carbon is part of a carboxyl group. In glut-  *r amine, the carboxyl group has formed an amide, and in hydroxymethylglutaryl  CoA (HMG-CoA), it has formed a thioester with coenzyme A.    Fig. 38.9. Role of glutamate in urea production. Glutamate collects nitrogen from other  amino acids by transamination reactions. This nitrogen can be released as NH 4 + by glutamate  dehydrogenase (GDH). NH 4 + is also produced by other reactions (see Fig. 38.5). NH 4 + pro-  vides one of the nitrogens for urea synthesis. The other nitrogen comes from aspartate and is  obtained from glutamate by transamination of oxaloacetate.   Glutamine is synthesized from glutamate by the fixation of ammonia, requiring  energy (adenosine triphosphate [ATP]) and the enzyme glutamine synthetase  (Fig. 38.11), which is a cytoplasmic enzyme found in all cells. Under conditions of  rapid amino acid degradation within a tissue, such that ammonia levels increase, the  glutamate that has been formed from transamination reactions will accept another  nitrogen molecule to form glutamine. The glutamine travels to the liver, kidney, or  intestines, where glutaminase (see Fig. 38.11) will remove the amide nitrogen to  form glutamate plus ammonia. In the kidney, the release of ammonia, and the for-  mation of ammonium ion, serves to form salts with metabolic acids in the urine. In  the intestine, the glutamine is used as a fuel (see Chapter 42). In the liver, the ammo-  nia is used for urea biosynthesis.   Glutamine synthetase in liver is  located in cells surrounding the  portal vein. Its major role is to con-  vert any ammonia that has escaped from  urea production into glutamine, such that  free ammonia does not leave the liver and  enter the circulation.   The normal human adult is in nitrogen balance; that is, the amount of nitrogen  ingested each day, mainly in the form of dietary protein, is equal to the amount  of nitrogen excreted. The major nitrogenous excretory product is urea, which  exits from the body in the urine. This innocuous compound, produced mainly in  the liver by the urea cycle, serves as the disposal form of ammonia, which is  toxic, particularly to the brain and central nervous system. Normally, little  ammonia (or NH 4 + ) is present in the blood. The concentration ranges between    Fig. 38.10. The glucose/alanine cycle. Within the muscle, amino acid degradation leads to the transfer of nitrogens to a-ketoglutarate and pyru-  vate. The alanine formed travels to the liver, where the carbons of alanine are used for gluconeogenesis and the alanine nitrogen is used for urea  biosynthesis. This could occur during exercise, when the muscle uses blood-borne glucose (see Chapter 47).    Fig. 38.11. Synthesis of glutamine in peripheral tissues and its transport to the liver. Within the liver, glutaminase converts glutamine to gluta-  mate. Note how a-ketoglutarate can accept two molecules of ammonia to form glutamine. GDH = glutamate dehydrogenase.   0 o Percy Veere's symptoms and labora-  ( /~oA ) tory abnormalities did not slowly  ^ subside over the next 6 weeks as  they usually do in uncomplicated viral hepati-  tis A infections. Instead, his serum total biliru-  bin, ALT, AST, and alkaline phosphatase levels  increased further. His vomiting became  intractable, and his friend noted jerking  motions of his arms (asterixis), facial grimac-  ing, restlessness, slowed mentation, and slight  disorientation. He was admitted to the hospital  with a diagnosis of hepatic failure with incipi-  ent hepatic encephalopathy (brain dysfunction  caused by accumulation of various toxins in  the blood), a rare complication of acute type A  viral hepatitis alone. The possibility of a super-  imposed acute hepatic toxicity caused by the  use of acetaminophen was considered.   0 o When ornithine transcarbamoylase  ( /^OA ) (OTC) is deficient, the carbamoyl  Y phosphate that normally would  enter the urea cycle accumulates and floods  the pathway for pyrimidine biosynthesis.  Under these conditions, excess orotic acid  (orotate), an intermediate in pyrimidine  biosynthesis, is excreted in the urine. It pro-  duces no ill effects but is indicative of a  problem in the urea cycle.   30 and 60 jxM. Ammonia is rapidly removed from the blood and converted to  urea by the liver. Nitrogen travels in the blood mainly in amino acids, particu-  larly alanine and glutamine.   Nitrogen enters the urea cycle as NH 4 + and aspartate (Fig. 38.12). NH 4 + forms car-  bamoyl phosphate, which reacts with ornithine to form citrulline. Ornithine is the  compound that both initiates and is regenerated by the cycle (similar to oxaloacetate  in the TCA cycle). Aspartate reacts with citrulline, eventually donating its nitrogen  for urea formation. Arginine is formed in two successive steps. Cleavage of arginine  by arginase releases urea and regenerates ornithine.   In the first step of the urea cycle, NH 4 + , bicarbonate, and ATP react to form car-  bamoyl phosphate (see Fig. 38.12). The cleavage of 2 ATPs is required to form the  high-energy phosphate bond of carbamoyl phosphate. Carbamoyl phosphate syn-  thetase I (CPSI), the enzyme that catalyzes this first step of the urea cycle, is found  mainly in mitochondria of the liver and intestine. The Roman numeral suggests that  another carbamoyl phosphate synthetase exists, and indeed, CPSII, located in the  cytosol, produces carbamoyl phosphate for pyrimidine biosynthesis, using nitrogen  from glutamine (see Chapter 41).   Carbamoyl phosphate reacts with ornithine to form citrulline (see Fig. 38.12). The  high- energy phosphate bond of carbamoyl phosphate provides the energy required  for this reaction, which occurs in mitochondria and is catalyzed by ornithine tran-  scarbamoylase. The product citrulline is transported across the mitochondrial mem-  branes in exchange for cytoplasmic ornithine and enters the cytosol. The carrier for  this transport reaction catalyzes an electroneutral exchange of the two compounds.   In the cytosol, citrulline reacts with aspartate, the second source of nitrogen for  urea synthesis, to produce argininosuccinate (see Fig. 38.12). This reaction, cat-  alyzed by argininosuccinate synthetase, is driven by the hydrolysis of ATP to adeno-  sine monophosphate (AMP) and pyrophosphate. Aspartate is produced by transam-  ination of oxaloacetate.   The urea cycle was proposed in 1932 by Hans Krebs and a medical student, Kurt  Kj ) Henseleit, based on their laboratory observations. It was originally called the  Krebs-Henseleit cycle. Subsequently, Krebs used this concept of metabolic  cycling to explain a second process that also bears his name, the Krebs (or TCA) cycle.   Fig. 38.12. Urea cycle. The steps of the cycle are numbered 1 to 5.   Argininosuccinate is cleaved by argininosuccinate lyase to form fumarate and  arginine (see Fig. 38.12). Fumarate is produced from the carbons of argininosucci-  nate provided by aspartate. Fumarate is converted to malate (using cytoplasmic  fumarase), which is used either for the synthesis of glucose by the gluconeogenic  pathway or for the regeneration of oxaloacetate by cytoplasmic reactions similar to  those observed in the TCA cycle (Fig. 38.13). The oxaloacetate that is formed is  transaminated to generate the aspartate that carries nitrogen into the urea cycle.  Thus, the carbons of fumarate can be recycled to aspartate.   Arginine, which contains nitrogens derived from NH 4 + and aspartate, is cleaved by  arginase, producing urea and regenerating ornithine (see Fig. 38.12). Urea is pro-  duced from the guanidinium group on the side chain of arginine. The portion of  arginine originally derived from ornithine is reconverted to ornithine.   The reactions by which citrulline is converted to arginine and arginine is cleaved  to produce urea occur in the cytosol. Ornithine, the other product of the arginase   Fig. 38.14. The ornithine aminotransferase  reaction. This is a reversible reaction depend-  ent on pyridoxal phosphate, which normally  favors ornithine degradation.   0 o The precise pathogenesis of the  /\A ) central nervous system (CNS)  ™ signs and symptoms that accom-  pany liver failure (hepatic encephalopathy)  in patients such as Percy Veere is not com-  pletely understood. These changes are,  however, attributable in part to toxic materi-  als that are derived from the metabolism of  nitrogenous substrates by bacteria in the gut  that circulate to the liver in the portal vein.  These materials "bypass" their normal  metabolism by the liver cells, however,  because the acute inflammatory process of  viral hepatitis severely limits the ability of  liver cells to degrade these compounds to  harmless metabolites. As a result, these tox-  ins are "shunted" into the hepatic veins  unaltered and eventually reach the brain  through the systemic circulation ("portal-  systemic encephalopathy").   Fig. 38.13. The Krebs bicycle, indicating the common steps between the TCA and urea  cycles. All reactions shown occur in the cytoplasm except for the synthesis of citrulline,  which occurs within the mitochondria.   reaction, is transported into the mitochondrion in exchange for citrulline, where it  can react with carbamoyl phosphate, initiating another round of the cycle.   Ornithine is an amino acid. However, it is not incorporated into proteins during the  process of protein synthesis because no genetic codon exists for this amino acid.  Although ornithine is normally regenerated by the urea cycle (one of the products  of the arginase reaction), ornithine also can be synthesized de novo if needed. The  reaction is an unusual transamination reaction catalyzed by ornithine aminotrans-  ferase under specific conditions in the intestine (Fig. 38.14). The usual direction of  this reaction is the formation of glutamate semialdehyde, which is the first step of  the degradation pathway for ornithine.   The human liver has a vast capacity to convert amino acid nitrogen to urea, thereby  preventing toxic effects from ammonia, which would otherwise accumulate. In gen-  eral, the urea cycle is regulated by substrate availability; the higher the rate of  ammonia production, the higher the rate of urea formation. Regulation by substrate  availability is a general characteristic of disposal pathways, such as the urea cycle,  which remove toxic compounds from the body. This is a type of "feed-forward" reg-  ulation, in contrast to the "feedback" regulation characteristic of pathways that pro-  duce functional endproducts.   Two other types of regulation control the urea cycle: allosteric activation of CPSI  by Af-acetylglutamate (NAG) and induction/repression of the synthesis of urea cycle  enzymes. NAG is formed specifically to activate CPSI; it has no other known func-  tion in mammals. The synthesis of NAG from acetyl Co A and glutamate is stimu-  lated by arginine (Fig. 38.15). Thus, as arginine levels increase within the liver, two  important reactions are stimulated. The first is the synthesis of NAG, which will  increase the rate at which carbamoyl phosphate is produced. The second is to pro-  duce more ornithine (via the arginase reaction), such that the cycle can operate more  rapidly.   The induction of urea cycle enzymes occurs in response to conditions that  require increased protein metabolism, such as a high-protein diet or prolonged  fasting. In both of these physiologic states, as amino acid carbon is converted to  glucose, amino acid nitrogen is converted to urea. The induction of the synthesis  of urea cycle enzymes under these conditions occurs even though the uninduced  enzyme levels are far in excess of the capacity required. The ability of a high-pro-  tein diet to increase urea cycle enzyme levels is another type of "feed-forward"  regulation.   Fig. 38.15. Activation of carbamoyl phosphate synthetase I (CPSI). Arginine stimulates the  synthesis of Af-acetylglutamate, which activates CPSI.   During fasting, the liver maintains blood glucose levels. Amino acids from muscle  protein are a major carbon source for the production of glucose by the pathway of  gluconeogenesis. As amino acid carbons are converted to glucose, the nitrogens are  converted to urea. Thus, the urinary excretion of urea is high during fasting (Fig.  38.16). As fasting progresses, however, the brain begins to use ketone bodies, spar-  ing blood glucose. Less muscle protein is cleaved to provide amino acids for glu-  coneogenesis, and decreased production of glucose from amino acids is accompa-  nied by decreased production of urea (see Chapter 31).   The major amino acid substrate for gluconeogenesis is alanine, which is syn-  thesized in peripheral tissues to act as a nitrogen carrier (see Fig. 38.10).  Glucagon release, which is expected during fasting, stimulates alanine transport   Fig. 38.16. Nitrogen excretion during fasting. Human subjects were initially given intra-  venous (IV) glucose as indicated, then fasted. Total nitrogen excretion was measured as well  as the nitrogen in urea (dark area). Based on Ruderman NB, et al. Gluconeogenesis and its  disorders in man. In: Hanson RW, Mehlman MA, eds. Gluconeogenesis: Its Regulation in  Mammalian Species. New York: John Wiley, 1976:518.   NH 4 + is one of the toxins that results  from the degradation of urea or pro-  teins by intestinal bacteria and is not  metabolized by the infected liver. The subse-  quent elevation of ammonia concentrations in  the fluid bathing the brain causes depletion of  tricarboxylic acid (TCA) cycle intermediates  and ATP in the central nervous system, a-  Ketoglutarate, a TCA cycle intermediate, com-  bines with ammonia to form glutamate in a  reaction catalyzed by glutamate dehydroge-  nase. Glutamate subsequently reacts with  ammonia to form glutamine.   The absolute level of ammonia and its  metabolites, such as glutamine, in the blood  or cerebrospinal fluid in patients with hepatic  encephalopathy correlates only roughly with  the presence or severity of the neurologic  signs and symptoms. 7-Aminobutyric acid  (GABA), an important inhibitory neurotrans-  mitter in the brain, is also produced in the gut  lumen and is shunted into the systemic circu-  lation in increased amounts in patients with  hepatic failure. In addition, other compounds  (such as aromatic amino acids, false neuro-  transmitters, and certain short-chain fatty  acids) bypass liver metabolism and accumu-  late in the systemic circulation, adversely  affecting central nervous system function.  Their relative importance in the pathogenesis  of hepatic encephalopathy remains to be  determined.   In addition to producing urea, the  reactions of the urea cycle also  serve as the pathway for the  biosynthesis of arginine. Therefore, this  amino acid is not required in the diet of the  adult; however, it is required in the diet for  growth.   Urea is not cleaved by human  enzymes. However, bacteria,  including those in the human  digestive tract, can cleave urea to ammonia  and C0 2 , using the enzyme urease. Urease is  not produced by humans.   To some extent, humans excrete urea  into the gut and saliva. Intestinal bacteria  convert urea to ammonia. This ammonia, as  well as ammonia produced by other bacter-  ial reactions in the gut, is absorbed into the  hepatic portal vein. It is normally extracted  by the liver and converted to urea. Approxi-  mately one fourth of the total urea released  by the liver each day is recycled by bacteria.   Fig. 38.17. Conversion of alanine to glucose and urea. 1. Alanine, the key gluconeogenic  amino acid, is transaminated to form pyruvate, which is converted to glucose. The nitrogen,  now in glutamate, can be released as NH 4 + (2) or transferred to oxaloacetate to form aspar-  tate (3). NH 4 + and aspartate enter the urea cycle, which produces urea. In summary, the car-  bons of alanine form glucose and the nitrogens form urea. Two molecules of alanine are  required to produce one molecule of glucose and one molecule of urea.   into the liver by activating the transcription of transport systems for alanine.  Two molecules of alanine are required to generate one molecule of glucose. The  nitrogen from the two molecules of alanine is converted to one molecule of urea  (Fig. 38.17).   The two most serious complications of acute viral hepatitis found in  patients such as Percy Veere are massive hepatic necrosis leading to ful-  minant liver failure and the eventual development of chronic hepatitis.  Both complications are rare in acute viral hepatitis type A, however, suggesting that  acetaminophen toxicity may have contributed to Percy's otherwise unexpectedly  severe hepatocellular dysfunction and early hepatic encephalopathy.   Fortunately, bed rest, rehydration, parenteral nutrition, and therapy directed at  decreasing the production of toxins that result from bacterial degradation of  nitrogenous substrates in the gut lumen (e.g., administration of lactulose, which  reduces gut ammonia levels by a variety of mechanisms, the use of enemas and  antibiotics to decrease the intestinal flora, a low-protein diet) prevented Percy  Veere from progressing to the later stages of hepatic encephalopathy. As with  most patients who survive an episode of fulminant hepatic failure, recovery to his  previous state of health occurred over the next 3 months. Percy's liver function  studies returned to normal, and a follow-up liver biopsy showed no histologic  abnormalities.   Disorders of the urea cycle are dangerous because of the accumulation of  ammonia in the circulation. Ammonia is toxic to the nervous system, and  its concentration in the body must be carefully controlled. Under normal  conditions, free ammonia is rapidly fixed into either a-ketoglutarate (by glutamate  dehydrogenase, to form glutamate) or glutamate (by glutamine synthease, to form  glutamine). The glutamine can be used by many tissues, including the liver; the glu-  tamate donates nitrogens to pyruvate to form alanine, which travels to the liver.  Within the liver, as the nitrogens are removed from their carriers, carbamoyl phos-  phate synthetase I fixes the ammonia into carbamoyl phosphate to initiate the urea   cycle. However, when a urea cycle enzyme is defective, the cycle is interrupted,  which leads to an accumulation of urea cycle intermediates before the block.  Because of the block in the urea cycle, glutamine levels increase in the circulation,  and because a-ketoglutarate is no longer being regenerated by removal of nitrogen  from glutamine, the a-ketoglutarate levels are too low to fix more free ammonia,  leading to elevated ammonia levels in the blood. So how does one reduce ammonia  and glutamine levels in such patients?   The key to treating patients with urea cycle defects is to diagnose the disease  early and then aggressively treat with compounds that can aid in nitrogen removal   CH 2 -C-NH-C- CH 2 CH 2 -C - NH 2   Fig. 38.18. The metabolism of benzoic acid (A) and phenylbutyrate (B), two agents used to  reduce nitrogen levels in patients with urea cycle defects.   from the patient. Low-protein diets are essential to reduce the potential for exces-  sive amino acid degradation. If the enzyme defect in the urea cycle comes after the  synthesis of argininosuccinate, massive arginine supplementation has proved bene-  ficial. Once argininosuccinate has been synthesized, the two nitrogen molecules  destined for excretion have been incorporated into the substrate; the problem is that  ornithine cannot be regenerated. If ornithine could be replenished to allow the cycle  to continue, argininosuccinate could be used as the carrier for nitrogen excretion  from the body. Thus, ingesting large levels of arginine leads to ornithine production  by the arginase reaction, and nitrogen excretion via argininosuccinate in the urine  can be enhanced.   Arginine therapy will not work for enzyme defects that exist in steps before the  synthesis of argininosuccinate. For these disorders, drugs are used that form conju-  gates with amino acids. The conjugated amino acids are excreted, and the body then  has to use its nitrogen to resynthesize the excreted amino acid. The two compounds  most frequently used are benzoic acid and phenylbutyrate (the active component of  pheylbutyrate is pheylacetate, its oxidation product. Phenylacetate has a bad odor,  which makes it difficult to take orally). As indicated in Figure 38. 18 A, benzoic acid,  after activation, reacts with glycine to form hippuric acid, which is excreted. As  glycine is synthesized from serine, the body now uses nitrogens to synthesize ser-  ine, so more glycine can be produced. Phenylacetate (see Fig. 38.18B) forms a con-  jugate with glutamine, which is excreted. This conjugate removes two nitrogens per  molecule and requires the body to resynthesize glutamine from glucose, thereby  using another two nitrogen molecules.   Urea cycle defects are excellent candidates for treatment by gene therapy. This  is because the defect only has to be repaired in one cell type (in this case, the hepa-  tocyte), which makes it easier to target the vector carrying the replacement gene.  Preliminary gene therapy experiments had been carried out on individuals with  ornithine transcarbamolyase deficiency (the most common inherited defect in the  urea cycle), but the experiments came to a halt when one of the patients died of a  severe immunologic reaction to the vector used to deliver the gene. This incident  has placed gene replacement therapy in the United States "on hold" for the fore-  seeable future.   Brusilow S, Horwich A. Urea cycle enzymes. In: Scriver CR, Beaudet AL, Valle D, Sly WS, et al., eds.  The Metabolic and Molecular Bases of Inherited Disease, vol II, 8th Ed. New York: McGraw-Hill,  2001:1909-1963.   Marshall E. Gene therapy death prompts review of adenovirus vector. Science 1999;286:2244-2245.  Morris SM Jr. Regulation of enzymes of the urea cycle and arginine metabolism. Annu Rev Nutr  2002;22:87-105.   1. Deficiency diseases have been described that involve each of the five enzymes of the urea cycle. Clinical manifestations may  appear in the neonatal period. Infants with defects in the first four enzymes usually appear normal at birth, but after 24 hours  progressively develop lethargy, hypothermia, and apnea. They have high blood ammonia levels, and the brain becomes  swollen. One possible explanation for the swelling is the osmotic effect of the accumulation of glutamine in the brain pro-  duced by the reactions of ammonia with a-ketoglutarate and glutamate. Arginase deficiency is not as severe as deficiencies of  the other urea cycle enzymes.   Given the following information about five newborn infants (identified as I to V) who appeared normal at birth but developed  hyperammonemia after 24 hours, determine which urea cycle enzyme might be defective in each case (for each infant, choose  from the same five answers, lettered A through E). All infants had low levels of blood urea nitrogen (BUN). (Normal citrulline  levels are 10-20 jxM.)   * = Value not determined; low = below normal; high = above normal.   2. The nitrogens in urea are directly derived from which of the following compounds?   3. Which one of the following enzymes can fix ammonia into an organic molecule?   4. Pyridoxal phosphate, which is required for transaminations, is also required for which of the following pathways?   5. The major regulated step of the urea cycle is which of the following?   As a general rule, genetic codons  exist for 20 amino acids. Only  these 20 common amino acids are  incorporated into proteins during the  process of protein synthesis. Modifications  to these amino acids occur after they are  incorporated into proteins (such as the syn-  thesis of hydroxyproline in collagen). The  major exception to this rule is selenocys-  teine, which is an essential component of  enzymes involved in scavenging free radi-  cals (such as glutathione peroxidase-1; see  Chapter 24). Selenocysteine has a selenium  atom in the place of the oxygen atom in ser-  ine and is synthesized enzymatically in a  reaction that requires adenosine triphos-  phate (ATP), selenium, and serine attached  to a tRNA specific for selenocysteine. This  reaction uses two high-energy bonds. The  codon recognized by the tRNA-selenocys-  teine is a stop codon in the mRNA (UGA).  The secondary structure of the mRNA allows  the ribosomes and tRNA to understand  which UGA is a stop codon and which  requires the insertion of selenocysteine.    Because each of the 20 common amino acids has a unique structure, their meta-  bolic pathways differ Despite this, some generalities do apply to both the synthe-  sis and degradation of all amino acids. These are summarized in the following  sections. Because a number of the amino acid pathways are clinically relevant, we  present most of the diverse pathways occurring in humans. However, we will be as  succinct as possible.   Important coenzymes: Pyridoxal phosphate (derived from vitamin B6) is the  quintessential coenzyme of amino acid metabolism. In degradation, it is involved in  the removal of amino groups, principally through transamination reactions and in  donation of amino groups for various amino acid biosynthetic pathways. It is also  required for certain reactions involving the carbon skeleton of amino acids.  Tetrahydrofolate (FH 4 ) is a coenzyme used to transfer one-carbon groups at vari-  ous oxidation states. FH 4 is used both in amino acid degradation (e.g., serine and  histidine) and biosynthesis (e.g„ glycine). Tetrahydrobiopterin (BH 4 ) is a cof actor  required for ring hydroxy lation reactions (e.g., phenylalanine to tyrosine).   Synthesis of the amino acids: Eleven of the twenty common amino acids can be  synthesized in the body (Fig. 39.1 ). The other nine are considered "essential" and  must be obtained from the diet. Almost all of the amino acids that can be synthe-  sized by humans are amino acids used for the synthesis of additional nitrogen-con-  taining compounds. Examples include glycine, which is used for porphyrin and  purine synthesis; glutamate, which is required for neurotransmitter and purine syn-  thesis; and aspartate, which is required for both purine and pyrimidine biosynthesis.   Nine of the eleven "nonessential" amino acids can be produced from glucose  plus, of course, a source of nitrogen, such as another amino acid or ammonia.  The other two nonessential amino acids, tyrosine and cysteine, require an essen-  tial amino acid for their synthesis (phenylalanine for tyrosine, and methionine for  cysteine). The carbons for cysteine synthesis come from glucose; the methionine  only donates the sulfur.   The carbon skeletons of the 10 nonessential amino acids derived from glucose  are produced from intermediates of glycolysis and the tricarboxylic acid (TCA)  cycle (see Fig 39.1 ). Four amino acids (serine, glycine, cysteine, and alanine) are  produced from glucose through components of the glycolytic pathway. TCA cycle  intermediates (which can be produced from glucose) provide carbon for synthesis  of the six remaining nonessential amino acids. a-Ketoglutarate is the precursor  for the synthesis of glutamate, glutamine, proline, and arginine. Oxaloacetate pro-  vides carbon for the synthesis of aspartate and asparagine.   The regulation of individual amino acid biosynthesis can be quite complex, but  the overriding feature is that the pathways are feedback regulated such that as  the concentration of free amino acid increases, a key biosynthetic enzyme is  allosterically or transcriptionally inhibited. Amino acid levels, however, are   Fig. 39.1. Overview of the synthesis of the nonessential amino acids. The carbons of 10 amino acids may be produced from glucose through  intermediates of glycolysis or the TCA cycle. The 11th nonessential amino acid, tyrosine, is synthesized by hydroxylation of the essential amino  acid phenylalanine. Only the sulfur of cysteine comes from the essential amino acid methionine; its carbons and nitrogen come from serine.  Transamination (TA) reactions involve pyridoxal phosphate (PLP) and another amino acid/a-keto acid pair.   always maintained at a level such that the aminoacyl-tRNA synthetases can  remain active, and protein synthesis can continue.   Degradation of amino acids: The degradation pathways for amino acids are, in  general, distinct from biosynthetic pathways. This allows for separate regulation  of the anabolic and catabolic pathways. Because protein is a fuel, almost every  amino acid will have a degradative pathway that can generate NADH, which is  used as an electron source for oxidative phosphorylation. However, the energy-  generating pathway may involve direct oxidation, oxidation in the TCA cycle, con-  version to glucose and then oxidation, or conversion to ketone bodies, which are  then oxidized.   The fate of the carbons of the amino acids depends on the physiologic state of  the individual and the tissue where the degradation occurs. For example, in the  liver during fasting, the carbon skeletons of the amino acids produce glucose,  ketone bodies, and C0 2 . In the fed state, the liver can convert intermediates of  amino acid metabolism to glycogen and triacy I glycerols. Thus, the fate of the car-  bons of the amino acids parallels that of glucose and fatty acids. The liver is the  only tissue that has all of the pathways of amino acid synthesis and degradation.   As amino acids are degraded, their carbons are converted to (a) C0 2 , (b) com-  pounds that produce glucose in the liver (pyruvate and the TCA cycle intermedi-  ates a-ketoglutarate, succinyl CoA,fumarate, and oxaloacetate), and (c) ketone  bodies or their precursors (acetoacetate and acetyl CoA) (Fig. 39.2). For simplic-  ity, amino acids are considered to be glucogenic if their carbon skeletons can be  converted to a precursor of glucose and ketogenic if their carbon skeletons can be  directly converted to acetyl CoA or acetoacetate. Some amino acids contain car-  bons that produce a glucose precursor and other carbons that produce acetyl CoA  or acetoacetate. These amino acids are both glucogenic and ketogenic.   The amino acids synthesized from intermediates of glycolysis (serine, alanine,  and cysteine) plus certain other amino acids (threonine, glycine, and tryptophan)   Fig. 39.2. Degradation of amino acids. A. Amino acids that produce pyruvate or intermediates of the TCA cycle. These amino acids are consid-  ered glucogenic because they can produce glucose in the liver. The fumarate group of amino acids produces cytoplasmic fumarate. Potential  mechanisms whereby the cytoplasmic fumarate can be oxidized are presented in section III.C.l. B. Amino acids that produce acetyl CoA or  ketone bodies. These amino acids are considered ketogenic.   produce pyruvate when they are degraded. The amino acids synthesized from TCA  cycle intermediates (aspartate, asparagines, glutamate, glutamine, proline, and  arginine) are reconverted to these intermediates during degradation. Histidine is  converted to glutamate and then to the TCA cycle intermediate a-ketoglutarate.  Methionine, threonine, valine, and isoleucine form succinyl CoA, and pheny-  lalanine (after conversion to tyrosine) forms fumarate. Because pyruvate and the  TCA cycle intermediates can produce glucose in the liver, these amino acids are  glucogenic.   Some amino acids with carbons that produce glucose also contain other car-  bons that produce ketone bodies. Tryptophan, isoleucine, and threonine produce  acetyl CoA, and phenylalanine and tyrosine produce acetoacetate. These amino  acids are both glucogenic and ketogenic.   Two of the essential amino acids (lysine and leucine) are strictly ketogenic.  They do not produce glucose, only acetoacetate and acetyl-CoA.    Piquet Yuria, a 4-month-old female infant, emigrated from the Soviet  Union with her French mother and Russian father 1 month ago. She was  normal at birth but in the last several weeks was less than normally   attentive to her surroundings. Her psychomotor maturation seemed delayed, and a  tremor of her extremities had recently appeared. When her mother found her hav-  ing gross twitching movements in her crib, she brought the infant to the hospital  emergency room. A pediatrician examined Piquet and immediately noted a musty  odor to the baby's wet diaper. A drop of her blood was obtained from a heel prick  and used to perform a Guthrie bacterial inhibition assay using a special type of fil-  ter paper. This screening procedure was positive for the presence of an excess of  phenylalanine in Piquet's blood.   Homer Sistine, a 14-year-old boy, had a sudden grand mal seizure (with  jerking movements of the torso and head) in his eighth grade classroom.  The school physician noted mild weakness of the muscles of the left side  of Homer's face and of his left arm and leg. Homer was hospitalized with a tenta-  tive diagnosis of a cerebrovascular accident involving the right cerebral hemisphere,  which presumably triggered the seizure.   Homer's past medical history was normal, except for slight mental retardation  requiring placement in a special education group. He also had a downward partial  dislocation of the lenses of both eyes for which he had had a surgical procedure (a  peripheral iridectomy).   Homer's left-sided neurologic deficits cleared spontaneously within 3 days, but  a computerized axial tomogram (CAT) showed changes consistent with a small  infarction (damaged area caused by a temporary or permanent loss of adequate arte-  rial blood flow) in the right cerebral hemisphere. A neurologist noted that Homer  had a slight waddling gait, which his mother said began several years earlier and  was progressing with time. Further studies confirmed the presence of decreased  mineralization (decreased calcification) of the skeleton (called osteopenia if mild  and osteoporosis if more severe) and high methionine and homocysteine but low  cystine levels in the blood.   All of this information, plus the increased length of the long bones of Homer's  extremities and a slight curvature of his spine (scoliosis), caused his physician to  suspect that Homer might have an inborn error of metabolism.   Amino acid metabolism requires the participation of three important cof actors.  Pyridoxal phosphate is the quintessential coenzyme of amino acid metabolism (see  Chapter 38). All amino acid reactions requiring pyridoxal phosphate occur with the  amino group of the amino acid covalently bound to the aldehyde carbon of the coen-  zyme (Fig. 39.3). The pyridoxal phosphate then pulls electrons away from the bonds  around the a-carbon. The result is transamination, deamination, decarboxylation,  (3 -elimination, racemization, and 7-elimination, depending on which enzyme and  amino acid are involved.   The coenzyme FH 4 is required in certain amino acid pathways to either accept or  donate a one-carbon group. The carbon can be in various states of oxidation.  Chapter 40 describes the reactions of FH 4 in much more detail.   Fig. 39.3. Pyridoxal phosphate covalently attached to an amino acid substrate. The arrows  indicate which bonds are broken for the various types of reactions in which pyridoxal phos-  phate is involved. The X and Y represent leaving groups that may be present on the amino  acid (such as the hydroxy 1 group on serine or threonine).   The coenzyme BH 4 is required for ring hydroxylations. The reactions involve  molecular oxygen, and one atom of oxygen is incorporated into the product. The  second is found in water (see Chapter 24). BH 4 is important for the synthesis of  tyrosine and neurotransmitters (see Chapter 48).   Four amino acids are synthesized from intermediates of glycolysis: serine, glycine,  cysteine, and alanine. Serine, which produces glycine and cysteine, is synthesized  from 3-phosphoglycerate, and alanine is formed by transamination of pyruvate, the  product of glycolysis (Fig. 39.4). When these amino acids are degraded, their car-  bon atoms are converted to pyruvate or to intermediates of the glycolytic/gluco-  neogenic pathway and, therefore, can produce glucose or be oxidized to C0 2 .   Fig. 39.4. Amino acids derived from interme-  diates of glycolysis. These amino acids can be  synthesized from glucose. Their carbons can  be reconverted to glucose in the liver.   In the biosynthesis of serine from glucose, 3-phosphoglycerate is first oxidized to a  2-keto compound (3-phosphohydroxypyruvate), which is then transaminated to  form phosphoserine (Fig. 39.5). Phosphoserine phosphatase removes the phosphate,  forming serine. The major sites of serine synthesis are the liver and kidney.   Serine can be used by many tissues and is generally degraded by transamination  to hydroxypyruvate followed by reduction and phosphorylation to form 2-phospho-  glycerate, an intermediate of gycolysis that forms PEP and, subsequently, pyruvate.  Serine also can undergo ^-elimination of its hydroxyl group, catalyzed by serine  dehydratase, to form pyruvate.   Regulatory mechanisms maintain serine levels in the body. When serine levels fall,  serine synthesis is increased by induction of 3-phosphoglycerate dehydrogenase and by  release of the feedback inhibition of phosphoserine phosphatase (caused by higher lev-  els of serine). When serine levels rise, synthesis of serine decreases because synthesis  of the dehydrogenase is repressed and the phosphatase is inhibited (see Fig. 39.5).   Glycine can be synthesized from serine and, to a minor extent, threonine. The major  route from serine is by a reversible reaction that involves FH 4 and pyridoxal phos-  phate (Fig. 39.6). Tetrahydrofolate is a coenzyme that transfers one-carbon groups  at different levels of oxidation. It is derived from the vitamin folate and is discussed  in more detail in Chapter 40. The minor pathway for glycine production involves  threonine degradation (this is an aldolase-like reaction because threonine contains a  hydroxyl group located two carbons from the carbonyl group).   Fig. 39.5. The major pathway for serine synthesis from glucose is on the left, and for serine degradation on the right. Serine levels are main-  tained because serine causes repression (circled i) of 3-phosphoglycerate dehydrogenase synthesis. Serine also inhibits (circled - ) phosphoser-  ine phosphatase.   Fig. 39.6. Metabolism of glycine. Glycine can be synthesized from serine (major route) or threonine. Glycine forms serine or C0 2 and NH 4 + by  reactions that require tetrahydrofolate (FH 4 ). Glycine also forms glyoxylate, which is converted to oxalate or to C0 2 and H 2 0.   Oxalate, produced from glycine or  obtained from the diet, forms pre-  cipitates with calcium. Kidney  stones (renal calculi) are often composed of  calcium oxalate. A lack of the transaminase  that can convert glyoxylate to glycine (see  Fig 39.6) leads to the disease primary oxaluria  type I (PH 1). This disease has a consequence  of renal failure attributable to excessive accu-  mulation of oxalate in the kidney.   Cystathionuria, the presence of cys-  tathionine in the urine, is relatively  common in premature infants. As  they mature, cystathionase levels rise, and the  levels of cystathionine in the urine decrease.   In adults, a genetic deficiency of cys-  tathionase causes cystathionuria. Individu-  als with a genetically normal cystathionase  can also develop cystathionuria from a  dietary deficiency of pyridoxine (vitamin B6),  because cystathionase requires the cofactor  pyridoxal phosphate. No characteristic clini-  cal abnormalities have been observed in  individuals with cystathionase deficiency,  and it is probably a benign disorder.   Cystinuria and cystinosis are disor-  ders involving two different trans-  port proteins for cystine, the disul-  fide formed from two molecules of cysteine.  Cystinuria is caused by a defect in the trans-  port protein that carries cystine, lysine, argi-  nine, and ornithine into intestinal epithelial  cells and that permits resorption of these  amino acids by renal tubular cells. Cystine,  which is not very soluble in the urine, forms  renal calculi (stones). Cal Kulis, a patient  with cystinuria, developed cystine stones  (see Chapter 37).   Cystinosis is a rare disorder caused by a  defective carrier that normally transports  cystine across the lysosomal membrane  from lysosomal vesicles to the cytosol. Cys-  tine accumulates in the lysosomes in many  tissues and forms crystals, impairing their  function. Children with this disorder develop  renal failure by 6-12 years of age.   Alanine aminotransferase (ALT)  and aspartate amino transferase  (AST) are released from the liver  when liver cells are injured. Measurement of  these two transaminases in the serum (ALT  and AST) is one of the standard laboratory  tests for liver damage caused by a variety of  conditions.   The conversion of glycine to glyoxylate by the enzyme D-amino acid oxidase is  a degradative pathway of glycine that is medically important. Once glyoxylate is  formed, it can be oxidized to oxalate, which is sparingly soluble and tends to pre-  cipitate in kidney tubules, leading to kidney stone formation. Approximately 40%  of oxalate formation in the liver comes from glycine metabolism. Dietary oxalate  accumulation has been estimated to be a low contributor to excreted oxalate in the  urine because of poor absorption of oxalate in the intestine.   Although glyoxalate can be transaminated back to glycine, this is not really con-  sidered a biosynthetic route for "new" glycine, because the primary route for gly-  oxylate formation is from glycine oxidation.   Generation of energy from glycine occurs through a dehydrogenase (glycine  cleavage enzyme) that oxidizes glycine to C0 2 , ammonia, and a carbon that is  donated to FH 4 .   The carbons and nitrogen for cysteine synthesis are provided by serine, and the sulfur  is provided by methionine (Fig. 39.7). Serine reacts with homocysteine (which is  produced from methionine) to form cystathionine. This reaction is catalyzed by cys-  tathionine p-synthase. Cleavage of cystathionine by cystathionase produces cysteine  and a-ketobutyrate, which forms succinyl CoA via propionyl CoA. Both cystathionine  P-synthase (p -elimination) and cystathionase (7 -elimination) require PLR   Cysteine inhibits cystathionine p -synthase and, therefore, regulates its own pro-  duction to adjust for the dietary supply of cysteine. Because cysteine derives its sul-  fur from the essential amino acid methionine, cysteine becomes essential if the sup-  ply of methionine is inadequate for cysteine synthesis. Conversely, an adequate  dietary source of cysteine "spares" methionine; that is, it decreases the amount that  must be degraded to produce cysteine.   When cysteine is degraded, the nitrogen is converted to urea, the carbons to pyru-  vate, and the sulfur to sulfate, which has two potential fates (see Fig. 39.7; see also  Chapter 43). Sulfate generation, in an aqueous media, is essentially generating sul-  furic acid, and both the acid and sulfate need to be disposed of in the urine. Sulfate  is also used in most cells to generate an activated form of sulfate known as PAPS  (3'-phosphoadenosine 5'-phosphosulfate), which is used as a sulfate donor in mod-  ifying carbohydrates or amino acids in various structures (glycosaminoglycans) and  proteins in the body.   The conversion of methionine to homocysteine and homocysteine to cysteine is  the major degradative route for these two amino acids. Because this is the only  degradative route for homocysteine, vitamin B6 deficiency or congenital cystathi-  none p-synthase deficiency can result in homocystinemia, which is associated with  cardiovascular disease.   Alanine is produced from pyruvate by a transamination reaction catalyzed by ala-  nine aminotransaminase (ALT) and may be converted back to pyruvate by a rever-  sal of the same reaction (see Fig. 39.4). Alanine is the major gluconeogenic amino  acid because it is produced in many tissues for the transport of nitrogen to the liver.   Two groups of amino acids are synthesized from TCA cycle intermediates; one  group from a-ketoglutarate and one from oxaloacetate (see Fig. 39.2). During  degradation, four groups of amino acids are converted to the TCA cycle intermedi-  ates a-ketoglutarate, oxaloacetate, succinyl CoA, and fumarate (see Fig. 39. 3A).   Fig. 39.7. Synthesis and degradation of cysteine. Cysteine is synthesized from the carbons  and nitrogen of serine and the sulfur of homocysteine (which is derived from methionine).  During the degradation of cysteine, the sulfur is converted to sulfate and either excreted in  the urine or converted to PAPS (universal sulfate donor; 3'-phosphoadenosine 5'-phospho-  sulfate), and the carbons are converted to pyruvate.   Homocysteine is oxidized to a  disulfide, homocystine. To indicate  that both forms are being consid-  ered, the term homocyst(e)ine is used.   Because a colorimetric screening test for uri-  nary homocystine was positive, the doctor  ordered several biochemical studies on  Homer Sistine's serum, which included tests  for methionine, homocyst(e)ine (both free  and protein-bound), cystine, vitamin B12,  and folate. The level of homocystine in a 24-  hour urine collection was also measured.   The results were as follows: the serum  methionine level was 980 fxM (reference  range, <30); serum homocysteine (both  free and protein bound) was markedly ele-  vated; cystine was not detected in the  serum; the serum B12 and folate levels were  normal. A 24-hour urine homocystine level  was elevated.   Based on these measurements, Homer  Sistine's doctor concluded that he had  homocystinuria caused by an enzyme defi-  ciency. What was the rationale for this con-  clusion?   v^—^v If the blood levels of methionine  /\ # ) and homocysteine are very ele-  vated and cystine is low, cystathio-  nine p-synthase could be defective,  but a cystathionase deficiency is also a pos-  sibility. With a deficiency of either of these  enzymes, cysteine could not be synthesized,  and levels of homocysteine would rise.  Homocysteine would be converted to  methionine by reactions that require B12  and tetrahydrofolate (see Chapter 40). In  addition, it would be oxidized to homocys-  tine, which would appear in the urine. The  levels of cysteine (measured as its oxidation  product cystine) would be low. A measure-  ment of serum cystathionine levels would  help to distinguish between a cystathionase  or cystathionine (3-synthase deficiency.   The five carbons of glutamate are derived from a-ketoglutarate either by transami-  nation or by the glutamate dehydrogenase reaction (see Chapter 38). Because a-  ketoglutarate can be synthesized from glucose, all of the carbons of glutamate can  be obtained from glucose (see Fig. 39.2). When glutamate is degraded, it is likewise  converted back to a-ketoglutarate either by transamination or by glutamate dehy-  drogenase. In the liver, a-ketoglutarate leads to the formation of malate, which pro-  duces glucose via gluconeogenesis. Thus, glutamate can be derived from glucose  and reconverted to glucose (Fig. 39.8).   Glutamate is used for the synthesis of a number of other amino acids (glutamine,  proline, ornithine, and arginine) (see Fig. 39.8) and for providing the glutamyl moi-  ety of glutathionine (7-glutamyl-cysteinyl-glycine; see Biochemical Comments of  Chapter 37). Glutathione is an important antioxidant, as has been described previ-  ously (see Chapter 24).   Glutamine is produced from glutamate by glutamine synthetase, which adds  NH 4 + to the carboxyl group of the side chain, forming an amide (Fig. 39.9). This is  one of only three human enzymes that can fix free ammonia into an organic mole-  cule; the other two are glutamate dehydrogenase and carbamoyl-phosphate syn-  thetase I (see Chapter 38). Glutamine is reconverted to glutamate by a different  enzyme, glutaminase, which is particularly important in the kidney. The ammonia  it produces enters the urine and can be used as an expendable cation to aid in the  excretion of metabolic acids (NH 3 + H + — > NH 4 + ).   Fig. 39.9. Synthesis and degradation of gluta-  mine. Different enzymes catalyze the addition and  the removal of the amide nitrogen of glutamine.   In the synthesis of proline, glutamate is first phosphorylated and then converted to  glutamate 5 -semialdehyde by reduction of the side chain carboxyl group to an   Fig. 39.8. Amino acids related through glutamate. These amino acids contain carbons that  can be reconverted to glutamate, which can be converted to glucose in the liver. All of these  amino acids except histidine can be synthesized from glucose.   aldehyde (Fig. 39.10). This semialdehyde spontaneously cyclizes (forming an inter-  nal Schiff base between the aldehyde and the a-amino group). Reduction of this  cyclic compound yields proline. Hydroxyproline is only formed after proline has  been incorporated into collagen (see Chapter 49) by the prolyl hydroxylase system,  which uses molecular oxygen, iron, a-ketoglutarate, and ascorbic acid (vitamin C).   Proline is converted back to glutamate semialdehyde, which is reduced to form  glutamate. The synthesis and degradation of proline use different enzymes even  though the intermediates are the same. Hydroxyproline, however, has an entirely  different degradative pathway (not shown). The presence of the hydroxyl group in  hydroxyproline will allow an aldolase-like reaction to occur once the ring has been  hydrolyzed, which is not possible with proline.   Arginine is synthesized from glutamate via glutamate semialdehyde, which is transam-  inated to form ornithine, an intermediate of the urea cycle (see Chapter 38 and Fig.  39.11). This activity (ornithine aminotransferase) appears to be greatest in the epithe-  lial cells of the small intestine (see Chapter 42). The reactions of the urea cycle then  produce arginine. However, the quantities of arginine generated by the urea cycle are  adequate only for the adult and are insufficient to support growth. Therefore, during  periods of growth, arginine becomes an essential amino acid. It is important to realize  that if arginine is used for protein synthesis, the levels of ornithine will drop, thereby  slowing the urea cycle. This will stimulate the formation of ornithine from glutamate.   Arginine is cleaved by arginase to form urea and ornithine. If ornithine is pres-  ent in amounts in excess of those required for the urea cycle, it is transaminated to  glutamate semialdehyde, which is reduced to glutamate. The conversion of an alde-  hyde to a primary amine is a unique form of a transamination reaction and requires  pyridoxal phosphate (PLP).   Although histidine cannot be synthesized in humans, five of its carbons form gluta-  mate when it is degraded. In a series of steps, histidine is converted to formiminog-  lutamate (FIGLU). The subsequent reactions transfer one carbon of FIGLU to the  FH 4 pool (see Chapter 40) and release NH 4 + and glutamate (Fig. 39.12).   Aspartate is produced by transamination of oxaloacetate. This reaction is readily  reversible, so aspartate can be reconverted to oxaloacetate (Fig. 39.13).   Asparagine is formed from aspartate by a reaction in which glutamine provides  the nitrogen for formation of the amide group. Thus, this reaction differs from the  synthesis of glutamine from glutamate, in which NH 4 + provides the nitrogen. How-  ever, the reaction catalyzed by asparaginase, which hydrolyzes asparagine to NH 4 +  and aspartate, is analogous to the reaction catalyzed by glutaminase.   Although the major route for aspartate degradation involves its conversion to  oxaloacetate, carbons from aspartate can form fumarate in the urea cycle (see Chap-  ter 38). This reaction generates cytosolic fumarate, which must be converted to  malate (using cytoplasmic fumarase) for transport into the mitochondria for oxida-  tive or anaplerotic purposes. An analogous sequence of reactions occurs in the  purine nucleotide cycle. Aspartate reacts with inosine monophosphate (IMP) to   H - C - CH 2 - CH 2 - CH - COO"  II   Fig. 39.10. Synthesis and degradation of pro-  line. Reactions 1,3, and 4 occur in mitochon-  dria. Reaction 2 occurs in the cytosol. Synthe-  sis and degradation involve different enzymes.  The cyclization reaction (formation of a Schiff  base) is nonenzymatic, i.e., spontaneous.   Certain types of tumor cells, partic-  ularly leukemic cells, require  asparagine for their growth. There-  fore, asparaginase has been used as an anti-  tumor agent. It acts by converting  asparagine to aspartate in the blood,  decreasing the amount of asparagine avail-  able for tumor cell growth.   H - C -rCH 2 - CH 2 - CH - COO"  II -   I H 3 N 7 CH 2 - CH 2 - CH 2 - CH - COO"  r Ornithine   H 2 N - C - CH 2 - CH 2 - CH 2 - CH - COO"  Arginine   Fig. 39.11. Synthesis and degradation of argi-  nine. The carbons of ornithine are derived from  glutamate semialdehyde, which is derived from  glutamate. Reactions of the urea cycle convert  ornithine to arginine. Arginase converts argi-  nine back to ornithine by releasing urea.   Fig. 39.13. Synthesis and degradation of aspar-  tate and asparagine. Note that the amide nitro-  gen of asparagine is derived from glutamine.  (The amide nitrogen of glutamine comes from  NH 4 + , see Fig. 39.9.)   form an intermediate (adenylosuccinate) which is cleaved, forming adenosine  monophosphate (AMP) and fumarate (see Chapter 41).   Phenylalanine is converted to tyrosine by a hydroxylation reaction. Tyrosine, pro-  duced from phenylalanine or obtained from the diet, is oxidized, ultimately form-  ing acetoacetate and fumarate. The oxidative steps required to reach this point are,  surprisingly, not energy-generating. The conversion of fumarate to malate, followed  by the action of malic enzyme, allows the carbons to be used for gluconeogenesis.  The conversion of phenylalanine to tyrosine and the production of acetoacetate are  considered further in section IV of this chapter.   The essential amino acids methionine, valine, isoleucine, and threonine are  degraded to form propionyl-CoA. The conversion of propionyl CoA to succinyl  CoA is common to their degradative pathways. Propionyl CoA is also generated  from the oxidation of odd-chain fatty acids.   : "ooc - ch - ch 2 - ch 2 - coo" :   Fig. 39.12. Degradation of histidine. The highlighted portion of histidine forms glutamate.  The remainder of the molecule provides one carbon for the tetrahydrofolate (FH 4 ) pool (see  Chapter 40) and releases NH 4 + .   Propionyl CoA is carboxylated in a reaction that requires biotin and forms D-  methylmalonyl CoA. The D-methylmalonyl CoA is racemized to L-methylmalonyl  CoA, which is rearranged in a vitamin B12-requiring reaction to produce succinyl  CoA, a TCA cycle intermediate (see Fig. 23.11).   Methionine is converted to S-adenosylmethionine (SAM), which donates its methyl  group to other compounds to form S-adenosylhomocysteine (SAH). SAH is then  converted to homocysteine (Fig. 39.14). Methionine can be regenerated from homo-  cysteine by a reaction requiring both FH 4 and vitamin B12 (a topic that is consid-  ered in more detail in Chapter 40). Alternatively, by reactions requiring PLP, homo-  cysteine can provide the sulfur required for the synthesis of cysteine (see Fig. 39.7).  Carbons of homocysteine are then metabolized to a-ketobutyrate, which undergoes  oxidative decarboxylation to propionyl- CoA. The propionyl-CoA is then converted   to succinyl CoA (see Fig. 39.14). The conversion of a-ketobutyrate to    In humans threonine is primarily degraded by a PLP-requiring dehydratase to  ammonia and a-ketobutyrate, which subsequently undergoes oxidative decarboxy-  lation to form propionyl CoA, just as in the case for methionine (see Fig. 39.14).   as well as by deficiencies of methyltetrahy-  drofolate (CH 3 -FH 4 ) or of methyl-B12. The  deficiencies of CH 3 -FH 4 or of methyl-B12 are  due either to an inadequate dietary intake of  folate or B12 or to defective enzymes  involved in joining methyl groups to tetrahy-  drofolate (FH 4 ), transferring methyl groups  from FH 4 to B12, or passing them from B12  to homocysteine to form methionine (see  Chapter 40).   Fig. 39.14. Conversion of amino acids to succinyl CoA. The amino acids methionine, threo-  nine, isoleucine, and valine, all of which form succinyl CoA via methylmalonyl CoA, are  essential in the diet. The carbons of serine are converted to cysteine and do not form succinyl  CoA by this pathway. SAM = 5-adenosylmethionine.   \ ^ folate levels are normal. Therefore,  he does not have a deficiency of dietary  folate or B12 or of the enzymes that transfer  methyl groups from tetrahydrofolate to  homocysteine to form methionine. In these  cases, homocysteine levels are elevated but  methionine levels are low.   A biopsy specimen from Homer Sistine's  liver was sent to the hospital's biochemistry  research laboratory for enzyme assays. Cys-  tathionine (3-synthase activity was reported  to be 7% of that found in normal liver.   0 © Thiamine deficiency will lead to an  ( /^oA } accumulation of a-keto acids in the  ^ blood because of an inability of  pyruvate dehydrogenase, a-ketog-  lutarate dehydrogenase, and branched-chain  a-keto acid dehydrogenase to catalyze their  reactions (see Chapter 8). Al Martini had a thi-  amine deficiency resulting from his chronic  alcoholism. His ketoacidosis resulted partly  from the accumulation of these a-ketoacids in  his blood and partly from the accumulation of  ketone bodies used for energy production.   The branched-chain amino acids (valine, isoleucine, and leucine) are a universal  fuel, and the degradation of these amino acids occurs at low levels in the mito-  chondria of most tissues, but the muscle carries out the highest level of branched-  chain amino acid oxidation. The branched-chain amino acids make up almost 25%  of the content of the average protein, so their use as fuel is quite significant. The  degradative pathway for valine and isoleucine has two major functions, the first  being energy generation and the second to provide precursors to replenish TCA  cycle intermediates (anaplerosis). Valine and isoleucine, two of the three branched-  chain amino acids, contain carbons that form succinyl Co A. The initial step in the  degradation of the branched-chain amino acids is a transamination reaction.  Although the enzyme that catalyzes this reaction is present in most tissues, the  level of activity varies from tissue to tissue. Its activity is particularly high in mus-  cle, however. In the second step of the degradative pathway, the a-keto analogs of  these amino acids undergo oxidative decarboxylation by the a-keto acid dehydro-  genase complex in a reaction similar in its mechanism and cofactor requirements  to pyruvate dehydrogenase and a-ketoglutarate dehydrogenase (see Chapter 20).  As with the first enzyme of the pathway, the highest level of activity for this dehy-  drogenase is found in muscle tissue. Subsequently, the pathways for degradation of  these amino acids follow parallel routes (Fig. 39.15). The steps are analogous to  those for (3 -oxidation of fatty acids so NADH and FAD(2H) are generated for  energy production.   Valine and isoleucine are converted to succinyl CoA (see Fig. 39.14).  Isoleucine also forms acetyl CoA. Leucine, the third branched-chain amino acid,   What compounds form succinyl  CoA via propionyl CoA and methyl-  malonyl CoA?    0 © In maple syrup urine disease, the  /^oA J branched-chain a-keto acid dehy-  ™ drogenase that oxidatively decar-  boxylates the branched-chain amino acids is  defective. As a result, the branched-chain  amino acids and their a-keto analogs (pro-  duced by transamination) accumulate. They  appear in the urine, giving it the odor of  maple syrup or burnt sugar. The accumula-  tion of a-keto analogs leads to neurologic  complications. This condition is difficult to  treat by dietary restriction, because abnor-  malities in the metabolism of three essential  amino acids contribute to the disease.   Fig. 39.15. Degradation of the branched-chain amino acids. Valine forms propionyl CoA.  Isoleucine forms propionyl CoA and acetyl CoA. Leucine forms acetoacetate and acetyl CoA.   does not produce succinyl CoA. It forms acetoacetate and acetyl CoA and is  strictly ketogenic .   Seven amino acids produce acetyl CoA or acetoacetate and therefore are catego-  rized as ketogenic. Of these, isoleucine, threonine, and the aromatic amino acids  (phenylalanine, tyrosine, and tryptophan) are converted to compounds that produce  both glucose and acetyl CoA or acetoacetate (Fig. 39.16). Leucine and lysine do not  produce glucose; they produce acetyl CoA and acetoacetate.   Phenylalanine is converted to tyrosine, which undergoes oxidative degradation  (Fig. 39.17). The last step in the pathway produces both fumarate and the ketone  body acetoacetate. Deficiencies of different enzymes in the pathway result in  phenylketonuria, tyrosinemia, and alcaptonuria.   Phenylalanine is hydroxylated to form tyrosine by a mixed function oxidase,  phenylalanine hydroxylase (PAH), which requires molecular oxygen and tetrahy-  drobiopterin (Fig. 39.18). The cofactor tetrahydrobiopterin is converted to quininoid  dihydrobiopterin by this reaction. Tetrahydrobiopterin is not synthesized from a  vitamin; it can be synthesized in the body from GTP. However, as is the case with  other cof actors, the body contains limited amounts. Therefore, dihydrobiopterin  must be reconverted to tetrahydrobiopterin for the reaction to continue to produce  tyrosine.   Alcaptonuria occurs when  homogentisate, an intermediate in  tyrosine metabolism, cannot be  further oxidized because the next enzyme in  the pathway, homogentisate oxidase, is  defective. Homogentisate accumulates and  auto-oxidizes, forming a dark pigment,  which discolors the urine and stains the dia-  pers of affected infants. Later in life, the  chronic accumulation of this pigment in car-  tilage may cause arthritic joint pain.   Transient tyrosinemia is frequently  observed in newborn infants, espe-  cially those that are premature. For  the most part, the condition appears to be  benign, and dietary restriction of protein  returns plasma tyrosine levels to normal. The  biochemical defect is most likely a low level,  attributable to immaturity, of 4-hydrox-  yphenylpyruvate dioxygenase. Because this  enzyme requires ascorbate, ascorbate sup-  plementation also aids in reducing circulat-  ing tyrosine levels.   Other types of tyrosinemia are related to  specific enzyme defects (see Fig. 39.17).  Tyrosinemia II is caused by a genetic defi-  ciency of tyrosine aminotransferase (TAT) and  may lead to lesions of the eye and skin as well  as neurologic problems. Patients are treated  with a low-tyrosine, low-phenylalanine diet.   Tyrosinemia I (also called tyrosinosis) is  caused by a genetic deficiency of fumary-  lacetoacetate hydrolase. The acute form is  associated with liver failure, a cabbagelike  odor, and death within the first year of life.   Fig. 39.16. Ketogenic amino acids. Some of these amino acids (tryptophan, phenylalanine,  and tyrosine) also contain carbons that can form glucose. Leucine and lysine are strictly keto-  genic; they do not form glucose.   ®ln addition to methionine, threo-  nine, isoleucine, and valine (see  Fig. 39.14), the last three carbons at  the o)-end of odd-chain fatty acids,  form succinyl CoA by this route (see Chapter  23)   0 o A small subset of patients with  J /\A ) hyperphenylalaninemia show an  ^ appropriate reduction in plasma  phenylalanine levels with dietary restriction  of this amino acid; however, these patients  still develop progressive neurologic symp-  toms and seizures and usually die within the  first 2 years of life ("malignant" hyper-  phenylalaninemia). These infants exhibit  normal phenylalanine hydroxylase (PAH)  activity but have a deficiency in dihy-  dropteridine reductase (DHPR), an enzyme  required for the regeneration of tetrahydro-  biopterin (BH 4 ), a cofactor of PAH (see Fig.  39.18). Less frequently, DHPR activity is nor-  mal but a defect in the biosynthesis of BH 4  exists. In either case, dietary therapy cor-  rects the hyperphenylalaninemia. However,  BH 4 is also a cofactor for two other hydroxy-  lations required in the synthesis of neuro-  transmitters in the brain: the hydroxylation  of tryptophan to 5-hydroxytryptophan and  of tyrosine to L-dopa (see Chapter 48). It has  been suggested that the resulting deficit in  central nervous system neurotransmitter  activity is, at least in part, responsible for the  neurologic manifestations and eventual  death of these patients.   "OOC - CH = CH -COO" !CjH 3 -iC J-CH 2 - COO"  Fumarate Acetoacetate   Fig. 39.17. Degradation of phenylalanine and tyrosine. The carboxyl carbon forms C0 2 , and  the other carbons form fumarate or acetoacetate as indicated. Deficiencies of enzymes (gray  bars) result in the indicated diseases. PKU = phenylketonuria.   0 o If the dietary levels of niacin and  / s~\Jl ) tryptophan are insufficient, the  ™ condition known as pellagra  results. The symptoms of pellagra are der-  matitis, diarrhea, dementia, and, finally,  death. In addition, abnormal metabolism of  tryptophan occurs in a vitamin B6 deficiency.  Kynurenine intermediates in tryptophan  degradation cannot be cleaved because  kynureninase requires PLP derived from  vitamin B6. Consequently, these intermedi-  ates enter a minor pathway for tryptophan  metabolism that produces xanthurenic acid,  which is excreted in the urine.   Tryptophan is oxidized to produce alanine (from the non-ring carbons), formate, and  acetyl CoA. Tryptophan is, therefore, both glucogenic and ketogenic (Fig. 39.19).   NAD and NADP can be produced from the ring structure of tryptophan. There-  fore, tryptophan "spares" the dietary requirement for niacin. The higher the dietary  levels of tryptophan, the lower the levels of niacin required to prevent symptoms of  deficiency.   As discussed previously, the major route of threonine degradation in humans is by  threonine dehydratase (see section III.D.2.). In a minor pathway, threonine degra-  dation by threonine aldolase produces glycine and acetyl CoA in the liver.   Fig. 39.18. Hydroxylation of phenylalanine. Phenylalanine hydroxylase (PAH) is a mixed-function oxidase; i.e., molecular oxygen (0 2 )  donates one atom to water and one to the product, tyrosine. The cofactor, tetrahydrobiopterin (BH 4 ), is oxidized to dihydrobiopterin (BH 2 ),  and must be reduced back to BH 4 for the phenylalanine to continue forming tyrosine. BH 4 is synthesized in the body from GTP. PKU results  from deficiencies of PAH (the classic form), dihydropteridine reductase, or enzymes in the biosynthetic pathway for BH 4 .   Fig. 39.19. Degradation of tryptophan. One of the ring carbons produces formate. The non-  ring portion forms alanine. Kynurenine is an intermediate, which can be converted to a num-  ber of urinary excretion products (e.g., xanthurenate), degraded to C0 2 and acetyl CoA, or  converted to the nicotinamide moiety of NAD and NADP, which also can be formed from the  vitamin niacin.   On more definitive testing of  Piquet Yuria's blood, the plasma  level of phenylalanine was  elevated at 18 mg/dL (reference range,  <1.2). Several phenyl ketones and other  products of phenylalanine metabolism,  which give the urine a characteristic odor,  were found in significant quantities in the  baby's urine.   A liver biopsy was sent to the special  chemistry research laboratory, where it was  determined that the level of activity of  phenylalanine hydroxylase (PAH) in Piquet's  blood was less than 1% of that found in nor-  mal infants. A diagnosis of "classic"  phenylketonuria (PKU) was made.   Until gene therapy allows substitution of  the defective PAH gene with its normal coun-  terpart in vivo, the mainstay of therapy in  classic PKU is to maintain levels of pheny-  lalanine in the blood between 3 and 12  mg/dL through dietary restriction of this  essential amino acid.   Isoleucine produces both succinyl CoA and acetyl CoA (see section IILD.3.).   Leucine is purely ketogenic and produces hydroxymethylglutaryl CoA (HMG-  CoA), which is cleaved to form acetyl CoA and the ketone body acetoacetate (see  Figs. 39.15 and 39.16). Most of the tissues in which it is oxidized can use ketone  bodies, with the exception of the liver. As with valine and isoleucine, leucine is a  universal fuel, with its primary metabolism occurring in muscle.   Lysine cannot be directly transaminated at either of its two amino groups. Lysine  is degraded by a complex pathway in which saccharopine, a-ketoadipate, and  crotonyl CoA are intermediates. During the degradation pathway NADH and FADH 2  are generated for energy. Ultimately, lysine generates acetyl CoA (see Fig. 39.16)  and is strictly ketogenic.   ^ Piquet Yuria. The overall incidence of hyperphenylalaninemia is  l^W ) approximately 100 per million births, with a wide geographic and ethnic  variation. PKU occurs by autosomal recessive transmission of a defec-  tive PAH gene, causing accumulation of phenylalanine in the blood well above  the normal concentration in young children and adults (less than 1-2 mg/dL). In  the newborn, the upper limit of normal is almost twice this value. Values above  16 mg/dL are usually found in patients, such as Piquet Yuria, with "classic" PKU.   Patients with classic PKU usually appear normal at birth. If the disease is not rec-  ognized and treated within the first month of life, the infant gradually develops vary-  ing degrees of irreversible mental retardation (IQ scores frequently under 50),  delayed psychomotor maturation, tremors, seizures, eczema, and hyperactivity. The  neurologic sequelae may result in part from the competitive interaction of pheny-  lalanine with brain amino acid transport systems and inhibition of neurotransmitter  synthesis. These biochemical alterations lead to impaired myelin synthesis and  delayed neuronal development, which result in the clinical picture in patients such  as Piquet Yuria. Because of the simplicity of the test for PKU (elevated phenylala-  nine levels in the blood), all newborns in the United States are required to have a  PKU test at birth. Early detection of the disease can lead to early treatment, and the  neurologic consequences of the disease can be bypassed.   To restrict dietary levels of phenylalanine, special semisynthetic preparations  such as Lofenalac or PKUaid are used in the United States. Use of these prepara-  tions reduces dietary intake of phenylalanine to 250-500 mg/day while maintain-  ing normal intake of all other dietary nutrients. Although it is generally agreed that  scrupulous adherence to this regimen is mandatory for the first decade of life, less  consensus exists regarding its indefinite use. Evidence suggests, however, that  without lifelong compliance with dietary restriction of phenylalanine, even adults  will develop at least neurologic sequelae of PKU. A pregnant woman with PKU  must be particularly careful to maintain satisfactory plasma levels of phenylala-  nine throughout gestation to avoid the adverse effects of hyperphenylalaninemia  on the fetus.   Piquet's parents were given thorough dietary instruction, which they followed  carefully. Although her pediatrician was not optimistic, it was hoped that the dam-  age done to her nervous system before dietary therapy was minimal and that her sub-  sequent psychomotor development would allow her to lead a relatively normal life.   ^ Homer Sistine. The most characteristic biochemical features of the disor-  ) der affecting Homer Sistine, a cystathionine p-synthase deficiency, are the  presence of an accumulation of both homocysteine and methionine in the  blood. Because renal tubular reabsorption of methionine is highly efficient, this   amino acid may not appear in the urine. Homocystine, the disulfide of homocys-  teine, is less efficiently reabsorbed, and amounts in excess of 1 mmol may be  excreted in the urine each day.   In the type of homocystinuria in which the patient is deficient in cystathione p-  synthase, the elevation in serum methionine levels is presumed to be the result of  enhanced rates of conversion of homocysteine to methionine, caused by increased  availability of homocysteine (see Fig. 39.14). In type II and type III homocystinuria,  in which there is a deficiency in the synthesis of methyl cobalamin and of N 5 -  methyltetrahydrofolate, respectively (both required for the methylation of homo-  cysteine to form methionine), serum homocysteine levels are elevated but serum  methionine levels are low (see Fig. 39.14).   Acute vascular events are common in these patients. Thrombi (blood clots) and  emboli (clots that have broken off and traveled to a distant site in the vascular sys-  tem) have been reported in almost every major artery and vein as well as in smaller  vessels. These clots result in infarcts in vital organs such as the liver, the  myocardium (heart muscle), the lungs, the kidneys, and many other tissues.  Although increased serum levels of homocysteine have been implicated in enhanced  platelet aggregation and damage to vascular endothelial cells (leading to clotting  and accelerated atherosclerosis), no generally accepted mechanism for these vascu-  lar events has yet emerged.   Treatment is directed toward early reduction of the elevated levels of homocys-  teine and methionine in the blood. In addition to a diet low in methionine, very high  oral doses of pyridoxine (vitamin B6) have significantly decreased the plasma lev-  els of homocysteine and methionine in some patients with cystathionine p- synthase  deficiency. (Genetically determined "responders" to pyridoxine treatment make up  approximately 50% of type I homocystinurics.) PLP serves as a cof actor for cys-  tathionine p-synthase; however, the molecular properties of the defective enzyme  that confer the responsiveness to B6 therapy are not known.   The terms hypermethioninemia, homocystinuria (or -emia), and cystathionin-  uria (or -emia) designate biochemical abnormalities and are not specific clinical  diseases. Each may be caused by more than one specific genetic defect. For exam-  ple, at least seven distinct genetic alterations can cause increased excretion of  homocystine in the urine. A deficiency of cystathionine p-synthase is the most  common cause of homocystinuria; more than 600 such proven cases have been  studied.    The pathologic findings that under-  lie the clinical features manifested  by Homer Sistine are presumed  (but not proved) to be the consequence of  chronic elevations of homocysteine (and  perhaps other compounds, e.g., methionine)  in the blood and tissues. The zonular fibers  that normally hold the lens of the eye in  place become frayed and break, causing dis-  location of the lens. The skeleton reveals a  loss of bone ground substance (i.e., osteo-  porosis), which may explain the curvature of  the spine. The elongation of the long bones  beyond their normal genetically determined  length leads to tall stature.   Animal experiments suggest that  increased concentrations of homocysteine  and methionine in the brain may trap adeno-  sine as S-adenosylhomocysteine, diminish-  ing adenosine levels. Because adenosine  normally acts as a central nervous system  depressant, its deficiency may be associated  with a lowering of the seizure threshold as  well as a reduction in cognitive function.   Phenylketonuria. Many enzyme deficiency diseases have been discovered  that affect the pathways of amino acid metabolism. These deficiency dis-  eases have helped researchers to elucidate the pathways in humans, in  whom experimental manipulation is, at best, unethical. These spontaneous muta-  tions ("experiments" of nature), although devastating to patients, have resulted in an  understanding of these diseases that now permit treatment of inborn errors of  metabolism that were once considered to be untreatable.   Classic PKU is caused by mutations in the gene located on chromosome 12 that  encodes the enzyme phenylalanine hydroxylase (PAH). This enzyme normally cat-  alyzes the hydroxylation of phenylalanine to tyrosine, the rate-limiting step in the  major pathway by which phenylalanine is catabolized.   In early experiments, sequence analysis of mutant clones indicated a single base  substitution in the gene with a G to A transition at the canonical 5 ' donor splice site  of intron 12 and expression of a truncated unstable protein product. This protein  lacked the C-terminal region, a structural change that yielded less than 1% of the  normal activity of PAH.   Since these initial studies, DNA analysis has shown over 100 mutations (mis-  sense, nonsense, insertions, and deletions) in the PAH gene, associated with PKU  and non-PKU hyperphenylalaninemia. That PKU is a heterogeneous phenotype is  supported by studies measuring PAH activity in needle biopsy samples taken from  the livers of a large group of patients with varying degrees of hyperphenylalanine-  mia. PAH activity varied from below 1% of normal in patients with classic PKU to  up to 35% of normal in those with a non-PKU form of hyperphenylalaninemia (such  as a defect in BH 4 production; see Chapter 48).   The genetic diseases affecting amino acid degradation that have been discussed  in this chapter are summarized in Table 39.1.   Burton BK. Inborn errors of metabolism in infancy: a guide to diagnosis. Pediatrics 1998; 102:e69.   Mudd S, Levy H, Klaus, JP. Disorders of trans sulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D,  eds. The Metabolic and Molecular Bases of Inherited Disease, vol. II, 8th Ed. New York: McGraw-  Hill, 2001:2007-2056.   St. Joer ST, Howard BV, et al. Dietary protein and weight reduction. A statement for healthcare profes-  sionals from the nutrition committee of the Council on Nutrition, Physical Activity, and Metabolism  of the American Heart Association. Circulation 2001;104:1869-1874.   Scriver C, Kaufman S,. Hyperphenylalanemia: Phenylalanine hydroxylase deficiency. In: Scriver CR,  Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease, vol. II,  8th Ed. New York: McGraw-Hill, 2001:1667-1724.   1. If an individual has a vitamin B6 deficiency, which of the following amino acids could still be synthesized and be considered  nonessential?   2. The degradation of amino acids can be classified into families, which are named after the end product of the degradative path-  way. Which of the following is such an end product?   3. A newborn infant has elevated levels of phenylalanine and phenylpyruvate in her blood. Which of the following enzymes  might be deficient in this baby?   4. Pyridoxal phosphate is required for which of the following reaction pathways or individual reactions?   5. A folic acid deficiency would interfere with the synthesis of which of the following amino acids from the indicated precur-  sors?   Fig. 40.1. Overview of the one-carbon pool.  FH 4 »C indicates tetrahydrofolate (FH 4 ) con-  taining a one-carbon unit that is at the formyl,  methylene, or methyl level of oxidation (see  Fig. 40.3). The origin of the carbons is indi-  cated, as are the final products after a one-car-  bon transfer.   Groups containing a single carbon atom can be transferred from one compound  to another These carbon atoms may be in a number of different oxidation states.  The most oxidized form, C0 2 , is transferred by biotin. One-carbon groups at  lower levels of oxidation than C0 2 are transferred by reactions involving tetrahy-  drofolate (FH 4 ), vitamin B12, and S-adenosylmethionine (SAM).   Tetrahydrofolate: Tetrahydrofolate, which is produced from the vitamin folate , is   the primary one-carbon carrier in the body. This vitamin obtains one-carbon units  from serine, glycine, histidine, formaldehyde, and formate (Fig. 40.1). While these  carbons are attached to FH 4 they can be either oxidized or reduced. Because of  this, folate can exist in a variety of chemical forms . Once a carbon has been  reduced to the methyl level (methyl-FH 4 ), however, it cannot be re-oxidized. Col-  lectively, these one-carbon groups attached to their carrier FH 4 are known as the  one-carbon pool. The term folate is used to represent a water-soluble B-complex  vitamin that functions in transferring single-carbon groups at various stages of  oxidation.   The one-carbon groups carried by FH 4 are used for many biosynthetic reac-  tions. For example, one-carbon units are transferred to the pyrimidine base of  deoxyuridine monophosphate (dUMP) to form deoxy thymidine monophosphate  (dTMP), to the amino acid glycine to form serine, to precursors of the purine  bases to produce carbons C2 and C8 of the purine ring, and to vitamin B12.   Vitamin B12: Vitamin B12 is involved in two reactions in the body. It participates  in the rearrangement of the methyl group of L-methylmalonyl-CoA to form succinyl-  CoA, and it transfers a methyl group, obtained from FH 4 , to homocysteine, forming  methionine.   S-adenosylmethionine (SAM): SAM, produced from methionine and adenosine  triphosphate (ATP), transfers the methyl group to precursors forming a number of  compounds, including creatine, phosphatidylcholine , epinephrine, melatonin,  methylated nucleotides, and methylated DNA.   Methionine metabolism is very dependent on both FH 4 and vitamin B12.  Homocysteine is derived from methionine metabolism and can be converted back  into methionine by using both methyl-FH 4 and vitamin B12. This is the only reac-  tion in which methyl-FH 4 can donate the methyl group. If the enzyme that cat-  alyzes this reaction is defective, or if vitamin B12 or FH 4 levels are insufficient,  homocysteine will accumulate. Elevated homocysteine levels have been linked to  cardiovascular and neurologic disease. A vitamin B12 deficiency can be brought  about by the lack of intrinsic factor , a gastric protein required for the absorption  of dietary B12. A consequence of vitamin B12 deficiency is the accumulation of  methyl-FH 4 and a decrease in other folate derivatives. This is known as the   methyl-trap hypothesis, in which, because of the B12 deficiency, most of the  carbons in the FH 4 pool are trapped in the methyl-FH 4 form, which is the most  stable. The carbons cannot be released from the folate, because the one reaction in  which they participate cannot occur because of the B12 deficiency. This will therefore  lead to a functional folate deficiency, even though total levels of folate are nor-  mal. A folate deficiency (whether functional or actual) will lead to megaloblastic  anemia caused by an inability of blood cell precursors to synthesize DNA and  therefore to divide. This leads to large, partially replicated cells being released  into the blood to attempt to replenish the cells that have died. Folate deficiencies  also have been linked to an increased incidence of neural-tube defects, such as  spina bifida, in mothers who become pregnant while folate deficient.   • After resection of the cancer in his large intestine and completion of a  Hfl ) course of postoperative chemotherapy with 5-fluorouracil (5-FU), Colin   " Tuma returned to his gastroenterologist for a routine follow-up  colonoscopy. His colon was completely normal, with excellent healing at the site of  the anastomosis. His physician expressed great optimism about a cure of Colin's  previous malignancy but cautioned him about the need for regular colonoscopic  examinations over the next few years.   Bea Twelvlow, a 75 -year-old woman, went to see her physician because  of a numbness and tingling in her arms. A diet history indicated a normal  and healthy diet, but Bea was not taking any supplemental vitamin pills.  Laboratory results indicated a slight elevation of methylmalonic acid, and this led  the physician to suspect a vitamin B12 deficiency. Direct measurement of serum  B12 levels did indicate a deficiency, but the results of a Schilling test were normal.   The initial laboratory profile, determined when Jean Ann Tonich first pre-  sented to her physician with evidence of early alcohol-induced hepatitis,  included a hematologic analysis that showed that Jean Ann was anemic.  Her hemoglobin was 1 1.0 g/dL (reference range = 12-16 for an adult female). The  erythrocyte (red blood cell) count was 3.6 million cells/mm3 (reference range =  4.0-5.2 for an adult female). The average volume of her red blood cells (mean cor-  puscular volume, or MCV) was 108 fL (reference range = 80-100), and the hema-  tology laboratory reported a striking variation in the size and shape of the red blood  cells in a smear of her peripheral blood (see Chapter 44). The nuclei of the circu-  lating granulocytic leukocytes had increased nuclear segmentation (polysegmented  neutrophils). Because these findings are suggestive of a macrocytic anemia (in  which blood cells are larger than normal), measurements of serum folate and vita-  min B12 (cobalamin) levels were ordered.   I. TETRAHYDROFOLATE (FH 4 )  A. Structure and Forms of FH 4   Folates exist in many chemical forms. The coenzyme form that functions in accept-  ing one-carbon groups is tetrahydrofolate polyglutamate (Fig. 40.2), generally just  referred to as tetrahydrofolate or FH 4 . It has three major structural components, a  bicyclic pteridine ring, para-aminobenzoic acid, and a polyglutamate tail consisting   The Schilling test involves the  patient ingesting radioactive (Co 60 )  crystalline vitamin B12 after which  a 24-hour urine sample is collected. The  radioactivity in the urine sample is com-  pared with the input radioactivity, and the  difference represents the amount of B12  absorbed through the digestive tract.   Folate deficiencies frequently  occur in individuals with chronic  alcoholism. A number of factors  are involved: inadequate dietary intake of  folate; direct damage to intestinal cells and  brush border enzymes, which interferes with  absorption of dietary folate; a defect in the  enterohepatic circulation, which reduces the  absorption of folate; liver damage causing  decreased hepatic production of plasma pro-  teins; and interference with kidney resorp-  tion of folate.   Because of the possibility of a direct toxic  effect of alcohol on hematopoietic tissues in  Jean Ann Tonich, a bone marrow aspirate  was performed. The aspirate contained a  greater than normal number of red and  white blood cell precursors, most of which  were larger than normal. The large red blood  cells are called megaloblasts.   These hematopoietic precursor cells  when exposed to too little folate and/or vita-  min B12 show slowed cell division, but cyto-  plasmic development occurs at a normal  rate. Hence, the megaloblastic cells tend to  be large, with an increased ratio of RNA to  DNA. Megaloblastic erythroid progenitors  are usually destroyed in the bone marrow  (although some reach the circulation). Thus,  marrow cellularity is often increased but pro-  duction of red blood cells is decreased, a  condition called "ineffective erythropoiesis."   Therefore, Jean Ann had a megaloblastic  anemia, characteristic of a folate or B12 defi-  ciency.   Jean Ann's serum folic acid level was 3.1  ng/mL (reference range =6-15), and her  serum B12 level was 154 pg/mL (reference  range = 150-750). Her serum iron level was  normal. It was clear, therefore, that Jean  Ann's megaloblastic anemia was caused by  a folate deficiency (although her B12 levels  were in the low range of normal). The man-  agement of a pure folate deficiency in an  alcoholic patient includes cessation of alco-  hol intake and a diet rich in folate.   ^The abbreviation fL stands for fem-  toliters. One fL is 10" 12 milliliters  (mL).   Sulfa drugs, which are used to treat  certain bacterial infections, are  analogs of para-aminobenzoic  acid. They prevent growth and cell division  in bacteria by interfering with the synthesis  of folate. Because we cannot synthesize  folate, sulfa drugs do not affect human cells  in this way.   The current recommended dietary  allowance (RDA) for folate equiva-  lents is approximately 400 fig for  adult men and women. In addition to being  prevalent in green leafy vegetables, other  good sources of this vitamin are liver, yeast,  legumes, and some fruits. Protracted cook-  ing of these foods, however, can destroy up  to 90% of their folate content. A standard US  diet provides 50 to 500 |xg absorbable folate  each day. Folate deficiency in pregnant  women, especially during the month before  conception and the month after, increases  the risk of neural tube defects, such as spina  bifida, in the fetus. To reduce the potential  risk of neural tube defects for women capa-  ble of becoming pregnant, the recommenda-  tion is to take 400 fxg of folic acid daily from  a multivitamin pill. If the women has a his-  tory of having a child with a neural tube  defect, this amount is increased to 4000  |xg/day for the month before and the month  after conception. Flour-containing products  in the United States are now supplemented  with folate to reduce the risk of neural tube  defects in newborns.   Fig. 40.2. Reduction of folate to tetrahydrofolate (FH 4 ). The same enzyme, dihydrofolate  reductase, catalyzes both reactions. Multiple glutamate residues are added within cells  (n ~ 5). Plants can synthesize folate, but humans cannot. Therefore, folate is a dietary require-  ment. R is the portion of the folate molecule shown to the right of N 10 . The different precur-  sors of FH 4 are indicated in the figure. PABA = para-aminobenzoic acid.   of several glutamate residues joined in amide linkage. The one-carbon group that is  accepted by the coenzyme and then transferred to another compound is bound to N 5 ,  to N 10 , or to both.   Different forms of folate may differ in the oxidation state of the one-carbon group,  in the number of glutamate residues attached, or in the degree of oxidation of the  pteridine ring. When the term folate or folic acid is applied to a specific chemical  form, it is the most oxidized form of the pteridine ring (see Fig. 40.2). Folate is  reduced to dihydrofolate and then to tetrahydrofolate by dihydrofolate reductase  present in cells. Reduction is the favored direction of the reaction; therefore, most of  the folate present in the body is present as the reduced coenzyme form, FH 4 .   Folates are synthesized in bacteria and higher plants and ingested in green leafy  vegetables, fruits, and legumes in our diet. The vitamin was named for its presence  in green, leafy vegetables (foliage). Most of the dietary folate derived from natural  food sources is present in the reduced coenzyme form. However, vitamin supple-  ments and fortified foods contain principally the oxidized form of the pteridine ring.   As dietary folates pass into the proximal third of the small intestine, folate con-  jugases in the brush border of the lumen cleave off glutamate residues to produce  the monoglutamate form of folate, which is then absorbed (see Fig. 40.2, upper   structure, when n = 1). Within the intestinal cells, folate is converted principally to  N 5 -methyl FH 4 , which enters the portal vein and goes to the liver. Smaller amounts  of other forms of folate also follow this route.   The liver, which stores half of the body's folate, takes up much of the folate from the  portal circulation; uptake may be through active transport or receptor-mediated endocy-  tosis. Within the liver, FH 4 is reconjugated to the polyglutamate form before being used  in reactions. A small amount of the folate is partially degraded, and the components  enter the urine. A relatively large portion of the folate enters the bile and is subsequently  reabsorbed (very similar to the fate of bile salts in the enterohepatic circulation).   N 5 -Methyl-FH 4 , the major form of folate in the blood, is loosely bound to plasma  proteins, particularly serum albumin.   One-carbon groups transferred by FH 4 are attached either to nitrogen N 5 or N 10 or  they form a bridge between N 5 and N 10 . The collection of one-carbon groups  attached to FH 4 is known as the one-carbon pool. While attached to FH 4 , these one-  carbon units can be oxidized and reduced (Fig. 40.3). Thus, reactions requiring a  carbon at a particular oxidation state may use carbon from the one-carbon pool that  was donated at a different oxidation state.   The individual steps for reduction of the one-carbon group are shown in Fig.  40.4. The most oxidized form is N 10 -formyl FH 4 . The most reduced form is N 5 -  methyl-FH 4 . Once the methyl group is formed, it is not readily reoxidized back to  N 5 , N 10 methylene FH 4 , and thus N 5 -methyl-FH 4 will tend to accumulate in the cell.   Carbon sources for the one-carbon pool include serine, glycine, formaldehyde, his-  tidine, and formate (see Fig. 40.4). These donors transfer the carbons to folate at dif-  ferent oxidation states. Serine is the major carbon source of one-carbon groups in  the human. Its hydroxymethyl group is transferred to FH 4 in a reversible reaction,  catalyzed by the enzyme serine hydroxymethyltransf erase. This reaction produces  glycine and N 5 , N 10 -methylene-FH 4 . Because serine can be synthesized from  3-phosphoglycerate, an intermediate of glycolysis, dietary carbohydrate can serve  as a source of carbon for the one-carbon pool. The glycine that is produced may be  further degraded by donation of a carbon to folate. Additional donors that form N 5 ,  N 10 methylene-FH 4 are listed in Table 40.1.   N 5 -formimino FH 4 is  converted to N 5 , N 10  methylene FH 4   b The carbon unit attached to FH 4 can be oxidized and reduced (see Fig. 40.3). At the methyl level, reoxida-  tion does not occur.   Fig. 40.3. One-carbon units attached to FH 4 . A. The active form of FH 4 . For definition of R,  see Figure 40.2. B. Interconversions of one-carbon units of FH 4 . Only the portion of FH 4  from N 5 to N 10 is shown, which is indicated by the dashed line in Part A. After a formyl group  forms a bridge between N 5 and N 10 , two reductions can occur. Note that N 5 -methyl-FH 4 can-  not be reoxidized. The most oxidized form of FH 4 is at the top of the figure, whereas the most  reduced form is at the bottom.   0 o A deficiency of folate results in the  ( /vA ) accumulation of FIGLU, which is  V excreted in the urine. A histidine  load test can be used for detecting folate  deficiencies. Patients were given a test dose  of histidine (a histidine load), and the  amount of FIGLU that appeared in the urine  was measured.   Histidine and formate provide examples of compounds that donate carbon at dif-  ferent oxidation levels (see Fig. 40.4). Degradation of histidine produces  formiminoglutamate (FIGLU), which reacts with FH 4 to donate a carbon and nitro-  gen (generating N 5 -formimino-FH 4 ), thereby releasing glutamate. Formate, pro-  duced from tryptophan oxidation, can react with FH 4 and generate N 10 -formyl-FH 4 ,  the most oxidized folate derivative.   Fig. 40.4. Sources of carbon (reactions l-4) for the FH 4 pool and the recipients of carbon (reactions 5-8) from the pool. See Figure 40.3 to view  the FH 4 derivatives involved in each reaction.   The one-carbon groups on FH 4 may be oxidized or reduced (see Fig. 40.3) and then  transferred to other compounds (see Fig. 40.4 and Table 40. 1). Transfers of this sort  are involved in the synthesis of glycine from serine, the synthesis of the base  thymine required for DNA synthesis, the purine bases required for both DNA and  RNA synthesis, and the transfer of methyl groups to vitamin Bl2.   Because the conversion of serine to glycine is readily reversible, glycine can be  converted to serine by drawing carbon from the one-carbon pool.   The nucleotide deoxythymidine monophosphate (dTMP) is produced from  deoxyuridine monophosphate (dUMP) by a reaction in which dUMP is methylated  to form dTMP (Fig. 40.5). The source of carbon is N 5 , N l0 -methylene FH 4 . Two  hydrogen atoms from FH 4 are used to reduce the donated carbon to the methyl level.  Consequently, dihydrofolate (FH 2 ) is produced. Reduction of FH 2 by NADPH in a   Fig. 40.5. Transfer of a one-carbon unit from N 5 , N 10 methhylene FH 4 to dUMP to form  dTMP. FH 4 is oxidized to FH 2 (dihydrofolate) in this reaction. FH 2 is reduced to FH 4 by  dihydrofolate reductase and FH 4 is converted to N 5 , N 10 methylene FH 4 using serine as a car-  bon donor. Shaded bars indicate the steps at which the antimetabolites 5-fluorouracil (5-FU)  and methotrexate act. 5-FU inhibits thymidylate synthase. Methotrexate inhibits dihydrofo-  late reductase.   0 © FH 4 is required for the synthesis of  I /^OA J deoxythymidine monophosphate  V and the purine bases used to pro-  duce the precursors for DNA replication.  Therefore, FH 4 is required for cell division.  Blockage of the synthesis of thymine and the  purine bases either by a dietary deficiency of  folate or by drugs that interfere with folate  metabolism results in a decreased rate of  cell division and growth.   A better understanding of the  structure and function of the purine  and pyrimidine bases and of folate  metabolism led to the development of com-  pounds having antimetabolic and antifolate  action useful for treatment of neoplastic dis-  ease. For example, Colin Tuma was suc-  cessfully treated for colon cancer with 5-flu-  orouracil (5-FU) (see Chapter 12 and Fig.  40.5). 5-FU is a pyrimidine analog, which is  converted in cells to the nucleotide fluo-  rodeoxyuridylate (FdUMP). FdUMP causes a  "thymineless death," especially for tumor  cells having a rapid turnover rate. It prevents  the growth of cancer cells by blocking the  thymidylate synthase reaction, i.e., the con-  version of dUMP to dTMP.   Jean Ann Tonich's megaloblastic  anemia was treated, in part, with  folate supplements (see Clinical  Comments). Within 48 hours of the initiation  of folate therapy, megaloblastic or "ineffec-  tive" erythropoiesis usually subsides, and  effective erythropoiesis begins.   A megaloblastic anemia is caused by a  decrease in the synthesis of thymine and the  purine bases. These deficiencies lead to an  inability of hematopoietic (and other) cells to  synthesize DNA and, therefore, to divide.  Their persistently thwarted attempts at nor-  mal DNA replication, DNA repair, and cell  division produce abnormally large cells  (called megaloblasts) with abundant cyto-  plasm capable of RNA and protein synthesis,  but with clumping and fragmentation of  nuclear chromatin (see Chapter 44). Some of  these large cells, although immature, are  released early from the marrow in an attempt  to compensate for the anemia. Thus, periph-  eral blood smears will also contain mega-  loblasts. Many of the large immature cells,  however, are destroyed in the marrow and  never reach the circulation.   The average daily diet in Western  countries contains 5-30 |xg vitamin  B12, of which 1-5 /jlq is absorbed  into the blood. (The RDA is 2.4  fig/day.) Total body content of this vitamin in  an adult is approximately 2-5 mg, of which 1  mg is present in the liver. As a result, a  dietary deficiency of B12 is uncommon and  is only observed after a number of years on  a diet deficient in this vitamin.   In spite of Jean Ann Tonich's relatively  malnourished state because of chronic alco-  holism, her serum cobalamin level was still  within the low-to-normal range. If her under-  nourished state had continued, a cobalamin  deficiency would eventually have developed.   reaction catalyzed by dihydrofolate reductase (DHFR) regenerates FH 4 . This is the  only reaction involving FH 4 in which the folate group is oxidized as the one-carbon  group is donated to the recipient. Recall that DHFR is also required to reduce the  oxidized form of the vitamin, which is obtained from the diet (see Fig. 40.2). Thus,  DHFR is essential for both regenerating FH 2 in the tissues and from the diet. These  reactions contribute to the effect of folate deficiency on DNA synthesis because  dTMP is only required for the synthesis of DNA.   During the synthesis of the purine bases, carbons 2 and 8 are obtained from the  one-carbon pool (see Chapter 41). Af 70 -Formyl-FH 4 provides both carbons. Folate  deficiency would also hinder these reactions, contributing to an inability to replicate  DNA because of the lack of precursors.   After the carbon group carried by FH 4 is reduced to the methyl level, it is trans-  ferred to vitamin B 12. This is the only reaction through which the methyl group can  leave FH 4 (recall that the reaction creating N 5 -methyl FH 4 is not reversible).   The structure of vitamin B12 (also known as cobalamin) is complex (Fig. 40.6). It  contains a corrin ring, which is similar to the porphyrin ring found in heme. The  corrin ring differs from heme, however, in that two of the four pyrrole rings are  joined directly rather than by a methylene bridge. Its most unusual feature is the  presence of cobalt, coordinated with the corrin ring (similar to the iron coordinated  with the porphyrin ring). This cobalt can form a bond with a carbon atom. In the  body, it reacts with the carbon of a methyl group, forming methylcobalamin, or  with the 5' -carbon of 5 '-deoxy adenosine, forming 5'-deoxyadenosylcobalamin  (note that in this case the deoxy designation refers to the 5' carbon, not the 2' car-  bon as is the case in the sugar found in DNA). The form of B12 found in vitamin  supplements is cyanocobalamin, in which a CN group is linked to the cobalt.   Although vitamin B12 is produced by bacteria, it cannot be synthesized by higher  plants or animals. The major source of vitamin B12 is dietary meat, eggs, dairy  products, fish, poultry, and seafood. The animals that serve as the source of these  foods obtain B12 mainly from the bacteria in their food supply. The absorption of  B12 from the diet is a complex process (Fig. 40.7).   Ingested B12 can exist in two forms, either free or bound to dietary proteins. If  free, the B12 binds to proteins known as R-binders (haptocorrins, also known as  transcobalamin I), which are secreted by the salivary glands and the gastric mucosa,  in either the saliva or the stomach. If the ingested B12 is bound to proteins, it must  be released from the proteins by the action of digestive proteases both in the stomach   Individuals with non-Hodgkin's lymphoma receive a number of drugs to treat  the tumor, including the use of methotrexate. The structure of methotrexate is  shown below.   Fig. 40.6. Vitamin B12. X = 5 '-deoxy adenosine in deoxyadenosylcobalamin; X = CH 3 in  methylcobalamin; X = CN in cyanocobalamin (the commercial form found in vitamin  tablets).   ©Methotrexate has the same struc-  ture as folate except that it has an  amino group on C4 and a methyl  group on N10. Anticancer drugs such as  methotrexate are folate analogs that act by  inhibiting dihydrofolate reductase, thereby  preventing the conversion of FH 2 to FH 4 (see  Fig. 40.5). Thus, the cellular pools of FH 4 are  not replenished, and reactions requiring FH 4  cannot proceed.   Q o Pernicious anemia, a deficiency of  f ) intrinsic factor, is a relatively com-  ™ mon problem caused by malab-  sorption of dietary cobalamin. It may result  from an inherited defect that leads to a  decreased ability of gastric parietal cells to  synthesize intrinsic factor or from partial  resection of the stomach or of the ileum.  Production of intrinsic factor often declines  with age and may be low in elderly individu-  als. An alternative circumstance that leads to  the development of a B12 deficiency is pan-  creatic insufficiency or a high intestinal pH,  which would result from too little acid being  produced by the stomach. Both of these  conditions prevent the degradation of the  R-binder-B12 complex; as a result, B12 will  not be released from the R-binder protein  and, therefore, cannot bind to intrinsic  factor.    and small intestine. Once the B12 is released from its bound protein, it will bind to  the haptocorrins. In the small intestine, the pancreatic proteases digest the hapto-  corrins, and the released B12 then binds to intrinsic factor, a glycoprotein secreted  by the parietal cells of the stomach when food enters the stomach. The intrinsic fac-  tor-B12 complex attaches to specific receptors in the terminal segment of the small  intestine known as the ileum, after which the complex is internalized.   The B12 within the enterocyte complexes with transcobalamin II and then is  released into circulation. The transcobalamin II-B12 complex delivers B12 to the  tissues, which contain specific receptors for this complex. The liver takes up  approximately 50% of the vitamin B12, and the remainder is transported to other  tissues. The amount of the vitamin stored in the liver is large enough that 3 to 6  years pass before symptoms of a dietary deficiency occur.   Vitamin B 12 is involved in two reactions in the body: the transfer of a methyl group  from N 5 -methyl FH 4 to homocysteine to form methionine and the rearrangement of  the methyl group of L-methylmalonyl CoA to form succinyl CoA (Fig. 40.8).   Tetrahydrofolate receives a one-carbon group from serine or from other sources.  This carbon is reduced to the methyl level and transferred to vitamin B12, forming   Because the problem in pernicious  anemia is a lack of intrinsic factor,  which results in an inability to  absorb vitamin B12 from the gastrointestinal  tract, B12 cannot be administered orally to  treat this condition. In the past, it was usu-  ally given by injection. An effective nasal  spray containing B12 has recently been mar-  keted, however, and its use precludes the  need for lifelong injections of this vitamin.    Fig. 40.7. Absorption, transport, and storage of vitamin B12. Dietary B12 binds to R-binders  (haptocorrins) in the stomach and travels to the intestine, where the R-binders are destroyed  by pancreatic proteases. The freed B 12 then binds to intrinsic factor (IF). B12 is absorbed in  the ileum and carried by called transcobalamins (TC) to the liver, where B12 is stored.   methyl-B12 (or methylcobalamin). Methylcobalamin transfers the methyl group to  homocysteine, which is converted to methionine by the enzyme methionine syn-  thase. Methionine can then be activated to SAM to transfer the methyl group to  other compounds (Fig. 40.9).   Vitamin B 12 also participates in the conversion of L-methylmalonyl Co A to suc-  cinyl CoA. In this case, the active form of the coenzyme is 5'-deoxyadenosylcobal-  amin. This reaction is part of the metabolic route for the conversion of carbons from  valine, isoleucine, threonine, thymine, and the last three carbons of odd-chain fatty  acids, all of which form propionyl CoA, to the TCA cycle intermediate succinyl  CoA (see Chapter 39).    There are two major clinical manifestations of cobalamin (B12) deficiency. One such presentation is hematopoietic (caused by  the adverse effects of a B12 deficiency on folate metabolism), and the other is neurologic (caused by hypomethylation in the  nervous system).   The hemopoietic problems associated with a B12 deficiency are identical to those observed in a folate deficiency and, in fact, result from a  folate deficiency secondary to (i.e., caused by) the B12 deficiency (i.e., the methyl trap hypothesis). As the FH 4 pool is exhausted, deficiencies  of the tetrahydrofolate derivatives needed for purine and dTMP biosynthesis develop, leading to the characteristic megaloblastic anemia.   The classical clinical presentation of the neurologic dysfunction associated with a B12 deficiency includes symmetric numbness and  tingling of the hands and feet, diminishing vibratory and position sense, and progression to a spastic gait disturbance. The patient may  become somnolent or may become extremely irritable ("megaloblastic madness"). Eventually, blind spots in the central portions of the  visual fields develop, accompanied by alterations in gustatory (taste) and olfactory (smell) function. This is believed to be caused by  hypomethylation within the nervous system, brought about by an inability to recycle homocysteine to methionine and from there to S-  adenosylmethionine. The latter is the required methyl donor in these reactions. The nervous system lacks the betaine pathway of methio-  nine regeneration and is dependent on the B12 system. With a B12 deficiency, this pathway is inoperable in the nervous system.   S-Adenosylmethionine (SAM) participates in the synthesis of many compounds  that contain methyl groups. It is used in reactions that add methyl groups to either  oxygen or nitrogen atoms in the acceptor (contrast that to folate derivatives, which  can add one-carbon groups to sulfur or to carbon). As examples, SAM is required  for the conversion of phosphatidylethanolamine to phosphatidylcholine, guanidi-  noacetate to creatine, norepinephrine to epinephrine, acetylserotonin to melatonin,  and nucleotides to methylated nucleotides (see Fig. 40. 9B). It is also required for  the inactivation of catecholamines and serotonin (see Chapter 48). More than 35  reactions in humans require methyl donation from SAM.   SAM is synthesized from methionine and ATP. As with the activation of vitamin  B12, ATP donates the adenosine. With the transfer of its methyl group, SAM forms  S-adenosylhomocysteine, which is subsequently hydrolyzed to form homocysteine  and adenosine.   Methionine, required for the synthesis of SAM, is obtained from the diet or  produced from homocysteine, which accepts a methyl group from vitamin B12   Many health food stores now sell SAMe, a stabilized version of S-adenosylme-  thionine. SAMe has been hypothesized to relieve depression because the syn-  thesis of certain neurotransmitters requires methylation by SAM (see Chapter  47). This has led to the hypothesis that by increasing SAM levels within the nervous sys-  tem, the biosynthesis of these neurotransmitters will be accelerated. This in turn might  alleviate the feelings of depression. There have been reports in the literature indicating  that this may occur, but its efficacy as an antidepressant must be confirmed. The major  questions that must be addressed include the stability of SAMe in the digestive system  and the level of uptake of SAMe by cells of the nervous system.   (see Fig. 40. 9 A). Thus, the methyl group of methionine is regenerated. The portion  of methionine that is essential in the diet is the homocysteine moiety. If we had an  adequate dietary source of homocysteine, methionine would not be required in the  diet. However, there is no good dietary source of homocysteine, whereas methion-  ine is plentiful in the diet.   Homocysteine provides the sulfur atom for the synthesis of cysteine (see Chap-  ter 39). In this case, homocysteine reacts with serine to form cystathionine, which  is cleaved, yielding cysteine and a-ketobutyrate. The first reaction in this sequence  is inhibited by cysteine. Thus, methionine, via homocysteine, is not used for cys-  teine synthesis unless the levels of cysteine in the body are lower than required for  its metabolic functions. An adequate dietary supply of cysteine, therefore, can  "spare" (or reduce) the dietary requirement for methionine.   If one analyzes the flow of carbon in the folate cycle, the equilibrium lies in the  direction of the N 5 -methyl FH 4 form. This appears to be the most stable form of car-  bon attached to the vitamin. However, in only one reaction can the methyl group be  removed from N 5 -methyl FH 4 , and that is the methionine synthase reaction, which  requires vitamin B12. Thus, if vitamin B12 is deficient, or if the methionine syn-  thase enzyme is defective, N 5 -methyl FH 4 will accumulate. Eventually most folate  forms in the body will become "trapped" in the N 5 -methyl form. A functional folate  deficiency results because the carbons cannot be removed from the folate. The  appearance of a functional folate deficiency caused by a lack of vitamin B12 is  known as the "methyl-trap" hypothesis, and its clinical implications are discussed  in following sections.   Other compounds involved in one-carbon metabolism are derived from degradation products of choline. Choline, an essential  KJ ) component of certain phospholipids, is oxidized to form betaine aldehyde, which is further oxidized to betaine (trimethylglycine).   In the liver, betaine can donate a methyl group to homocysteine to form methionine and dimethyl glycine. This allows the liver  to have two routes for homocysteine conversion to methionine. Under conditions in which SAM accumulates, glycine can be methylated  to form sarcosine (N-methyl glycine). This route is used when methionine levels are high and excess methionine needs to be metabolized.   HO - CH 2 - CH 2 - N - (CH 3 ) 3 ^ ^- ► X - CH 2 - N - (CH 3 ) 3 ^- ► X - CH 2 - N - (CH 3 ) 3   Elevated homocysteine levels have been linked to cardiovascular and neurologic  disease. Homocysteine levels can accumulate in a number of ways, which are  related to both folic acid and vitamin B12 metabolism. Homocysteine is derived  from S-adenosyl homocysteine, which arises when SAM donates a methyl group  (Fig. 40.10). Because SAM is frequently donating methyl groups, there is a constant  production of S-adenosyl homocysteine, which leads to a constant production of  homocysteine. Recall from Chapter 39 that homocysteine has two biochemical  fates. The homocysteine produced can either be remethylated to methionine or con-  densed with serine to form cystathionine. There are two routes to methionine pro-  duction. The major one is methylation by N 5 -methyl FH 4 , requiring vitamin B12.  The liver also contains a second pathway in which betaine (a degradation product  of choline) can donate a methyl group to homocysteine to form methionine, but this  is a minor pathway. The conversion of homocysteine to cystathionine requires pyri-  doxal phosphate. Thus, if an individual is deficient in vitamin B12, the conversion  of homocysteine to methionine by the major route is inhibited. This will direct  homocysteine to produce cystathionine, which eventually produces cysteine. As  cysteine levels accumulate, the enzyme that makes cystathinonine undergoes feed-  back inhibition, and that pathway is also inhibited (see Fig. 40.10). This, overall,  leads to an accumulation of homocysteine, which is released into the blood.   Homocysteine also accumulates in the blood if a mutation is present in the  enzyme that converts N 5 , N 10 methylene FH 4 to N 5 -methyl FH 4 . When this occurs,  the levels of N 5 -methyl FH 4 are too low to allow homocysteine to be converted to  methionine. The loss of this pathway, coupled with the feedback inhibition by cys-  teine on cystathionine formation, will also lead to elevated homocysteine levels in  the blood.   A third way in which serum homocysteine levels can be elevated is by a mutated  cystathinone-p-synthase or a deficiency in vitamin B6, the required cofactor for that  enzyme. These defects block the ability of homocysteine to be converted to cys-  tathionine, and the homocysteine that does accumulate cannot all be accommodated  by conversion to methionine. Thus, an accumulation of homocysteine results.   Fig. 40.10. Reaction pathways involving homocysteine. Defects in numbered enzymes (1 =  methionine synthase, 2 = N 5 , N 10 methylene FH 4 reductase, 3 = cystathionine- p-synthase)  lead to elevated homocysteine. Recall that as cysteine accumulates, there is feedback inhibi-  tion on cystathionine- p-synthase to stop further cysteine production.   Folate deficiency during pregnancy has been associated with an increased risk for  neural tube defects in the developing fetus. This risk is significantly reduced if  women take folic acid supplements periconceptually. The link between folate defi-  ciency and neural tube defects was first observed in women with hyperhomocys-  teinemia brought about by a thermolabile variant of N 5 , N 10 methylene tetrahydrofo-  late reductase. This form of the enzyme, which results from a single nucleotide  change (C to T) in position 677 of the gene encoding the protein, is less active at  body temperature than at lower temperatures. This results in a reduced level of N 5 -  methyl tetrahydrofolate being generated and, therefore, an increase in the levels of  homocysteine. Along with the elevated homocysteine, the women were also folate  deficient. The folate deficiency and the subsequent inhibition of DNA synthesis leads  to neural tube defects. The elevated homocysteine is one indication that such a deficit  is present. These findings have led to the recommendation that women considering  getting pregnant begin taking folate supplements before conception occurs, and for  at least 1 month after conception. The Department of Agriculture has, in fact, man-  dated that folate be added to flour-containing products in the United States.   Jean Ann Tonich developed a folate deficiency and is on the verge of devel-  oping a cobalamin (vitamin B12) deficiency as a consequence of prolonged  moderately severe malnutrition related to chronic alcoholism. Before folate  therapy is started, the physician must ascertain that the megaloblastic anemia is not  caused by a pure B12 deficiency or a combined deficiency of folate and B12.   If folate is given without cobalamin to a B12-deficient patient, the drug only par-  tially corrects the megaloblastic anemia because it will "bypass" the methyl-folate  trap and provide adequate FH 4 coenzyme for the conversion of dUMP to dTMP and  for a resurgence of purine synthesis. As a result, normal DNA synthesis, DNA  repair, and cell division occur. However, the neurologic syndrome, resulting from  hypomethylation in nervous tissue, may progress unless the physician realizes that  B12 supplementation is required. In Jean Ann's case, in which the serum B12 con-  centration was borderline low and in which the dietary history supported the possi-  bility of a B 12 deficiency, a combination of folate and B12 supplements is required  to avoid this potential therapeutic trap.   Bea Twelvlow was diagnosed with an inability to absorb dietary B12 but  not crystalline B12 (the Schilling test results were normal). One of the con-  sequences of aging is a reduced acid production by the gastric mucosa   (atrophic gastritis), which limits the ability of pepsin to work on dietary protein. A  reduced pepsin efficiency would then reduce the amount of bound B12 released  from dietary protein, as a result of which the B 12 would be unavailable for absorp-  tion. Because Bea absorbs crystalline B12 without a problem, her condition can be  easily treated by taking vitamin B12 supplements orally.   Folate Deficiencies and DNA Synthesis. Folate deficiencies result in  decreased availability of the deoxythymidine and purine nucleotides that  serve as precursors for DNA synthesis. The decreased concentrations of  these precursors affect not only the DNA synthesis that occurs during replication   before cell division, but also the DNA synthesis that occurs as a step in the  processes that repair damaged DNA.   Decreased methylation of deoxyuridine monophosphate (dUMP) to form  deoxythymidine monophosphate (dTMP), a reaction that requires N 5 , N 10 -methyl-  ene tetrahydrofolate as a coenzyme (see Fig. 40.5), leads to an increase in the intra-  cellular dUTP/dTTP ratio. This change causes a significant increase in the incorpo-  ration of uracil into DNA. Although much of this uracil can be removed by DNA  repair enzymes, the lack of available dTTP blocks the step of DNA repair catalyzed  by DNA polymerase. The result is fragmentation of DNA as well as blockade of  normal DNA replication.   These abnormal nuclear processes are responsible for the clumping and poly-  segmentation seen in the nuclei of neutrophilic leukocytes in the bone marrow and  in the peripheral blood of patients with a megaloblastic anemia caused either by a  primary folate deficiency or one that is secondary to a B 12 deficiency. The abnor-  malities in DNA synthesis and repair lead to an irreversible loss of the capacity for  cell division and eventually to cell death.   Fenech M. The role of folic acid and vitamin B12 in genomic stability of human cells. Mutation   Research 2000;475:57-67.  Herbert V. Folic acid. In: Shils ME, Olson JA, Shike M, Ross AC, eds. Modern Nutrition in Health and   Disease. Philadelphia: Lippincott, Williams & Wilkins, 1999:433^46.  Selhub J. Homocysteine metabolism. Annu Rev Nutr 1999;19:217-246.   Van der Put NMJ, Van Straaten HWM, Trijbels FJM, Blom HJ. Folate, homocysteine and neural tube  defects: An overview. Exp Biol Med 2001;226:243-270.   Weir DG, Scott JM. Vitamin B12 "cobalamin". In: Shils ME, Olson JA, Shike M, Ross AC, eds. Mod-  ern Nutrition in Health and Disease. Philadelphia: Lippincott, Williams & Wilkins, 1999:447^458.   1. Which of the following reactions requires N 5 , N 10 -methylene FH 4 as a carbon donor?   2. Propionic acid accumulation from amino acid degradation would result from a deficiency of which of the following vitamins?   3. A dietary vitamin B12 deficiency can result from which of the following?   4. Which of the following forms of tetrahydrofolate is required for the synthesis of methionine from homocysteine?   5. An alternative method to methylate homocysteine to form methionine is which of the following?   Purines and pyrimidine s are required for synthesizing nucleotides and nucleic  acids. These molecules can be synthesized either from scratch, de novo, or sal-  vaged from existing bases. Dietary uptake of purine and pyrimidine bases is low,  because most of the ingested nucleic acids are metabolized by the intestinal  epithelial cells.   The de novo pathway of purine synthesis is complex, consisting of 11 steps,  and requiring 6 molecules of ATP for every purine synthesized. The precursors  that donate components to produce purine nucleotides include glycine, ribose  5 -phosphate, glutamine, aspartate, carbon dioxide, and N 10 -formyl FH 4 (Fig.  41.1). Purines are synthesized as ribonucleotides, with the initial purine synthe-  sized being inosine monophosphate (IMP). Adenosine monophosphate (AMP)  and guanosine monophosphate (GMP) are each derived from IMP in two-step  reaction pathways.   The purine nucleotide salvage pathway allows free purine bases to be converted  into nucleotides, nucleotides into nucleosides, and nucleosides into free bases.  Enzymes included in this pathway are AMP and adenosine deaminase, adenosine  kinase, purine nucleoside phosphorylase , adenine phosphoribosyltransf erase  (APRT), and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Muta-  tions in a number of these enzymes lead to serious diseases. Deficiencies in purine  nucleoside phosphorylase and adenosine deaminase lead to immunodeficiency  disorders. A deficiency in HGPRT leads to Lesch-Nyhan syndrome. The purine  nucleotide cycle, in which aspartate carbons are converted to fumarate to replen-  ish TCA cycle intermediates in working muscle, and the aspartate nitrogen is  released as ammonia, uses components of the purine nucleotide salvage pathway.   Pyrimidine bases are first synthesized as the free base and then converted to a  nucleotide. Aspartate and carbamoyl phosphate form all components of the  pyrimidine ring. Ribose 5-phosphate, which is converted to phosphoribosyl  pyrophosphate (PRPP), is required to donate the sugar phosphate to form a  nucleotide. The first pyrimidine nucleotide produced is orotate monophosphate  (OMR). The OMR is converted to uridine monophosphate (UMP), which will  become the precursor for both cytidine triphosphate (CTP) and deoxy thymidine  monophosphate (dTMP) production.   The formation of deoxy ribonucleotides requires ribonucleotide reductase  activity, which catalyzes the reduction of ribose on nucleotide diphosphate sub-  strates to T -deoxyribose. Substrates for the enzyme include adenosine diphos-  phate (ADR), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and  uridine diphosphate (UDP). Regulation of the enzyme is complex. There are two  major allosteric sites. One controls the overall activity of the enzyme, whereas the  other determines the substrate specificity of the enzyme. All deoxy ribonucleotides  are synthesized using this one enzyme.   The regulation of purine nucleotide biosynthesis occurs at four points in the  pathway. The enzymes PRPP synthetase, amidophosphoribosyl transferase, IMP   dehydrogenase, and adenylosuccinate synthetase are regulated by allosteric  modifiers, as they occur at key branch points through the pathway. Pyrimidine  synthesis is regulated at the first committed step, which is the synthesis of cyto-  plasmic carbamoyl-phosphate, by the enzyme carbamoyl phosphate synthetase II  (CPS -II).   Purines, when degraded, cannot generate energy, nor can the purine ring be  substantially modified. The end product of purine ring degradation is uric acid,  which is excreted in the urine. Uric acid has a limited solubility, and if it were to  accumulate, uric acid crystals would precipitate in tissues of the body with a  reduced temperature (such as the big toe). This condition of acute painful inflam-  mation of specific soft tissues and joints is called gout. Pyrimidines, when  degraded, however, give rise to water-soluble compounds, such as urea, carbon  dioxide, and water and do not lead to a disease state if pyrimidine catabolism is  increased.   The initial acute inflammatory process that caused Lotta Topaigne to expe-  rience a painful attack of gouty arthritis responded quickly to colchicine  therapy (see Chapter 10). Several weeks after the inflammatory signs and  symptoms in her right great toe subsided, Lotta was placed on allopurinol, a drug that  reduces uric acid synthesis. Her serum uric acid level gradually fell from a pretreat-  ment level of 9.2 mg/dL into the normal range (2.5-8.0 mg/dL). She remained free  of gouty symptoms when she returned to her physician for a follow-up office visit.   As has been seen in previous chapters of this text, nucleotides serve numerous func-  tions in different reaction pathways. For example, nucleotides are the activated pre-  cursors of DNA and RNA. Nucleotides form the structural moieties of many coen-  zymes (examples include NADH, FAD, and coenzyme A). Nucleotides are critical  elements in energy metabolism (ATP, GTP). Nucleotide derivatives are frequently  activated intermediates in many biosyntheses. For example, UDP-glucose and  CDP-diacylglycerol are precursors of glycogen and phosphoglycerides, respec-  tively. S-Adenosylmethionine carries an activated methyl group. In addition,  nucleotides act as second messengers in intracellular signaling (e.g., cAMP,  cGMP). Finally, nucleotides and nucleosides act as metabolic allosteric regulators.  Think about all of the enzymes that have been studied that are regulated by levels  of ATP, ADP, and AMP.   Dietary uptake of purine and pyrimidine bases is minimal. The diet contains  nucleic acids and the exocrine pancreas secretes deoxyribonuclease and ribonucle-  ase, along with the proteolytic and lipolytic enzymes. This enables digested nucleic  acids to be converted to nucleotides. The intestinal epithelial cells contain alkaline  phosphatase activity, which will convert nucleotides to nucleosides. Other enzymes  within the epithelial cells tend to metabolize the nucleosides to uric acid, or to sal-  vage them for their own needs. Approximately 5% of ingested nucleotides will  make it into the circulation, either as the free base or as a nucleoside. Because of  the minimal dietary uptake of these important molecules, de novo synthesis of  purines and pyrimidines is required.   The purine bases are produced de novo by pathways that use amino acids as pre-  cursors and produce nucleotides. Most de novo synthesis occurs in the liver  (Fig. 41.2), and the nitrogenous bases and nucleosides are then transported to other  tissues by red blood cells. The brain also synthesizes significant amounts of  nucleotides. Because the de novo pathway requires six high-energy bonds per  purine produced, a salvage pathway, which is used by many cell types, can convert  free bases and nucleosides to nucleotides.   As purines are built on a ribose base (see Fig. 41.2), an activated form of ribose is  used to initiate the purine biosynthetic pathway. 5-Phosphoribosyl-l -pyrophosphate  (PRPP) is the activated source of the ribose moiety. It is synthesized from ATP and  ribose 5' -phosphate (Fig. 41.3), which is produced from glucose through the pen-  tose phosphate pathway (see Chapter 29). The enzyme that catalyzes this reaction,  PRPP synthetase, is a regulated enzyme (see section II.A.5); however, this step is  not the committed step of purine biosynthesis. PRPP has many other uses, which  are described as the chapter progresses.   In the first committed step of the purine biosynthetic pathway, PRPP reacts with  glutamine to form phosphoribosylamine (Fig. 41.4). This reaction, which produces  nitrogen 9 of the purine ring, is catalyzed by glutamine phosphoribosyl amido-  transferase, a highly regulated enzyme.   In the next step of the pathway, the entire glycine molecule is added to the grow-  ing precursor. Glycine provides carbons 4 and 5 and nitrogen 7 of the purine ring  (Fig. 41.5).   Subsequently, carbon 8 is provided by N 10 -formyl FH 4 , nitrogen 3 by glutamine,  carbon 6 by C0 2 , nitrogen 1 by aspartate, and carbon 2 by formyl tetrahydrofolate  (see Fig. 41.1). Note that six molecules of ATP are required (starting with ribose  5 -phosphate) to synthesize the first purine nucleotide, inosine monophosphate  (IMP). This nucleotide contains the base hypoxanthine joined by an Af-glycosidic  bond from nitrogen 9 of the purine ring to carbon 1 of the ribose (Fig. 41.6).   Fig. 41.3. Synthesis of PRPP. Ribose 5-phosphate is produced from glucose by the pentose  phosphate pathway.   Glutamine (N 3 )  J^C0 2 (C 6 )  ^Aspartate (N^  ^ A/ 10 -Formyl-FH 4 (C 2 )   Fig. 41.2. Overview of purine production,  starting with glutamine, ribose 5-phosphate,  and ATP. The steps that require ATP are also  indicated in this figure. RR = ribonucleotide  reductase. FH 4 = tetrahydrofolate. PRPP =  5-phosphoribosyl 1 -pyrophosphate.   Cellular concentrations of PRPP  \ \ ) and glutamine are usually below  *r their K m for glutamine phosphori-  bosyl amidotransferase. Thus, any situation  which leads to an increase in their concen-  tration can lead to an increase in de novo  purine biosynthesis.   The base hypoxanthine is found in  KJ ) the anticodon of tRNA molecules  (it is formed by the deamination of  an adenine base). Hypoxanthine's role in  tRNA is to allow wobble base-pairs to form,  as the base hypoxanthine can base pair with  adenine, cytosine, or uracil. The wobbling  allows one tRNA molecule to potentially  form base pairs with three different codons.   Fig. 41.4. The first step in purine biosynthe-  sis. The purine base is built on the ribose moi-  ety. The availability of the substrate PRPP is a  major determinant of the rate of this reaction.   IMP serves as the branchpoint from which both adenine and guanine nucleotides  can be produced (see Fig. 41.2). Adenosine monophosphate (AMP) is derived from  IMP in two steps (Fig. 41.7). In the first step, aspartate is added to IMP to form  adenylosuccinate, a reaction similar to the one catalyzed by argininosuccinate syn-  thetase in the urea cycle. Note how this reaction requires a high-energy bond,  donated by GTP. Fumarate is then released from the adenylosuccinate by the  enzyme adenylosuccinase to form AMP.   GMP is also synthesized from IMP in two steps (Fig. 41.8). In the first step, the  hypoxanthine base is oxidized by IMP dehydrogenase to produce the base xanthine  and the nucleotide xanthosine monophosphate (XMP). Glutamine then donates the  amide nitrogen to XMP to form GMP in a reaction catalyzed by GMP synthetase.  This second reaction requires energy, in the form of ATP.   AMP and GMP can be phosphorylated to the di- and triphosphate levels. The pro-  duction of nucleoside diphosphates requires specific nucleoside monophosphate  kinases, whereas the production of nucleoside triphosphates requires nucleoside  diphosphate kinases, which are active with a wide range of nucleoside diphos-  phates. The purine nucleoside triphosphates are also used for energy-requiring  processes in the cell and also as precursors for RNA synthesis (see Fig. 41.2).   Regulation of purine synthesis occurs at several sites (Fig. 41.9). Four key  enzymes are regulated: PRPP synthetase, amidophosphoribosyl transferase,   The aspartate to fumarate conver-  r| ) sion also occurs in the urea cycle.  In both cases, aspartate donates a  nitrogen to the product, while the carbons of  aspartate are released as fumarate.   Fig. 41.6. Structure of inosine monophos-  phate (IMP). The base is hypoxanthine.   Fig. 41.5. Incorporation of glycine into the purine precursor. The ATP is required for the con-  densation of the glycine carboxylic acid group with the 1' -amino group of phosphoribosyl  1 -amine.   adenylosuccinate synthetase, and IMP dehydrogenase. The first two enzymes  regulate IMP synthesis; the last two regulate the production of AMP and GMP,  respectively.   A primary site of regulation is the synthesis of PRPP. PRPP synthetase is nega-  tively affected by GDP and, at a distinct allosteric site, by ADR Thus, the simulta-  neous binding of an oxypurine (eg., GDP) and an aminopurine (eg., ADP) can occur  with the result being a synergistic inhibition of the enzyme. This enzyme is not the  committed step of purine biosynthesis; PRPP is also used in pyrimidine synthesis  and both the purine and pyrimidine salvage pathways.   The committed step of purine synthesis is the formation of 5-phosphoribosyl 1-  amine by glutamine phosphoribosyl amidotransf erase. This enzyme is strongly  inhibited by GMP and AMP (the end products of the purine biosynthetic pathway).  The enzyme is also inhibited by the corresponding nucleoside di- and triphos-  phates, but under cellular conditions, these compounds probably do not play a cen-  tral role in regulation. The active enzyme is a monomer of 133,000 daltons but is  converted to an inactive dimer (270,000 daltons) by binding of the end products.   The enzymes that convert IMP to XMP and adenylosuccinate are both regulated.  GMP inhibits the activity of IMP dehydrogenase, and AMP inhibits adenylosucci-  nate synthetase. Note that the synthesis of AMP is dependent on GTP (of which  GMP is a precursor), whereas the synthesis of GMP is dependent on ATP (which is  made from AMP). This serves as a type of positive regulatory mechanism to bal-  ance the pools of these precursors: when the levels of ATP are high, GMP will be   Fig. 41.8. The conversion of IMP to GMP. Note that ATP is required for the synthesis of  GMP.   Fig. 41.7. The conversion of IMP to AMR  Note that GTP is required for the synthesis of  AMR   Fig. 41.9. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric  sites, one for ADP, the other for GDP. Glutamine phosphoribosyl amidotransferase con-  tains adenine nucleotide and guanine nucleotide binding sites; the monophosphates are  the most important, although the di- and tri-phosphates will also bind to and inhibit  the enzyme. Adenylosuccinate synthetase is inhibited by AMP; IMP dehydrogenase is  inhibited by GMP.   made; when the levels of GTP are high, AMP synthesis will take place. GMP and  AMP act as negative effectors at these branch points, a classic example of feedback  inhibition.   A deficiency in purine nucleoside  phosphorylase activity leads to an  immune disorder in which T-cell  immunity is compromised. B-cell immunity,  conversely, may be only slightly compro-  mised or even normal. Children lacking this  activity have recurrent infections, and more  than half display neurologic complications.  Symptoms of the disorder first appear at  between 6 months and 4 years of age.   Most of the de novo synthesis of the bases of nucleotides occurs in the liver, and  to some extent in the brain, neutrophils, and other cells of the immune system.  Within the liver, nucleotides can be converted to nucleosides or free bases,  which can be transported to other tissues via the red blood cell in the circula-  tion. In addition, the small amounts of dietary bases or nucleosides that  are absorbed also enter cells in this form. Thus, most cells can salvage these  bases to generate nucleotides for RNA and DNA synthesis. For certain cell  types, such as the lymphocytes, the salvage of bases is the major form of  nucleotide generation.   The overall picture of salvage is shown in Figure 41.10. The pathways allow free  bases, nucleosides, and nucleotides to be easily interconverted. The major enzymes  required are purine nucleoside phosphorylase, phosphoribosyl transferases, and  deaminases.   Purine nucleoside phosphorylase catalyzes a phosphorolysis reaction of the N-  glycosidic bond that attaches the base to the sugar moiety in the nucleosides guano-  sine and inosine (Fig. 41.11). Thus, guanosine and inosine are converted to guanine  and hypoxanthine, respectively, along with ribose 1 -phosphate. The ribose  1 -phosphate can be isomerized to ribose 5-phosphate, and the free bases then sal-  vaged or degraded, depending on cellular needs.   Fig. 41.10. Salvage of bases. The purine bases hypoxanthine and guanine react with PRPP to  form the nucleotides inosine and guanosine monophosphate, respectively. The enzyme that cat-  alyzes the reaction is hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Adenine  forms AMP in a reaction catalyzed by adenine phosphoribosyltransferase (APRT). Nucleotides  are converted to nucleosides by 5 '-nucleotidase. Free bases are generated from nucleosides by  purine nucleoside phosphorylase. Deamination of the base adenine occurs with AMP and adeno-  sine deaminase. Of the purines, only adenosine can be directly phosphorylated back to a  nucleotide, by adenosine kinase.   Fig. 41.11. The purine nucleoside phosphory-  lase reaction, converting guanosine or inosine  to ribose 1 -phosphate plus the free bases gua-  nine or hypoxanthine.   The phosphoribosyl transferase enzymes catalyze the addition of a ribose 5-  phosphate group from PRPP to a free base, generating a nucleotide and  pyrophosphate (Fig. 41.12). Two enzymes do this: adenine phosphoribosyl  transferase (APRT) and hypoxanthine-guanine phosphoribosyl transferase  (HGPRT). The reactions they catalyze are the same, differing only in their substrate  specificity.   Adenosine and AMP can be deaminated by adenosine deaminase and AMP  deaminase, respectively, to form inosine and IMP (see Fig. 41.10). Adenosine is  also the only nucleoside to be directly phosphorylated to a nucleotide by adenosine  kinase. Guanosine and inosine must be converted to free bases by purine nucleoside  phosphorylase before they can be converted to nucleotides by HGPRT   A portion of the salvage pathway that is important in muscle is the purine  nucleotide cycle (Fig. 41.13). The net effect of these reactions is the deamination of  aspartate to fumarate (as AMP is synthesized from IMP and then deaminated back  to IMP by AMP deaminase). Under conditions in which the muscle must generate  energy, the fumarate derived from the purine nucleotide cycle is used anapleroti-  cally to replenish TCA cycle intermediates and to allow the cycle to operate at a  high speed. Deficiencies in enzymes of this cycle lead to muscle fatigue during  exercise.   Lesch-Nyhan syndrome is caused by a defective hypoxanthine-guanine phos-  phoribosyltransferase (HGPRT) (see Fig. 41.12). In this condition, purine bases  cannot be salvaged. Instead, they are degraded, forming excessive amounts of  uric acid. Individuals with this syndrome suffer from mental retardation. They are also  prone to chewing off their fingers and performing other acts of self-mutilation.   Fig. 41.12. The phosphoribosyltransferase  reaction. APRT uses the free base adenine;  HGPRT can use either hypoxanthine or gua-  nine as a substrate.    Fig. 41.13. The purine nucleotide cycle.  Using a combination of biosynthetic and sal-  vage enzymes, the net effect is the conversion  of aspartate to fumarate plus ammonia, with  the fumarate playing an anaplerotic role in the  muscle.   In bacteria, aspartate transcar-  \ \ ) bamoylase is the regulated step of  *r pyrimidine production. This is a  very complex enzyme and was a model sys-  tem for understanding how allosteric  enzymes were regulated. In humans, how-  ever, this enzyme is not regulated.   In the synthesis of the pyrimidine nucleotides, the base is synthesized first, and then  it is attached to the ribose 5' -phosphate moiety (Fig. 41.14). The origin of the atoms  of the ring (aspartate and carbamoyl-phosphate, which is derived from carbon diox-  ide and glutamine) is shown in Fig. 41. 15. In the initial reaction of the pathway, glu-  tamine combines with bicarbonate and ATP to form carbamoyl phosphate. This  reaction is analogous to the first reaction of the urea cycle, except that it uses glut-  amine as the source of the nitrogen (rather than ammonia) and it occurs in the  cytosol (rather than in mitochondria). The reaction is catalyzed by carbamoyl phos-  phate synthetase II, which is the regulated step of the pathway. The analogous reac-  tion in urea synthesis is catalyzed by carbamoyl phosphate synthetase I, which is  activated by TV-acetylglutamate. The similarities and differences between these two  carbamoyl phosphate synthetase enzymes is described in Table 41.1.   In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to  carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The  molecule subsequently closes to produce a ring (catalyzed by dihydroorotase),  which is oxidized to form orotic acid (or its anion, orotate) through the actions of  dihydroorotate dehydrogenase . The enzyme orotate phosphoribosyl transferase cat-  alyzes the transfer of ribose 5 -phosphate from PRPP to orotate, producing orotidine  5 '-phosphate, which is decarboxylated by orotidylic acid dehydrogenase to form   Fig. 41.14. Synthesis of the pyrimidine bases. CPSII = carbamoyl phosphate synthetase n. RR  = ribonucleotide reductase; ® = stimulated by; 0 = inhibited by; FH 2 and FH 4 = forms of  folate.   Fig. 41.15. The origin of the bases in the  pyrimidine ring.    uridine monophosphate (UMP) (Fig. 41.16). In mammals, the first three enzymes  of the pathway (carbamoyl phosphate synthetase II, aspartate transcarbamoylase,  and dihydroorotase) are located on the same polypeptide, designated as CAD. The  last two enzymes of the pathway are similarly located on a polypeptide known as  UMP synthase (the orotate phosphoribosyl transferase and orotidylic acid dehydro-  genase activities).   UMP is phosphorylated to UTP. An amino group, derived from the amide of glu-  tamine, is added to carbon 4 to produce CTP by the enzyme CTP synthetase (this  reaction cannot occur at the nucleotide monophosphate level). UTP and CTP are  precursors for the synthesis of RNA (see Fig. 41.14). The synthesis of thymidine  triphosphate (TTP) will be described in section IV.   Pyrimidine bases are normally salvaged by a two-step route. First, a relatively non-  specific pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their  respective nucleosides (Fig. 41.17). Notice that the preferred direction for this reac-  tion is the reverse phosphorylase reaction, in which phosphate is being released and  is not being used as a nucleophile to release the pyrimidine base from the nucleo-  side. The more specific nucleoside kinases then react with the nucleosides, forming  nucleotides (Table 41.2). As with purines, further phosphorylation is carried out by  increasingly more specific kinases. The nucleoside phosphorylase-nucleoside  kinase route for synthesis of pyrimidine nucleoside monophosphates is relatively  inefficient for salvage of pyrimidine bases because of the very low concentration of  the bases in plasma and tissues.   Pyrimidine phosphorylase can use all of the pyrimidines but has a preference for  uracil and is sometimes called uridine phosphorylase. The phosphorylase uses cyto-  sine fairly well but has a very, very low affinity for thymine; therefore, a ribonucle-  oside containing thymine is almost never made in vivo. A second phosphorylase,  thymine phosphorylase, has a much higher affinity for thymine and adds a deoxyri-  bose residue (see Fig. 41.17).   Of the various ribonucleosides and deoxyribonucleoside kinases, one that merits  special mention is thymidine kinase (TK). This enzyme is allosterically inhibited by  dTTP. Activity of thymidine kinase in a given cell is closely related to the prolifer-  ative state of that cell. During the cell cycle, the activity of TK rises dramatically as  cells enter S phase, and in general rapidly dividing cells have high levels of this  enzyme. Radiolabeled thymidine is widely used for isotopic labeling of DNA, for  example, in radioautographic investigations or to estimate rates of intracellular  DNA synthesis.   In hereditary orotic aciduria, orotic  acid is excreted in the urine  because the enzymes that convert  it to uridine monophosphate, orotate phos-  phoribosyltransferase and orotidine 5'-phos-  phate decarboxylase, are defective (see  Fig. 41.16). Pyrimidines cannot be synthe-  sized, and, therefore, normal growth does  not occur. Oral administration of uridine is  used to treat this condition. Uridine, which is  converted to UMP, bypasses the metabolic  block and provides the body with a source of  pyrimidines, as both CTP and dTMP can be  produced from UMP.   Table 41.2. Salvage Reactions for Conversion of Pyrimidine Nucleosides  to Nucleotides.   Fig. 41.17. Salvage reactions for pyrimidine  nucleoside production. Thymine phosphory-  lase uses deoxyribose 1 -phosphate as a  substrate, such that ribothymidine is rarely  formed.   Fig. 41.16. Conversion of carbamoyl phosphate and aspartate to UMP. The defective  enzymes in hereditary orotic aciduria are indicated (^m).   The regulated step of pyrimidine synthesis in humans is carbamoyl phosphate syn-  thetase II. The enzyme is inhibited by UTP and activated by PRPP (see Fig. 41.14).  Thus, as pyrimidines decrease in concentration (as indicated by UTP levels), CPS-  II is activated and pyrimidines are synthesized. The activity is also regulated by the  cell cycle. As cells approach S-phase, CPS-II becomes more sensitive to PRPP acti-  vation and less sensitive to UTP inhibition. At the end of S-phase, the inhibition by  UTP is more pronounced, and the activation by PRPP is reduced. These changes in  the allosteric properties of CPS-II are related to its phosphorylation state. Phospho-  rylation of the enzyme at a specific site by a MAP kinase leads to a more easily acti-  vated enzyme. Phosphorylation at a second site by the cAMP-dependent protein  kinase leads to a more easily inhibited enzyme.   For DNA synthesis to occur, the ribose moiety must be reduced to deoxyribose (Fig.  41.18). This reduction occurs at the dinucleotide level and is catalyzed by ribonu-  cleotide reductase, which requires the protein thioredoxin. The deoxyribonucleo-  side diphosphates can be phosphorylated to the triphosphate level and used as pre-  cursors for DNA synthesis (see Figs. 41.2 and 41.14).   The regulation of ribonucleotide reductase is quite complex. The enzyme con-  tains two allosteric sites, one controlling the activity of the enzyme and the other  controlling the substrate specificity of the enzyme. ATP bound to the activity site  activates the enzyme; dATP bound to this site inhibits the enzyme. Substrate speci-  ficity is more complex. ATP bound to the substrate site activates the reduction of  pyrimidines (CDP and UDP), to form dCDP and dUDP. The dUDP is not used for  DNA synthesis; rather, it is used to produce dTMP (see below). Once dTMP is pro-  duced, it is phosphorylated to dTTP, which then binds to the substrate site and  induces the reduction of GDP. As dGTP accumulates, it replaces dTTP in the sub-  strate site and allows ADP to be reduced to dADP. This leads to the accumulation  of dATP, which will inhibit the overall activity of the enzyme. These allosteric  changes are summarized in Table 41.3.   dUDP can be dephosphorylated to form dUMP, or, alternatively, dCMP can be  deaminated to form dUMP. Methylene tetrahydrofolate transfers a methyl group to  dUMP to form dTMP (see Figure 40.5). Phosphorylation reactions produce dTTP,  a precursor for DNA synthesis and a regulator of ribonucleotide reductase.   The degradation of the purine nucleotides (AMP and GMP) occurs mainly in the  liver (Fig. 41.19). Salvage enzymes are used for most of these reactions. AMP is  first deaminated to produce IMP (AMP deaminase). Then IMP and GMP are  dephosphorylated (5 '-nucleotidase), and the ribose is cleaved from the base by  purine nucleoside phosphorylase. Hypoxanthine, the base produced by cleavage of  IMP, is converted by xanthine oxidase to xanthine, and guanine is deaminated by   Fig. 41.18. Reduction of ribose to deoxyri-  bose. Reduction occurs at the nucleoside  diphosphate level. A ribonucleoside diphos-  phate (NDP) is converted to a deoxyribonucle-  oside diphosphate (dNDP). Thioredoxin is oxi-  dized to a disulfide, which must be reduced for  the reaction to continue producing dNDP.  N = a nitrogenous base.    When ornithine transcarbamoylase  is deficient (urea cycle disorder),  excess carbamoyl phosphate from  the mitochondria leaks into the cytoplasm.  The elevated levels of cytoplasmic car-  bamoyl phosphate lead to pyrimidine pro-  duction, as the regulated step of the path-  way, the reaction catalyzed by carbamoyl  synthetase II, is being bypassed. Thus, orotic  aciduria results.    Gout is caused by excessive uric  acid levels in the blood and tissues.  To determine whether a person  with gout has developed this problem  because of overproduction of purine  nucleotides or because of a decreased ability  to excrete uric acid, an oral dose of an ^re-  labeled amino acid is sometimes used.  Which amino acid would be most appropri-  ate to use for this purpose?   The entire glycine molecule is  incorporated into the precursor of  the purine nucleotides. The nitro-  gen of this glycine also appears in uric acid,  the product of purine degradation, ^re-  labeled glycine could be used, therefore, to  determine whether purines are being over-  produced.    Uric acid has a p/C of 5.4. It is ion-  ized in the body to form urate.  Urate is not very soluble in an   aqueous environment. The quantity in nor-  mal human blood is very close to the solu-  bility constant.   Normally, as cells die, their purine  nucleotides are degraded to  hypoxanthine and xanthine, which  are converted to uric acid by xanthine oxi-  dase (see Fig. 41.15). Allopurinol (a struc-  tural analog of hypoxanthine) is a substrate  for xanthine oxidase. It is converted to oxy-  purinol (also called alloxanthine), which  remains tightly bound to the enzyme, pre-  venting further catalytic activity (see Fig.  8.19). Thus, allopurinol is a suicide inhibitor.  It reduces the production of uric acid and  hence its concentration in the blood and tis-  sues (e.g., the synovial lining of the joints in  Lotta Topaigne's great toe). Xanthine and  hypoxanthine accumulate, and urate levels  decrease. Overall, the amount of purine  being degraded is spread over three prod-  ucts rather than appearing in only one.  Therefore, none of the compounds exceeds  its solubility constant, precipitation does not  occur, and the symptoms of gout gradually  subside.   Fig. 41.19. Degradation of the purine bases. The reactions inhibited by allopurinol are indi-  cated. A second form of xanthine oxidase exists that uses NAD + instead of 0 2 as the electron  acceptor.   the enzyme guanase to produce xanthine. The pathways for the degradation of ade-  nine and guanine merge at this point. Xanthine is converted by xanthine oxidase to  uric acid, which is excreted in the urine. Xanthine oxidase is a molybdenum-requir-  ing enzyme that uses molecular oxygen and produces hydrogen peroxide (H 2 0 2 ).  Another form of xanthine oxidase exists that uses NAD + as the electron acceptor  (see Chapter 24).   Note how little energy is derived from the degradation of the purine ring. Thus,  it is to the cell's advantage to recycle and salvage the ring, because it costs energy  to produce and not much is obtained in return.   The pyrimidine nucleotides are dephosphorylated, and the nucleosides are cleaved  to produce ribose 1 -phosphate and the free pyrimidine bases cytosine, uracil, and  thymine. Cytosine is deaminated, forming uracil, which is converted to C0 2 , NH 4 + ,   and p-alanine. Thymine is converted to C0 2 , NH 4 + , and p-aminoisobutyrate  (Fig. 41.20). These products of pyrimidine degradation are excreted in the urine or  converted to C0 2 , H 2 0, and NH 4 + (which forms urea). They do not cause any prob-  lems for the body, in contrast to urate, which is produced from the purines and can  precipitate, causing gout. As with the purine degradation pathway, little energy can  be generated by pyrimidine degradation.   ^ Hyperuricemia in Lotta Topaigne's case arose as a consequence of over-  ) production of uric acid. Treatment with allopurinol not only inhibits xan-  thine oxidase, lowering the formation of uric acid with an increase in the  excretion of hypoxanthine and xanthine, but also decreases the overall synthesis of  purine nucleotides. Hypoxanthine and xanthine produced by purine degradation are  salvaged (i.e., converted to nucleotides) by a process that requires the consumption  of PRPP. PRPP is a substrate for the glutamine phosphoribosyl amidotransferase  reaction that initiates purine biosynthesis. Because the normal cellular levels of  PRPP and glutamine are below the K m of the enzyme, changes in the level of either  substrate can accelerate or reduce the rate of the reaction. Therefore, decreased lev-  els of PRPP cause decreased synthesis of purine nucleotides.   flX A deficiency in adenosine deaminase activity leads to severe combined  ) immunodeficiency disease, or SCID. In the severe form of combined  immunodeficiency, both T cells (which provide cell-based immunity, see  Chapter 44) and B -cells (which produce antibodies) are deficient, leaving the indi-  vidual without a functional immune system. Children born with this disorder lack a  thymus gland and suffer from many opportunistic infections because of the lack of  a functional immune system. Death results if the child is not placed in a sterile envi-  ronment. Administration of polyethylene glycol-modified adenosine deaminase has  been successful in treating the disorder, and the ADA gene was the first to be used  in gene therapy in treating the disorder. The question that remains, however, is that  even though all cells of the body are lacking ADA activity, why are the immune  cells specifically targeted for destruction?   The specific immune disorder is not caused by any defect in purine salvage path-  ways, as children with Lesch-Nyhan syndrome have a functional immune system,  although there are other major problems in those children. This suggests that per-  haps the accumulation of precursors to ADA lead to toxic effects. Three hypotheses  have been proposed and are outlined below.   In the absence of ADA activity, both adenosine and deoxy adenosine will accu-  mulate. When deoxyadenosine accumulates, adenosine kinase can convert it to  dAMP. Other kinases will allow dATP to then accumulate within the lymphocyte.  Why specifically the lymphocyte? The other cells of the body are secreting the  deoxyadenosine they cannot use, and it is accumulating in the circulation. As the  lymphocytes are present in the circulation, they tend to accumulate this compound  more so than cells not constantly present within the blood. As dATP accumulates,  ribonucleotide reductase becomes inhibited, and the cells can no longer produce  deoxyribonucleotides for DNA synthesis. Thus, when cells are supposed to grow  and differentiate in response to cytokines, they cannot, and they die.   A second hypothesis suggests that the accumulation of deoxyadenosine in lym-  phocytes leads to an inhibition of S-adenosylhomocysteine hydrolase, the enzyme  that converts S-adenosylhomocysteine to homocysteine and adenosine. This leads    Once nucleotide biosynthesis and  salvage was understood at the  pathway level, it was quickly real-  ized that one way to inhibit cell proliferation  would be to block purine or pyrimidine syn-  thesis. Thus, drugs were developed that  would interfere with a cell's ability to gener-  ate precursors for DNA synthesis, thereby  inhibiting cell growth. This is particularly  important for cancer cells, which have lost  their normal growth regulatory properties.  Such drugs have been introduced previously  with a number of different patients. Colin  Tuma was treated with 5-fluorouracil, which  inhibits thymidylate synthase (dUMP to TMP  synthesis). Arlyn Foma was treated with  methotrexate for his leukemia; methotrexate  inhibits dihydrofolate reductase, thereby  blocking the regeneration of tetrahydrofo-  late and de novo purine synthesis and  thymidine synthesis. Mannie Weitzels was  treated with hydroxyurea to block ribonu-  cleotide reductase activity, with the goal of  inhibiting DNA synthesis in the leukemic  cells. Development of these drugs would not  have been possible without an understand-  ing of the biochemistry of purine and pyrim-  idine salvage and synthesis. Such drugs also  affect rapidly dividing normal cells, which  brings about a number of the side effects of  chemotherapeutic regimens.    Painful joints  Loss of immune  system, including  no T or B cells   to hypo-methylation in the cell and an accumulation of S-adenosylhomocysteine.  S-adenosylhomocysteine accumulation has been linked to the triggering of apoptosis.   The third hypothesis suggested is that elevated adenosine levels lead to inappro-  priate activation of adenosine receptors. Adenosine is also a signaling molecule, and  stimulation of the adenosine receptors results in activation of protein kinase A and  elevated cAMP levels in thymocytes. Elevated levels of cAMP in these cells trig-  gers both apoptosis and developmental arrest of the cell.   Although it is still not clear which potential mechanism best explains the arrested  development of immune cells, it is clear that elevated levels of adenosine and  deoxyadenosine are toxic. The biochemical disorders of purine and pyrimidine  metabolism discussed in this chapter are summarized in Table 41.4.   Becker MA. Hyperuricemia and gout. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic  and Molecular Bases of Inherited Disease, vol II, 8th Ed. New York: McGraw-Hill, 2001:2513-2535.   Hershfield MS, Mitchell BS. Immunodeficiency diseases caused by adenosine deaminase deficiency and  purine nucleoside phosphorylase deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The  Metabolic and Molecular Bases of Inherited Disease, vol II, 8th Ed. New York: McGraw-Hill,  2001:2585-2625.   Webster DR, Becroft DMO, Van Gennip AH, Van Kuilenberg ABP. Hereditary orotic aciduria and other  disorders of pyrimidine metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Meta-  bolic and Molecular Bases of Inherited Disease, vol II, 8th Ed. New York: McGraw-Hill,  2001:2663-2702.   Similarities between carbamoyl phosphate synthetase I and carbamoyl phosphate synthetase II include which ONE of the fol-  lowing?   2. Gout can result from a reduction in activity of which one of the following enzymes?   3. Lesch-Nyhan syndrome is due to an inability to catalyze which of the following reactions?   4. Allopurinol can be used to treat gout because of its ability to inhibit which one of the following reactions?   5. The regulation of ribonucleotide reductase is quite complex. Assuming that an enzyme deficiency leads to highly elevated lev-  els of dGTP, what effect would you predict on the reduction of ribonucleotides to deoxyribonucleotides under these condi-  tions?   (D) Reduced thioredoxin would become rate-limiting, thereby reducing the activity of ribonucleotide reductase.   (E) Deoxy-GTP would bind to the overall activity site and inhibit the functioning of the enzyme.    Fig. 42.1. Amino acid flux in sepsis and  trauma. In sepsis and traumatic injury, gluta-  mine and other amino acids are released from  skeletal muscle for uptake by tissues involved  in the immune response and tissue repair, such  as macrophages, lymphocytes, fibroblasts, and  the liver. Nitrogen excretion as urea and NH 4 +  results in negative nitrogen balance.   The body maintains a relatively large free amino acid pool in the blood, even  during fasting. As a result, tissues have continuous access to individual amino  acids for the synthesis of proteins and essential amino acid derivatives, such as  neurotransmitters. The amino acid pool also provides the liver with amino acid  substrates for gluconeogenesis and provides several other cell types with a source  of fuel. The free amino acid pool is derived from dietary amino acids and the  turnover of proteins in the body. During an overnight fast and during hypercata-  bolic states, degradation of labile protein, particularly that in skeletal muscle, is  the major source of free amino acids.   The liver is the major site of amino acid metabolism in the body and the major  site of urea synthesis. The liver is also the major site of amino acid degradation.  Hepatocytes partially oxidize most amino acids, converting the carbon skeleton to  glucose, ketone bodies, or C0 2 . Because ammonia is toxic, the liver converts most  of the nitrogen from amino acid degradation to urea, which is excreted in the  urine. The nitrogen derived from amino acid catabolism in other tissues is trans-  ported to the liver as alanine or glutamine and converted to urea.   The branched-chain amino acids, or BCAA (valine, isoleucine, and leucine) are  oxidized principally in skeletal muscle and other tissues and not in the liver. In  skeletal muscle, the carbon skeletons and some of the nitrogen are converted to glut-  amine, which is released into the blood. The remainder of the nitrogen is incorpo-  rated into alanine, which is taken up by the liver and converted to urea and glucose.   The formation and release of glutamine from skeletal muscle and other tissues  serves several functions. In the kidney, the NH 4 + carried by glutamine is excreted  into the urine. This process removes protons formed during fuel oxidation and  helps to maintain the body s pH, especially during metabolic acidosis. Glutamine  also provides a fuel for the kidney and gut. In rapidly dividing cells (e.g., lympho-  cytes and macrophages), glutamine is required as a fuel, as a nitrogen donor for  biosynthetic reactions, and as a substrate for protein synthesis.   During conditions of sepsis (the presence of various pathogenic organisms, or  their toxins, in the blood or tissues), trauma, injury, or burns, the body enters a  catabolic state characterized by a negative nitrogen balance (Fig. 42.1). Increased  net protein degradation in skeletal muscle increases the availability of glutamine  and other amino acids for cell division and protein synthesis in cells involved in  the immune response and wound healing. In these conditions, an increased release  of glucocorticoids from the adrenal cortex stimulates proteolysis.   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   Katta Bolic, a 62-year-old homeless woman, was found by a neighbor-  hood child who heard Katta's moans coming from an abandoned building.  The child's mother called the police, who took Katta to the hospital emer-  gency room. The patient was semicomatose, incontinent of urine, and her clothes  were stained with vomitus. She had a fever of 103°F, was trembling uncontrollably,  appeared to be severely dehydrated, and had marked muscle wasting. Her heart rate  was very rapid, and her blood pressure was low (85/46 mm Hg). Her abdomen was  distended and without bowel sounds. She responded to moderate pressure on her  abdomen with moaning and grimacing.   Blood was sent for a broad laboratory profile, and cultures of her urine, stool,  throat, and blood were taken. Intravenous glucose, saline, and parenteral broad-  spectrum antibiotics were begun. X-rays performed after her vital signs were stabi-  lized suggested a bowel perforation. These findings were compatible with a diagno-  sis of a ruptured viscus (e.g., an infected colonic diverticulum that perforated,  allowing colonic bacteria to infect the tissues of the peritoneal cavity, causing peri-  tonitis). Further studies confirmed that a diverticulum had ruptured, and appropriate  surgery was performed. All of the arterial blood cultures grew out Escherichia coli,  indicating that Katta also had a Gram-negative infection of her blood (septicemia)  that had been seeded by the proliferating organisms in her peritoneal cavity. Inten-  sive fluid and electrolyte therapy and antibiotic coverage were continued. The med-  ical team (surgeons, internists, and nutritionists) began developing a complex thera-  peutic plan to reverse Katta's severely catabolic state.   The body maintains a relatively large free amino acid pool in the blood, even in the  absence of an intake of dietary protein. The large free amino acid pool ensures the  continuous availability of individual amino acids to tissues for the synthesis of pro-  teins, neurotransmitters, and other nitrogen-containing compounds (Fig. 42.2). In a  normal, well-fed, healthy individual, approximately 300 to 600 g body protein is  degraded per day. At the same time, roughly 100 g protein is consumed in the diet  per day, which adds additional amino acids. From this pool, tissues use amino acids  for the continuous synthesis of new proteins (300-600 g) to replace those degraded.  The continuous turnover of proteins in the body makes the complete complement of  amino acids available for the synthesis of new and different proteins, such as anti-  bodies. Protein turnover allows shifts in the quantities of different proteins produced  in tissues in response to changes in physiologic state and continuously removes mod-  ified or damaged proteins. It also provides a complete pool of specific amino acids  that can be used as oxidizable substrates; precursors for gluconeogenesis and for  heme, creatine phosphate, purine, pyrimidine, and neurotransmitter synthesis; for  ammoniagenesis to maintain blood pH levels; and for numerous other functions.   The fasting state provides an example of the interorgan flux of amino acids neces-  sary to maintain the free amino acid pool in the blood and supply tissues with their  required amino acids (Fig. 42.3). During an overnight fast, protein synthesis in the  liver and other tissues continues, but at a diminished rate compared with the    The concentration of free amino  acids in the blood is not nearly as  rigidly controlled as blood glucose  levels. The free amino acid pool in the blood  is only a small part (0.5%) of the total amino  acid pool in whole body protein. Because of  the large skeletal muscle mass, approxi-  mately 80% of the body's total protein is in  skeletal muscle. Consequently, the concen-  tration of individual amino acids in the blood  is strongly affected by the rates of protein  synthesis and degradation in skeletal mus-  cle, as well as the rate of uptake and utiliza-  tion of individual amino acids for metabo-  lism in liver and other tissues. For the most  part, changes in the rate of protein synthesis  and degradation take place over a span of  hours.   What changes in hormone levels  and fuel metabolism occur during  an overnight fast?   ^"*Sl The hormonal changes that occur  £^ •) during an overnight fast include a  \ ^ decrease of blood insulin levels  and an increase of glucagon relative to lev-  els after a high-carbohydrate meal. Gluco-  corticoid levels also increase in the blood.  These hormones coordinate the changes of  fat, carbohydrate, and amino acid metabo-  lism. Fatty acids are released from adipose  triacylglycerols and are used as the major  fuel by heart, skeletal muscle, liver, and  other tissues. The liver converts some of the  fatty acids to ketone bodies. Liver glycogen  stores are diminished and gluconeogenesis  becomes the major support of blood glucose  levels for glucose-dependent tissues. The  major precursors of gluconeogenesis  include amino acids released from skeletal  muscle, lactate, and glycerol.   Fig. 42.2. Maintenance of the blood amino acid pool. Dietary protein (1) and degradation of  endogenous protein (2) provide a source of essential amino acids (those that cannot be syn-  thesized in the human). 3. The synthesis of new protein is the major use of amino acids from  the free amino acid pool. 4. Compounds synthesized from amino acid precursors are essential  for physiologic functions. Many of these compounds are degraded to N-containing urinary  metabolites and do not return to the free amino acid pool. 5. In tissues, the nitrogen is removed  from amino acids by transamination and deamination reactions. 6. The nitrogen from amino  acid degradation appears in the urine primarily as urea or NH 4 + , the ammonium ion. Ammo-  nia excretion is necessary to maintain the pH of the blood. 7. Amino acids are used as fuels  either directly or after being converted to glucose by gluconeogenesis. 8. Some amino acids  can be synthesized in the human, provided that glucose and a nitrogen source are available.   postprandial state. Net degradation of labile protein occurs in skeletal muscle  (which contains the body's largest protein mass) and other tissues.   The efflux of amino acids from skeletal muscle supports the essential amino acid  pool in the blood (see Fig. 42.3). Skeletal muscle oxidizes the BCAA (valine,  leucine, isoleucine) to produce energy and glutamine. The amino groups of the  BCAA, and of aspartate and glutamate, are transferred out of skeletal muscle in ala-  nine and glutamine. Alanine and glutamine account for approximately 50% of the  total a-amino nitrogen released by skeletal muscle (Fig. 42.4).   The release of amino acids from skeletal muscle is stimulated during an  overnight fast by the decrease of insulin and increase of glucocorticoid levels in the  blood (see Chapters 31 and 43). Insulin promotes the uptake of amino acids and the  general synthesis of proteins. The mechanisms for the stimulation of protein syn-  thesis in human skeletal muscle are not all known, but probably include an activa-  tion of the A system for amino acid transport (a modest effect), a general effect on  initiation of translation, and an inhibition of lysosomal proteolysis. The fall of blood  insulin levels during an overnight fast results in net proteolysis and release of amino  acids. As glucocorticoid release from the adrenal cortex increases, an induction of  ubiquitin synthesis and an increase of ubiquitin-dependent proteolysis also occur.   The major site of alanine uptake is the liver, which disposes of the amino nitrogen  by incorporating it into urea (see Fig. 42.3). The liver also extracts free amino acids,   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS    Fig. 42.3. Interorgan amino acid exchange after an overnight fast. After an overnight fast (the postabsorptive state), the utilization of amino acids  for protein synthesis, for fuels, and for the synthesis of essential functional compounds continues. The free amino acid pool is supported largely  by net degradation of skeletal muscle protein. Glutamine and alanine serve as amino group carriers from skeletal muscle to other tissues. Glut-  amine brings NH 4 + to the kidney for the excretion of protons and serves as a fuel for the kidney, gut, and cells of the immune system. Alanine  transfers amino groups from skeletal muscle, the kidney, and the gut to the liver, where they are converted to urea for excretion. The brain con-  tinues to use amino acids for neurotransmitter synthesis.   Fig. 42.4. Amino acid release from skeletal muscle. The arteriovenous difference (concentration in arterial blood minus the concentration in  venous blood) across the human forearm has been measured for many amino acids. This graph compares the amount of alanine, glutamine, and  BCAA released with their composition in the average protein. Alanine and glutamine represent a much higher percentage of total nitrogen  released than originally present in the degraded protein, evidence that they are being synthesized in the skeletal muscle. The BCAA (leucine,  valine, and isoleucine) are released in much lower amounts than those present in the degraded protein, evidence that they are being catabolized.  Aspartate and glutamate also contribute nitrogen to the formation of alanine and glutamine   Fig. 42.5. Hormonal regulation of hepatic  amino acid metabolism in the postabsorptive  state. Circled + = glucagon-mediated activa-  tion of enzymes or proteins; circled T = induc-  tion of enzyme synthesis mediated by  glucagon and glucocorticoids. Induction of  urea cycle enzymes occurs both during fasting  and after a high-protein meal. Because many  individuals in the United States normally have  a high-protein diet, the levels of urea cycle  enzymes may not fluctuate to any great extent.   The body normally produces  approximately 1 mmol of protons  per kilogram of body weight per  day. Nevertheless, the pH of the blood and  extracellular fluid is normally maintained  between 7.36 and 7.44. The narrow range is  maintained principally by the bicarbonate  (HC0 3 ~), phosphate (HP0 4 ~), and hemoglo-  bin buffering systems, and by the excretion  of an amount of acid equal to that produced.  The excretion of protons by the kidney  regenerates bicarbonate, which can be  reclaimed from the glomerular filtrate.   The acids are produced from normal fuel  metabolism. The major acid is carbonic acid,  which is formed from water and C0 2 pro-  duced in the TCA cycle and other oxidative  pathways. The oxidation of sulfur-containing  amino acids (methionine and cysteine) ulti-  mately produces sulfuric acid (H 2 S0 4 ), which  dissociates into 2H + + S0 4 2 ", and the protons  and sulfate are excreted. The hydrolysis of  phosphate esters produces the equivalent of  phosphoric acid. What other acids produced  during metabolism appear in the blood?    a-keto acids, and some glutamine from the blood. Alanine and other amino acids are  oxidized and their carbon skeletons converted principally to glucose. Glucagon and  glucocorticoids stimulate the uptake of amino acids into liver and increase gluconeo-  genesis and ureagenesis (Fig. 42.5). Alanine transport into the liver, in particular, is  enhanced by glucagon. The induction of the synthesis of gluconeogenic enzymes by  glucagon and glucocorticoids during the overnight fast correlates with an induction of  many of the enzymes of amino acid degradation (e.g., tyrosine aminotransferase) and  an induction of urea cycle enzymes (see Chapter 38). Urea synthesis also increases  because of the increased supply of NH 4 + from amino acid degradation in the liver.   Glucose, produced by the liver, is used for energy by the brain and other glucose-  dependent tissues, such as erythrocytes. The muscle, under conditions of exercise,  when the AMP- activated protein kinase is active, also oxidizes some of this glucose  to pyruvate, which is used for the carbon skeleton of alanine (the glucose-alanine  cycle; see Chapter 38).   Glutamine is generated in skeletal muscle from the oxidation of BCAA, and by  the lungs and brain for the removal of NH 4 + formed from amino acid catabolism  or entering from the blood. The kidney, the gut, and cells with rapid turnover rates  such as those of the immune system are the major sites of glutamine uptake (see  Fig. 42.3). Glutamine serves as a fuel for these tissues, as a nitrogen donor for  purine synthesis, and as a substrate for ammoniagenesis in the kidney. Much of  the unused nitrogen from glutamine is transferred to pyruvate to form alanine in  these tissues. Alanine then carries the unused nitrogen back to the liver.   The brain is glucose dependent, but, like many cells in the body, can use BCAA  for energy. The BCAA also provide a source of nitrogen for neurotransmitter syn-  thesis during fasting. Other amino acids released from skeletal muscle protein  degradation also serve as precursors of neurotransmitters.   The pattern of interorgan flux of amino acids is strongly affected by conditions that  change the supply of fuels (for example, the overnight fast, a mixed meal, a high-  protein meal) and by conditions that increase the demand for amino acids (meta-  bolic acidosis, surgical stress, traumatic injury, burns, wound healing, and sepsis).  The flux of amino acid carbon and nitrogen in these different conditions is dictated  by several considerations:   1. Ammonia (NH 3 ) is toxic. Consequently, it is transported between tissues as ala-  nine or glutamine. Alanine is the principal carrier of amino acid nitrogen from   Katta Bolic was in a severe stage of negative nitrogen balance on admission,  which was caused by both her malnourished state and her intra-abdominal  infection complicated by sepsis. The physiologic response to her advanced  catabolic status includes a degradation of muscle protein with the release of amino acids  into the blood. This release is coupled with an increased uptake of amino acids for "acute  phase" protein synthesis by the liver (systemic response) and other cells involved in the  immune response to general and severe infection.   ^The differences in amino acid metabolism between tissues are dictated by the  I types and amounts of different enzyme and transport proteins present in each  tissue and the ability of each tissue to respond to different regulatory messages  (hormones and neural signals).   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   other tissues back to the liver, where the nitrogen is converted to urea and subse-  quently excreted into the urine by the kidneys. The amount of urea synthesized is  proportional to the amount of amino acid carbon that is being oxidized as a fuel.   2. The pool of glutamine in the blood serves several essential metabolic functions  (Table 42.1). It provides ammonia for excretion of protons in the urine as NH 4 + . It  serves as a fuel for the gut, the kidney, and the cells of the immune system. Glut-  amine is also required by the cells of the immune system and other rapidly divid-  ing cells in which its amide group serves as the source of nitrogen for biosynthetic  reactions. In the brain, the formation of glutamine from glutamate and NH 4 + pro-  vides a means of removing ammonia and of transporting glutamate between dif-  ferent cell types within the brain. The utilization of the blood glutamine pool is pri-  oritized. During metabolic acidosis, the kidney becomes the predominant site of  glutamine uptake, at the expense of glutamine utilization in other tissues. Conver-  sly, during sepsis, in the absence of acidosis, cells involved in the immune response  (macrophages, hepatocytes) become the preferential sites of glutamine uptake.   3. The BCAA (valine, leucine, and isoleucine) form a significant portion of the  composition of the average protein and can be converted to tricarboxylic acid  (TCA) cycle intermediates and used as fuels by almost all tissues. They are also  the major precursors of glutamine. Except for the BCAA and alanine, aspartate,  and glutamate, the catabolism of amino acids occurs principally in the liver.   4. Amino acids are major gluconeogenic substrates, and most of the energy obtained  from their oxidation is derived from oxidation of the glucose formed from their car-  bon skeletons. A much smaller percentage of amino acid carbon is converted to  acetyl CoA or to ketone bodies and oxidized. The utilization of amino acids for glu-  cose synthesis for the brain and other glucose-requiring tissues is subject to the hor-  monal regulatory mechanisms of glucose homeostasis (see Chapters 31 and 36).   5. The relative rates of protein synthesis and degradation (protein turnover) deter-  mine the size of the free amino acid pools available for the synthesis of new pro-  teins and for other essential functions. For example, the synthesis of new proteins  to mount an immune response is supported by the net degradation of other pro-  teins in the body.   Amino sugars  Asparagine  Other compounds  Glutamate donor for synthesis of:  Glutathione  GABA  Ornithine  Arginine  Proline   The ability to convert 4-carbon  intermediates of the TCA cycle to  pyruvate is required for oxidation  of both BCAA and glutamine. This sequence  of reactions requires PEP carboxykinase, or  decarboxylating malate dehydrogenase  (malic enzyme). Most tissues have one, or  both, of these enzymes.   Lactic acid is produced from glu-  1 cose and amino acid metabolism.  The ketone bodies (acetoacetate  and p-hydroxybutyrate) produced  during fatty acid oxidation are also acids.  Many a-keto acids, formed from transamina-  tion reactions, are also found in the blood.   Because tissues differ in their physiologic functions, they have different amino acid  requirements and contribute differently to whole body nitrogen metabolism. How-  ever, all tissues share a common requirement for essential amino acids for protein  synthesis, and protein turnover is an ongoing process in all cells.   One of the primary roles of amino acid nitrogen is to provide ammonia in the kid-  ney for the excretion of protons in the urine. NH 4 + is released from glutamine by  glutaminase and by glutamate dehydrogenase, resulting in the formation of a-  ketoglutarate (Fig. 42.6). a-Ketoglutarate is used as a fuel by the kidney and is oxi-  dized to C0 2 , converted to glucose for use in cells in the renal medulla, or converted  to alanine to return ammonia to the liver for urea synthesis.   The rate of glutamine uptake from the blood and its utilization by the kidney  depends principally on the amount of acid that must be excreted to maintain a  normal pH in the blood. During a metabolic acidosis, the excretion of NH 4 + by the  kidney increases severalfold (Table 42.2). Because glutamine nitrogen provides    Fig. 42.6. Renal glutamine metabolism. Renal  tubule cells preferentially oxidize glutamine.  During metabolic acidosis, it is the major fuel  for the kidney. Conversion of glutamine to a-  ketoglutarate generates NH 4 + . Ammonium ion  excretion helps to buffer systemic acidemia.   H 2 0 1,000  S0 4 " 2 2-5  P0 4 " 2 2-5  K + 1-2   approximately two thirds of the NH 4 + excreted by the kidney, glutamine uptake by  the kidney also increases. Renal glutamine utilization for proton excretion takes  precedence over the requirements of other tissues for glutamine.   Ammonia increases proton excretion by providing a buffer for protons that are  transported into the renal tubular fluid (which is transformed into urine as it passes  through the tubules of the kidney) (Fig. 42.7). Specific transporters in the mem-  branes of the renal tubular cells transport protons from these cells into the tubular  lumen in exchange for Na + . The protons in the tubular fluid are buffered by   Glomerular filtrate  in the renal tubule  lumen: H 2 0, urea,   Fig. 42.7. Ammonia excretion by the kidney. Ammonia increases proton excretion by combining with a proton to form ammonium ion in the  renal tubular fluid, which is transformed into urine as it passes through the tubules of the kidney. As blood is filtered in the capillary bed of the  glomerulus, urea, sugars, amino acids, ions, and H 2 0 enter the renal tubular fluid (glomerular filtrate). As this fluid passes through a progression  of tubules (the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule, and the collecting duct) on its way to becoming urine,  various components are reabsorbed or added to the filtrate by the epithelial cells lining the tubules. Specific transporters in the membranes of the  renal tubule cells transport protons into the tubule lumen in exchange for Na + so that the glomerular filtrate becomes more acidic as it is trans-  formed into urine. The protons in the tubule fluid are buffered by phosphate, by bicarbonate, and by NH 3 . The ammonia, which is uncharged, is  able to diffuse through the membrane of the renal tubule cells into the urine. As it combines with a proton in the urine, it forms NH 4 + , which  cannot be transported back into the cells. The removal of protons as NH 4 + decreases the requirement for bicarbonate excretion to buffer the urine.   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   a Glucose used in the renal medulla is produced in the renal cortex.   phosphate, by bicarbonate, and by ammonia. Ammonia (NH 3 ), which is uncharged,  enters the urine by free diffusion through the cell membrane. As it combines with a  proton in the fluid, it forms ammonium ion (NH 4 + ), which cannot be transported  back into the cells and is excreted in the urine.   Glutamine is used as a fuel by the kidney in the normal fed state and, to a greater  extent, during fasting and metabolic acidosis (Table 42.3). The carbon skeleton forms  a-ketoglutarate, which is oxidized to C0 2 , converted to glucose, or released as the car-  bon skeleton of serine or alanine (Fig. 42.8). a-Ketoglutarate can be converted to  oxaloacetate by TCA cycle reactions, and oxaloacetate is converted to phospho-  enolpyruvate (PEP) by PEP carboxykinase. PEP can then be converted to pyruvate and  subsequently acetyl CoA, alanine, serine, or glucose. The glucose is used principally  by the cells of the renal medulla, which have a relatively high dependence on anaero-  bic glycolysis because of their lower oxygen supply and mitochondrial capacity. The  lactate released from anaerobic glycolysis in these cells is taken up and oxidized in the  renal cortical cells, which have a higher mitochondrial capacity and a greater blood  supply.   Fig. 42.8. Metabolism of glutamine and other fuels in the kidney. To completely oxidize glu-  tamate carbon to C0 2 , it must enter the TCA cycle as acetyl CoA. Carbon entering the TCA  cycle as a-Ketoglutarate (a-KG) exits as oxaloacetate and is converted to phosphoenolpyru-  vate (PEP) by PEP carboxykinase. PEP is converted to pyruvate, which may be oxidized to  acetyl CoA. PEP also can be converted to serine, glucose, or alanine. GDH = glutamate  dehydrogenase; PEPCK = phosphoenolpyruvate carboxykinase; TA = transaminase;  OAA = oxaloacetate.   When the carbon skeleton of ala-  nine is derived from glucose, the  efflux of alanine from skeletal mus-  cle and its uptake by liver provide no net  transfer of amino acid carbon to the liver for  gluconeogenesis. However, some of the ala-  nine carbon is derived from sources other  than glucose. Which amino acids can pro-  vide carbon for alanine formation? (Hint: See  Fig. 42.9.)   Skeletal muscle, because of its large mass, is a major site of protein synthesis and  degradation in the human. After a high-protein meal, insulin promotes the uptake of  certain amino acids and stimulates net protein synthesis. The insulin stimulation of  protein synthesis is dependent on an adequate supply of amino acids to undergo pro-  tein synthesis. During fasting and other catabolic states, a net degradation of skele-  tal muscle protein and release of amino acids occur (see Fig. 42.3). The net degra-  dation of protein affects functional proteins, such as myosin, which are sacrificed to  meet more urgent demands for amino acids in other tissues. During sepsis, degra-  dation of skeletal muscle protein is stimulated by the glucocorticoid Cortisol. The  effect of Cortisol is exerted through the activation of ubiquitin-dependent proteoly-  sis. During fasting, the decrease of blood insulin levels and the increase of blood  Cortisol levels increase net protein degradation.   Skeletal muscle is a major site of glutamine synthesis, thereby satisfying the  demand for glutamine during the postabsorptive state, during metabolic acidosis,  and during septic stress and trauma. The carbon skeleton and nitrogen of glutamine  are derived principally from the metabolism of BCAA. Amino acid degradation in  skeletal muscle is also accompanied by the formation of alanine, which transfers  amino groups from skeletal muscle to the liver in the glucose-alanine cycle.   The BCAA play a special role in muscle and most other tissues because they are the  major amino acids that can be oxidized in tissues other than the liver. However, all  tissues can interconvert amino acids and TCA cycle intermediates through transam-  inase reactions, i.e., alanine <-» pyruvate, aspartate <-» oxaloacetate, and a-ketoglu-  tarate <-» glutamate. The first step of the pathway, transamination of the BCAA to  a-keto acids, occurs principally in brain, heart, kidney, and skeletal muscles. These  tissues have a high content of BCAA transaminase relative to the low levels in liver.  The a-keto acids of the BCAA are then either released into the blood and taken up  by liver, or oxidized to C0 2 or glutamine within the muscle or other tissue  (Fig. 42.9). They can be oxidized by all tissues that contain mitochondria.   The oxidative pathways of the BCAA convert the carbon skeleton to either suc-  cinyl CoA or acetyl CoA (see Chapter 39 and Fig. 42.9). The pathways generate  NADH and FAD(2H) for ATP synthesis before the conversion of carbon into inter-  mediates of the TCA cycle, thus providing the muscle with energy without loss of  carbon as C0 2 . Leucine is "ketogenic" in that it is converted to acetyl CoA and ace-  toacetate. Skeletal muscle, adipocytes, and most other tissues are able to use these  products and, therefore, directly oxidize leucine to C0 2 . The portion of isoleucine  converted to acetyl CoA is also oxidized directly to C0 2 . For the portion of valine  and isoleucine that enters the TCA cycle as succinyl CoA to be completely oxidized  to C0 2 , it must first be converted to acetyl CoA. To form acetyl CoA, succinyl CoA  is oxidized to malate in the TCA cycle, and malate is then converted to pyruvate by  malic enzyme (malate + NADP + -> pyruvate + NADPH + H + ) (see Fig. 42.9).  Pyruvate can then be oxidized to acetyl CoA. Alternatively, pyruvate can form ala-  nine or lactate.   The major route of valine and isoleucine catabolism in skeletal muscle is to enter  the TCA cycle as succinyl CoA and exit as a-ketoglutarate to provide the carbon  skeleton for glutamine formation (see Fig. 42.9). Some of the glutamine and C0 2  that is formed from net protein degradation in skeletal muscle may also arise from   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   Fig. 42.9. Metabolism of the carbon skeletons of BCAA in skeletal muscle. 1. The first step  in the metabolism of BCAA is transamination (TA). 2. Carbon from valine and isoleucine  enters the TCA cycle as succinyl CoA and is converted to pyruvate by decarboxylating  malate dehydrogenase (malic enzyme). 3. The oxidative pathways generate NADH and  FAD(2H) even before the carbon skeleton enters the TCA cycle. The rate-limiting enzyme in  the oxidative pathways is the a-keto acid dehydrogenase complex. The carbon skeleton also  can be converted to glutamate and alanine, shown in blue.   ®Some of the alanine released from  skeletal muscle is derived directly  from protein degradation. The car-  bon skeletons of valine, isoleucine, aspar-  tate, and glutamate, which are converted to  malate and oxaloacetate in the TCA cycle,  can be converted to pyruvate and subse-  quently transaminated to alanine. The extent  to which these amino acids contribute car-  bon to alanine efflux differs between differ-  ent types of muscles in the human. These  amino acids also may contribute to alanine  efflux from the gut.   the carbon skeletons of aspartate and glutamate. These amino acids are transami-  nated and become part of the pool of 4-carbon intermediates of the TCA cycle.   Glutamine nitrogen is derived principally from the BCAA (Fig. 42.10). The  a-amino group arises from transamination reactions that form glutamate from  a-ketoglutarate, and the amide nitrogen is formed from the addition of free ammo-  nia to glutamate by glutamine synthetase. Free ammonia in skeletal muscle arises  principally from the deamination of glutamate by glutamate dehydrogenase or from  the purine nucleotide cycle.   In the purine nucleotide cycle (Fig. 42.11), the deamination of AMP to IMP  releases NH 4 + . AMP is resynthesized with amino groups provided from aspartate.  The aspartate amino groups can arise from the BCAA through transamination reac-  tions. The fumarate can be used to replenish TCA cycle intermediates.   The nitrogen arising from the oxidation of BCAA in skeletal muscle can also be  transferred back to the liver as alanine in the glucose-alanine cycle (Fig. 42.12, see  also Fig. 41.13). The amino group of the BCAA is first transferred to a-ketoglutarate   The purine nucleotide cycle is  KJ ] found in skeletal muscle and brain  *r but is absent in liver and many  other tissues. One of its functions in skeletal  muscle is to respond to the rapid utilization  of ATP during exercise. During exercise, the  rapid hydrolysis of ATP increases AMP lev-  els, resulting in an activation of AMP deami-  nase (see Fig. 42.11). As a consequence, the  cellular concentration of IMP increases and  ammonia is generated. IMP, like AMP, acti-  vates muscle glycogen phosphorylase dur-  ing exercise (see Chapter 22). The ammonia  that is generated may help to buffer the  increased lactic acid production occurring in  skeletal muscles during strenuous exercise.   Fig. 42.10. Formation of glutamine from the amino groups of BCAA. The BCAA are first  transaminated with a-ketoglutarate to form glutamate and the branched chain a-keto acids.  The glutamate nitrogen can then follow either of two paths leading to glutamine formation.  TA = transamination; OAA = oxaloacetate; a-KG = a-ketoglutarate.   to form glutamate and then transferred to pyruvate to form alanine by sequential  transamination reactions. The pyruvate arises principally from glucose via the gly-  colytic pathway. The alanine released from skeletal muscle is taken up principally  by the liver, where the amino group is incorporated into urea, and the carbon skele-  ton can be converted back to glucose through gluconeogenesis. Although the  amount of alanine formed varies with dietary intake and physiologic state, the  transport of nitrogen from skeletal muscle to liver as alanine occurs almost contin-  uously throughout our daily fasting-feeding cycle.   Fig. 42.11. Purine nucleotide cycle. In skele-  tal muscle, the purine nucleotide cycle can  convert the amino groups of the BCAA to  NH 3 , which is incorporated into glutamine.  The compounds containing the amino group  released in the purine nucleotide cycle are  shown in blue.   Amino acids are an important fuel for the intestinal mucosal cells after a protein-con-  taining meal and in catabolic states such as fasting or surgical trauma (Fig. 42. 13). Dur-  ing fasting, glutamine is one of the major amino acids used by the gut. The principal  fates of glutamine carbon in the gut are oxidation to C0 2 and conversion to the carbon    Fig. 42.12. Glucose-alanine cycle. The pathway for transfer of the amino groups from  BCAA in skeletal muscle to urea in the liver is shown in blue.   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   Fig. 42.13. Amino acid metabolism in the gut. The pathways of glutamine metabolism in the  gut are the same whether it is supplied by the diet (postprandial state) or from the blood  (postabsorptive state). Cells of the gut also metabolize aspartate, glutamate, and BCAA. Glu-  cose is converted principally to the carbon skeleton of alanine. a-KG = a-ketoglutarate;  GDH = glutamate dehydrogenase; TA = transaminase.   skeletons of lactate, citrulline, and ornithine. The gut also oxidizes BCAA. Nitrogen  derived from amino acid degradation is converted to citrulline, alanine, NH 4 + , and  other compounds that are released into the blood and taken up by the liver. Although  most of the carbon in this alanine is derived from glucose, the oxidation of glucose to  C0 2 is not a major fuel pathway for the gut. Fatty acids are also not a significant source  of fuel for the intestinal mucosal cells, although they do use ketone bodies.   After a protein meal, dietary glutamine is a major fuel for the gut, and the prod-  ucts of glutamine metabolism are similar to those seen in the postabsorptive state.  The gut also uses dietary aspartate and glutamate, which enter the TCA cycle.  Colonocytes (the cells of the colon) also use short-chain fatty acids, derived from  bacterial action in the lumen.   The importance of the gut in whole body nitrogen metabolism arises from the  high rate of division and death of intestinal mucosal cells and the need to continu-  ously provide these cells with amino acids to sustain the high rates of protein syn-  thesis required for cellular division. Not only are these cells important for the uptake  of nutrients, but they maintain a barrier against invading bacteria from the gut lumen  and are, therefore, part of our passive defense system. As a result of these important  functions, the intestinal mucosal cells are supplied with the amino acids required for  protein synthesis and fuel oxidation at the expense of the more expendable skeletal  muscle protein.   The intestine contains the enzymes  \m ) for the urea cycle, but the V max for  argininosuccinate synthetase and  argininosuccinate lyase are very low, sug-  gesting that the primary role of the urea  cycle enzymes in the gut is to produce cit-  rulline from the carbons of glutamine (gluta-  mine — » glutamate — » glutamate semialde-  hyde — » ornithine — > citrulline). The citrulline  is released in the circulation for use by the  liver.   Glutamine utilization by the gut is  \ \ ) diminished by a metabolic acidosis  *S compared with the postabsorptive  or postprandial states. During metabolic aci-  dosis, the uptake of glutamine by the kidney  is increased, and blood glutamine levels  decrease. As a consequence, the gut takes  up less glutamine.   The liver is the major site of amino acid metabolism. It is the major site of amino  acid catabolism and converts most of the carbon in amino acids to intermediates of  the TCA cycle or the glycolytic pathway (which can be converted to glucose or oxi-  dized to C0 2 ), or to acetyl CoA and ketone bodies. The liver is also the major site  for urea synthesis. It can take up both glutamine and alanine and convert the   nitrogen to urea for disposal (see Chapter 38). Other pathways in the liver provide  it with an unusually high amino acid requirement. The liver synthesizes plasma pro-  teins, such as serum albumin, transferrin, and the proteins of the blood coagulation  cascade. It is a major site for the synthesis of nonessential amino acids, the conju-  gation of xenobiotic compounds with glycine, the synthesis of heme and purine  nucleotides, and the synthesis of glutathione.   A major function of amino acid metabolism in neural tissue is the synthesis of neu-  rotransmitters. More than 40 compounds are believed to function as neurotransmit-  ters, and all of these contain nitrogen derived from precursor amino acids. They  include amino acids, which are themselves neurotransmitters (e.g., glutamate,  glycine), the catecholamines derived from tyrosine (dopamine and norepinephrine),  serotonin (derived from tryptophan), GAB A (derived from glutamate), acetyl-  choline (derived from choline synthesized in the liver and acetyl Co A), and many  peptides. In general, neurotransmitters are formed in the presynaptic terminals of  axons and stored in vesicles until released by a transient change in electrochemical  potential along the axon. Subsequent catabolism of some of the neurotransmitter  results in the formation of a urinary excretion product. The rapid metabolism of  neurotransmitters requires the continuous availability of a precursor pool of amino  acids for de novo neurotransmitter synthesis (see Chapter 47).   The brain is a net glutamine producer owing principally to the presence of gluta-  mine synthetase in astroglial cells (see Chapter 47). Glutamate and aspartate are  synthesized in these cells, using amino groups donated by the BCAA (principally  valine) and TCA cycle intermediates formed from glucose and from the carbon  skeletons of BCAA (Fig. 42.14) The glutamate is converted to glutamine by gluta-  mine synthetase, which incorporates NH 4 + released from deamination of amino  acids and deamination of AMP in the purine nucleotide cycle in the brain. This glu-  tamine may efflux from the brain, carrying excess NH 4 + into the blood, or serve as  a precursor of glutamate in neuronal cells.   Fig. 42.14. Role of glutamine in the brain. Glutamine serves as a nitrogen transporter in  the brain for the synthesis of many different neurotransmitters. Different neurons convert  glutamine to y-aminobutyric acid (GABA) or to glutamate. Glutamine also transports  excess NH 4 + from the brain into the blood. BCKA = branched-chain keto acids; a-KG =  a-ketoglutarate.   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   Glutamine synthesized in the astroglial cells is a precursor of glutamate (an exci-  tatory neurotransmitter) and GABA (an inhibitory neurotransmitter) in the neuronal  cells (see Fig. 42.14). It is converted to glutamate by a neuronal glutaminase  isozyme. In GABAergic neurons, glutamate is then decarboxylated to GABA,  which is released during excitation of the neuron. GABA is one of the neurotrans-  mitters that is recycled; a transaminase converts it to succinaldehyde, which is then  oxidized to succinate. Succinate enters the TCA cycle.   III. CHANGES IN AMINO ACID METABOLISM WITH  DIETARY AND PHYSIOLOGIC STATE   The rate and pattern of amino acid utilization by different tissues change with  dietary and physiologic state. Two such states, the postprandial period following a  high-protein meal and the hypercatabolic state produced by sepsis or surgical  trauma, differ from the postabsorptive state with respect to the availability of amino  acids and other fuels and the levels of different hormones in the blood. As a result,  the pattern of amino acid utilization is somewhat different.   After the ingestion of a high-protein meal, the gut and the liver use most of the  absorbed amino acids (Fig. 42.15). Glutamate and aspartate are used as fuels by the  gut, and very little enters the portal vein. The gut also may use some BCAA. The  liver takes up 60 to 70% of the amino acids present in the portal vein. These amino  acids, for the most part, are converted to glucose in the gluconeogenic pathway.   After a pure protein meal, the increased levels of dietary amino acids reaching  the pancreas stimulate the release of glucagon above fasting levels, thereby increas-  ing amino acid uptake into liver and stimulating gluconeogenesis. Insulin release is  also stimulated, but not nearly to the levels found after a high-carbohydrate meal.   During hyperammonemia, ammo-  \m ) n 'a (NH 3 ) can diffuse into the brain  from the blood. The ammonia is  able to inhibit the neural isozyme of glutam-  inase, thereby decreasing additional ammo-  nia formation in the brain and inhibiting the  formation of glutamate and its subsequent  metabolism to GABA. This effect of ammo-  nia might contribute to the lethargy associ-  ated with the hyperammonemia found in  patients with hepatic disease.   The levels of transthyretin (binds  to vitamin A and thyroid hormones  in the blood) and serum albumin in  the blood may be used as indicators of the  degree of protein malnutrition. In the  absence of hepatic disease, decreased levels  of these proteins in the blood indicate insuf-  ficient availability of amino acids to the liver  for synthesis of serum proteins.   In what ways does liver metabo-  lism after a high-protein meal  resemble liver metabolism in the  fasting state?   y^-^v Both of these dietary states are  /\ # ) characterized by an elevation of  glucagon. Glucagon stimulates  amino acid transport into the liver, stimu-  lates gluconeogenesis through decreasing  levels of fructose 2,6-bisphosphate, and  induces the synthesis of enzymes in the urea  cycle, the gluconeogenic pathway, and the  pathways for degradation of some of the  amino acids.   Q o The Atkins high-protein diet is  / /^oA ) based on the premise that ingest-  ™ ing high-protein, low-carbohydrate  meals will keep circulating insulin levels low,  such that energy storage is not induced,  and glucagon release will point the  insulin/glucagon ratio to energy mobiliza-  tion, particularly fatty acid release from the  adipocyte and oxidation by the tissues. The  lack of energy storage, coupled with the loss  of fat, leads to weight loss.   In general, the insulin released after a high-protein meal is sufficiently high that the  uptake of BCAA into skeletal muscle and net protein synthesis is stimulated, but  gluconeogenesis in the liver is not inhibited. The higher the carbohydrate content of  the meal, the higher the insulin/glucagon ratio and the greater the shift of amino  acids away from gluconeogenesis into biosynthetic pathways in the liver such as the  synthesis of plasma proteins.   Most of the amino acid nitrogen entering the peripheral circulation after a high-  protein meal or a mixed meal is present as the BCAA. Because the liver has low  levels of transaminases for these amino acids, it cannot oxidize them to a significant  extent, and they enter the systemic circulation. The BCAA are slowly taken up by  skeletal muscle and other tissues. These peripheral nonhepatic tissues use the amino  acids derived from the diet principally for net protein synthesis.   Surgery, trauma, burns, and septic stress are examples of hypercatabolic states char-  acterized by increased fuel utilization and a negative nitrogen balance (Fig. 42.16).  The mobilization of body protein, fat, and carbohydrate stores serves to maintain  normal tissue function in the presence of a limited dietary intake, as well as to sup-  port the energy and amino acid requirements for the immune response and wound  healing. The negative nitrogen balance that occurs in these hypercatabolic states   Fig. 42.16. Negative nitrogen balance during infection. The effects of experimentally  induced infections on nitrogen balance were determined in human volunteers. After inocula-  tion with sandfly fever, increased amino acid catabolism produced a negative nitrogen bal-  ance. A few days after exposure, the daily nitrogen balance became positive until the volun-  teers returned to their original state. Experiments with patients exposed to tularemia showed  that the negative nitrogen balance was much larger than could be expected from a decreased  appetite alone. Volunteers who ate the same amount of food as the infected individuals (pair-  fed nonexposed controls) had a much smaller cumulative negative nitrogen balance than the  infected volunteers. From Beisel WR. Am J Clin Nutr 1977;30:1236-1247. © 1977 American  Society for Clinical Nutrition.   CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   results from an accelerated protein turnover and an increased rate of net protein  degradation, primarily in skeletal muscle.   The catabolic state of sepsis (acute, generalized, febrile infection) is one of  enhanced mobilization of fuels and amino acids to provide the energy and precur-  sors required by cells of the immune system, host defense mechanisms, and wound  healing. The amino acids must provide the substrates for new protein synthesis and  cell division. Glucose synthesis and release are enhanced to provide fuel for these  cells, and the patient may become mildly hyperglycemic.   In these hypercatabolic states, skeletal muscle protein synthesis decreases, and  protein degradation increases. Oxidation of BCAA is increased and glutamine pro-  duction enhanced. Amino acid uptake is diminished. Cortisol is the major hormonal  mediator of these responses, although certain cytokines may also have direct effects  on skeletal muscle metabolism. As occurs during fasting and metabolic acidosis,  increased levels of Cortisol stimulate ubiquitin-mediated proteolysis, induce the  synthesis of glutamine synthetase, and enhance release of amino acids and gluta-  mine from the muscle cells.   The amino acids released from skeletal muscle during periods of hypercatabolic  stress are used in a prioritized manner, with the cellular components of the immune  system receiving top priority. For example, the uptake of amino acids by the liver  for the synthesis of acute phase proteins, which are part of the immune system, is  greatly increased. Conversly, during the early phase of the acute response, the syn-  thesis of other plasma proteins (e.g., albumin) is decreased. The increased avail-  ability of amino acids and the increased Cortisol levels also stimulate gluconeogen-  esis, thereby providing fuel for the glucose-dependent cells of the immune system  (e.g., lymphocytes). An increase of urea synthesis accompanies the acceleration of  amino acid degradation.   The increased efflux of glutamine from skeletal muscle during sepsis serves sev-  eral functions (see Fig. 42.1). It provides the rapidly dividing cells of the immune  system with an energy source. Glutamine is available as a nitrogen donor for purine  synthesis, for NAD + synthesis, and for other biosynthetic functions essential to  growth and division of the cells. An increased production of metabolic acids may  accompany stress such as sepsis, so there is an increased utilization of glutamine by  the kidney.   Under the influence of elevated levels of glucocorticoids, epinephrine, and  glucagon, fatty acids are mobilized from adipose tissue to provide alternate fuels for  other tissues and spare glucose. Under these conditions, fatty acids are the major  energy source for skeletal muscle, and glucose uptake is decreased. These changes  may lead to a mild hyperglycemia.    The degree of the body's hyper-  catabolic response depends on the  severity and duration of the trauma  or stress. After an uncomplicated surgical  procedure in an otherwise healthy patient,  the net negative nitrogen balance may be  limited to about 1 week. The mild nitrogen  losses are usually reversed by dietary pro-  tein supplementation as the patient recov-  ers. With more severe traumatic injury or  septic stress, the body may catabolize body  protein and adipose tissue lipids for a pro-  longed period, and the negative nitrogen  balance may not be corrected for weeks.   Katta Bolic's severe negative  nitrogen balance was caused by  both her malnourished state and  her intra-abdominal infection complicated  by sepsis. The systemic and diverse  responses the body makes to insults such as  an acute febrile illness are termed the "acute  phase response." An early event in this  response is the stimulation of phagocytic  activity (see Fig. 42.17). Stimulated  macrophages release cytokines, which are  regulatory proteins that stimulate the  release of Cortisol, insulin, and growth hor-  mone. Cytokines also directly mediate the  acute phase response of the liver and skele-  tal muscle to sepsis.   ^ mining whether several unique acute phase proteins are being secreted by  the liver. C-reactive protein, so named because of its ability to interact with the C-  polysaccharide of pneumococci, and serum amyloid A protein, a precursor of the  amyloid fibril found in secondary amyloidosis, are elevated in patients undergoing  the acute phase response and as compared with healthy individuals. Other proteins  normally found in the blood of healthy individuals are present in increased concen-  trations in patients undergoing an acute phase response. These include haptoglobin,  certain protease inhibitors, complement components, ceruloplasmin, and fibrino-  gen. The elevated concentration of these proteins in the blood increases the ery-  throcyte sedimentation rate (ESR), another laboratory measure of the presence of an  acute phase response.   To determine the ESR, the patients' blood is placed vertically in a small-bore  glass tube. The speed with which the red blood cells sediment toward the bottom of  the tube depends on what percentage of the red blood cells clump together and,  thereby, become more dense. The degree of clumping is directly correlated with the  presence of one or more of the first-phase proteins listed previously. These proteins  interfere with what is known as the zeta-potential of the red blood cells, which nor-  mally prevents the red blood cells from clumping. Because many different proteins  can individually alter the zeta-potential, the ESR is a nonspecific test for the pres-  ence of acute inflammation.   The weight loss often noted in septic patients is primarily caused by a loss of  appetite resulting from the effect of certain cytokines on the medullary appetite cen-  ter. Other causes include increased energy expenditure from fever and enhanced  muscle proteolysis.   After a catabolic insult such as injury, trauma, infection, or cancer, the  interorgan flow of glutamine and fuels is dramatically altered. Teleologi-  cally, the changes in metabolism that occur give first priority to cells that  are part of the immune system. Evidence suggests that the changes in glutamine and    Fig. 42.17. Cytokines and hormones mediate amino acid flux during sepsis. Bacterial prod-  ucts act on macrophages to stimulate the release of cytokines and on the brain to stimulate the  sympathoadrenal response. The result is a stimulation of the release of the insulin counter-  regulatory hormones, epinephrine, glucagon, and glucocorticoids. The glucocorticoid Cortisol  may be the principal mediator of net muscle protein degradation during sepsis. Hepatic pro-  tein synthesis, particularly that of acute phase proteins, is stimulated both by Cortisol and  cytokines. Amino acid metabolism in the gut is also probably affected by glucocorticoids and  cytokines. Because of the release of the counterregulatory hormones, muscle and other tissues  become resistant to insulin action, as indicated by the bar on the figure. Adapted with per-  mission from Fisher J. Am J Surg 1991; 161:270.    CHAPTER 42 / INTERTISSUE RELATIONSHIPS IN THE METABOLISM OF AMINO ACIDS   fuel metabolism are mediated by the insulin counterregulatory hormones, such as  Cortisol and epinephrine, and several different cytokines (see Chapter 11 for a  review of cytokines). Cytokines appear to play a more important role than hormones  during sepsis, although they exert their effects, in part, through hormones (Fig. 42.17).  Although cytokines can be released from a variety of cells, macrophages are the  principal source during trauma and sepsis.   Two cytokines that are important in sepsis are interleukin-1 (IL-1) and tumor  necrosis factor (TNF). IL-1 and TNF affect amino acid metabolism both through  regulation of the release of glucocorticoids and through direct effects on tissues.  Although cytokines are generally considered to be paracrine, with their effects  being exerted over cells in the immediate vicinity, TNF and IL-1 increase in the  blood during sepsis. Other cytokines, such as IL-6, also may be involved.   During sepsis, TNF, IL-1, and possibly other cytokines, bacterial products, or  mediators act on the brain to stimulate the release of glucocorticoids from the adre-  nal cortex (a process mediated by adrenocorticotropic hormone [ACTH]), epineph-  rine from the adrenal medulla, and both insulin and glucagon from the pancreas.  Although insulin is elevated during sepsis, the tissues exhibit an insulin resistance  that is similar to that of the non-insulin-dependent diabetes mellitus patient, possi-  bly resulting from the elevated levels of the insulin counterregulatory hormones  (glucocorticoids, epinephrine, and glucagon). Changes in the rate of acute phase  protein synthesis are mediated, at least in part, by effects of TNF, IL-1, and IL-6 on  the synthesis of groups of proteins in the liver.   Hypercatabolic states may be  \ \ ) accompanied by varying degrees   of insulin resistance caused, in  part, by the release of counterregulatory  hormones into the blood. Thus, patients  with diabetes mellitus may require higher  levels of exogenous insulin during sepsis.   Abcouwer SF, Bode BP, Souba WW. Glutamine as a metabolic intermediate. In: Fischer JE, ed. Nutri-  tion and Metabolism of the Surgical Patient. 2nd Ed. Boston: Little, Brown & Company,  1996:353-384.   Abumrad NN, Williams P, Frexes-Steed M, et al. Inter-organ metabolism of amino acids in vivo. Dia-  betes Metabol Rev 1989;5:213-226.   Bessey PQ. Metabolic response to trauma and infection. In: Fischer JE, ed. Nutrition and Metabolism of  the Surgical Patient. 2nd Ed. Boston: Little, Brown & Company, 1996:1577-1600.   Fischer J, Hasselgren P-O. Cytokines and glucocorticoids in the regulation of the "hepato- skeletal mus-  cle axis" in sepsis. Am J Surg 1991:161:266-271.   Newsholme P, Procopio J, Lima, MMR, Pithon-Curu TC, Cori R. Glutamine and glutamate — their cen-  tral role in cell metabolism and function. Cell Biochem Funct 2003;21:1-9.   Wolfe RR. Effects of insulin on muscle tissue. Curr Opin Clin Nutr Metab Care 2000;3:67-71.   1. Which of the profiles indicated below would occur within 2 hours after eating a meal very high in protein and low in  carbohydrates?   2. The gut uses glutamine as an energy source, but can also secrete citrulline, synthesized from the carbons of glutamine. Which  of the following compounds is an obligatory intermediate in this conversion (consider only the carbon atoms of glutamine  while answering this question)?   3. The signal that indicates to muscle that protein degradation needs to be initiated is which of the following?   4. The skeletal muscles convert BCAA carbons to glutamine for export to the rest of the body. An obligatory intermediate, which  carries carbons originally from the BCAA, in the conversion of BCAA to glutamine, is which of the following?   5. An individual in sepsis would display which of the following metabolic patterns?   Although many of the pathways described previously are present in all  tissues of the body, many tissues also carry out specialized functions  and contain unique biochemical pathways. This section describes  a number of such tissues and, in some cases, how the tissues inter-  act with the rest of the body to coordinate their functions.  The previous chapters of this text have focused primarily on  insulin and glucagon as the major mediators for regulating metabolic pathways;  however, a large number of other hormones also regulate the storage and utilization  of metabolic fuels (see Chapter 43). These hormones primarily counteract the  effects of insulin and are called counter-regulatory hormones. They include growth  hormone; thyroid hormone; glucocorticoids, such as Cortisol; small peptides, such  as the somatostatins; and small molecules, such as the catecholamines. Growth hor-  mone works, in part, by inducing the synthesis of the insulin-like growth factors.  These hormones can exert their effects rapidly (through covalent modification of  selected enzymes) or long-term (through alterations in the rate of synthesis of  selected enzymes). The interplay of these hormones with insulin and glucagon is  discussed, as are the synthesis, secretion, and conditions leading to secretion of each  hormone.   The proteins and cells in the blood form their own tissue system (see Chapter  44). All blood cells are derived from a common precursor, the stem cell, in the bone  marrow. Different cytokine signals trigger differentiation of a particular blood cell  lineage. For example, when there is a decreased supply of oxygen to the tissues, the  kidney responds by releasing erythropoietin. This hormone specifically stimulates  the production of red blood cells.   The red blood cell has limited metabolic functions, owing to its lack of internal  organelles. Its main function is to deliver oxygen to the tissues through the binding  of oxygen to hemoglobin. When the number of red blood cells is reduced, an anemia  is said to have developed. This can be attributable to many causes, including nutri-  tional deficiencies or mutations (hereditary anemias). The morphology of the red  blood cell can sometimes aid in distinguishing between the various types of anemia.   Red blood cell metabolism is geared toward preserving the ability of these cells  to transport oxygen, as well as to regulate oxygen binding to hemoglobin. Glycol-  ysis provides energy and NADH to protect the oxidation state of the heme-bound  iron. The hexose monophosphate shunt pathway generates NADPH to protect red  blood cell membranes from oxidation. Heme synthesis, which uses succinyl CoA  and glycine for all of the carbon and nitrogen atoms in the structure, occurs in the  precursors of red blood cells. Inherited defects in heme synthesis lead to a class of  diseases known as the porphyrias. Because the red blood cell normally passes  through the very narrow capillaries, its membrane must be easily deformable. This  deformability is, in part, attributable to the complex cytoskeletal structure that sur-  rounds the erythrocyte. Mutations in these structural proteins can lead to less  deformable cells. This, in turn, can result in a hemolytic anemia.   Among other functions, the hematologic system is responsible for hemostasis as  well as for maintaining a constant blood volume (see Chapter 45). A tear in the wall   of a vessel can lead to blood loss, which, when extensive, can be fatal. Repairing ves-  sel damage, whether internal or external, is accomplished by a complicated series of  zymogen activations of circulating blood proteins resulting in the formation of a fibrin  clot (the coagulation cascade). Platelets play a critical role in hemostasis not only  through their release of procoagulants but through their ability to form aggregates  within the thrombus (clot) as well. Clots function as a plug, allowing vessel walls to  repair and preventing further blood loss. Conversely, inappropriate clot formation in  vessels that supply blood to vital organs or tissues can have devastating consequences,  such as an acute cerebral or myocardial infarction. Because clotting must be tightly con-  trolled, intricate mechanisms exist that regulate this important hematologic function.   The liver is an altruistic organ that provides multiple services for other tissues (see  Chapter 46). It supplies glucose and ketone bodies to the rest of the body when fuel  stores are limiting. It disposes of ammonia as urea when amino acid degradation  occurs. It is the site of detoxification of xenobiotics, and it synthesizes many of the  proteins found in the blood. The liver synthesizes fatty acids and cholesterol and dis-  tributes them to other tissues in the form of very-low-density lipoprotein (VLDL).  The liver also synthesizes bile acids for fat digestion in the intestine. The liver recy-  cles cholesterol and triglyceride through its uptake of intermediate density lipopro-  tein (IDL), chylomicron and VLDL remnants, and low-density lipoprotein (LDL)  particles. Because of its protective nature and its strategic location between the gut  and the systemic circulation, the liver has "first crack" at all compounds that enter  the blood through the enterohepatic circulation. Thus, xenobiotic compounds can be  detoxified as they enter the liver before they have a chance to reach other tissues.   Muscle cells contain unique pathways that allow them to store energy as creatine  phosphate and to closely regulate their use of fatty acids as an energy source (see  Chapter 47). The adenosine monophosphate (AMP) -activated protein kinase is an  important regulator of muscle energy metabolism. Muscle is comprised of different  types of contractile fibers that derive their energy from different sources. For exam-  ple, the slow-twitch type I fibers use oxidative energy pathways, whereas the type  II fast-twitch fibers use the glycolytic pathway for their energy requirements.   The nervous system consists of various cell types that are functionally intercon-  nected so as to allow efficient signal transmission throughout the system (see Chapter  48). The cells of the central nervous system are protected from potentially toxic com-  pounds by the blood-brain barrier, which restricts entry of compounds into the nerv-  ous system (ammonia, however, is a notable exception). The brain cells communicate  with each other and with other organs, through the synthesis of neurotransmitters and  neuropeptides. Many of the neurotransmitters are derived from amino acids, most of  which are synthesized within the nerve cell. Because the pathways of amino acid and  neurotransmitter biosynthesis require cofactors (such as pyridoxal phosphate, thi-  amine pyrophosphate, and vitamin B12), deficiencies of these cofactors can lead to  neuropathies (dysfunction of specific neurons within the nervous system).   Because of the restrictions imposed by the blood-brain barrier, the brain also  must synthesize its own lipids. An adequate supply of lipids is vital to the central  nervous system because they are constituents of the myelin sheath that surrounds  the neurons and allows them to conduct impulses normally. The neurodegenerative  disorders, such as multiple sclerosis, are a consequence of varying degrees of  demyelination of the neurons.   Connective tissue, which consists primarily of fibroblasts, produces extracellular  matrix materials that surround cells and tissues, determining their appropriate posi-  tion within the organ (see Chapter 49). These materials include structural proteins  (collagen and elastin), adhesive proteins (fibronectin), and glycosaminoglycans  (heparan sulfate, chondroitin sulfate). The unique structures of the proteins and car-  bohydrates found within the extracellular matrix allow tissues and organs to carry  out their many functions. A loss of these supportive and barrier functions of con-  nective tissue sometimes leads to significant clinical consequences, such as those  that result from the microvascular alterations that lead to blindness or renal failure,  or peripheral neuropathies in patients with diabetes mellitus.   Many hormones affect fuel metabolism, including those that regulate appetite as  well as those that influence absorption, transport, and oxidation of foodstuff s . The  major hormones that influence nutrient metabolism and their actions on muscle,  liver, and adipose tissue are listed in Table 43.1.   Insulin is the major anabolic hormone. It promotes the storage of nutrients as  glycogen in liver and muscle, and as triacylglycerols in adipose tissue. It also  stimulates the synthesis of proteins in tissues such as muscle. At the same time,  insulin acts to inhibit fuel mobilization.   Glucagon is the major counterregulatory hormone. The term counter regula-  tory means that its actions are generally opposed to those of insulin (contrainsu-  lar). The major action of glucagon is to mobilize fuel reserves by stimulating  glycogenolysis and gluconeogenesis. These actions ensure that glucose will be  available to glucose -dependent tissues between meals.   Epinephrine, norepinephrine, Cortisol, somatostatin, and growth hormone  also have contrainsular activity. Thyroid hormone also must be classified as an  insulin counterregulatory hormone because it increases the rate of fuel consump-  tion and also increases the sensitivity of the target cells to other insulin counter-  regulatory hormones.   Insulin and the counterregulatory hormones exert two types of metabolic regu-  lation (see Chapter 26). The first type of control occurs within minutes to hours of  the hormone-receptor interaction and usually results from changes in the cat-  alytic activity or kinetics of key preexisting enzymes, caused by phosphorylation  or dephosphorylation of these enzymes. The second type of control involves regu-  lation of the synthesis of key enzymes by mechanisms that stimulate or inhibit  transcription and translation ofmRNA. These processes are slow and require  hours to days.   Otto Shape, now a third-year medical student, was assigned to do a history and  physical examination on a newly admitted 47-year-old patient named Corti  Solemia. Mr. Solemia had consulted his physician for increasing weakness and  fatigue and was found to have a severely elevated serum glucose level. While examin-  ing the patient, Otto noted marked redness of the patient's facial skin as well as reddish-  purple stripes (striae) in the skin of the patient's lower abdomen and thighs. The  patient's body fat was unusually distributed in that it appeared to be excessively   a Hormones with actions that are generally opposed to those of insulin.   Somatostatin's effects on metabolism are indirect via suppression of secretion of insulin, glucagon, growth hormone, and thyroid hormone and by effects on gastric acid secretion, gastric emptying time, and  pancreatic exocrine secretion (see text).   deposited centrally in his face, neck, upper back, chest and abdomen, while the dis-  tal portions of his arms and legs appeared to be almost devoid of fat. The patient's  skin appeared thinned, and large bruises were present over many areas of his body,  for which Mr. Solemia had no explanation. The neurologic examination showed  severe muscle weakness, especially in the proximal arms and legs, where the mus-  cles seemed atrophied.   Sam Atotrope, a 42-year-old jeweler, noted increasingly severe headaches  behind his eyes sometimes associated with a "flash of light" in his visual  fields. At times his vision seemed blurred, making it difficult to perform  some of the intricate work required of a jeweler. He consulted his ophthalmologist,  who was impressed with the striking change in Sam's facial features that had  occurred since he last saw the patient 5 years earlier. The normal skin creases in  Sam's face had grown much deeper, his skin appeared to be thickened, his nose and  lips appeared more bulbous, and his jaw seemed more prominent. The doctor also  noted that Sam's hands appeared bulky, and his voice had deepened. An eye exam-  ination showed that Sam's optic nerves appeared slightly atrophied, and there was  a loss of the upper outer quadrants of his visual fields.   The effects of insulin on fuel metabolism and substrate flux were considered in  many of the earlier chapters of this book, particularly in Chapter 26. Insulin stimu-  lates the storage of glycogen in liver and muscle and the synthesis of fatty acids and  triacylglycerols and their storage in adipose tissue. In addition, insulin stimulates  the synthesis in various tissues of more than 50 proteins, some of which contribute  to the growth of the organism. In fact, it is difficult to separate the effects of insulin  on cell growth from those of a family of proteins known as the somatomedins or the  insulin-like growth factors I and II (IGF-I and IGF-II) (see Section III.B.6. of this  chapter).   Finally, insulin has paracrine actions within the pancreatic islet cells. When  insulin is released from the p cells, it suppresses glucagon release from the a cells.   Glucagon is one of several counterregulatory (contrainsular) hormones. It is syn-  thesized as part of a large precursor protein, proglucagon. Proglucagon is produced  in the a cells of the islets of Langerhans in the pancreas and in the L cells of the  intestine. It contains a number of peptides linked in tandem: glicentin-related pep-  tide, glucagon, glucagon-related peptide 1 (GLP-1), and glucagon-related peptide 2  (GLP-2). Proteolytic cleavage of proglucagon releases various combinations of its  constituent peptides. Glucagon is cleaved from proglucagon in the pancreas and  constitutes 30 to 40% of the immunoreactive glucagon in the blood. The remaining  immunoreactivity is caused by other cleavage products of proglucagon released  from the pancreas and the intestine. Pancreatic glucagon has a plasma half -life of 3  to 6 minutes and is removed mainly by the liver and kidney.   Glucagon promotes glycogenolysis, gluconeogenesis, and ketogenesis by stimu-  lating the generation of cyclic adenosine monophosphate (cAMP) in target cells.  The liver is the major target organ for glucagon, in part because the concentrations  of this hormone bathing the liver cells in the portal blood are higher than in the  peripheral circulation. Portal vein levels of glucagon may reach concentrations as  high as 500 pg/mL.   Finally, glucagon stimulates insulin release from the p cells of the pancreas.  Whether this is a paracrine effect or an endocrine effect has not been established.  The pattern of blood flow in the pancreatic islet cells is believed to bathe the p cells   first and then the a-cells. Therefore, the p cells may influence a-cell function by an  endocrine mechanism, whereas the influence of p-cell hormone on a-cell function  is more likely to be paracrine.   Preprosomatostatin, a 1 16-amino acid peptide, is encoded on the long arm of chro-  mosome 3. Somatostatin (SS-14), a cyclic peptide with a molecular weight of  1,600, is produced from the 14 amino acids at the C-terminus of this precursor mol-  ecule. SS-14 was first isolated from the hypothalamus and named for its ability to  inhibit the release of growth hormone (GH, somatotropin) from the anterior pitu-  itary. It also inhibits the release of insulin. In addition to the hypothalamus, somato-  statin is also secreted from the D cells (8 cells) of the pancreatic islets, many areas  of the central nervous system outside of the hypothalamus, and in gastric and duo-  denal mucosal cells. SS-14 predominates in the central nervous system (CNS) and  is the sole form secreted by the 8 cells of the pancreas. In the gut, however, proso-  matostatin (SS-28), which has 14 additional amino acids extending from the  C-terminal portion of the precursor, makes up 70 to 75% of the immunoreactivity (the  amount of hormone that reacts with antibodies to SS-14). The prohormone SS-28 is  7 to 10 times more potent in inhibiting the release of GH and insulin than is SS-14.   Tolbutamide, a sulfonylurea drug  rl ] that increases insulin secretion,  *r also increases the secretion of pan-  creatic somatostatin.   The secretagogues for somatostatin are similar to those that cause secretion of  insulin. The metabolites that increase somatostatin release include glucose, argi-  nine, and leucine. The hormones that stimulate somatostatin secretion include  glucagon, vasoactive intestinal polypeptide (VIP), and cholecystokinin (CCK).  Insulin, however, does not directly influence somatostatin secretion.   In addition to its effects on normal  \\ ) GH secretion, somatostatin also   suppresses the pathologic increase  in GH that occurs in acromegaly (caused by  a GH-secreting pituitary tumor), diabetes  mellitus, and carcinoid tumors (tumors that  secrete serotonin). Somatostatin also sup-  presses the basal secretion of TSH, TRH,  insulin, and glucagon. The hormone also  has a suppressive effect on a wide variety of  nonendocrine secretions.   The major limitation in the clinical use of  native somatostatin is its short half-life of  less than 3 minutes in the circulation.  Analogs of native somatostatin, however,  have been developed that are resistant to  degradation and, therefore, have a longer  half-life. One such analog is octreotide, an  octapeptide variant of somatostatin with a  half-life of approximately 110 minutes.   Five somatostatin receptors have been identified and characterized, all of which are  members of the G protein-coupled receptor superfamily. Four of the five receptors  do not distinguish between SS-14 and SS-28. Somatostatin binds to its plasma  membrane receptors on target cells. These "activated" receptors interact with  inhibitory G proteins of adenylate cyclase. As a result, the production of cAMP is  inhibited, and protein kinase A is not activated. This inhibitory effect suppresses  secretion of GH and thyroid- stimulating hormone (TSH) from the anterior pituitary  gland as well as the secretion of insulin and glucagon from the pancreatic islets. If  one were to summarize the action of somatostatin in one phrase, it would be  "somatostatin inhibits the secretion of many other hormones." As such, it acts to  regulate the effects of those other hormones. In addition to these effects on hor-  mones that regulate fuel metabolism, somatostatin also reduces nutrient absorption  from the gut by prolonging gastric emptying time (through a decrease in the secre-  tion of gastrin, which reduces gastric acid secretion), by diminishing pancreatic  exocrine secretions (i.e., digestive enzymes, bicarbonate, and water), and by  decreasing visceral blood flow. Thus, somatostatin exerts a broad, albeit indirect,  influence on nutrient absorption and, therefore, the utilization of fuels.   Somatostatin and its synthetic analogs are used clinically to treat a variety of  secretory neoplasms such as GH-secreting tumors of the pituitary. Such tumors can  cause gigantism if growth hormone is secreted in excess before the closure of the   growth centers of the ends of long bones or acromegaly if excess growth hormone  is chronically secreted after the closure of these centers (as in Sam Atrope).   Growth hormone is a polypeptide that, as its name implies, stimulates growth. Many  of its effects are mediated by insulin-like growth factors (IGFs, also known as  somatomedins) that are produced by cells in response to the binding of GH to its  cell membrane receptors (see Section 6 below). However, GH also has direct effects  on fuel metabolism.   Human growth hormone is a water-soluble 22-kDa polypeptide with a plasma  half-life of 20 to 50 minutes. It is composed of a single chain of 191 amino acids  having two intramolecular disulfide bonds (Fig. 43.1). The gene for GH is located  on chromosome 17. It is secreted by the somatotroph cells (the cells that synthesize  and release GH) present in the lateral areas of the anterior pituitary. GH is struc-  turally related to human prolactin and to human chorionic somatomammotropin  (hCS) from the placenta, a polypeptide that stimulates growth of the developing  fetus. Yet the hCS peptide has only 0.1% of the growth-inducing potency of GH.  Growth hormone is the most abundant trophic hormone in the anterior pituitary,  being present in concentrations of 5 to 15 mg per gram of tissue. The other anterior  pituitary hormones are present in microgram-per-gram of tissue quantities.   The actions of GH can be classified as those that occur as a consequence of the  hormone's direct effect on target cells and those that occur indirectly through the  ability of GH to generate other factors, particularly IGF-I.   The IGF-I-independent actions of GH are exerted primarily in hepatocytes. GH  administration is followed by an early increase in the synthesis of 8 to 10 proteins,  among which are IGF-I, a 2 -macroglobulin, and the serine protease inhibitors Spi  2.1 and Spi 2.3. Expression of the gene for ornithine decarboxylase, an enzyme  active in polyamine synthesis (and, therefore, in the regulation of cell proliferation),  is also significantly increased by GH.   Muscle and adipocyte cell membranes contain GH receptors that mediate direct,  rapid metabolic effects on glucose and amino acid transport as well as on lipolysis.  These receptors use associated cytoplasmic tyrosine kinases for signal transduction  (such as the janus kinases; see Chapter 11, section III.C.). For example, in adipose  tissue, GH has acute insulinlike effects followed by increased lipolysis, inhibition  of lipoprotein lipase, stimulation of hormone-sensitive lipase, decreased glucose  transport, and decreased lipogenesis. In muscle, GH causes increased amino acid  transport, increased nitrogen retention, increased fat-free (lean) tissue, and  increased energy expenditure. GH also has growth-promoting effects. GH receptors   brain showed a macroadenoma (a  tumor greater than 10 mm in diam-  eter) in the pituitary gland, with superior  extension that compressed the optic nerve  as it crossed above the sella turcica, causing  his visual problems. The skeletal and vis-  ceral changes noted by the ophthalmologist  are characteristic of acromegalic patients  with chronically elevated serum levels of GH  and IGF-I.   Therapeutic alternatives for acromegaly  caused by a GH-secreting tumor of the ante-  rior pituitary gland include lifelong medical  therapy with the somatostatin analog  octreotide or the GH receptor antagonist  pegvisomant. Other therapeutic options  include stereotactic radiation therapy or sur-  gical resection of the neoplasm. If the exces-  sive secretion of GH is controlled success-  fully, some of the visceral or soft tissue  changes of acromegaly may slowly subside  to varying degrees. The skeletal changes,  however, cannot be reversed.   The ophthalmologist ordered a  morning fasting serum GH level on  Sam Atotrope, which was elevated  at 56 ng/mL (normal = 0-5 ng/mL)   Sam Atotrope was given an oral  dose of 100 g glucose syrup. This  dose would suppress serum GH  levels to less than 2 ng/mL in normal sub-  jects, but not in patients with acromegaly  who have an autonomously secreting pitu-  itary tumor making GH. Because Sam's  serum GH level was 43 ng/mL after the oral  glucose load, a diagnosis of acromegaly was  made. The patient was referred to an  endocrinologist for further evaluation.    Fig. 43.1. Structure of human growth hormone. From Murray, RK, et al, Harper's Biochemistry,  23rd Ed. Stamford, CT: Appleton & Lange, 1993:502.   GH not only stimulates IGF-I gene  expression in the liver but in a  number of extrahepatic tissues as  well. In acromegalics, rising levels of IGF-I  cause a gradual generalized increase in  skeletal, muscular, and visceral growth. As a  consequence, a diffuse increase occurs in  the bulk of all tissues (enlargement =  "megaly") especially in the "acral" (most  peripheral) tissues of the body, such as the  face, the hands, and the feet, hence the term  "acromegaly."   Sam Atotrope's coarse facial features  and bulky hands are typical of patients with  acromegaly.   Fig. 43.2. Control of growth hormone secre-  tion. Various factors stimulate the release of  GHRH from the hypothalamus. The hypothal-  amus also releases somatostatin in response to  other stimuli. GHRH stimulates and somato-  statin inhibits the release of growth hormone  from the anterior pituitary. Growth hormone  causes the release of IGF-I from liver and other  tissues. IGF-I inhibits GHRH release and stim-  ulates somatostatin release.   are present on a variety of tissues in which GH increases IGF-I gene expression.  The subsequent rise in IGF-I levels contributes to cell multiplication and differenti-  ation by autocrine or paracrine mechanisms. These, in turn, lead to skeletal, mus-  cular, and visceral growth. These actions are accompanied by a direct anabolic  influence of GH on protein metabolism with a diversion of amino acids from oxi-  dation to protein synthesis and a shift to a positive nitrogen balance.   Although the regulation of GH secretion is complex, the major influence is hormonal  (Fig. 43.2). The pulsatile pattern of GH secretion reflects the interplay of two hypo-  thalamic regulation peptides. Release is stimulated by growth hormone-releasing hor-  mone (GHRH, also called somatocrinin). The structure of GHRH was identified in  1982 (Fig. 43.3). It exists as both a 40- and a 44-amino acid peptide encoded on chro-  mosome 20 and produced exclusively in cells of the arcuate nucleus. Its C-terminal  leucine residue is amidated. Full biologic activity of this releasing hormone resides in  the first 29 amino acids of the N-terminal portion of the molecule. GHRH interacts  with specific receptors on the plasma membranes of the somatotrophs. The intracel-  lular signaling mechanisms that result in GH synthesis and release appear to be mul-  tiple, as cAMP and calcium-calmodulin both stimulate GH release.   Conversely, GH secretion is suppressed by growth hormone release-inhibiting  hormone (GHRIH, also called somatostatin, which has already been discussed). In  addition, IGF-I, produced primarily in the liver in response to the action of GH on  hepatocytes, feeds back negatively on the somatotrophs to limit GH secretion. Other  physiologic factors (e.g., exercise and sleep) and many pathologic factors control its  release (Table 43.2).   In addition, GH release is modulated by plasma levels of all of the metabolic  fuels, including proteins, fats, and carbohydrates. A rising level of glucose in  the blood normally suppresses GH release, whereas hypoglycemia increases GH   Fig. 43.3. Structures of growth hormone-releasing hormone (GHRH) and growth hormone  release-inhibiting hormone (GHRIH, also called somatostatin). GHRH has an amide at the  C-terminal.   a-Adrenergic agonists  (3-Adrenergic antagonists  Dopamine agonists  Serotonin precursors  K + infusion   secretion in normal subjects. Amino acids, such as arginine, stimulate release of GH  when their concentrations rise in the blood. Rising levels of fatty acids may blunt  the GH response to arginine or a rapidly dropping blood glucose level. However,  prolonged fasting, in which fatty acids are mobilized in an effort to spare protein, is  associated with a rise in GH secretion. Some of the physiologic, pharmacologic, and  pathologic influences on GH secretion are given in Table 43.2.   These modulators of GH secretion  \ \ ) provide the basis for clinical sup-  pression and stimulation tests in  patients suspected of having excessive or  deficient GH secretion.   GH affects the uptake and oxidation of fuels in adipose tissue, muscle, and liver  and indirectly influences energy metabolism through its actions on the islet cells  of the pancreas. In summary, GH increases the availability of fatty acids, which are  oxidized for energy. This and other effects of GH spare glucose and protein; that  is, GH indirectly decreases the oxidation of glucose and amino acids (Fig. 43.4).    While Sam Atotrope was trying to  decide which of the major alterna-  tives for the treatment of his  growth hormone (GH)-secreting pituitary  tumor to choose, he noted progressive  fatigue and the onset of increasing urinary  frequency associated with a marked  increase in thirst. In addition, he had lost 4 lb  over the course of the last 6 weeks in spite of  a good appetite. His physician suspected  that Mr. Atotrope had developed diabetes  mellitus, perhaps related to the chronic  hypersecretion of GH. This suspicion was  confirmed when Sam's serum glucose level,  drawn before breakfast, was reported to be  236 mg/dL   As a result of the metabolic effects  of GH, the clinical course of acro-  megaly may be complicated by  impaired glucose tolerance or even overt  diabetes mellitus, as occurred with Sam  Atotrope.   Fig 43.5. Production and action of IGFs. The  hypothalamus produces growth hormone-  releasing hormone (GHRH), which stimulates  somatotrophs in the anterior pituitary to release  growth hormone (GH). Growth hormone  release-inhibiting hormone (GHRIH) inhibits  GH release. GH binds to cell surface receptors  and stimulates IGF production and release by  liver and other tissues. IGF binds to cell surface  receptors and stimulates the phosphorylation of  proteins that cause mitosis and growth.   High levels of circulating IGF-1 has  been linked to the development of  breast, prostate, colon, and lung  cancer. Additionally, experimental modula-  tion of IGF-1 receptor activity can alter the  growth of different types of tumor cells. Cur-  rent research is aimed at targeting the inter-  action of IGF-1 and its receptor to reduce  tumor cell proliferation.   Growth hormone increases the sensitivity of the adipocyte to the lipolytic action of  the catecholamines and decreases its sensitivity to the lipogenic action of insulin.  These actions lead to the release of free fatty acids and glycerol into the blood to be  metabolized by the liver. GH also decreases esterification of fatty acids, thereby  reducing triacylglycerol synthesis within the fat cell. Recent evidence suggests that  GH may impair glucose uptake by both fat and muscle cells by a postreceptor inhi-  bition of insulin action.   The lipolytic effects of GH increase free fatty acid levels in the blood bathing mus-  cle. These fatty acids are preferentially used as fuel, indirectly suppressing glucose  uptake by muscle cells. Through the effects on glucose uptake, the rate of glycoly-  sis is proportionately reduced.   GH increases the transport of amino acids into muscle cells, providing substrate  for protein synthesis. Through a separate mechanism, GH increases the synthesis of  DNA and RNA. The positive effect on nitrogen balance is reinforced by the protein-  sparing effect of GH-induced lipolysis that makes fatty acids available to muscle as  an alternative fuel source.   When plasma insulin levels are low, as in the fasting state, GH enhances fatty acid  oxidation to acetyl CoA. This effect in concert with the increased flow of fatty acids  from adipose tissue enhances ketogenesis. The increased amount of glycerol reach-  ing the liver as a consequence of enhanced lipolysis acts as a substrate for gluco-  neogenesis.   Hepatic glycogen synthesis is also stimulated by GH in part because of the  increased gluconeogenesis in the liver. Finally, glucose metabolism is suppressed by  GH at several steps in the glycolytic pathway.   A major effect of GH on liver is to stimulate production and release of IGFs. The  IGFs are also known as somatomedins. The two somatomedins in humans share  structural homologies with proinsulin, and both have substantial insulin-like growth  activity; hence the designations, insulin-like growth factor I (human IGF-I, or  somatomedin-C) and insulin-like growth factor II (human IGF-II, or somatomedin  A). IGF-I is a single-chain basic peptide having 70 amino acids, and IGF-II is  slightly acidic with 67 amino acids. These two peptides are identical to insulin in  half of their residues. In addition, they contain a structural domain that is homolo-  gous to the C-peptide of proinsulin.   A broad spectrum of normal cells respond to high doses of insulin by increasing  thymidine uptake and initiating cell propagation. In most instances, IGF-I causes  the same response as insulin in these cells but at significantly smaller, more physi-  ologic concentrations. Thus, the IGFs are more potent than insulin in their growth-  promoting actions.   Evidence suggests that the IGFs exert their effects through either an endocrine or  a paracrine/autocrine mechanism. IGF-I appears to stimulate cell propagation and  growth by binding to specific IGF-I receptors on the plasma membrane of target  cells, rather than binding to GH receptors (Fig. 43.5).   Like insulin, the intracellular portion of the plasma membrane receptor for IGF-I  (but not IGF-II) has intrinsic tyrosine kinase activity. The fact that the receptors for  insulin and a number of other growth factors have intrinsic tyrosine kinase activity  indicates that tyrosine phosphorylation initiates the process of cellular replication  and growth. Subsequently, a chain of kinases is activated, which include a number of  proto-oncogene products (see Chapters 11 and 18).   Most cells of the body have mRNA for IGF, but the liver has the greatest con-  centration of these messengers, followed by kidney and heart. The synthesis of IGF-  I is regulated, for the most part, by GH, whereas hepatic production of IGF-II is  independent of GH levels in the blood.   The catecholamines belong to a family of bioamines and are secretory products of  the sympathoadrenal system, which are required for the body to adapt to a great  variety of acute and chronic stresses. Epinephrine (80-85% of stored cate-  cholamines) is synthesized primarily in the cells of the adrenal medulla, whereas  norepinephrine (15-20% of stored catecholamines) is synthesized and stored not  only in the adrenal medulla but also in various areas of the central nervous system  (CNS) and in the nerve endings of the adrenergic nervous system. Dopamine,  another catecholamine, acts primarily as a neurotransmitter and has little effect on  fuel metabolism.   The first total chemical synthesis of epinephrine was accomplished by F. Stolz et  al in 1904. In 1950, Earl Sutherland was the first to demonstrate that epinephrine  (and glucagon) induces glycogenolysis. This marked the beginning of our under-  standing of the molecular mechanisms through which hormones act.   Tyrosine is the precursor of the catecholamines. The pathway for the biosynthesis  of these molecules is described in Chapter 48.   Secretion of epinephrine and norepinephrine from the adrenal medulla is stimu-  lated by a variety of stresses, including pain, hemorrhage, exercise, hypo-  glycemia, and hypoxia. Release is mediated by stress-induced transmission of  nerve impulses emanating from adrenergic nuclei in the hypothalamus. These  impulses stimulate the release of the neurotransmitter acetylcholine from pregan-  glionic neurons that innervate the adrenomedullary cells. Acetylcholine depolar-  izes the plasma membranes of these cells, allowing the rapid entry of extracellu-  lar calcium (Ca 2+ ) into the cytosol. Ca 2+ stimulates the synthesis and release of  epinephrine and norepinephrine from the chromaffin granules into the extracellu-  lar space by exocytosis.   The catecholamines act through two major types of receptors on the plasma mem-  brane of target cells, the a-adrenergic and the p-adrenergic receptors (see Chapter  26, section IV.C).   The actions of epinephrine and norepinephrine in the liver, the adipocyte, the  skeletal muscle cell, and the a and p cells of the pancreas directly influence fuel  metabolism (Fig. 43.6). These catecholamines are counterregulatory hormones that  have metabolic effects directed toward mobilization of fuels from their storage sites  for oxidation by cells to meet the increased energy requirements of acute and  chronic stress. They simultaneously suppress insulin secretion, which ensures that  fuel fluxes will continue in the direction of fuel utilization rather than storage as  long as the stressful stimulus persists.   In addition, norepinephrine works as a neurotransmitter and affects the sympa-  thetic nervous system in the heart, lungs, blood vessels, bladder, gut, and other   In patients suspected of having a  neoplasm of the adrenal medulla  that is secreting excessive quanti-  ties of epinephrine or norepinephrine (a  pheochromocytoma), either the cate-  cholamines themselves (epinephrine, norepi-  nephrine, and dopamine) or their metabolites  (the metanephrines and vanillylmandelic  acid, VMA) may be measured in a 24-hour  urine collection, or the level of cate-  cholamines in the blood may be measured. A  patient who has consistently elevated levels  in the blood or urine should be considered to  have a pheochromocytoma, particularly if the  patient has signs and symptoms of cate-  cholamine excess, such as excessive sweat-  ing, palpitations, tremulousness, and hyper-  tension.   ®A relatively rare form of second-  ary hypertension (high blood pres-  sure) is caused by a cate-  cholamine-secreting neoplasm of the  adrenal medulla, known as a pheochromo-  cytoma. Patients with the tumor periodi-  cally secrete large amounts of epinephrine  and norepinephrine into the bloodstream.  Symptoms related to this secretion include  a sudden and often severe increase in  blood pressure, heart palpitations, a throb-  bing headache, and inappropriate and dif-  fuse sweating. In addition, chronic hyper-  secretion of these catecholamines may lead  to impaired glucose tolerance or even overt  diabetes mellitus. Describe the actions of  these hormones that lead to the significant  rise in glucose levels.    Glucocorticoids, such as Cortisol,  were named for their ability to  raise blood glucose levels. These   steroids are among the "counterregulatory"  hormones that protect the body from  insulin-induced hypoglycemia.    Fig. 43.6. Effects of epinephrine on fuel metabolism and pancreatic endocrine function. Epi-  nephrine (Epi) stimulates glycogen breakdown in muscle and liver, gluconeogenesis in liver,  and lipolysis in adipose tissue. Epinephrine further reinforces these effects because it  increases the secretion of glucagon, a hormone that shares many of the same effects as epi-  nephrine. Epi also inhibits insulin release but stimulates glucagon release from the pancreas.   organs. These effects of catecholamines on the heart and blood vessels serve to  increase cardiac output and systemic blood pressure, hemodynamic changes that  facilitate the delivery of blood borne fuels to metabolically active tissues.   Epinephrine has a short half-life in the blood and to be effective pharmacologi-  cally must be administered parenterally. It may be used clinically to support the  beating of the heart, to dilate inflamed bronchial muscles, and even to decrease  bleeding from organs during surgery.   Catecholamines have a relatively low affinity for both a- and p -receptors. After  binding, the catecholamine disassociates from its receptor quickly, causing the  duration of the biologic response to be brief. The free hormone is degraded in sev-  eral ways, as outlined in Chapter 48.   Cortisol (hydrocortisone) is the major physiologic glucocorticoid (GC) in humans,  although corticosterone also has some glucocorticoid activity. The biosynthesis of  steroid hormones and their basic mechanism of action has been described in  Chapters 34 and 17.   The synthesis and secretion of Cortisol is controlled by a cascade of neural and  endocrine signals linked in tandem in the cerebrocortical-hypothalamic-pituitary-  adrenocortical axis. Cerebrocortical signals to the midbrain are initiated in the  cerebral cortex by "stressful" signals such as pain, hypoglycemia, hemorrhage,  and exercise (Fig. 43.7). These nonspecific "stresses" elicit the production of  monoamines in the cell bodies of neurons of the midbrain. Those that stimulate  the synthesis and release of corticotropin-releasing hormone (CRH) are acetyl-  choline and serotonin. These neurotransmitters then induce the production of  CRH by neurons originating in the paraventricular nucleus. These neurons dis-  charge CRH into the hypothalamico-hypophyseal portal blood. CRH is delivered  through these portal vessels to specific receptors on the cell membrane of the  adrenocorticotropic hormone (ACTH)- secreting cells of the anterior pituitary  gland (corticotrophs). This hormone-receptor interaction causes ACTH to be  released into the general circulation to eventually interact with specific receptors  for ACTH on the plasma membranes of cells in the zona fasciculata and zona  reticulosum of the adrenal cortex. The major trophic influence of ACTH on Corti-  sol synthesis is at the level of the conversion of cholesterol to pregnenolone, from  which the adrenal steroid hormones are derived (see Chapter 34 for the biosyn-  thesis of the steroid hormones).   Cortisol is secreted from the adrenal cortex in response to ACTH. The concen-  tration of free (unbound) Cortisol that bathes the CRH-producing cells of the hypo-  thalamus and the ACTH-producing cells of the anterior pituitary acts as a negative  feedback signal that has a regulatory influence on the release of CRH and ACTH  (see Fig. 43.7). High Cortisol levels in the blood suppress CRH and ACTH secre-  tion, and low Cortisol levels stimulate secretion. In severe stress, however, the neg-  ative feedback signal on ACTH secretion exerted by high Cortisol levels in the  blood is overridden by the stress-induced activity of the higher portions of the axis  (see Fig. 43.7).   The effects of glucocorticoids on fuel metabolism in liver, skeletal muscle, and  adipose tissue are outlined in Table 43.1 and in Figure 43.8. Their effects on other  tissues are diverse and, in many instances, essential for life. Some of the nonmeta-  bolic actions of GCs are listed in Table 43.3.   Glucocorticoids have diverse actions that affect most tissues of the body. At first  glance, some of these effects may appear to be contradictory (such as inhibition of   When Otto was writing his list of differential diagnoses to explain the clinical  presentation of Corti Solemia, he suddenly thought of a relatively rare  endocrine disorder that could explain all of the presenting signs and symp-  toms. He made a provisional diagnosis of excessive secretion of Cortisol secondary to an  excess secretion of ACTH (Cushing's "disease") or by a primary increase of Cortisol pro-  duction by an adrenocortical tumor (Cushing's syndrome).   Otto suggested that resting, fasting plasma Cortisol and ACTH levels be measured at  8:00 the next morning. These studies showed that Mr. Solemia's morning plasma ACTH  and Cortisol levels were both significantly above the reference range. Therefore, Otto  concluded that Mr. Solemia probably had a tumor that was producing ACTH  autonomously (i.e., not subject to normal feedback inhibition by Cortisol). The high  plasma levels of ACTH were stimulating the adrenal cortex to produce excessive  amounts of Cortisol. Additional laboratory and imaging studies indicated that the hyper-  cortisolemia was caused by a benign ACTH-secreting adenoma of the anterior pituitary  gland (Cushing's "disease").   Fig. 43.7. Regulation of Cortisol secretion. Vari-  ous factors act on the hypothalamus to stimulate  the release of corticotrophin-releasing hormone  (CRH). CRH stimulates the release of ACTH  from the anterior pituitary, which stimulates the  release of Cortisol from the adrenal cortex. Cor-  tisol inhibits the release of CRH and ACTH.   ®The catecholamines are counterreg-  ulatory hormones that mobilize  fuels from their storage sites for oxi-  dation in target cells to meet the increased  energy requirements that occur during acute  or chronic stress, or, in this case, the release  of catecholamines by a tumor in the adrenal  medulla. These actions provide the liver, for  example, with increased levels of substrate  needed for gluconeogenesis. Although in  normal individuals most of the glucose gen-  erated through this mechanism is oxidized,  blood glucose levels rise in the process. In  addition, the catecholamines suppress insulin  secretion to ensure that fuels will continue to  flow in the direction of utilization rather than  storage under these circumstances. Hence,  blood glucose levels may rise in patients who  have a pheochromocytoma.   On electrolyte and water balance:  Increase sodium and water retention (1/3,000 the potency of aldosterone)  Increase renal glomerular filtration rate to maintain water excretion rate  Suppress arginine vasopressin (ADH) release from posterior pituitary    On cardiovascular system:  Indirect effect of glucocorticoid actions on sodium and water metabolism (above)  Maintain volume of microcirculation to tissues (cardiac output)  Maintain normal vasomotor response to vasoconstricting agents   On skeletal muscle:  Maintain muscle function by providing normal microcirculation to muscle  Influence muscle mass by enhancing protein catabolism and suppressing protein synthesis   On formed elements in blood:  Increase red blood cell mass and granulocyte proliferation  Decrease lymphocyte, monocyte, and basophil proliferation   Inhibit early inflammatory process (i.e., edema, fibrin deposition, capillary dilation, leuko   cyte migration, and phagocytic action)  Inhibit late inflammatory process (proliferation of capillaries and fibroblasts, deposition of   Immune suppressant actions (of questionable significance at physiologic levels):  Prevent manifestations of humoral and cellular immunity   Interfere with production of cytokines needed for immune competence via cell-to-cell  communication   Fig. 43.8. Effects of glucocorticoids (GC) on fuel metabolism. Glucocorticoids stimulate  lipolysis in adipose tissue and the release of amino acids from muscle protein. In liver, glu-  cocorticoids stimulate gluconeogenesis and the synthesis of glycogen. The breakdown of  liver glycogen is stimulated by epinephrine.   glucose uptake by certain tissues), but taken together they promote survival in times  of stress.   In many tissues, GCs inhibit DNA, RNA, and protein synthesis and stimulate  the degradation of these macromolecules. In response to chronic stress, GCs act  to make fuels available, so that when the acute alarm sounds and epinephrine is  released, the organism can fight or flee. When GCs are elevated, glucose uptake  by the cells of many tissues is inhibited, lipolysis occurs in peripheral adipose  tissue, and proteolysis occurs in skin, lymphoid cells, and muscle. The fatty acids  that are released are oxidized by the liver for energy, and the glycerol and amino  acids serve in the liver as substrates for the production of glucose, which is con-  verted to glycogen and stored. The alarm signal of epinephrine stimulates liver  glycogen breakdown, making glucose available as fuel to combat the acute  stress.   The mechanism by which GCs exert these effects involves binding of the steroid  to intracellular receptors, interaction of the steroid-receptor complex with GC  response elements on DNA, transcription of genes, and synthesis of specific pro-  teins (see Chapter 16, section III.C.2.). In some cases, the specific proteins respon-  sible for the GC effect are known (e.g., the induction of phosphoenolpyruvate car-  boxykinase that stimulates gluconeogenesis). In other cases, the proteins  responsible for the GC effect have not yet been identified.   The secretory products of the thyroid acinar cells are tetraiodothyronine (thyroxine,  T 4 ) and triiodothyronine (T 3 ). Their structures are shown in Figure 43.9. The basic  steps in the synthesis of T 3 and T 4 in these cells involve the transport or trapping of  iodide from the blood into the thyroid acinar cell against an electrochemical gradi-  ent; the oxidation of iodide to form an iodinating species; the iodination of tyrosyl  residues on the protein, thyroglobulin, to form iodotyrosines; and the coupling of  residues of monoiodo- and diiodotyrosine in thyroglobulin to form residues of T 3  and T 4 (Fig. 43.10). Proteolytic cleavage of thyroglobulin releases free T 3 and T 4 .  The steps in thyroid hormone synthesis are stimulated by thyroid- stimulating hor-  mone (TSH), a glycoprotein produced by the anterior pituitary.   Iodide transport from the blood into the thyroid acinar cell is accomplished  through an energy-requiring, iodide-trapping mechanism that is poorly defined but  may involve the Na + ,K + -ATPase coupled to a cotransporter for Na + and iodide in  the plasma membrane of the acinar cell.    Otto was now able to explain the  mechanism for most of Corti  Solemia's signs and symptoms. For  example, Otto knew the metabolic explana-  tion for the patient's hyperglycemia. Some of  Mr. Solemia's muscle wasting and weakness  were caused by the catabolic effect of hyper-  cortisolemia on protein stores, such as those  in skeletal muscle, to provide amino acids as  precursors for gluconeogenesis. This cata-  bolic action also resulted in the degradation  of elastin, a major supportive protein of the  skin, as well as an increased fragility of the  walls of the capillaries of the cutaneous tis-  sues. These changes resulted in the easy  bruisability and the torn subcutaneous tis-  sues of the lower abdomen, which resulted  in red striae or stripes. The plethora (red-  ness) of Mr. Solemia's facial skin was also  caused in part by the thinning of the skin as  well as by a cortisol-induced increase in the  bone marrow production of red blood cells,  which enhanced the "redness" of the subcu-  taneous tissues.   If Corti Solemia's problem had  ■ been caused by a neoplasm of the  adrenal cortex, what would his lev-  els of blood ACTH and Cortisol have been?   Normally, the thyroid gland  secretes 80-100 |xg T 4 and approxi-  mately 5 juug T 3 per day. The addi-  tional 22-25 (jug T 3 "produced"  daily is the result of the deiodination of the  5'-carbon of T 4 in peripheral tissues. T 3 is  believed to be the predominant biologically  active form of thyroid hormone in the body.   The "central" deposition of fat in patients, such as Corti Solemia, with Cushing's  "disease" or syndrome is not readily explained because GCs actually cause  lipolysis in adipose tissue. The increased appetite caused by an excess of GC  and the lipogenic effects of the hyperinsulinemia that accompanies the GC-induced  chronic increase in blood glucose levels have been suggested as possible causes. Why  the fat is deposited centrally under these circumstances, however, is not understood. This  central deposition leads to the development of a large fat pad at the center of the upper  back ("buffalo hump"), to accumulation of fat in the cheeks and jowls ("moon fades") and  neck area, as well as a marked increase in abdominal fat. Simultaneously, there is a loss  of adipose and muscle tissue below the elbows and knees, exaggerating the appearance  of "central obesity" in Cushing's "disease" or syndrome.   Approximately 35% of T 4 is deiodinated at the 5' position to form T 3 , and 43%  \\ ) is deiodinated at the 5 position to form the inactive "reverse" T 3 . Further deio-  dination or oxidative deamination leads to formation of compounds that have  no biologic activity.   y^-^v If Corti Solemia's problem had  /\ # ) resulted from primary hypersecre-  tion of Cortisol by a neoplasm of  the adrenal cortex, his blood Cortisol levels  would have been elevated. The Cortisol  would have acted on the CRH-producing  cells of the hypothalamus and the ACTH-  secreting cells of the anterior pituitary by a  negative feedback mechanism to decrease  ACTH levels in the blood.   Because his Cortisol and ACTH levels  were both high, Mr. Solemia's tumor was  most likely in the pituitary gland or possibly  in neoplastic extrapituitary tissue that was  secreting ACTH "ectopically." (Ectopic  means that the tumor or neoplasm is pro-  ducing and secreting a substance that is not  ordinarily made or secreted by the tissue  from which the tumor developed.) Mr.  Solemia's tumor was in the anterior pituitary,  not in an extrapituitary ACTH-producing site.   The iodide concentrating or trap-  ping process present in the plasma  membrane of thyroid acinar cells  creates iodide levels within the thyroid cell  that are several hundredfold greater than  those in the blood, depending on the current  size of the total body iodide pool and the  present need for new hormone synthesis.    In areas of the world in which the  soil is deficient in iodide, hypothy-  roidism is prevalent. The thyroid   gland enlarges (forms a goiter) in an attempt  to produce more thyroid hormone. In the  United States, table salt (NaCI) enriched with  iodide (iodized salt) is used to prevent  hypothyroidism caused by iodine deficiency.   The thyroid gland is unique in that  it has the capacity to store large  amounts of hormone as amino  acid residues in thyroglobulin within its col-  loid space. This storage accounts for the low  overall turnover rate of T 3 and T 4 in the body.    Fig. 43.10. Synthesis of the thyroid hormones (T 3 and T 4 ). The protein thyroglobulin (Tgb)  is synthesized in thyroid follicular cells and secreted into the colloid. Iodination and coupling  of tyrosine residues in Tgb produce T 3 and T 4 residues, which are released from Tgb by  pinocytosis (endocytosis) and lysosomal action. The coupling of a monoiodotyrosine with a  diiodotyrosine (DIT) to form triiodothyronine (T 3 ) is not depicted here.   The rate of iodide transport is influenced by the absolute concentration of iodide  within the thyroid cell. An internal autoregulatory mechanism decreases transport  of iodide into the cell when the intracellular iodide concentration exceeds a certain  threshold and increases transport when intracellular iodide falls below this thresh-  old level.   The oxidation of intracellular iodide is catalyzed by thyroid peroxidase (located at  the apical border of the thyroid acinar cell) in what may be a two-electron oxidation  step forming I + (iodinium ion). Iodinium ion may react with a tyrosine residue in the  protein thyroglobulin to form a tyrosine quinoid and then a 3 '-monoiodotyrosine  (MIT) residue. It has been suggested that a second iodide is added to the ring by sim-  ilar mechanisms to form a 3, 5 -diiodotyrosine (DIT) residue. Because iodide is added  to these organic compounds, iodination is also referred to as the "organification of  iodide."   The biosynthesis of thyroid hormone proceeds with the coupling of an MIT and  a DIT residue to form a triiodothyronine (T 3 ) residue or of two DIT residues to form  a tetraiodothyronine (T 4 ) residue. T 3 and T 4 are stored in the thyroid follicle as  amino acid residues in thyroglobulin. Under most circumstances, the T 4 /T 3 ratio in  thyroglobulin is approximately 13:1.   The plasma half-life of T 4 is approximately 7 days, and that of T 3 is 1 to 1.5 days.  These relatively long plasma half-lives result from binding of T 3 and T 4 to several  transport proteins in the blood. Of these transport proteins, thyroid-binding globulin  (TBG) has the highest affinity for these hormones and carries approximately 70% of   bound T 3 and T 4 . Only 0.03% of total T 4 and 0.3% of total T 3 in the blood are in the  unbound state. This free fraction of hormone has biologic activity because it is the  only form that is capable of diffusing across target cell membranes to interact with  intracellular receptors. The transport proteins, therefore, serve as a large reservoir of  hormone that can release additional free hormone as the metabolic need arises.   The thyroid hormones are degraded in liver, kidney, muscle, and other tissues by  deiodination, which produces compounds with no biologic activity.   The release of T 3 and T 4 from thyroglobulin is controlled by thyroid- stimulating  hormone (TSH) from the anterior pituitary. TSH stimulates the endocytosis of thy-  roglobulin to form endocytic vesicles within the thyroid acinar cells (see  Fig. 43.10). Lysosomes fuse with these vesicles, and lysosomal proteases hydrolyze  thyroglobulin, releasing free T 4 and T 3 into the blood in a 10:1 ratio. In various tis-  sues, T 4 is deiodinated, forming T 3 , which is the active form of the hormone.   TSH is synthesized in the thyrotropic cells of the anterior pituitary. Its secretion  is primarily regulated by a balance between the stimulatory action of hypothalamic  thyroid-releasing hormone (TRH) and the inhibitory (negative feedback) influence  of thyroid hormone (primarily T 3 ) at levels above a critical threshold in the blood  bathing the pituitary thyrotrophs. TSH secretion occurs in a circadian pattern, a surge  beginning late in the afternoon and peaking before the onset of sleep. In addition,  TSH is secreted in a pulsatile fashion with intervals of 2 to 6 hours between peaks.   TSH stimulates all phases of thyroid hormone synthesis by the thyroid gland,  including iodide trapping from the plasma, organification of iodide, coupling of  monoiodotyrosine and diiodotyrosine, endocytosis of thyroglobulin, and proteoly-  sis of thyroglobulin to release triiodothyronine (T 3 ) and tetraiodothyronine (T 4 ) (see  Fig. 43.10). In addition, the vascularity of the thyroid gland increases as TSH stim-  ulates hypertrophy and hyperplasia of the thyroid acinar cells.   The predominant mechanism of action of TSH is mediated by binding of TSH to  its specific receptor on the plasma membrane of the thyroid acinar cell, leading to  an increase in the concentration of cytosolic cAMP. Recent evidence indicates,  however, that TSH also increases the cellular levels of inositol trisphosphate and  diacylglycerol, causing a rise in cytosolic Ca 2+ within the thyroid cell.   The large protein thyroglobulin, which contains T 3 and T 4 in peptide linkage, is  stored extracellularly in the colloid that fills the central space of each thyroid folli-  cle. Each of the biochemical reactions that leads to the release and eventual secre-  tion of T 3 and T 4 , such as those that lead to their formation in thyroglobulins, is  TSH-dependent. Rising levels of serum TSH stimulate the endocytosis of stored  thyroglobulin into the thyroid acinar cell. Lysosomal enzymes then cleave T 3 and T 4  from thyroglobulin. T 3 and T 4 are secreted into the bloodstream in response to ris-  ing levels of TSH.   As the free T 3 level in the blood bathing the thyrotrophs of the anterior pituitary  gland rises, the feedback loop is closed. Secretion of TSH is inhibited until the free  T 3 levels in the systemic circulation fall just below a critical level, which once again  signals the release of TSH. This feedback mechanism ensures an uninterrupted sup-  ply of biologically active free T 3 in the blood (Fig. 43.11). High levels of T 3 also  inhibit the release of TRH from the hypothalamus.   Only those physiologic actions of thyroid hormone that influence fuel metabolism  are considered here. It is important to stress the term physiologic, because the  effects of supraphysiologic concentrations of thyroid hormone on fuel metabolism  may not be simple extensions of their physiologic effects. In general, the following   Fig. 43.11. Feedback regulation of thyroid  hormone levels. TRH from the hypothalamus  stimulates the release of TSH from the anterior  pituitary, which stimulates the release of T 3  and T 4 from the thyroid. T 4 is converted to T 3  in the liver and other cells. T 3 and T 4 inhibit the  release of TSH from the anterior pituitary and  of TRH from the hypothalamus.   A patient presents with the follow-  I ■•) ing clinical and laboratory profile:  X^v/ the serum free and total T 3 and T 4  and the serum TSH levels are elevated, but  the patient has symptoms of mild hypothy-  roidism, including a diffuse, palpable goiter.  What single abnormality in the pituitary-thy-  roid-thyroid hormone target cell axis would  explain all of these findings?   A generalized (i.e., involving all of  the target cells for thyroid hor-  mone in the body), but incomplete  resistance of cells to the actions of thyroid  hormone could explain the profile of the  patient. In Refetoffs disorder, a mutation in  the portion of the gene that encodes the lig-  and binding domain of the (3-subunit of the  thyroid hormone receptor protein causes a  relative resistance to the suppressive action  of thyroid hormone on the secretion of TSH  by the thyrotrophs of the anterior pituitary  gland. Therefore, the gland releases more  TSH than normal into the blood. The ele-  vated level of TSH causes an enlargement of  the thyroid gland (goiter) as well as an  increase in the secretion of thyroid hormone  into the blood. As a result, the serum levels  of both T 3 and T 4 rise in the blood. The  increase in the secretion of thyroid hormone  may or may not be adequate to fully com-  pensate for the relative resistance of the  peripheral tissues to thyroid hormone. If the  compensatory increase in the secretion of  thyroid hormone is inadequate, the patient  may develop the signs and symptoms of  hypothyroidism.    When present in excess, T 3 has  severe catabolic effects that  increase the flow of amino acids    comments apply to the effects of thyroid hormone on energy metabolism in indi-  viduals with normal thyroid hormone levels in the blood.   Several of the actions of thyroid hormone affect carbohydrate and lipid metabolism  in the liver. Thyroid hormone increases glycolysis and cholesterol synthesis and  increases the conversion of cholesterol to bile salts. Through its action of increas-  ing the sensitivity of the hepatocyte to the gluconeogenic and glycogenolytic  actions of epinephrine, T 3 indirectly increases hepatic glucose production (permis-  sive or facilitatory action). Because of its ability to sensitize the adipocyte to the  lipolytic action of epinephrine, T 3 increases the flow of fatty acids to the liver and  thereby indirectly increases hepatic triacylglycerol synthesis. The concurrent  increase in the flow of glycerol to the liver (as a result of increased lipolysis) fur-  ther enhances hepatic gluconeogenesis.   T 3 has an amplifying or facilitatory effect on the lipolytic action of epinephrine on  the fat cell. Yet thyroid hormone has a bipolar effect on lipid storage, because it  increases the availability of glucose to the fat cell, which serves as a precursor for  fatty acid and glycerol 3 -phosphate synthesis. The major determinant of the rate of  lipogenesis, however, is not T 3 , but rather the amount of glucose and insulin avail-  able to the adipocyte for triacylglycerol synthesis.   In physiologic concentrations, T 3 increases glucose uptake by muscle cells. It also  stimulates protein synthesis, and, therefore, growth of muscle, through its stimula-  tory actions on gene expression.   In physiologic concentrations, thyroid hormone sensitizes the muscle cell to the  glycogenolytic actions of epinephrine. Glycolysis in muscle is increased by this  action of T 3 .   Thyroid hormone increases the sensitivity of the (3 cells of the pancreas to those stim-  uli that normally promote insulin release and is required for optimal insulin secretion.   The oxidation of fuels converts approximately 25% of the potential energy pres-  ent in the foods ingested by humans to ATP. This relative inefficiency of the  human "engine" leads to the production of heat as a consequence of fuel utiliza-  tion. This inefficiency, in part, allows homeothermic animals to maintain a con-  stant body temperature in spite of rapidly changing environmental conditions. The  acute response to cold exposure is shivering, which is probably secondary to  increased activity of the sympathetic nervous system in response to this "stress-  ful" stimulus.   Thyroid hormone participates in this acute response by sensitizing the sympa-  thetic nervous system to the stimulatory effect of cold exposure. The ability of T 3 to  increase heat production is related to its effects on the pathways of fuel oxidation,  which both generate ATP and release energy as heat. The effects of T 3 on the sym-  pathetic nervous system increase the release of norepinephrine. Norepinephrine  stimulates the uncoupling protein thermogenin in brown adipose tissue (BAT),  resulting in increased heat production from the uncoupling of oxidative phosphory-   lation (see Chapter 21). Very little residual brown fat persists in normal adult human  beings, however.   Norepinephrine also increases the permeability of BAT and skeletal muscle to  sodium. Because an increase of intracellular Na + is potentially toxic to cells,  Na + ,K + -ATPase is stimulated to transport Na+ out of the cell in exchange for K + .  The increased hydrolysis of ATP by Na + ,K + -ATPase stimulates the oxidation of  fuels and the regeneration of more ATP and heat from oxidative phosphorylation.  Over a longer time course, thyroid hormone also increases the level of Na + ,K + -  ATPase and many of the enzymes of fuel oxidation. Because even at normal room  temperature ATP utilization by Na + ,K + -ATPase accounts for 20% or more of our  basal metabolic rate (BMR), changes in its activity can cause relatively large  increases in heat production.   Thyroid hormone also may increase heat production by stimulating ATP utiliza-  tion in futile cycles (in which reversible ATP-consuming conversions of substrate to  product and back to substrate use fuels and, therefore, produce heat).   In addition to insulin and the counterregulatory hormones discussed, a variety of  peptides synthesized in the endocrine cells of the pancreatic islets, or the cells of  the enteric nervous system, or the endocrine cells of the stomach, small bowel and  large bowel, as well as certain cells of the central and peripheral nervous system,  influence fuel metabolism directly. Some of these peptides and their tissue of ori-  gin, their actions on fuel metabolism, and the factors that stimulate (or suppress)  their secretion are listed in Table 43.4. In addition to these peptides, others such as  gastrin, motilin, pancreatic polypeptide (PP), peptide YY (PYY), and secretin may  also influence fuel metabolism but by indirect effects on the synthesis or secretion  of insulin or the counterregulatory hormones (Table 43.5). For example, gastrin  induces gastric acid secretion, which ultimately affects nutrient absorption and  metabolism. Motilin, secreted by enteroendocrine M cells of the proximal small  bowel, stimulates gastric and pancreatic enzyme secretion, which, in turn, influ-  ences nutrient digestion. Pancreatic polypeptide (PP) from the pancreatic islets  reduces gastric emptying and slows upper intestinal motility. Peptide YY (PYY)  from the alpha cells in the mature pancreatic islets inhibits gastric acid secretion.  Finally, secretin, produced by the enteroendocrine S cells in the proximal small  bowel, regulates pancreatic enzyme secretion and inhibits gastrin release and gas-  tric acid secretion. Although not directly influencing fuel metabolism, these "gut"  hormones have a significant impact on how ingested nutrients are digested and pre-  pared for absorption. If digestion or absorption of fuels is altered through a distur-  bance in the delicate interplay of all of the peptides, fuel metabolism will be  altered as well.   Several of these gastrointestinal peptides such as GLP-1 and GIP do not act  as direct insulin secretagogues when blood glucose levels are normal but do  so after a meal large enough to cause an increase in the blood glucose concen-  tration.   The release of these peptides may explain why the modest postprandial increase  in serum glucose seen in normal subjects has a relatively robust stimulatory effect  on insulin release, whereas a similar glucose concentration in vitro elicits a signifi-  cantly smaller increase in insulin secretion. Likewise, this effect (certain factors  potentiating insulin release), known as the "incretin effect," could account for the  greater beta cell response seen after an oral glucose load as opposed to that seen  after the administration of glucose intravenously.   In hypothyroid patients, insulin  release may be suboptimal,  although glucose intolerance on  this basis alone is uncommon.   In hyperthyroidism, the degradation and  the clearance of insulin are increased. These  effects, plus the increased demand for  insulin caused by the changes in glucose  metabolism, may lead to varying degrees of  glucose intolerance in these patients (a con-  dition called metathyroid diabetes mellitus).  The patient with uncomplicated hyperthy-  roidism, however, rarely develops signifi-  cant diabetes mellitus.    Nervous system, pituitary,  neurons of gut, pancreas,  thyroid, and adrenal gland   Small intestinal N cells  (especially ileum), enteric  nervous system, adrenal  gland, pancreas   Stimulates release of  cholecystokinin; GIP, gastrin,  glucagon, GLP-1, GLP-2, and  somatostatin   Primary counter-regulatory hormone  that restores glucose levels in  hypoglycemic state (increases  glycogenolysis and  gluconeogenesis as well as  protein-lipid flux in liver and  muscle)   1. Enhances glucose disposal after  meals by inhibiting glucagon  secretion and stimulating insulin  secretion   2. Acts through second  messengers in beta cells to  increase sensitivity of these cells  to glucose (an incretin)   1 . Inhibits secretion of insulin,  glucagon and PP (islets), and gas-  trin, secretin, GLP-1, and   Enteroendocrine G cells of the  stomach, duodenal bulb, and  other cells   Endocrine cells in periphery of  islets in the head of the  pancreas   Neurons localized in the sub-  mucous and myenteric  plexuses; enterochromaffin  cells in gut epithelium   Enteric nervous system, colon,  G cells of stomach, and  pancreatic beta cell   3. Inhibits postprandial gastric emptying,  gastrin release, and gastric acid  secretion   1 . Luminal contents, especially  aromatic amino acids, cal-  cium, coffee, and ethanol   1 . Gastric acid, bile salts, fatty  acids, peptides, and ethanol   Direct and indirect activation of  neurons in submucosa and  myenteric plexuses in gut  epithelium   Although beyond the scope of this text, the gastrointestinal neuroendocrine system  is briefly described with regard to its effects on fuel metabolism. The pancreatic  islet cells are innervated by both the adrenergic and the cholinergic limbs of the  autonomic nervous system. Although stimulation of both the sympathetic and the  parasympathetic systems increases glucagon secretion, insulin secretion is  increased by vagus nerve fibers and suppressed by sympathetic fibers via the alpha-  adrenoreceptors. Evidence also suggests that the sympathetic nervous system indi-  rectly regulates pancreatic beta cell function through stimulation or suppression of  the secretion of somatostatin, beta 2 -adrenergic receptor number, and the neuropep-  tides, neuropeptide Y and galanin.   A tightly controlled interaction between the hormonal and neural factors that  control nutrient metabolism is necessary to maintain normal fuel and, hence, energy  homeostasis.   To establish the diagnosis of a  secretory tumor of an endocrine  gland, one must first demonstrate  that basal serum levels of the hormone in  question are regularly elevated. More impor-  tantly, one must show that the hypersecre-  tion of the hormone (and, hence, its elevated  level in the peripheral blood) cannot be ade-  quately inhibited by "maneuvers" that are  known to suppress secretion from a nor-  mally functioning gland (i.e., one must show  that the hypersecretion is "autonomous").   To ensure that both the basal and the  postsuppression levels of the specific hor-  mone to be tested will reflect the true secre-  tory rate of the suspected endocrine tumor,  all of the known factors that can stimulate  the synthesis of the hormone must be elimi-  nated. For GH, for example, the secreta-  gogues (stimulants to secretion) include  nutritional factors; the patient's level of  activity, consciousness, and stress; and cer-  tain drugs. GH secretion is stimulated by a  high-protein meal or by a low level of fatty  acids or of glucose in the blood. Vigorous  exercise, stage lll-IV sleep, psychological  and physical stress, and levodopa, clonidine,  and estrogens also increase GH release.   The suppression test used to demon-  strate the autonomous hypersecretion of GH  involves giving the patient an oral glucose  load and, subsequently, measuring GH lev-  els. A sudden rise in blood glucose sup-  presses serum GH to 2 ng/mL or less in nor-  mal subjects, but not in patients with active  acromegaly.   If one attempts to demonstrate auto-  nomous hypersecretion of GH in a patient  suspected of having acromegaly, therefore,  before drawing the blood for both the basal  (pre-glucose load) serum GH level and the  post-glucose load serum GH level, one must  be certain that the patient has not eaten for  6-8 hours, has not done vigorous exercise  for at least 4 hours, remains fully awake dur-  ing the entire testing period (in a non-  stressed state to the extent possible), and  has not taken any drugs known to increase  GH secretion for at least 1 week.   Under these carefully controlled circum-  stances, if both the basal and postsuppres-  sion serum levels of the suspect hormone  are elevated, one can conclude that  autonomous hypersecretion is probably  present. At this point, localization proce-  dures (such as an MRI of the pituitary gland  in an acromegalic suspect) are performed to  further confirm the diagnosis.   One of the functions of Cortisol is to prepare the body to deal with periods  of stress. In response to Cortisol, the body re-sorts its fuel stores so that  they can rapidly be made available for the "fight or flight" response to the  alarm signal sounded by epinephrine. Cortisol causes gluconeogenic substrates to  move from peripheral tissues to the liver, where they are converted to glucose and  stored as glycogen. The release of epinephrine stimulates the breakdown of glyco-  gen, increasing the supply of glucose to the blood. Thus fuel becomes available for  muscle to fight or flee.   Cushing's "disease," the cause of Corti Solemia's current problems, results from  prolonged hypersecretion of ACTH from a benign pituitary tumor. ACTH stimulates  the adrenal cortex to produce Cortisol, and blood levels of this steroid hormone rise.   Other nonpituitary causes of Cushing's syndrome, however, include a primary  tumor of the adrenal cortex secreting excessive amounts of Cortisol directly into the  bloodstream. This disorder also can result from the release of ACTH from secretory  nonendocrine nonpituitary neoplasms ("ectopic" ACTH syndrome). Cushing's syn-  drome is often caused by excessive doses of synthetic GCs used to treat a variety of  disorders because of their potent anti-inflammatory effects (iatrogenic Cushing's  syndrome).   The diabetogenic potential of chronically elevated GH levels in the blood is mani-  fest by the significant incidence of diabetes mellitus (25%) and impaired glucose tol-  erance (33%) in patients with acromegaly, such as Sam Atotrope. Yet, under normal  circumstances, physiologic concentrations of GH (as well as Cortisol and thyroid hor-  mone) have a facilitatory or permissive effect on the quantity of insulin released in  response to hyperglycemia and other insulin secretagogues. This "proinsular" effect is  probably intended to act as a "brake" to dampen any potentially excessive "contrain-  sular" effects that increments in GH and the other counterregulatory hormones exert.   Most hormones are present in body fluids in picomolar to nanomolar  amounts, requiring highly sensitive assays to determine their concentration in  the blood or urine. Radioimmunoassays (RIAs), developed in the 1960s, use  an antibody, generated in animals, against a specific antigen (the hormone to be meas-  ured). Determining the concentration of the hormone in the sample involves incubat-  ing the plasma or urine sample with the antibody and then quantifying the level of anti-  gen-antibody complex formed during the incubation by one of several techniques.   The classic RIA uses very high-affinity antibodies, which have been fixed  (immobilized) on the inner surface of a test tube, a Teflon bead, or a magnetized  particle. A standard curve is prepared, using a set amount of the antibody and vari-  ous known concentrations of the unlabeled hormone to be measured. In addition to  a known concentration of the unlabeled hormone, each tube contains the same  small, carefully measured amount of radiolabeled hormone. The labeled hormone  and the unlabeled hormone compete for binding to the antibody. The higher the  amount of unlabeled hormone in the sample, the less radiolabeled hormone is  bound. A standard curve is plotted (Fig. 43.12). The unknown sample from the  patient's blood or urine, containing the unlabeled hormone to be measured, is incu-  bated with the immobilized antibody in the presence of the same small, carefully  measured amount of radiolabeled hormone. The amount of radiolabeled hormone  bound to the antibody is determined, and the standard curve is used to quantitate the  amount of unlabeled hormone in the patient sample.   The same principle is used in immunoradiometric assays (IRMAs), but with  this technique the antibody, rather than the antigen to be measured, is radiola-  beled.   The sensitivity of RIAs can be enhanced using a "sandwich technique." This  method uses two different monoclonal antibodies (antibodies generated by a single  clone of plasma cells rather than multiple clones), each of which recognizes a dif-  ferent specific portion of the hormone's structure. The first antibody, attached to a  solid support matrix such as a plastic culture dish, binds the hormone to be assayed.  After exposure of the patient sample to this first antibody, the excess plasma is  washed away, and the second antibody (which is radiolabeled) is then incubated  with the first antibody-hormone complex. The amount of binding of the second  (labeled) antibody to the first complex is proportional to the concentration of the  hormone in the sample.   The sandwich technique can be improved even further if the second antibody is  attached to an enzyme, such as alkaline phosphatase. The enzyme rapidly converts  an added colorless substrate into a colored product, or a nonfluorescent substrate  into a highly fluorescent product. These changes can be quantitated if the degree of  change in color or fluorescence is proportional to the amount of hormone present in  the patient sample. Less than a nanogram (1CT 9 g) of a protein can be measured by  such an enzyme-linked immunosorbent assay (ELISA).   Fig. 43.12. Standard curve for a radioim-  munoassay. A constant amount of radioactive  T 4 is added to a series of tubes, each of which  contains a different amount of nonradioactive  T 4 . The amount of radioactive hormone that  binds to an antibody specific for the hormone  is measured and plotted against the nonra-  dioactive hormone concentration. When more  nonradioactive T 4 is present in the tube, less  radioactive T 4 binds to the antibody.   Bloomgarden ZT. Fat metabolism and diabetes: 2003 American Diabetes Association Postgraduate   Course. Diabetes Care, 2003 (July);26:2198-2203.  Davis SN, Shavers C, Costa F, Mosqueda-Garcia R. Role of Cortisol in the pathogenesis of deficient   counterregulation after antecedent hypoglycemia in normal humans. J Clin Invest 1996; 98:680-691.  Goodman HM. Hormonal regulation of fuel metabolism. In: Basic Medical Endocrinology. New York:   Raven Press, 1994:203-224.  Itani SI, Ruderman NB, Schmieder F, Boden G. Lipid-induced insulin resistance in human muscle is   associated with changes in diacylglycerol, protein kinase C, and I kappa B-a. Diabetes 2002;   Laurent D, Peterson KF, Russell RR, et al. Effect of epinephrine on muscle glycogenolysis and insulin-  stimulated muscle glycogen synthesis in humans. Am J Physiol 1998;274:E130-E138.   Cryer PE. Glucose homeostasis and hypoglycemia. In: Larsen PF, Kronenberg HM, Melmed S, Polon-  sky KS, eds. Williams Textbook of Endocrinology, 10th Ed. Philadelphia: Saunders Publishing,  2003;1585-1591.   Zander M., Madsbad S, Madsen JL, Hoist JJ. Effect of 6- week course of glucagon-like peptide one on  glycemic conrol, insulin sensitivity, and beta cell function in type 2 diabetes. Lancet  2002;359:824-830.   1. As a third-year medical student, you examine your first patient. You find that he is 52 years old, has a round face, acne, and  a large hump of fat on the back of his neck. He complains that he is too weak to mow his lawn. His fasting blood glucose level  is 170 mg/dL (reference range = 80-100 mg/dL). Plasma Cortisol levels are 62 jxg/mL (reference range = 3-31 jxg/mL).  Plasma ACTH levels are 0 pg/mL (reference range = 0-100 pg/mL).   Based on the information given above, if the patient's problem is attributable to a single cause, the most likely diagnosis  is which of the following?   2. A woman was scheduled for a growth hormone suppression test. If each of the following events occurred the morning of the  test, which one of the events would be most likely to cause a decrease in growth hormone levels?   (B) She was on estrogen replacement therapy and took her tablets after breakfast.   (C) While unlocking her car, she was chased by the neighbor's vicious dog.   (D) She fell asleep at the start of the test and slept soundly until it was completed 1.5 hours later.   3. A dietary deficiency of iodine would lead to which of the following?   (A) A direct effect on the synthesis of thyroglobulin on ribosomes   4. A woman whose thyroid gland was surgically removed was treated with 0. 10 mg thyroxine daily (tablet form). After 3 months  of treatment, serial serum TSH levels ranged between 10 and 15 MIU/mL (reference range = 0.3 - 5.0 MIU/mL). She com-  plained of fatigue, weight gain, and hoarseness. Her dose of thyroid hormone should be adjusted in which direction?   A patient complains of nervousness, palpitations, sweating, and weight loss without loss of appetite, and has a goiter. Sus-  pecting a defect in thyroid function, the physician orders a total serum T 4 . The test is performed by radioimmunoassay.  The standard curve for the assay, which measures T 4 in 0.1 mL serum, is shown in Fig. 43.12. Normal levels of T 4 =  4-10 |JLg/dL. In an assay of 0.1 mL of the patient's serum, 15% of the radioactive T 4 was bound by the antibody.   5. According to the radioimmunoassay, the approximate blood level of T 4 is is which of the following?   The cells of the blood are classified as erythrocytes, leukocytes, or thrombocytes.   The erythrocytes (red cells) carry oxygen to the tissues and are the most numerous  cells in the blood. The leukocytes (white cells) are involved in defense against  infection, and the thrombocytes (platelets) function in blood clotting. All of the  cells in the blood can be generated from hematopoietic stem cells in the bone mar-  row on demand. For example, in response to infection, leukocytes secrete cytokines  called interleukins that stimulate the production of additional leukocytes to fight  the infection. Decreased supply of oxygen to the tissues signals the kidney to  release erythropoietin, a hormone that stimulates the production of red cells.   The red cell has limited metabolic function, owing to its lack of internal  organelles. Glycolysis is the main energy- generating pathway, with lactate pro-  duction regenerating NAD + for glycolysis to continue. The NADH produced in  glycolysis is also used to reduce the ferric form of hemoglobin, methemoglobin,  to the normal ferrous state. Glycolysis also leads to a side pathway in which 2,3  bisphosphogly cerate is produced, which is a major allosteric effector for oxygen  binding to hemoglobin. The hexose monophosphate shunt pathway generates  NADPH to protect red cell membrane lipids and proteins from oxidation, through  regeneration of reduced glutathione. Heme synthesis occurs in the precursors of  red cells and is a complex pathway that originates from succinyl-CoA and glycine.  Mutations in any of the steps of heme synthesis lead to a group of diseases known  collectively as porphyrias.   The red cell membrane must be highly deformable to allow it to travel through-  out the capillary system in the body. This is because of a complex cytoskeletal  structure that consists of the major proteins spectrin, ankyrin, and band 3  protein. Mutations in these proteins lead to improper formation of the membrane  cytoskeleton, ultimately resulting in malformed red cells, spherocytes, in the cir-  culation. Spherocytes have a shortened life span, leading to loss of blood cells.   When the body does not have sufficient red cells, the patient is said to be ane-  mic. Anemia can result from many causes. Nutritional deficiencies of iron, folate,  or vitamin B12 prevent the formation of adequate numbers of red cells. Mutations  in the genes that encode red cell metabolic enzymes, membrane structural pro-  teins, and globins cause hereditary anemias. The appearance of red cells on a  blood smear frequently provides clues to the cause of an anemia. Because the  mutations that give rise to hereditary anemias also provide some protection against  malaria, hereditary anemias are some of the most common genetic diseases known.   The human alters globin gene expression during development, a process known  as hemoglobin switching. The switch between expression of one gene to another  is regulated by transcription factor binding to the promoter regions of these genes.  Current research is attempting to reactivate fetal hemoglobin genes to combat  sickle-cell disease and thalassemia.   • Anne Niemick, who has |3 + thalassemia, complains of pain in her lower  flP ) spine (see Chapters 14 and 15). A quantitative computed tomogram (CT)   " of the vertebral bodies of the lumbar spine shows evidence of an area of  early spinal cord compression in the upper lumbar region. She is suffering from  severe anemia, resulting in stimulation of production of red blood cell precursors  (the erythroid mass) from the stem cells in her bone marrow. This expansion of  marrow volume causes compression of tissues in this area, which, in turn, causes  pain. Local irradiation is considered, as is a program of regular blood transfusions  to maintain the oxygen- carrying capacity of circulating red blood cells. The  results of special studies related to the genetic defect underlying her thalassemia  are pending, although preliminary studies have shown that she has elevated levels  of fetal hemoglobin, which, in part, moderates the manifestations of her disease.  Anne Niemick's parents have returned to the clinic to discuss the results of these  tests.   • Spiro Site is a 21 -year-old college student who complains of feeling tired  H|I all the time. Two years previously he had had gallstones removed, which   " consisted mostly of bilirubin. His spleen is palpable, and jaundice is  evidenced by yellowing of the whites of his eyes. His hemoglobin was low (8 g/dL;  reference valuel 3.5-17.5 gm/dL). A blood smear showed dark, rounded, abnormally  small red cells called spherocytes as well as an increase in the number of circulating  immature red blood cells known as reticulocytes.   The blood, together with the bone marrow, makes up the organ system that  makes a significant contribution to achieving homeostasis, the maintenance of  the normal composition of the body's internal environment. Blood can be con-  sidered a liquid tissue consisting of water, proteins, and specialized cells. The  most abundant cell in the blood is the erythrocyte or red blood cell, which trans-  ports oxygen to the tissues and contributes to buffering of the blood through the  binding of protons by hemoglobin (see section IV of this chapter, and the mate-  rial in Chapter 4, section IV.D.2., and Chapter 7, section VII). Red blood cells  lose all internal organelles during the process of differentiation. The white blood  cells (leukocytes) are nucleated cells present in blood that function in the  defense against infection. The platelets (thrombocytes), which contain cyto-  plasmic organelles but no nucleus, are involved in the control of bleeding by con-  tributing to normal thrombus (clot) formation within the lumen of the blood ves-  sel. The average concentration of these cells in the blood of normal individuals  is presented in Table 44.1.   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   The leukocytes can be classified either as polymorphonuclear leukocytes (granulo-  cytes) or mononuclear leukocytes, depending on the morphology of the nucleus in  these cells. The mononuclear leukocyte has a rounded nucleus, whereas the poly-  morphonuclear leukocytes have a multilobed nucleus.   The granulocytes, so named because of the presence of secretory granules visible  on staining, are the neutrophils, eosinophils, and basophils. When these cells are  activated in response to chemical stimuli, the vesicle membranes fuse with the cell  plasma membrane, resulting in the release of the granule contents (degranulation).  The granules contain many cell-signaling molecules that mediate inflammatory  processes. The granulocytes, in addition to displaying segmented nuclei (are poly-  morphonuclear), can be distinguished from each other by their staining properties  (caused by different granular contents) in standard hematologic blood smears; neu-  trophils stain pink, eosinophils stain red, and basophils stain blue.   Neutrophils are phagocytic cells that rapidly migrate to areas of infection or tis-  sue damage. As part of the response to acute infection, neutrophils engulf foreign  bodies, and destroy them, in part, by initiating the respiratory burst (see Chapter  24). The respiratory burst creates oxygen radicals that rapidly destroy the foreign  material found at the site of infection.   A primary function of eosinophils is to destroy parasites such as worms. The  eosinophilic granules are lysosomes containing hydrolytic enzymes and cationic  proteins, which are toxic to parasitic worms. Eosinophils have also been implicated  in asthma and allergic responses, although their exact role in the development of  these disorders is still unknown, and this is an active area of research.   Basophils, the least abundant of the leukocytes, participate in hypersensitivity  reactions, such as allergic responses. Histamine, produced by the decarboxylation  of histidine, is stored in the secretory granules of basophils. Release of histamine  during basophil activation stimulates smooth muscle cell contraction and  increases vascular permeability. The granules also contain enzymes such as  proteases, p-glucuronidase, and lysophospholipase. These enzymes degrade  microbial structures and assist in the remodeling of damaged tissue.   The mononuclear leukocytes consist of various classes of lymphocytes and the  monocytes. Lymphocytes are small, round cells originally identified in lymph  fluid. These cells have a high ratio of nuclear volume to cytoplasmic volume and  are the primary antigen (foreign body) -recognizing cells. There are three major  types of lymphocytes: T cells, B cells, and NK cells. The precursors of T cells  (thymus -derived lymphocytes) are produced in the bone marrow and then migrate  to the thymus, where they mature before being released to the circulation. Several  subclasses of T cells exist. These subclasses are identified by different surface  membrane proteins, the presence of which correlate with the function of the sub-  class. Lymphocytes that mature in the bone marrow are the B cells, which secrete  antibodies in response to antigen binding. The third class of lymphocytes are the  natural killer cells (NK cells), which target virally infected and malignant cells for  destruction.   Circulatory monocytes are the precursors of tissue macrophages. Macrophages  (large eater) are phagocytic cells that enter inflammatory sites and consume  microorganisms and necrotic host cell debris left behind by granulocyte attack of  the foreign material. Macrophages in the spleen play an important role in maintaining   the oxygen-delivering capabilities of the blood by removing damaged red blood  cells that have a reduced oxygen-carrying capacity.   Platelets are heavily granulated disc-like cells that aid in intravascular clotting. Like  the erythrocyte, platelets lack a nucleus. Their function is discussed in the follow-  ing chapter. Platelets arise by budding of the cytoplasm of megakaryocytes, multi-  nucleated cells that reside in the bone marrow.   include the mean corpuscular vol-  ume (MCV) and the mean corpuscular  hemoglobin concentration (MCHC). The  MCV is the average volume of the red blood  cell, expressed in femto (10~ 15 ) liters. Nor-  mal MCV range from 80 to 100 fl_. The MCHC  is the average concentration of hemoglobin  in each individual erythrocyte, expressed in  g/L. The normal range is 32 to 37; a value of  less than 32 would indicate hypochromic  cells. Thus, microcytic, hypochromic red  blood cells have an MCV of less than 80 and  an MCHC of less than 32. Macrocytic, nor-  mochromic cells have an MCV of greater  than 100, with an MCHC between 32 and 37.   The trace amounts of 2,3 BPG  \\ ) found in cells other than erythro-  *r cytes is required for the phospho-  glycerate mutase reaction of glycolysis, in  which 3-phosphoglycerate is isomerized to  2-phosphoglycerate. As the 2,3 BPG is  regenerated during each reaction cycle, it is  only required in catalytic amounts.   The major function of erythrocytes is to deliver oxygen to the tissues. To do this, a  sufficient concentration of hemoglobin in the red blood cells is necessary for effi-  cient oxygen delivery to occur. When the hemoglobin concentration falls below nor-  mal values (Table 44.2), the patient is classified as anemic. Anemias can be catego-  rized based on red cell size and hemoglobin concentration. Red cells can be of  normal size (normocytic), small (microcytic), or large (macrocytic). Cells contain-  ing a normal hemoglobin concentration are termed normochromic; those with  decreased concentration are hypochromic. This classification system provides  important diagnostic tools (Table 44.3) that enable one to properly classify, diag-  nose, and treat the anemia.   To best understand how the erythrocyte can carry out its major function, a discus-  sion of erythrocyte metabolism is required. Mature erythrocytes contain no intra-  cellular organelles, so the metabolic enzymes of the red blood cell are limited to  those found in the cytoplasm. In addition to hemoglobin, the cytosol of the red  blood cell contains enzymes necessary for the prevention and repair of damage done  by reactive oxygen species (see Chapter 24) and the generation of energy  (Fig. 44.1). Erythrocytes can only generate adenosine triphosphate (ATP) by gly-  colysis (see Chapter 22). The ATP is used for ion transport across the cell membrane  (primarily Na + , K + , and Ca +2 ), the phosphorylation of membrane proteins, and the  priming reactions of glycolysis. Erythrocyte glycolysis also uses the Rapaport-Lue-  bering shunt to generate 2,3 -bisphosphogly cerate (2,3-BPG). Red cells contain 4 to  5 mM 2,3-BPG, compared with trace amounts in other cells. As discussed in more  detail in Section IV, 2,3-BPG is a modulator of oxygen binding to hemoglobin that  stabilizes the deoxy form of hemoglobin, thereby facilitating the release of oxygen  to the tissues.   To bind oxygen, the iron of hemoglobin must be in the ferrous ( + 2) state.  Reactive oxygen species can oxidize the iron to the ferric ( + 3) state, producing   Table 44.3. Classification of the Anemias on the Basis of Red Cell  Morphology   Acute bleeding, sickle cell  disease, red cell metabolic  defects, red cell membrane  defects   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.1. Overview of erythrocyte metabolism. Glycolysis is the major pathway, with branches for the hexose monophosphate shunt (for pro-  tection against oxidizing agents) and the Rapoport-Luebering shunt (which generates 2,3 bisphosphoglycerate, which moderates oxygen bind-  ing to hemoglobin). The NADH generated from glycolysis can be used to reduce methemoglobin (Fe +3 ) to normal hemoglobin (Fe +2 ), or to con-  vert pyruvate to lactate, such that NAD + can be regenerated and used for glycolysis. Pathways unique to the erythrocyte are indicated in blue.   methemoglobin. Some of the NADH produced by glycolysis is used to regenerate  hemoglobin from methemoglobin by the NADH-cytochrome b 5 methemoglobin  reductase system. Cytochrome b 5 reduces the Fe +3 of methemoglobin. The oxi-  dized cytochrome b 5 is then reduced by a flavin-containing enzyme, cytochrome b 5  reductase (also called methemoglobin reductase), using NADH as the reducing  agent.   An inherited deficiency in pyruvate kinase leads to hemolytic anemia (an anemia caused by the destruction of red blood cells; hemo-  globin values typically drop to 4 to 10 g/dL in this condition). Because the amount of ATP formed from glycolysis is decreased by 50%,  red blood cell ion transporters cannot function effectively. The red blood cells tend to gain Ca 2+ and lose K + and water. The water loss  increases the intracellular hemoglobin concentration. With the increase in intracellular hemoglobin concentration, the internal viscosity of the  cell is increased to the point that the cell becomes rigid and, therefore, more susceptible to damage by shear forces in the circulation. Once  damaged, the red blood cells are removed from circulation, leading to the anemia. However, the effects of the anemia are frequently moder-  ated by the twofold to threefold elevation in 2,3-BPG concentration that results from the blockage of the conversion of phosphoenol pyruvate  to pyruvate. Because 2,3-BPG binding to hemoglobin decreases the affinity of hemoglobin of oxygen, the red blood cells that remain in circu-  lation are highly efficient in releasing their bound oxygen to the tissues.   Congenital methemoglobinemia,  the presence of excess methemo-  globin, is found in people with an  enzymatic deficiency in cytochrome b 5 red-  uctase or in people who have inherited hem-  oglobin M. In hemoglobin M, a single amino  acid substitution in the heme-binding pocket  stabilizes the ferric (Fe +3 ) oxygen. Individu-  als with congenital methemoglobinemia  appear cyanotic but have few clinical prob-  lems. Methemoglobinemia can be acquired  by ingestion of certain oxidants such as  nitrites, quinones, aniline, and sulfon-  amides. Acquired methemoglobinemia can  be treated by the administration of reducing  agents, such as ascorbic acid or methylene  blue.   G6PD deficiency is the most com-  \\ ) mon enzyme deficiency known in   humans, probably, in part, because  individuals with G6PD deficiency are resist-  ant to malaria. The resistance to malaria  counterbalances the deleterious effects of  the deficiency. G6PD-deficient red cells have  a shorter life span and are more likely to lyse  under conditions of oxidative stress. When  soldiers during the Korean War were given  the antimalarial drug primaquine prophylac-  tically, approximately 10% of the soldiers of  African ancestry developed a spontaneous  anemia. Because the gene for G6PD is found  on the X chromosome, these men had only  one copy of a variant G6PD gene   All known G6PD variant genes contain  small in-frame deletions or missense muta-  tions. The corresponding proteins, therefore,  have decreased stability or lowered activity,  leading to a reduced half-life or lifespan for  the red cell. No mutations have been found  that result in complete absence of G6PD.  Based on studies with knockout mice, those  mutations would be expected to result in  embryonic lethality.   Heme, which is red, is responsible  \% J for the color of red blood cells and  *r of muscles that contain a large  number of mitochondria.   Chlorophyll, the major porphyrin in  plants, is similar to heme, except that it is  coordinated with magnesium rather than  iron, and it contains different substituents on  the rings, including a long-chain alcohol  (phytol). As a result of these structural dif-  ferences, chlorophyll is green.    Approximately 5 to 10% of the glucose metabolized by red blood cells is used  to generate NADPH by way of the hexose monophosphate shunt. The NADPH is  used to maintain glutathione in the reduced state. The glutathione cycle is the red  blood cell's chief defense against damage to proteins and lipids by reactive oxygen  species (see Chapter 24).   The enzyme that catalyzes the first step of the hexose monophosphate shunt is  glucose-6-phosphate dehydrogenase (G6PD). The lifetime of the red blood cell cor-  relates with G6PD activity. Lacking ribosomes, the red blood cell cannot synthesize  new G6PD protein. Consequently, as the G6PD activity decreases, oxidative damage  accumulates, leading to lysis of the erythrocyte. When red blood cell lysis (hemoly-  sis) substantially exceeds the normal rate of red blood cell production, the number of  erythrocytes in the blood drops below normal values, leading to a hemolytic anemia.   Heme consists of a porphyrin ring coordinated with an atom of iron (Fig. 44.2).  Four pyrrole rings are joined by methionyl bridges ( — CH — ) to form the porphyrin  ring (see Fig. 7.12). Eight side chains serve as substituents on the porphyrin ring,  two on each pyrrole. These side chains may be acetyl (A), propionyl (P), methyl  (M), or vinyl (V) groups. In heme, the order of these groups isMVMVMPPM.  This order, in which the position of the methyl group is reversed on the fourth ring,  is characteristic of the porphyrins of the type III series, the most abundant in nature.   Heme is the most common porphyrin found in the body. It is complexed with  proteins to form hemoglobin, myoglobin, and the cytochromes (see Chapters 7 and  21), including cytochrome P450 (see Chapter 24).   Heme is synthesized from glycine and succinyl Co A (Fig. 44.3), which condense in  the initial reaction to form 8 -aminolevulinic acid (8-ALA) (Fig 44.4). The enzyme  that catalyzes this reaction, 8-ALA synthase, requires the participation of pyridoxal  phosphate, as the reaction is an amino acid decarboxylation reaction (glycine is  decarboxylated; see Chapter 39).   The next reaction of heme synthesis is catalyzed by o-ALA dehydratase, in  which two molecules of 8-ALA condense to form the pyrrole, porphobilinogen  (Fig. 44.5). Four of these pyrrole rings condense to form a linear chain and then a  series of porphyrinogens. The side chains of these porphyrinogens initially contain   Fig. 44.2. Structure of heme. The side chains can be abbreviated as MVMVMPPM. M =  methyl (CH 3 ); V = vinyl (— CH=CH 2 ); P = propionyl (— CH 2 — CH 2 — COO").   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.3. Synthesis of heme. To produce one molecule of heme, 8 molecules each of glycine  and succinyl CoA are required. A series of porphyrinogens are generated in sequence.  Finally, iron is added to produce heme. Heme regulates its own production by repressing the  synthesis of 8-aminolevulinic acid (8-ALA) synthase (circled \) and by directly inhibiting  the activity of this enzyme (circled -). Deficiencies of enzymes in the pathway result in a  series of diseases known as porphyrias (listed on the right, beside the deficient enzyme).   acetyl (A) and propionyl (P) groups. The acetyl groups are decarboxylated to form  methyl groups. Then the first two propionyl side chains are decarboxylated and oxi-  dized to vinyl groups, forming a protoporphyrinogen. The methylene bridges are  subsequently oxidized to form protoporphyrin IX (see Fig. 44.3).   In the final step of the pathway, iron (as Fe 2+ ) is incorporated into protopor-  phyrin IX in a reaction catalyzed by ferrochelatase (also known as heme synthase).   Iron, which is obtained from the diet, has a Recommended Dietary Allowance  (RDA) of 10 mg for men and postmenopausal women, and 15 mg for pre-  menopausal women. The average American diet contains 10 to 50 mg of iron. How-  ever, only 10 to 15% is normally absorbed, and iron deficiencies are fairly common.   Fig. 44.4. Synthesis of 8-aminolevulinic acid  (8 -ALA). PLP = pyridoxal phosphate.   Pyridoxine (vitamin B6) deficiencies  are often associated with a micro-  cytic, hypochromic anemia. Why  would a B6 deficiency result in small (micro-  cytic), pale (hypochromic) red blood cells?   8-ALA dehydratase, which contains  zinc, and ferrochelatase are inacti-  vated by lead. Thus, in lead poison-  ing, 8-ALA and protoporphyrin IX accumulate,  and the production of heme is decreased.  Anemia results from a lack of hemoglobin,  and energy production decreases because of  the lack of cytochromes for the electron trans-  port chain.   Porphyrias are a group of rare inherited disorders resulting from deficiencies of enzymes in the pathway for heme biosynthesis (see  Fig. 44.3). Intermediates of the pathway accumulate and may have toxic effects on the nervous system that cause neuropsychiatric  symptoms. When porphyrinogens accumulate, they may be converted by light to porphyrins, which react with molecular oxygen to  form oxygen radicals. These radicals may cause severe damage to the skin. Thus, individuals with excessive production of porphyrins are pho-  tosensitive. The scarring and increased growth of facial hair seen in some porphyrias may have contributed to the development of the were-  wolf legends.   x*™^ In a B6 deficiency, the rate of heme  •] production is slow because the  first reaction in heme synthesis  requires pyridoxal phosphate (see Fig. 44.4).  Thus, less heme is synthesized, causing red  blood cells to be small and pale. Iron stores  are usually elevated.   The iron in meats is in the form of heme, which is readily absorbed. The non-heme  iron in plants is not as readily absorbed, in part because plants often contain  oxalates, phytates, tannins, and other phenolic compounds that chelate or form  insoluble precipitates with iron, preventing its absorption. Conversely, vitamin C  (ascorbic acid) increases the uptake of non-heme iron from the digestive tract. The  uptake of iron is also increased in times of need by mechanisms that are not yet  understood. Iron is absorbed in the ferrous (Fe 2+ ) state (Fig. 44.6), but is oxidized  to the ferric state by a ferroxidase known as ceruloplasmin (a copper-containing  enzyme) for transport through the body.   Because free iron is toxic, it is usually found in the body bound to proteins (see  Fig. 44.6). Iron is carried in the blood (as Fe 3+ ) by the protein apotransferrin, with  which it forms a complex known as transferrin. Transferrin is usually only one-third  saturated with iron. The total iron-binding capacity of blood, mainly due to its con-  tent of transferrin, is approximately 300 jxg/dL.   Storage of iron occurs in most cells but especially those of the liver, spleen, and  bone marrow. In these cells, the storage protein, apoferritin, forms a complex with  iron (Fe 3+ ) known as ferritin. Normally, little ferritin is present in the blood. This  amount increases, however, as iron stores increase. Therefore, the amount of ferritin  in the blood is the most sensitive indicator of the amount of iron in the body's stores.   Iron can be drawn from ferritin stores, transported in the blood as transferrin, and  taken up via receptor-mediated endocytosis by cells that require iron (e.g., by retic-  ulocytes that are synthesizing hemoglobin). When excess iron is absorbed from the  diet, it is stored as hemosiderin, a form of ferritin complexed with additional iron  that cannot be readily mobilized.   Heme regulates its own synthesis by mechanisms that affect the first enzyme in the  pathway, 8-ALA synthase (see Fig. 44.3). Heme represses the synthesis of this  enzyme, and also directly inhibits the activity of the enzyme (an allosteric modi-  fier). Thus, heme is synthesized when heme levels fall. As heme levels rise, the rate  of heme synthesis decreases.   Heme also regulates the synthesis of hemoglobin by stimulating synthesis of the  protein globin. Heme maintains the ribosomal initiation complex for globin synthe-  sis in an active state (see Chapter 15).   The iron lost by adult males  \\ (approximately 1 mg/day) by   desquamation of the skin and in  bile, feces, urine, and sweat is replaced by  iron absorbed from the diet. Men are not as  likely to suffer from iron deficiencies as pre-  menopausal adult women, who also lose iron  during menstruation and who must supply  iron to meet the needs of the growing fetus  during a pregnancy. If a man eating a Western  diet has iron-deficiency anemia, his physician  should suspect bleeding from the gastroin-  testinal tract due to ulcers or colon cancer.   Although spinach has been touted  \ \ as a wonderful source of iron  *S (mostly by the cartoon character  Popeye), this iron is not readily absorbed  because spinach has a high content of phy-  tate (inositol with a phosphate group  attached to each of its 6 hydroxyl groups).   Heme is degraded to form bilirubin, which is conjugated with glucuronic acid and  excreted in the bile (Fig. 44.7). Although heme from cytochromes and myoglobin  also undergoes conversion to bilirubin, the major source of this bile pigment is  hemoglobin. After red blood cells reach the end of their lifespan (approximately 120  days), they are phagocytosed by cells of the reticuloendothelial system. Globin is  cleaved to its constituent amino acids, and iron is returned to the body's iron stores.  Heme is oxidized and cleaved to produce carbon monoxide and biliverdin  (Fig. 44.8). Biliverdin is reduced to bilirubin, which is transported to the liver com-  plexed with serum albumin.   In the liver, bilirubin is converted to a more water-soluble compound by reacting  with UDP-glucuronate to form bilirubin monoglucuronide, which is converted to  the diglucuronide (see Fig. 30.5). This conjugated form of bilirubin is excreted into  the bile.   Drugs, such as phenobarbital, induce enzymes of the drug metabolizing sys-  \ \ ) terns of the endoplasmic reticulum that contain cytochrome P450. Because  «^ heme is used for synthesis of cytochrome P450, free heme levels will fall and  8-ALA synthase will be induced to increase the rate of heme synthesis.   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.6. Iron metabolism. Iron is absorbed from the diet, transported in the blood in transferrin, stored in ferritin, and used for the synthesis of  cytochromes, iron-containing enzymes, hemoglobin, and myoglobin. It is lost from the body with bleeding and sloughed-off cells, sweat, urine,  and feces. Hemosiderin is the protein in which excess iron is stored. Small amounts of ferritin enter the blood and can be used to measure the  adequacy of iron stores. RE = reticuloendothelial.   In the intestine, bacteria deconjugate bilirubin diglucuronide and convert the /^^\ ln an iron deficienc y' wnat charac-  bilirubin to urobilinogens (see Fig. 44.7). Some urobilinogen is absorbed into the (Ol) teristics would blood exhibit?  blood and excreted in the urine. However, most of the urobilinogen is oxidized to  urobilins, such as stercobilin, and excreted in the feces. These pigments give feces  their brown color.   Under the microscope, the red blood cell appears to be a red disc with a pale cen-  tral area (biconcave disc) (Fig. 44. 9). The biconcave disc shape (as opposed to a  spherical shape) serves to facilitate gas exchange across the cell membrane. The  membrane proteins that maintain the shape of the red blood cell also allow the red  blood cell to traverse the capillaries with very small luminal diameters to deliver  oxygen to the tissues. The interior diameters of many capillaries are smaller than  the approximately 7.5-|xm diameter of the red cell. Furthermore, in passing  through the kidney, red blood cells traverse hypertonic areas that are up to six times  the normal isotonicity, and back again, causing the red cell to shrink and expand  during its travels. The spleen is the organ responsible for determining the viability  of the red blood cells. Erythrocytes pass through the spleen 120 times per day. The  elliptical passageways through the spleen are approximately 3 jxm in diameter, and    Fig. 44.7. Overview of heme degradation. Heme is degraded to bilirubin, carried in the blood by albumin, conjugated to form the diglucuronide  in the liver, and excreted in the bile. The iron is returned to the body's iron stores. RES = reticuloendothelial system: RBC = red blood cells.   Iron deficiency would result in a  microcytic, hypochromic anemia.  Red blood cells would be small and  pale. In contrast to a vitamin B6 deficiency,  which also results in a microcytic, hypo-  chromic anemia, iron stores are low in an  iron-deficiency anemia.   ^> The unusual names for some ery-  \ m i throcyte membrane proteins, such  as band 4.1, arose through analysis  of red blood cell membranes by polyacry-  lamide gel electrophoresis. The stained  bands observed in the gel were numbered  according to molecular weight (band 1, band  2, and so on), and as functions were  assigned to the proteins, more common  names were assigned to the proteins (for  example, spectrin is actually band 1).   normal red cells traverse them in approximately 30 seconds. Thus, to survive in the  circulation, the red cell must be highly deformable. Damaged red cells that are no  longer deformable become trapped in the passages in the spleen, where they are  destroyed by macrophages. The reason for the erythrocyte's deformability lies in  its shape and in the organization of the proteins that make up the red blood cell  membrane.   The surface area of the red cell is approximately 140 |xm 2 , which is greater  than the surface of a sphere needed to enclose the contents of the red cell (98  |xm 2 ). The presence of this extra membrane and the cytoskeleton that supports it  allows the red cell to be stretched and deformed by mechanical stresses as the  cell passes through narrow vascular beds. On the cytoplasmic side of the mem-  brane, proteins form a two-dimensional lattice that gives the red cell its flexibil-  ity (Fig. 44.10). The major proteins are spectrin, actin, band 4.1, band 4.2, and  ankyrin. Spectrin, the major protein, is a heterodimer composed of a and p sub-  units wound around each other. The dimers self-associate at the heads. At the  opposite end of the spectrin dimers, actin and band 4.1 bind near to each other.  Multiple spectrins can bind to each actin filament, resulting in a branched mem-  brane cytoskeleton.   The spectrin cytoskeleton is connected to the membrane lipid bilayer by ankyrin,  which interacts with (3 -spectrin and the integral membrane protein, band 3. Band  4.2 helps to stabilize this connection. Band 4.1 anchors the spectrin skeleton with  the membrane by binding the integral membrane protein glycophorin C and the  actin complex, which has bound multiple spectrin dimers.   When the red blood cell is subjected to mechanical stress, the spectrin network  rearranges. Some spectrin molecules become uncoiled and extended; others   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.8. Conversion of heme to bilirubin. A methylene bridge in heme is cleaved, releasing  carbon monoxide (CO) and iron. Then, the center methylene bridge is reduced.   become compressed, thereby changing the shape of the cell, but not its surface  area.   The mature erythrocyte cannot synthesize new membrane proteins or lipids.  However, membrane lipids can be freely exchanged with circulating lipoprotein  lipids. The glutathione system protects the proteins and lipids from oxidative  damage.   The major agents that affect oxygen binding to hemoglobin are shown in Figure 44.1 1.  A. 2,3-Bisphosphoglycerate   2,3-Bisphosphoglycerate (2,3-BPG) is formed in red blood cells from the glycolytic  intermediate 1, 3 -bisphosphogly cerate, as indicated in Figure 44.1. 2,3-BPG binds  to hemoglobin in the central cavity formed by the four subunits, increasing the  energy required for the conformational changes that facilitate the binding of oxy-  gen. Thus, 2,3-BPG lowers the affinity of hemoglobin for oxygen. Therefore, oxy-  gen is less readily bound (i.e., more readily released in tissues) when hemoglobin  contains 2,3-BPG.   Fig. 44.9. The shape of the red blood cell. A.  Wright- stained cells, displaying the pale stain-  ing in the center. Three leukocytes also are  present in the preparation. The magnification  is 350 X. B. Scanning electron micrograph,  showing the biconcave disc structure of the  cells. The stacks of erythrocytes in this prepa-  ration (collected from a blood tube) is not  unusual. The magnification is 28,000 X. These  photographs were obtained, with permission,  from Ross et al, Histology, A Text and Atlas  with Cell and Molecular Biology, 4th Ed.  Philadelphia: Lippincott, 2003:216-217.   Defects in erythrocyte cytoskeletal  proteins lead to hemolytic anemia.  Shear stresses in the circulation  result in the loss of pieces of the red cell  membrane. As the membrane is lost, the red  blood cell becomes more spherical and loses  its deformability. As these cells become more  spherical, they are more likely to lyse in  response to mechanical stresses in the circu-  lation, or to be trapped and destroyed in the  spleen.   Fig. 44.11. Agents that affect oxygen binding  by hemoglobin. Binding of hydrogen ions, 2,3  bisphosphoglycerate, and carbon dioxide to  hemoglobin decrease its affinity for oxygen.    Fig. 44.10. A generalized view of the erythrocyte cytoskeleton. A. The major protein, spec-  trin, is linked to the plasma membrane either through interactions with ankyrin and band 3,  or with actin, band 4.1, and glycophorin. Other proteins in this complex, but not shown, are  tropomyosin and adducin. B. A view from inside the cell, looking up at the cytoskeleton. This  view displays the cross-linking of the spectrin dimers to actin and band 3 anchor sites.   Fig. 44.12. Effect of pH on oxygen saturation  curves. As the pH decreases, the affinity of  hemoglobin for oxygen decreases, producing  the Bohr effect.   The binding of protons by hemoglobin lowers its affinity for oxygen (Fig. 44.12),  contributing to a phenomenon known as the Bohr effect (Fig. 44.13). The pH of the  blood decreases as it enters the tissues (and the proton concentration rises) because  the C0 2 produced by metabolism is converted to carbonic acid by the reaction cat-  alyzed by carbonic anhydrase in red blood cells. Dissociation of carbonic acid pro-  duces protons that react with several amino acid residues in hemoglobin, causing  conformational changes that promote the release of oxygen.   In the lungs, this process is reversed. Oxygen binds to hemoglobin, causing a  release of protons, which combine with bicarbonate to form carbonic acid. This  decrease of protons causes the pH of the blood to rise. Carbonic anhydrase cleaves  the carbonic acid to H 2 0 and C0 2 , and the C0 2 is exhaled. Thus, in tissues in which  the pH of the blood is low because of the C0 2 produced by metabolism, oxygen is  released from hemoglobin. In the lungs, where the pH of the blood is higher because  C0 2 is being exhaled, oxygen binds to hemoglobin.   Although most of the C0 2 produced by metabolism in the tissues is carried to the  lungs as bicarbonate, some of the C0 2 is covalently bound to hemoglobin  (Fig. 44.14). In the tissues, C0 2 forms carbamate adducts with the N-terminal  amino groups of deoxyhemoglobin and stabilizes the deoxy conformation. In the   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.14. Binding of C0 2 to hemoglobin.  C0 2 forms carbamates with the N-terminal  amino groups of Hb chains. Approximately  15% of the C0 2 in blood is carried to the lungs  bound to Hb. The reaction releases protons,  which contribute to the Bohr effect. The over-  all effect is the stabilization of the deoxy form  of hemoglobin.    Fig. 44.13. Effect of H + on oxygen binding by hemoglobin (Hb). A. In the tissues, C0 2 is  released. In the red blood cell, this C0 2 forms carbonic acid, which releases protons. The pro-  tons bind to Hb, causing it to release oxygen to the tissues. B. In the lungs, the reactions are  reversed. 0 2 binds to protonated Hb, causing the release of protons. They bind to bicarbon-  ate (HC0 3 ~), forming carbonic acid, which is cleaved to water and C0 2 , which is exhaled.   lungs, where the p0 2 is high, oxygen binds to hemoglobin and this bound C0 2 is  released.   The various types of cells (lineages) that make up the blood are constantly being  produced in the bone marrow. All cell lineages are descended from hematopoietic  stem cells, cells that are renewable throughout the life of the host. The population  of hematopoietic stem cells is quite small. Estimates vary between 1 to 10 per 10 5  bone marrow cells. In the presence of the appropriate signals, hematopoietic stem    Populations of hematopoietic cells  enriched with stem cells can be iso-  lated by fluorescence activated cell  sorting, based on the expression of specific  cell surface markers. Increasing the popula-  tion of stem cells in cells used for a bone  marrow transplantation increases the  chances of success of the transplantation.   Fig. 44.15. The hematopoietic tree. All blood cells arise from the self-renewing pluripotent stem cell. Different cytokines are required at each  step for these events to occur. CFU = colony-forming unit; BFU = burst-forming unit.   Q o Leukemias, malignancies of the  /\A J blood, arise when a differentiating  ^ hematopoietic cell does not com-  plete its developmental program but  remains in an immature, proliferative state.  Leukemias have been found in every  hematopoietic lineage.   cells proliferate, differentiate, and mature into any of the types of cells that make up  the blood (Figure 44.15).   Hematopoietic differentiation is hierarchical. The number of fates a developing  blood cell may adopt becomes progressively restricted. Hematopoietic progenitors  are designated colony-forming unit-lineage, or colony-forming unit-erythroid  (CFU-E). Progenitors that form very large colonies are termed burst-forming units.   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Developing progenitor cells in the marrow grow in proximity with marrow stromal  cells. These include fibroblasts, endothelial cells, adipocytes, and macrophages.  The stromal cells form an extracellular matrix and secrete growth factors that regu-  late hematopoietic development.   The hematopoietic growth factors have multiple effects. An individual growth  factor may stimulate proliferation, differentiation, and maturation of the progenitor  cells and also may prevent apoptosis. These factors also may activate various func-  tions within the mature cell. Some hematopoietic growth factors act on multiple lin-  eages, whereas others have more limited targets.   Most hematopoietic growth factors are recognized by receptors belonging to the  cytokine receptor superfamily. Binding of ligand to receptor results in receptor  aggregation, which induces phosphorylation of Janus kinases (JAKs). The JAKs are  a family of cytoplasmic tyrosine kinases that are active when phosphorylated (see  Chapter 11, section III.C, and Fig. 11.15). The activated JAKs then phosphorylate  the cytokine receptor. Phosphorylation of the receptor creates docking regions  where additional signal transduction molecules bind, including members of the sig-  nal transducer and activator of transcription (STAT) family of transcription factors.  The JAKs phosphorylate the STATs, which dimerize and translocate to the nucleus,  where they activate target genes. Additional signal transduction proteins bind to the  phosphorylated cytokine receptor, leading to activation of the Ras/Raf/MAP kinase  pathways. Other pathways are also activated, some of which lead to an inhibition of  apoptosis (see Chapter 18).   The response to cytokine binding is usually transient because the cell contains  multiple negative regulators of cytokine signaling. The family of silencer of cytokine  signaling (SOCS) proteins are induced by cytokine binding. One member of the fam-  ily binds to the phosphorylated receptor and prevents the docking of signal trans-  duction proteins. Other SOCS proteins bind to JAKs and inhibit them. Whether  SOCS inhibition of JAKs is a consequence of steric inhibition or whether SOCS  recruit phosphatases that then dephosphorylate the JAKs (Figure 44.16) is uncertain.   SHP-1 is a tyrosine phosphatase found primarily in hematopoietic cells that is  necessary for proper development of myeloid and lymphoid lineages. Its function is  to dephosphorylate JAK2, thereby inactivating it.   STATs are also inactivated. The protein inhibitors of activated STAT (PI AS)  family of proteins bind to phosphorylated STATs and prevent their dimerization or  promote the dissociation of STAT dimers. STATs also may be inactivated by  dephosphorylation, although the specific phosphatases have not yet been identified,  or by targeting activated STATs for proteolytic degradation.   The production of red cells is regulated by the demands of oxygen delivery to the  tissues. In response to reduced tissue oxygenation, the kidney releases the hormone  erythropoietin, which stimulates the multiplication and maturation of erythroid pro-  genitors. The progression along the erythroid pathway begins with the stem cell and  passes through the mixed myeloid progenitor cell, (CFU-GEMM, colony-forming  unit-granulocyte, erythroid, monocyte, megakaryocyte), burst-forming unit-ery-  throid (BFU-E), colony-forming unit-erythroid (CFU-E), and to the first recogniz-  able red cell precursor, the normoblast. Each normoblast undergoes four more  cycles of cell division. During these four cycles, the nucleus becomes smaller and  more condensed. After the last division, the nucleus is extruded. The red cell at this  state is called a reticulocyte. Reticulocytes still retain ribosomes and mRNA and are  capable of synthesizing hemoglobin. They are released from the bone marrow and  circulate for 1 to 2 days. Reticulocytes mature in the spleen, where the ribosomes  and mRNA are lost (Fig. 44.17).   Bone marrow cells can be cultured  in semisolid media with the addi-  tion of the appropriate growth fac-  tors. After 14 to 18 days in culture, colonies  of blood cells can be seen. The type (lineage)  of these cells can be determined based on  morphological or staining properties. Most  colonies will be of single lineage, indicating  descent from a hematopoietic progenitor  that was committed to a lineage. Occasion-  ally a multilineage colony will be obtained,  indicating that it was derived from a more  primitive hematopoietic progenitor.   In X-linked severe combined  immunodeficiency disease (SCID),  the most common form of SCID,  circulating T lymphocytes are not formed,  and B lymphocytes are not active. The  affected gene encodes the gamma chain of  the interleukin 2 receptor. Mutant receptors  are unable to activate JAK3, and the cells are  unresponsive to the cytokines that stimulate  growth and differentiation. Recall also that  adenosine deaminase deficiency (see Chap-  ter 41), which is not X-linked, also leads to a  form of SCID, but for different reasons.   Families have been identified  whose members have a mutant  erythropoietin (epo) receptor that  is unable to bind SHP-1. Erythropoietin is the  hematopoietic cytokine that stimulates pro-  duction of red blood cells. Individuals with  the mutant epo receptor have a higher than  normal percentage of red blood cells in the  circulation, because the mutant epo receptor  cannot be deactivated by SHP-1. Erythropoi-  etin causes sustained activation of JAK2 and  STAT 5 in this case.   Perturbed JAK/STAT signaling is  associated with development of  lymphoid and myeloid leukemias,  severe congenital neutropenia, a condition  in which levels of circulating neutrophils are  severely reduced, and Fanconi anemia,  which is characterized by bone marrow fail-  ure and increased susceptibility to malig-  nancy.    Fig. 44.16. Cytokine signaling through the JAK/STAT pathway. 1. Cytokine binding to receptors initiates dimerization and activation of the JAK  kinase, which phosphorylates the receptor on tyrosine residues. 2. STAT proteins bind to the activated receptors and are themselves phosphorylated.  3. Phosphorylated STAT proteins dimerize, travel to the nucleus, and initiate gene transcription. 4. One of the proteins whose synthesis is stimulated  by STATs is SOCS (suppressor of cytokine signaling), which inhibits further activation of STAT proteins (circle 5) by a variety of mechanisms.   Each person produces approximately 10 12 red blood cells per day. Because so many  cells must be produced, nutritional deficiencies in iron, vitamin B 12, and folate prevent  adequate red blood cell formation. The physical appearance of the cells in the case of  a nutritional anemia frequently provides a clue as to the nature of the deficiency.   In the case of iron deficiency, the cells are smaller and paler than normal. The lack  of iron results in decreased heme synthesis, which in turn affects globin synthesis.  Maturing red cells following their normal developmental program divide until their  hemoglobin has reached the appropriate concentration. Iron- (and hemoglobin-) defi-  cient developing red blood cells continue dividing past their normal stopping point,  resulting in small (microcytic) red cells. The cells are also pale because of the lack of  hemoglobin, as compared with normal cells (thus, a pale, microcytic anemia results).   Fig. 44.17. Erythropoietin stimulation of erythrocyte maturation. The abbreviations are  described in the text.   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Deficiencies of folate or vitamin B12 can cause megaloblastic anemia, in which  the cells are larger than normal. Folate and B 12 are required for DNA synthesis (see  Chapters 40 and 41). When these vitamins are deficient, DNA replication and nuclear  division do not keep pace with the maturation of the cytoplasm. Consequently, the  nucleus is extruded before the requisite number of cell divisions has taken place, and  the cell volume is greater than it should be, and fewer blood cells are produced.   A. Hemoglobinopathies: Disorders in the Structure  or Amount of the Globin Chains   More than 700 different mutant hemoglobins have been discovered. Most arise from  a single base substitution, resulting in a single amino acid replacement. Many have  been discovered during population screenings and are not clinically significant.  However, in patients with hemoglobin S (HbS, sickle cell anemia), the most common  hemoglobin mutation, the amino acid substitution has a devastating effect in the  homozygote (see Will Sichel in Chapter 6). Another common hemoglobin variant,  HbC, results from a glu to lys replacement in the same position as the HbS mutation.  This mutation has two effects. It promotes water loss from the cell by activating the  K + transporter by an unknown mechanism, resulting in a higher than normal con-  centration of hemoglobin within the cell. The amino acid replacement also substan-  tially lowers the hemoglobin solubility in the homozygote, resulting in a tendency of  the mutant hemoglobin to precipitate within the red cell, although, unlike sickle cells,  the cell does not become deformed. Homozygotes for the HbC mutation have a mild  hemolytic anemia. Heterozygous individuals are clinically unaffected.    A registry of hemoglobin muta-  \ \ ) tions is found at the International  Hemoglobin Information Center  http://e20.manu.edu.mk/rcgeb/ihic/.   A complication of sickle cell dis-  ease is an increased formation of  gallstones. A sickle cell crisis  accompanied by the intravascular destruc-  tion of red blood cells (hemolysis) experi-  enced by patients with sickle cell disease,  such as Will Sichel, increases the amount of  unconjugated bilirubin that is transported to  the liver. If the concentration of this uncon-  jugated bilirubin exceeds the capacity of the  hepatocytes to conjugate it to the more sol-  uble diglucuronide through interaction with  hepatic UDP-glucuronate, both the total and  the unconjugated bilirubin levels would rise  in the blood. More unconjugated bilirubin  would be secreted by the liver into the bile.  The increase in unconjugated bilirubin  (which is not very water-soluble) results in  its precipitation within the gallbladder  lumen, leading to the formation of pig-  mented (calcium bilirubinate) gallstones.   For optimum function, the hemoglobin a and p -globin chains must have the proper  structure and be synthesized in a 1 : 1 ratio. A large excess of one subunit over the other  results in the class of diseases called thalassemias. These anemias are clinically very  heterogeneous, as they can arise by multiple mechanisms. Like sickle cell anemia, the  thalassemia mutations provide resistance to malaria in the heterozygous state.   Hemoglobin single amino acid replacement mutations that give rise to a globin  subunit of decreased stability is one mechanism by which thalassemia arises. More  common, however, are mutations that result in decreased synthesis of one subunit.  Alpha thalassemias usually arise from complete gene deletions. Two copies of the a-  globin gene are found on each chromosome 16, for a total of 4 a-globin genes per  precursor cell. If one copy of the gene is deleted, the size and hemoglobin concen-  tration of the individual red blood cells is minimally reduced. If two copies are  deleted, the red blood cells are of decreased size (microcytic) and reduced hemoglo-  bin concentration (hypochromic). However, the individual is usually not anemic. The  loss of three a-globin genes causes a moderately severe microcytic hypochromic  anemia (hemoglobin 7-10 g/dL) with splenomegaly (enlarged spleen). The absence  of four a-globin genes (hydrops fetalis) is usually fatal in utero.   There are two ways in which an individual could have two a-globin genes deleted.  \\ In one case, one copy of chromosome 16 could have both a-globin genes deleted,   whereas the other copy had two functional a genes. In the second case, both  chromosomes could have lost one of their two copies of the a-globin gene. The former pos-  sibility is more common among Asians; the latter among Africans.   HbC is found in high frequency in  West Africa, in regions with a high  frequency of HbS. Consequently,  compound heterozygotes for HbS and HbC  are not uncommon both in some African  regions and among African-Americans.  HbS/HbC individuals have significantly more  hematopathology than individuals with  sickle cell trait (HbA/HbS). Polymerization of  deoxygenated HbS is dependent on the HbS  concentration within the cell. The presence  of HbC in the compound heterozygote  increases the HbS concentration by stimulat-  ing K + and water efflux from the cell.  Because the HbC globin is produced more  slowly than HbA or HbS, the proportion of  HbS tends to be higher in HbS/HbC cells  than in the cells of individuals with sickle cell  trait (HbS/HbA). The way in which multiple  mutations ameliorate or exacerbate hemato-  logic diseases has provided insights into the  molecular mechanisms of hemoglobin func-  tion and developmental regulation.   The difference in amino acid com-  position between the p-chains of  HbA and 7-chains of HbF results in  structural changes that cause HbF to have a  lower affinity for 2,3-BPG than adult hemo-  globin (HbA) and, thus, a greater affinity for  oxygen. Therefore, the oxygen released  from the mother's hemoglobin (HbA) is  readily bound by HbF in the fetus. Thus, the  transfer of oxygen from the mother to the  fetus is facilitated by the structural differ-  ence between the hemoglobin molecule of  the mother and that of the fetus.   Individuals with sickle cell anemia or  beta thalassemia (usually) have  intact 7-globin loci. If a way could be  found to reactivate the 7-globin loci (the drug  hydroxyurea is a potential candidate for this),  it would be an attractive therapeutic option for  the treatment of these diseases.   An additional source of variation in  the levels of fetal hemoglobin is  the FCP (F-cell producing) locus on  the short arm of the X chromosome in a  region thought not to be susceptible to X  inactivation. Both normal individuals and  individuals with hemoglobinopathies vary in  the amount of hemoglobin F they produce.  In studies of normal individuals, a high level  of hemoglobin F appears to be inherited as  an X-linked dominant trait. The FCP locus is  responsible for a substantial amount of the  variation in Hemoglobin F seen among  sickle cell patients. The protein encoded at  the FCP locus has not been identified; cur-  rent speculations are that it is a transcription  factor involved in the regulation of the glo-  bin locus.    As discussed in Chapter 14, beta thalassemia is a very heterogeneous genetic dis-  ease. Insufficient p -globin synthesis can result from deletions, promoter mutations,  and splice junction mutations. Heterozygotes for P + (some globin chain synthesis)  or (3 null (p°, no globin chain synthesis) are generally asymptomatic, though they  typically have microcytic, hypochromic red blood cells and may have a mild anemia.  P + /p + homozygotes have an anemia of variable severity, p + /p° compound het-  erozygotes tend to be more severely affected, and p°/p° homozygotes have severe  disease. In general, diseases of p chain deficiency are more severe than diseases of  a chain deficiency. Excess p chains form a homotetramer, hemoglobin H (HbH),  which is useless for delivering oxygen to the tissues because of its high oxygen affin-  ity. As red blood cells age, HbH will precipitate in the cells, forming inclusion bod-  ies. Red blood cells with inclusion bodies have a shortened life span, because they  are more likely to be trapped and destroyed in the spleen. Excess a chains are unable  to form a stable tetramer. However, excess a chains precipitate in erythrocytes at  every developmental stage. The a chain precipitation in erythroid precursors results  in their widespread destruction, a process called ineffective erythropoiesis. The pre-  cipitated a chains also damage red blood cell membranes through the heme-facili-  tated lipid oxidation by reactive oxygen species. Both lipids and proteins, particu-  larly band 4.1, are damaged.   Fetal hemoglobin (HbF), the predominant hemoglobin of the fetal period, consists  of two alpha chains and two gamma chains, whereas adult Hb consists of two alpha  and two beta chains. The process that regulates the conversion of HbF to HbA is  called hemoglobin switching. Hb switching is not 100%; most individuals continue  to produce a small amount of HbF throughout life. However, some people, who are  clinically normal, produce abnormally high levels (up to 100%) of fetal hemoglo-  bin (Hemoglobin F) in place of HbA. Patients with hemoglobinopathies such as  (3 -thalassemia or sickle cell anemia frequently have less severe illnesses if their lev-  els of fetal hemoglobin are elevated. One goal of much research on hemoglobin  switching is to discover a way to reactivate transcription of the 7 -globin genes to  compensate for defective p -globin synthesis. Individuals who express fetal hemo-  globin past birth have hereditary persistence of fetal hemoglobin (HPFH).   The non-deletion forms of HPFH are those that derive from point mutations in the  A7 and G7 promoters. When these mutations are found with sickle cell or beta tha-  lassemia mutations, they have an ameliorating effect on the disease, because of the  increased production of gamma chains.   In deletion HPFH, both the entire delta and beta genes have been deleted from one  copy of chromosome 1 1 and only HbF can be produced. In some individuals the  fetal globins remain activated after birth, and enough HbF is produced that the  individuals are clinically normal. Other individuals with similar deletions that  remove the entire delta and beta genes do not produce enough fetal hemoglobin to  compensate for the deletion and are considered to have o°p° thalassemia. The dif-  ference between these two outcomes is believed to be the site at which the dele-  tions end within the p -globin gene cluster. In deletion HPFH, powerful enhancer  sequences 3' of the p-globin gene are resituated because of the deletion such that  they activate the gamma promoters. In individuals with 8°P° thalassemia, the  enhancer sequences have not been relocated such that they can interact with the  gamma promoters.   CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   In humans, embryonic megaloblasts (the embryonic red blood cell is large and is  termed a "blast" because it retains its nucleus) are first produced in the yolk sac  approximately 15 days after fertilization. After 6 weeks, the site of erythropoiesis  shifts to the liver. The liver and to a lesser extent the spleen are the major sites of  fetal erythropoiesis. In the last few weeks before birth, the bone marrow begins pro-  ducing red blood cells. By 8 to 10 weeks after birth, the bone marrow is the sole site  of erythrocyte production. The composition of the hemoglobin also changes with  development, because both the a-globin locus and the p -globin locus have multiple  genes that are differentially expressed during development (Figure 44.18).   E. Structure and Transcriptional Regulation of the Alpha  and Beta Globin Gene Loci   The a-globin locus on chromosome 16 contains the embryonic £ (zeta) gene  and two copies of the alpha gene, a 2 and o^. The p-globin locus on chromosome 1 1  contains the embryonic e gene, two copies of the fetal p -globin gene G 7 and A 7   Fig 44.18. Globin gene clusters and expression during development. A. The globin gene  clusters with the a genes on chromosome 16 and the p genes on chromosome 11. LCR =  locus control region. B. The switching of globin chain synthesis during development.   (which differ by one amino acid), and two adult genes, 8 and p. The order of the genes  along the chromosome parallels the order of expression of the genes during develop-  ment (see Fig. 44.18). The embryonic hemoglobins are £ 2 e 2 (Gower 1), £ 2 72 (Port-  land), and a 2 y 2 (Gower 2). Fetal hemoglobin is predominantly a 2 G 72 . The major adult  species is a 2 (3 2 (hemoglobin A); the minor adult species is a 2 8 2 (hemoglobin A 2 ). The  fetal hemoglobin found in adult cells is a 2 A 72 . The timing of hemoglobin switching  is controlled by a developmental clock not significantly altered by environmental  conditions and is related to changes in expression of specific transcription factors. Pre-  mature newborns convert from HbF to HbA on schedule with their gestational ages.   Spiro Site's red blood cells are deficient in spectrin. This deficiency impairs  the ability of his erythrocytes to maintain the redundant surface area neces-  sary to maintain deformability. Mechanical stresses in the circulation cause  progressive loss of pieces of membrane. As membrane components are lost, Spiro  Site's red blood cells become spherical and unable to deform. His spleen is enlarged  because of the large number of red blood cells that have become trapped within it. His  erythrocytes are lysed by mechanical stresses in the circulation and by macrophages  in the spleen. Consequently, this hemolytic process results in an anemia. His gall-  stones were the result of the large amounts of bilirubin that were produced and stored  in the gallbladder as a result of the hemolysis. The abnormally rounded red cells seen  on a blood smear are characteristic of hereditary spherocytosis.   Mutations in the genes for ankyrin, (3 -spectrin, or band 3 account for three quar-  ters of the cases of hereditary spherocytosis, whereas mutations in the genes for a-  spectrin or band 4.2 account for the remainder. The result of defective synthesis of  any of the membrane cytoskeletal proteins results in improper formation of the  membrane cytoskeleton. Excess membrane proteins are catabolized, resulting in a  net deficiency of spectrin. Spiro Site underwent a splenectomy. Because the spleen  was the major site of destruction of his red blood cells, his anemia significantly  improved after surgery. He was discharged with the recommendation to take a folate  supplement daily. It was explained to Mr. Site that because the spleen plays a major  role in protection against certain bacterial agents, he would require immunizations  against pneumococcus, meningococcus, and Haemophilus influenzae type b.   Anne Niemick was found to be a compound heterozygote for mutations in  the p-globin gene. On one gene, a mutation in position 6 of intron 1 con-  verted a T to a C. The presence of this mutation, for unknown reasons,  raises HbF production. The other (3-globin gene had a mutation in position 110 of  exon 1 (a C to T mutation). Both (3-globin chains have reduced activity, but com-  bined with the increased expression of HbF, results in a p + thalassemia.   How is hemoglobin switching controlled? Although there are still many  unanswered questions, some of the molecular mechanisms have been iden-  tified. The a-globin locus covers ~100 kb. The major regulatory element, HS  40, is a nuclease-sensitive region of DNA that lies 5' of the £ gene (see Fig. 44.18).  HS 40 acts as an erythroid-specific enhancer that interacts with the upstream regula-  tory regions of the £ and a genes, and stimulates their transcription. The region imme-  diately 5 ' of the £ gene contains the regulatory sequences responsible for silencing £  gene transcription. However, the exact sequences and transcription factors responsi-  ble for this silencing have not yet been identified. Even after silencing, low levels of    CHAPTER44 / THE BIOCHEMISTRY OF THE ERYTHROCYTE AND OTHER BLOOD CELLS   Fig. 44.19. The 7-globin gene promoter indicating some of the transcription factor binding  sites associated with hereditary persistence of fetal hemoglobin.   £ gene transcripts are still produced after the embryonic period; however, they are not  translated. This is because both the £ globin and a-globin transcripts have regions that  bind to a messenger ribonucleoprotein (mRNP) stability-determining complex. Bind-  ing to this complex prevents the mRNA from being degraded. The a-globin messen-  ger RNA has a much higher affinity for the mRNP than the £ -globin message, which  leads to the £-globin message being rapidly degraded.   The p-globin locus covers ~100 kb. From 5 to 25 kb upstream of the e gene is  the locus control region (LCR), containing five DNAse hypersensitive sites. The  LCR is necessary for the function of the p-globin locus. It maintains the chromatin  of the entire locus in an active configuration and acts as an enhancer and entry point  for the factors that transcribe the genes of the p-globin locus. One model of the con-  trol of hemoglobin switching postulates that proteins bound at the promoters of the  e-, 7-, and p-globin genes compete to interact with the enhancers of the LCR.   Each gene in the p-globin locus has individual regulatory elements — a promoter,  silencers, or enhancers that control its developmental regulation. The promoters  controlling the 7 and p-globin genes have been intensively studied because of their  clinical relevance.   The e-globin gene, like the £ globin gene, has silencers in the 5' regulatory  region. Binding of proteins to these regions turns off the e gene.   The proximal region of the 7-globin gene promoter has multiple transcription  factor binding sites (Fig. 44.19). Many HPFH mutations map to these transcrip-  tion factor-binding sites, either by destroying a site or by creating a new one, but  the exact mechanisms are still not understood. Two sites that appear to be signif-  icant in the control of hemoglobin switching are the stage selector protein bind-  ing (SSP) site and the CAAT box region. When the SSP complex is bound to the  promoter, the 7-globin gene has a competitive advantage over the p-globin pro-  moter for interaction with the LCR. A second transcription factor, Spl, also binds  at the SSP-binding site, where it may act as a repressor, and competition between  these two protein complexes for the SSP-binding site helps to determine the activ-  ity of the 7-globin gene. A similar mechanism appears to be operating at the  CAAT box. CP1, thought to be a transcription activator, binds at the CAAT box.  CAAT displacement protein (CDP) is a repressor that binds at the CAAT site and  displaces CP1. Part of the mechanism of hemoglobin switching appears to be the  binding of repressors at the e-globin and 7-globin upstream regulatory regions.   The p-globin gene also has binding sites for multiple transcription factors in its  regulatory regions. Mutations that affect binding of transcription factors can pro-  duce thalassemia by reducing the activity of the p-globin promoter. There is also an  enhancer 3' of the poly A signal that seems to be required for stage-specific activa-  tion of the p-globin promoter.   Transgenic mice are an invaluable  \\ ) tool for studying the roles of tran-  scription factors in developmental  processes in general and hemoglobin  switching in particular. Transgenic mice  were created with mutations in the silencer  regions of the e-globin genes. These mice  continued to express the e-globin gene into  adulthood.   Krebs DL, Hilton DJ. SOCS proteins: negative regulators of cytokine signaling. Stem Cells  2001;19:378-387.   Stamatoyannopoulos G, Grosveld F. Hemoglobin switching. In: Stamatoyannopoulos G, Majerus PW,  Perlmutter, RM Varmus H, eds. The Molecular Basis of Blood Diseases. 3rd Ed. Philadelphia: WB  Saunders, 2001:135-182   Ward AC, Touw I, Yoshimura A. The Jak-Stat pathway in normal and perturbed hematopoiesis. Blood  2000;95:19-29.   Weatherall DJ. The thalassemias In: Stamatoyannopoulos G, Majerus PW, Perlmutter, RM Varmus H,  eds. The Molecular Basis of Blood Diseases. 3rd Ed. Philadelphia: WB Saunders, 2001:183-226.   1. A compensatory mechanism to allow adequate oxygen delivery to the tissues at high altitudes, where oxygen concentrations  are low, would be which of the following?   2. A 2-year-old boy of normal weight and height is brought to a clinic because of excessive fatigue. Blood work indicates an  anemia, with microcytic hypochromic red cells. The boy lives in a 100-year-old apartment building and has been caught  ingesting paint chips. His parents indicate that the child eats a healthy diet and takes a Flintstones vitamin supplement every  day. His anemia is most likely attributable to a deficiency in which of the following?   3. Drugs are being developed that will induce the transcription of globin genes, which are normally silent in patients affected  with sickle cell disease. A good target gene for such therapy in this disease would be which of the following?   4. A mature blood cell that lacks a nucleus is which of the following?   5. A family has two children, one with a mild case of thalassemia, and a second with a severe case of thalassemia, requiring fre-  quent blood transfusions as part of the treatment plan. One parent is of Mediterranean descent, the other is of Asian descent.  Neither parent exhibits clinical signs of thalassemia. Both children express 20% of the expected level of p-globin; the more  severely affected child expresses normal levels of a-globin, whereas the less severely affected child only expresses 50% of  the normal levels of a-globin. Why is the child who has a deficiency in a-globin expression less severely affected?   (A) Thalassemia is caused by a mutation in the a gene, and the more severely affected child expresses more of it.   (B) The less severely affected child must be synthesizing the £ gene to make up for the deficiency in a a chain synthesis.   (D) The more severely affected child produces more inactive globin tetramers than the less severely affected child.   (E) Thalassemia is caused by an iron deficiency, and when the child is synthesizing normal levels of a-globin there is insuf-  ficient iron to populate all of the heme molecules synthesized.   The blood is the body's main transport system. Although the transport and deliv-  ery of oxygen to the cells of the tissues is carried out by specialized cells, other  vital components such as nutrients, metabolites, electrolytes, and hormones, are  all carried in the none ellular fraction of the blood, the plasma. Some components,  such as glucose, are dissolved in the plasma; others, for example, lipids and  steroid hormones, are bound to carrier proteins for transport. The osmotic pres-  sure of the plasma proteins regulates the distribution of water between the blood  and the tissues. Plasma proteins in conjunction with platelets maintain the  integrity of the circulatory system through the process of clotting.   Blood normally circulates through endothelium-lined blood vessels. When a  blood vessel is severed, a blood clot (called a thrombus, which is formed by the  process of thrombosis) forms as part of hemo stasis , the physiologic response that  stops bleeding. Blood clots also form to repair damage to the endothelial lining,  in which components of the subendothelial layer become accessible to plasma  proteins.   In hemostasis and thrombosis, a primary hemostatic plug, consisting of aggre-  gated platelets and a fibrin clot, forms at the site of injury. Platelets are attached  to the subendothelial layer of the vessel principally through the protein von Wille-  brand factor, and are activated to bind fibrinogen. Fibrinogen binds the platelets  to each other to form a platelet aggregate. The platelet aggregate is rapidly cov-  ered with layers of fibrin, which are formed from fibrinogen by the proteolytic  enzyme thrombin.   Injury to the endothelium and exposure of tissue factor on the subendothelial  layer to plasma proteins also activate the blood coagulation cascade, which ulti-  mately activates thrombin and Factor XIII. Factor XIII cross-links strands of  polymerized fibrin monomers to form a stable clot (the secondary hemostatic  plug). The blood coagulation cascade consists of a series of enzymes (such as  Factor X), which are inactive until proteolytic ally cleaved by the preceding  enzyme in the cascade. Other proteins (Factor V and Factor VIII) serve as bind-  ing proteins, which assemble factor complexes at the site of injury. Ca 2+ and y-  carboxyglutamate residues in the proteins (formed by a vitamin K-dependent  process in the liver) attach the factor complexes to phospholipids exposed on  platelet membranes. Consequently, thrombus formation is rapidly accelerated and  localized to the site of injury.   Regulatory mechanisms within the blood coagulation cascade and antifibri-  nolytic mechanisms prevent random coagulation within blood vessels that might  obstruct blood flow. Impairments in these mechanisms lead to thrombosis.   • Sloe Klotter, a 6-month-old male infant, was brought to his pediatrician's   ) office with a painful, expanding mass in his right upper thigh that was first  noted just hours after he fell down three uncarpeted steps in his home. The  child appeared to be in severe distress.   An x-ray showed no fractures, but a soft tissue swelling, consistent with a  hematoma (bleeding into the tissues), was noted. Sloe's mother related that soon  after he began to crawl, his knees occasionally became swollen and seemed painful.   The pediatrician suspected a disorder of coagulation. A screening coagulation  profile suggested a possible deficiency of Factor VIII, a protein involved in the for-  mation of blood clots. Sloe's plasma Factor VIII level was found to be only 3% of  the average level found in normal subjects. A diagnosis of hemophilia A was made.   J^5\ The hydrostatic pressure in an  \§ ) arteriole is the force that "pushes"  fluid out of the capillary and into  the interstitial spaces. The plasma protein  osmotic pressure, plus the tissue pressure,  is the force that "pulls" water from intersti-  tial spaces into the venular side of the capil-  lary. Thus, if the hydrostatic pressure is  greater than the osmotic pressure, fluid will  leave the circulation; if it is less, fluid will  enter the circulation.   0 o In cases of severe protein malnutri-  ( / r \A ) tion (kwashiorkor), the concentra-  ^ tion of the plasma proteins  decreases, as a result of which the osmotic  pressure of the blood decreases. As a result,  fluid is not drawn back to the blood and  instead accumulates in the interstitial space  (edema). The distended bellies of famine vic-  tims are the result of fluid accumulation in  the extravascular tissues because of the  severely decreased concentration of plasma  proteins, particularly albumin. Albumin syn-  thesis decreases fairly early under condi-  tions of protein malnutrition.   I. PLASMA PROTEINS MAINTAIN PROPER  DISTRIBUTION OF WATER BETWEEN  BLOOD AND TISSUES   When the cells are removed from the blood, the remaining plasma is composed of  water, nutrients, metabolites, hormones, electrolytes, and proteins. Plasma has  essentially the same electrolyte composition as other extracellular fluids and con-  stitutes approximately 25% of the body's total extracellular fluid. The plasma pro-  teins serve a number of functions, which include maintaining the proper distribution  of water between the blood and the tissues, transporting nutrients, metabolites, and  hormones throughout the body, defending against infection, and maintaining the  integrity of the circulation through clotting. Many diseases alter the amounts of  plasma proteins produced and, hence, their concentration in the blood. These  changes can be determined by electrophoresis of plasma proteins over the course of  a disease.   As the arterial blood enters the capillaries, fluid moves from the intravascular space  into the interstitial space (that surrounding the capillaries) because of what are  known as Starling's forces. The hydrostatic pressure in the arteriolar end of the cap-  illaries (~37 mm Hg) exceeds the sum of the tissue pressure (~1 mm Hg) and the  osmotic pressure of the plasma proteins (-25 mm Hg). Thus, water tends to leave  the capillaries and enter extravascular spaces. At the venous end of the capillaries,  the hydrostatic pressure falls to approximately 17 mm Hg while the osmotic pres-  sure and the tissue pressure remain constant, resulting in movement of fluid back  from the extravascular (interstitial) spaces and into the blood. Thus, most of the  force bringing water back from the tissues is the osmotic pressure mediated by the  presence of proteins in the plasma.   As indicated in Table 45.1, the liver produces a number of transport or binding pro-  teins and secretes them into the blood. The major protein synthesized is albumin,  which constitutes approximately 60% of the total plasma protein, but because of its  relatively small size (69 kDa) is thought to contribute 70 to 80% of the total osmotic  pressure of the plasma. Albumin, like most plasma proteins, is a glycoprotein and is  a carrier of free fatty acids, calcium, zinc, steroid hormones, copper, and bilirubin.    Binds thyroxine (T 4 ); also forms a complex with retinol-   Many drugs also bind to albumin, which may have important pharmacologic impli-  cations. For example, when a drug binds to albumin, such binding will likely lower  the effective concentration of that drug and may lengthen its lifetime in the circula-  tion. Drug dosimetry may need to be recalculated if a patient's plasma protein con-  centration is abnormal.   Two different sets of proteins aid in the immune response, the immunoglobulins and  the complement proteins. The immunoglobulins are secreted by a subset of differen-  tiated B lymphocytes termed plasma cells and bind antigens at binding sites formed  by the hypervariable regions of the proteins (see Chapter 7). Once the antibody-anti-  gen complex is formed, it must be cleared from the circulation. The complement sys-  tem participates in this function. The complement system consisting of approxi-  mately 20 proteins becomes activated in either of two ways. The first is interaction  with antigen-antibody complexes, and the second, specific for bacterial infections,  is through interaction of bacterial cell polysaccharides with complement protein  C3b. Activation of the complement system by either trigger results in a proteolytic  activation cascade of the proteins of the complement system, resulting in the release  of biologically active peptides or polypeptide fragments. These peptides mediate the  inflammatory response, attract phagocytic cells to the area, initiate degranulation of  granulocytes, and promote clearance of antigen-antibody complexes.   Protease inhibitors in plasma serve to carefully control the inflammatory  response. Activated neutrophils release lysosomal proteases from their granules that  can attack elastin, the various types of collagen, and other extracellular matrix pro-  teins. The plasma proteins al-antiproteinase (a 1 -antitrypsin) and a2-macroglobu-  lin limit proteolytic damage by forming noncovalent complexes with the proteases,  thereby inactivating them. However, the product of neutrophil myeloperoxidase,  HOC1, inactivates the protease inhibitors, thereby insuring that the proteases are  active at the site of infection.   Blood is lost from the circulation when the endothelial lining of the blood vessels  is damaged and the vessel wall is partly or wholly severed. When this occurs, the  subendothelial cell layer is exposed, consisting of the basement membrane of the  endothelial cells and smooth muscle cells. In response to the damage, a barrier (the  hemostatic plug, a fibrin clot), initiated by platelet binding to the damaged area, is  formed at the site of injury. Regulatory mechanisms limit clot formation to the site  of injury and control its size and stability. As the vessel heals, the clot is degraded  (fibrinolysis). Plasma proteins are required for these processes to occur.   In spite of the importance of albu-  min in the maintenance of osmotic  pressure in the blood, individuals  lacking albumin (analbuminemia) have only  moderate edema. Apparently, the concentra-  tion of other plasma proteins is increased to  compensate for the lack of albumin. The fre-  quency of analbuminemia is less than one  per million individuals.    a1-Antiproteinase (AAP) is the main  serine protease inhibitor of human  plasma. Individuals with a point  mutation in exon 5 of the AAP gene, which  results in a single amino acid substitution in  the protein, have diminished secretion of  AAP from the liver. These individuals are at  increased risk for developing emphysema.  When neutrophils degranulate in the lungs  as part of the body's defense against  microorganisms, insufficient levels of AAP  are present to neutralize the elastase and  other proteases released. The excess prote-  olytic activity damages lung tissue, leading  to loss of alveolar elasticity and emphysema.   Methionine 358 of AAP is necessary for  AAP binding to the proteases. Oxidation of  this methionine destroys AAP's protease-  binding capacity. Cigarette smoke oxidizes  Met-358, thereby increasing the risk for  emphysema.   row. Megakaryocytes differentiate  from the hematopoietic stem cell. As the  megakaryocyte matures, it undergoes syn-  chronous nuclear replication without cellular  division, to form a cell with a multilobed  nucleus and enlarged cytoplasm. At approx-  imately the 8-nucleus stage, the cytoplasm  becomes granular, and the platelets are bud-  ded off the cytoplasm. A single megakary-  ocyte gives rise to approximately 4,000  platelets.   0 o Idiopathic thrombocytopenic pur-  ( /~oA ) pura (ITP) is an autoimmune dis-  ^ ease in which antibodies to platelet  glycoproteins are produced. Antibody bind-  ing to platelets results in their clearance by  the spleen. An early symptom of the disorder  is the appearance of small red spots on the  skin (petechial hemorrhages) caused by  blood leakage from capillaries. Minor dam-  age to vascular endothelial cells is constantly  being caused by mechanical forces related to  blood flow. In patients with ITP, few platelets  are available to repair the damage.   ^> Von Willebrand factor is a large  KJ ] multimeric glycoprotein with a  *r subunit molecular weight of  220,000 daltons. Its size in the circulation  ranges between 500 and 20,000 kDa, and its  role in circulation is to stabilize Factor VIII  and protect it from degradation. The high-  molecular-weight forms are concentrated in  the endothelium of blood vessels and are  released in response to stress hormones and  endothelial damage. High-molecular-weight  vWF released by the endothelium is cleaved  by a specific metalloprotease in the serum,  reducing the size of the circulating vWF.  Large vWF multimers are more effective at  forming complexes with platelets than are  small vWF multimers.   Platelets are non-nucleated cells present in the blood whose major role is to form  mechanical plugs at the site of vessel injury and to secrete regulators of the clotting  process and vascular repair. In the absence of platelets, leakage of blood from vents  in small vessels is common.   In the nonactivated platelet (a platelet not involved in forming a clot), the plasma  membrane invaginates extensively into the interior of the cell, forming an open  membrane (canalicular) system. Because the plasma membrane contains receptors  and phospholipids that accelerate the clotting process, the canalicular structure sub-  stantially increases the membrane surface area potentially available for clotting  reactions. The platelet interior contains microfilaments and an extensive  actin/myosin system. Platelet activation in response to endothelial injury causes  Ca 2+ -dependent changes in the contractile elements, which, in turn, substantially  change the architecture of the plasma membrane. Long pseudopodia are generated,  increasing the surface area of the membrane as clot formation is initiated.   Platelets contain three types of granules. The first are electron-dense granules,  which contain calcium, adenosine diphosphate (ADP), adenosine triphosphate  (ATP), and serotonin. The second type of granule is the a granule, which contains  a heparin antagonist (heparin interferes with blood clotting; see biochemical com-  ments), platelet-derived growth factor, (3-thromboglobulin, fibrinogen, von Wille-  brand factor (vWF), and other clotting factors. The third type of granule is the lyso-  somal granule, which contains hydrolytic enzymes. During activation, the contents  of these granules, which modulate platelet aggregation and clotting, are secreted.   Three fundamental mechanisms are involved in platelet function during blood coag-  ulation: adhesion, aggregation, and secretion. Adhesion sets off a series of reactions  termed platelet activation, which leads to platelet aggregation and secretion of  platelet granule contents.   The adhesion step refers primarily to the platelet-subendothelial interaction that  occurs when platelets initially adhere to the sites of blood vessel injury (Fig. 45.1).  Blood vessel injury exposes collagen, subendothelial matrix-bound vWF, and  other matrix components. vWF is a protein synthesized in endothelial cells and  megakaryocytes, and is located in the subendothelial matrix, in specific platelet  granules, and in the circulation bound to Factor VIII. The platelet cell membrane  contains glycoproteins (GPs) that bind to collagen and to vWF, causing the platelet   Fig. 45.1. Adhesion of platelets to the subendothelial cell layer. 1. GPIa initially binds to the  exposed collagen, which results in changes in the three-dimensional configuration of  the complex, allowing GPIb to bind to vWF (2). 3. This second binding event exposes the  GPIIb/GPIIIa complex, which also can bind to vWF and fibrinogen.   to adhere to the subendothelium. Binding to collagen by GPIa (integrin a2(31)  causes the platelet to change its shape from a flat disc to a spherical cell. The latter  extrudes long pseudopods, which promote platelet/platelet interactions. Binding of  subendothelial vWF by GPIb causes changes in the platelet membrane that expose  GPIIb/IIIa (integrin allb|33) binding sites to fibrinogen and vWF.   The initial adherence of platelets sets off a series of reactions (platelet activation)  that results in more platelets being recruited and aggregated at the site of injury.  After initial adherence, some of the platelets release the contents of their dense  granules and their a granules, with ADP release being of particular importance  because ADP is a potent platelet activator. ADP released from the platelets and from  damaged red blood cells binds to a receptor on the platelet membrane, which leads  to the further unmasking of GPIIb/IIIa binding sites. Aggregation of platelets can-  not take place without ADP stimulation, because ADP induces swelling of the  activated platelets, promoting platelet/platelet contact and adherence.   Fibrinogen is a protein that circulates in the blood and is also found in platelet  granules. It consists of two triple helices held together with disulfide bonds.  (Fig.45.2) Binding of fibrinogen to activated platelets is necessary for aggregation,   vWF deficiency is the most com-  mon cause of inherited bleeding  disorders. Both platelet adherence  and the clotting cascade are affected  because levels of Factor VIII are low. In the  absence of vWF, Factor VIII is rapidly cleared  from the system. The vWF gene is large, cov-  ering approximately 180 kb, and contains 52  exons. Multiple mutations are known, with  varying clinical presentations.   Defects in GPIb cause a bleeding  disorder known as Bernard-Soulier  syndrome. Platelet aggregation is  affected, because of the inability of GPIb to  adhere to subendothelial vWF.    N-terminal regions of oc,p chains of two triple  helices held together with disulfide bonds   Fig. 45.2. Cleavage of fibrinogen results in clot formation. A. Fibrinogen, the precursor protein of fibrin, is formed from two triple helices joined  together at their N-terminal ends. The a, p peptides are held together by disulfide bonds, and the y-peptides are joined to each other by disul-  fide bonds. The terminal a, p peptide regions, shown in blue, contain negatively charged glutamate and aspartate residues that repel each other  and prevent aggregation. B. Thrombin, a serine protease, cleaves the terminal portions of fibrinogen containing negative charges. The fibrin  monomers can then aggregate and form a "soft" clot. The soft clot is then subsequently cross-linked by another enzyme.   Thrombotic thrombocytopenic pur-  pura (TTP) is a disease character-  ized by the formation in the circula-  tion of microdots (microthrombi) consisting  of aggregated platelets. The microthrombi  collect in the microvasculature and damage  red blood cells, resulting in hemolytic ane-  mia. They also damage vascular endothe-  lium, exposing collagen and releasing high-  molecular-weight vWF, promoting more  platelet aggregation. The subsequent deple-  tion of platelets renders the patient suscepti-  ble to internal hemorrhage. Mortality in  untreated TTP can approach 90%.   Familial TTP is associated with mutations  in the vWF-specific metalloprotease, although  not all individuals with defective protease  develop TTP. Sporadic cases of TTP are asso-  ciated with the development of an antibody to  the metalloprotease.   In response to collagen and throm-  bin, platelets release vasoconstric-  tors. Serotonin is released from the  dense granules of the platelets, and the syn-  thesis of thromboxane A2 is stimulated. This  will reduce blood flow to the damaged area.  Platelet-derived growth factor, which stimu-  lates proliferation of vascular cells, is also  released into the environment surrounding  the damage.   The utilization of an active site ser-  ine to cleave a peptide bond is  common to a variety of enzymes  referred to as serine proteases. Serine pro-  teases are essential for activating the forma-  tion of a blood clot from fibrin. Fibrin and  many of the other proteins involved in blood  coagulation are present in the blood as inac-  tive precursors or zymogens, which must be  activated by proteolytic cleavage. Thrombin,  the serine protease that converts fibrinogen  to fibrin, has the same aspartate-histidine-  serine catalytic triad found in chymotrypsin  and trypsin.   Thrombin is activated by proteolytic  cleavage of its precursor protein, prothrom-  bin. The sequence of proteolytic cleavages  leading to thrombin activation requires Fac-  tor VIII, the blood-clotting protein deficient in  Sloe Klotter.    providing, in part, the mechanism by which platelets adhere to one another.  Cleavage of fibrinogen by thrombin (a protease that is activated by the coagulation  cascade) produces fibrin monomers that polymerize and, together with platelets,  form a "soft clot". Thrombin itself is a potent activator of platelets, through binding  to a specific receptor on the platelet surface.   Thrombus (clot) formation is enhanced by thrombin activation, which is mediated by  the complex interaction that constitutes the blood coagulation cascade. This cascade  (Fig. 45.3) consists primarily of proteins that serve as enzymes or cofactors, which  function to accelerate thrombin formation and localize it at the site of injury. These  proteins are listed in Table 45.2. All of these proteins are present in the plasma as pro-  proteins (zymogens). These precursor proteins are activated by cleavage of the  polypeptide chain at one or more sites. The key to successful and appropriate throm-  bus formation is the regulation of the proteases that activate these zymogens.   The proenzymes (Factors VII, XI, IX, X, XII, and prothrombin) are serine pro-  teases that, when activated by cleavage, cleave the next proenzyme in the cascade.  Because of the sequential activation, a great acceleration and amplification of the  response is achieved. That cleavage and activation have occurred is indicated by the  addition of an "a" to the name of the proenzyme (e.g., Factor IX is cleaved to form  the active Factor IXa).   The cofactor proteins (tissue factor, Factors V and VIII) serve as binding sites for  other factors. Tissue factor is not related structurally to the other blood coagulation  cofactors and is an integral membrane protein that does not require cleavage for  active function. Factors V and VIII serve as procofactors, which, when activated by  cleavage, function as binding sites for other factors.   Two additional proteins that are considered part of the blood coagulation cas-  cade, protein S and protein C, are regulatory proteins. Only protein C is regulated  by proteolytic cleavage, and when activated, is itself a serine protease.   Activation of the blood coagulation cascade is triggered by the reaction of plasma  proteins with the subendothelium at the same time that platelets are adhering to the  subendothelial layer. Historically, two different pathways were discovered, one  dependent on external stimuli (such as blunt trauma, which initiates the extrinsic  pathway) and one using internal stimuli (the intrinsic pathway). As our understand-  ing of blood clotting has expanded, it has become obvious that these distinctions are  no longer correct, because there is overlap between the pathways, but the terms have  persisted in the description of the pathways.   In the case of external trauma, damaged tissues present tissue factor to the blood  at the injured site, thereby triggering the extrinsic phase of blood coagulation. Cir-  culating Factor VII binds to tissue factor, which autocatalyzes its own activation to  Factor Vila. Factor Vila then activates Factor X (to Xa) in the extrinsic pathway and  Factor IX (to IXa) in the intrinsic pathway. Factor IXa, as part of the intrinsic path-  way, also activates Factor X. Therefore, activation of both the extrinsic and intrin-  sic pathways result in the conversion of Factor X to Factor Xa. All of these conver-  sions require access to membranes and calcium; the platelet membrane, which had  adhered to the damaged site, is used as a scaffold for the activation reactions to  occur. The 7-carboxylated clotting proteins are chelated to membrane surfaces via  electrostatic interactions with calcium and negatively charged phospholipids of the  platelet membrane. The protein cofactors Villa and Va serve as sites for assembling  enzyme-cofactor complexes on the platelet surface, thereby accelerating and local-  izing the reaction. The result is thrombin formation, which augments its own  formation by converting Factors V, VIII, and XI into activated cofactors and   Fig. 45.3. Blood coagulation cascade. Activation of clot formation occurs through two separate but interlocking pathways, termed the intrinsic  and extrinsic pathways. The intrinsic pathway is activated when plasma proteins react with the exposed subendothelium of the damaged blood  vessel. Platelets and vWF bind to the exposed subendothelium, and the platelets, in turn, bind fibrinogen. The extrinsic pathway is activated by  tissue factor. The reactions designated by "PL, Ca" are occurring through cofactors bound to phospholipids (PL) on the cell surface in a Ca 2+ -  coordination complex. Factors Xlla, XIa, IXa, Vila, Xa, and thrombin are serine proteases. Note the positive feedback regulation of thrombin on  the activation of proteases earlier in the cascade sequence. HMWK = high-molecular- weight kininogen.   Serine protease  Receptor and cofactor  Cofactor  Cofactor  Serine protease  Cofactor  Serine protease  Serine protease  Serine protease  Serine protease   The initial activation of prothrom-  \\ J bin to thrombin is slow, because  *S the activator cofactors, Factors  Villa and Va, are only present in small  amounts. However, once a small amount of  thrombin is activated, it will accelerate its  own production by cleaving Factors V and  VIII to their active forms.   stimulating platelet degranulation. Note that these factors are in the intrinsic path-  way. The intrinsic pathway is thought to sustain the coagulation response initiated  by the extrinsic pathway. The major substrate of thrombin is fibrinogen, which is  hydrolyzed to form fibrin monomers that undergo spontaneous polymerization to  form the fibrin clot. This is considered a "soft" clot because the fibrin monomers  are not cross-linked. Cross-linking requires Factor XHIa, which is activated by  thrombin cleavage of Factor XIII.   Fig. 45.4. The transamidation reaction cat-  alyzed by Factor XHIa, transglutaminase. This  reaction cross-links fibrin monomers, allowing  "hard" clots to form.   Factor XHIa catalyzes a transamidation reaction between Gin and Lys side chains  on adjacent fibrin monomers. The covalent cross-linking takes place in three dimen-  sions, creating a strong network of fibers resistant to mechanical and proteolytic  damage. This network of fibrin fibers traps the aggregated platelets and other cells,  forming the clot that plugs the vent in the vascular wall. (Fig. 45.4) Factor XHIa is  the only enzyme in the blood coagulation cascade that is not a serine protease.   The classical intrinsic pathway begins with the assembly of prekallikrein, high-  molecular- weight kininogen (HMWK), Factor XII, and Factor XI on a negatively  charged surface, presumably an endothelial cell in vivo (see Fig. 45.3). High-  molecular- weight kininogen is a glycoprotein that binds prekallikrein and aids in its  assembly on the endothelial cell. Prekallikrein is the zymogen form of a serine pro-  tease. Factor XII autoactivates, forming Factor Xlla, which converts prekallikrein to  kallikrein. Kallikrein then enhances the activation of Factor XII, which leads to the  activation of Factors XI and VII.   How important these steps are in the initiation of the coagulation cascade is  unknown. Individuals lacking HMWK, prekallikrein, or Factor XII do not suffer  from bleeding disorders. Under usual conditions, activation of Factor VII with sub-  sequent activation of Factors IX and X is thought to be sufficient to activate the  coagulation pathway.   In several of the essential steps in the blood coagulation cascade, the activated pro-  tease is bound in a complex attached to the surface of the platelets that have aggre-  gated at the site of injury. Factors VII, IX, X, and prothrombin contain a domain in  which one or more glutamate residues are carboxylated to 7-carboxyglutamate in a  reaction requiring vitamin K (Fig 45.5). Prothrombin and Factor X both contain 10  or more 7 -carboxy glutamate residues that bind Ca 2+ . Ca 2+ forms a coordination  complex with the negatively charged platelet membrane phospholipids and the  7-carboxyglutamates, thereby localizing the complex assembly and thrombin for-  mation to the platelet surface.   Cofactor Va contains a binding site for both Factor Xa and prothrombin, the  zymogen substrate of Factor Xa. On binding to the Factor Va-platelet complex, pro-  thrombin undergoes a conformational change, rendering it more susceptible to  enzymatic cleavage. Binding of Factor Xa to the Factor Va-prothrombin-platelet  complex allows the prothrombin-to-thrombin conversion. Complex assembly accel-  erates the rate of this conversion 10,000- to 15,000-fold as compared with  non-complex formation.   Factor Villa forms a similar type of complex on the surface of activated platelets,  but binds Factor IXa and its zymogen substrate, Factor X. Tissue factor works  slightly differently because it is an integral membrane protein. However, once  exposed by injury, it also binds Factor Vila and initiates complex formation.   Fig. 45.5. A. Structures of vitamin K derivatives. Phylloquinone is found in green leaves, and intestinal bacteria synthesize menaquinone.  Humans will convert menadione to a vitamin K active form. B. Vitamin K-dependent formation of 7-carboxy glutamate residues. Thrombin, Fac-  tor VII, Factor IX, and Factor X are bound to their phospholipid activation sites on cell membranes by Ca 2+ . The vitamin K-dependent car-  boxylase, which adds the extra carboxyl group, uses a reduced form of vitamin K (KH 2 ) as the electron donor and converts vitamin K to an epox-  ide. Vitamin K epoxide is reduced, in two steps, back to its active form by the enzymes vitamin K epoxide reductase and vitamin K reductase.   Complex assembly has two physiologically important consequences. First, it  enhances the rate of thrombin formation by as much as several hundred thousand-  fold, enabling the clot to form rapidly enough to preserve hemostasis. Secondly,  such explosive thrombin formation is localized to the site of vascular injury at  which the negatively charged phospholipids are exposed. From our knowledge of  the location of such phospholipids in cellular and subcellular organelle membranes,  these surface-binding sites are only exposed at an injury site in which cell rupture  exposes the internal membrane surface (recall that certain phospholipids only face  the cytoplasm; if these lipids are now exposed to the environment, substantial cell  damage must have occurred).   The formation of the 7-carboxyglutamate residues on blood coagulation factors  takes place in the hepatocyte before release of the protein. Within the hepatocyte,  vitamin K (which is present in the quinone form) is reduced to form vitamin KH 2  by a microsomal quinone reductase (see Fig.45.5). Vitamin KH 2 is a cofactor for  carboxylases that add a carboxyl group to the appropriate glutamate residues in the  proenzyme to form the carboxylated proenzyme (e.g., prothrombin). In the same   Warfarin (Coumadin®) is a slow-  and long-acting blood anticoagu-  lant with a structure resembling that  of vitamin K. The structural similarity allows  the compound to compete with vitamin K and  prevent 7-carboxylation of glutamate residues  in Factors II, VII, IX, X, and proteins C and S.  The noncarboxylated blood clotting protein  precursors increase in both the blood and  plasma, but they are unable to promote blood  coagulation because they cannot bind cal-  cium and thus cannot bind to their phospho-  lipid membrane sites of activation.   Warfarin is a commonly used rat poison  and thus is occasionally encountered in  emergency departments in cases of acciden-  tal poisoning. It is effective as a rat poison  because it takes many hours to develop  pathologic symptoms, which allows one poi-  soned trap to kill more than one rat.   Deficiency in the amount or func-  tionality of protein C or protein S  increases the risk for venous throm-  boembolism. Homozygous individuals for  these mutations do not survive the neonatal  period unless given replacement therapy.   0 © In European populations, a point  I /~0A ) mutation in the Factor V gene (Fac-  ^ tor V Leiden) causes the replace-  ment of an arg with a gin in the preferred  site for cleavage by activated protein C, ren-  dering Factor Va Leiden resistant to APC.  Heterozygous individuals have a sixfold to  eightfold increased risk of deep vein throm-  boses, and homozygous individuals have a  30- to 140-fold increased risk. The Factor V  Leiden mutation does not appear to be asso-  ciated with increased risk for arterial throm-  bosis, such as myocardial infarction, except  in young women who smoke.   Genetic studies suggest that the Factor V  Leiden mutation arose after the separation  of the European, Asian, and African popula-  tions. The frequency of this variant indicates  that it conferred some selective advantage at  one time. In the developed world, inherited  APC resistance is the most prevalent risk fac-  tor for familial thrombotic disease.   Fig. 45.6. Anti-thrombotic effects of thrombin. Thrombin, bound to thrombomodulin on the  endothelial cell surface, activates protein C. Activated protein C, in complex with protein S,  binds to the platelet membrane, and the activated complex destroys Factors Va and Villa,  thereby inhibiting the coagulation cascade.   reaction, vitamin K is converted to vitamin K epoxide. To recover active vitamin  KH 2 , vitamin K is first reduced to the quinone form by vitamin K epoxide reduc-  tase, and then to the active hydroquinone form.   Once the formation of the clot (thrombus) begins, clot formation is accelerated in  an almost explosive manner by a number of processes collectively termed feedback  amplification.   Thrombin has both a prothrombotic regulatory role (feedback amplification) and an  antithrombotic regulatory role (feedback inhibition). The prothrombotic action is  initiated when thrombin stimulates its own formation by activating Factors V, VIII,  and XI, thereby accelerating the rate of clot formation (see Fig. 45.3). Thrombin  also promotes clot formation by activating platelet aggregation, stimulating the  release of Factor VIII from vWF, and cleaving Factor XIII to Factor XHIa.   Antithrombotic effects of thrombin result from its binding to an endothelial cell  receptor called thrombomodulin (Fig. 45.6). Thrombomodulin abolishes the clot-  ting function of thrombin and allows thrombin to activate protein C, which has anti-  coagulant effects.   Protein C and its cofactor protein S serve to suppress the activity of the coagu-  lation cascade. After activation, protein C forms a complex with protein S. Pro-  tein S anchors the activated protein C complex (APC) to the clot through  Ca ++ /7-carboxyglutamate binding to platelet phospholipids. The APC destroys  the active blood coagulation cofactors Factor Villa and Factor Va by proteolytic  cleavage, decreasing the production of thrombin. The APC also stimulates  endothelial cells to increase secretion of the prostaglandin PGI 2 , which reduces  platelet aggregation.   Many proteases of the blood coagulation enzyme system are serine proteases.  Because uncontrolled proteolytic activity would be destructive, modulating   mechanisms control and limit intravascular proteolysis. The serpins (serine pro-  tease inhibitors) are a group of naturally occurring inhibitory proteins present in the  plasma at high concentration (approximately 10% of the plasma proteins are ser-  pins). Eight major inhibitors have been found that share a common mechanism of  action and inhibit proteases involved in coagulation and clot dissolution (fibrinoly-  sis). Each inhibitor possesses a reactive site that appears to be an ideal substrate for  a specific serine protease and thus acts as a trap for that protease. The bound serine  protease attacks a peptide bond located at a critical amino acid residue within the  serpin and forms a tight enzyme-inhibitor complex.   The activity of thrombin is controlled by the serpin antithrombin III (ATIII).  Regulation of blood coagulation at the level of thrombin is critical because this  enzyme affects the pathways of both coagulation and fibrinolysis (see section F).  One molecule of ATIII irreversibly inactivates one molecule of thrombin through  reaction of an arginine residue in ATIII with the active- site serine residue of throm-  bin. ATIII-thrombin complex formation is markedly enhanced in the presence of  heparin. Heparin is a glycosaminoglycan (see Chapter 49) found in the secretory  granules of mast cells and in the loose connective tissue around small vascular beds.  Heparin binds to lysyl residues on ATIII and dramatically accelerates its rate of  binding to thrombin. This is because of an allosteric alteration in ATIII such that the  position of the critical arginine residue of ATIII is more readily available for inter-  action with thrombin. The formation of the ATIII-thrombin complex releases the  heparin molecule so that it can be reused, and therefore, the function of heparin is  catalytic. Thrombin that is attached to a surface, for example, to thrombomodulin  on the endothelial cell membrane, is no longer participating in clot formation and is  not readily attacked by ATIII or the ATIII-heparin complex. The ATIII-heparin  complex also can inactivate the serine protease Factors XHIa, XIa, IXa and Xa, but  has no effect on Factor Vila or activated protein C.   Endothelial cells of blood vessels provide a selectively permeable barrier between  the circulating blood and the tissues. The normal endothelial cell lining neither acti-  vates coagulation nor supports platelet adhesion; thus, it is called a nonthrombo-  genic surface. The thromboresistance of normal vascular endothelium is contributed  to by several properties. Endothelial cells are highly negatively charged, a feature  that may repel the negatively charged platelets. Endothelial cells synthesize  prostaglandin I 2 (PGI 2 ) and nitric oxide, vasodilators and powerful inhibitors of  platelet aggregation. PGI 2 synthesis is stimulated by thrombin, epinephrine, and  local vascular injury. Endothelial cells also synthesize two cofactors that each  inhibit the action of thrombin, thrombomodulin and heparan sulfate. Heparan sul-  fate is a glycosaminoglycan similar to heparin that potentiates antithrombin III, but  not as efficiently. The inactivation of thrombin is accelerated by heparan sulfate  present on the endothelial cell surface. Thus, the intact endothelium has the capa-  bility of modifying thrombin action and inhibiting platelet aggregation.   After successful formation of a hemostatic plug, further propagation of the clot  must be prevented. This is accomplished in part by switching off blood coagulation  and in part by turning on fibrinolysis. Fibrinolysis involves the degradation of fib-  rin in a clot by plasmin, a serine protease that is formed from its zymogen, plas-  minogen. Plasminogen is a circulating serum protein that has a high affinity for fib-  rin, promoting the incorporation of plasminogen in the developing clot. The activity  of plasminogen is mediated by proteins known as plasminogen activators. The con-  version of plasminogen to plasmin by plasminogen activators can occur both in the  liquid phase of the blood and at the clot surface; however, the latter process is by   Fig. 45.7. Regulation of plasmin activation.  Plasminogen can be activated by either t-PA or  scu-PA . PAI-1 blocks t-PA action .  Streptokinase binding to plasminogen allows  autocatalysis to form plasmin. Circulating a2-  antiplasmin blocks  the activity of any sol-  uble plasmin that may be in the blood.   0 o Both streptokinase and t-PA have  f /^OA J been approved for the treatment of  ™ myocardial infarction. Both reduce  mortality. Although there are more side  effects associated with the use of streptoki-  nase, it is substantially cheaper than t-PA.   far more efficient. Activated protein C (APC), in addition to turning off the blood  coagulation cascade, also stimulates the release of plasminogen activator from tis-  sues (t-PA, tissue plasminogen activator) and simultaneously inactivates an inhibitor  of plasminogen activator, PAI-1.   Plasminogen activator release can lead to plasmin formation in the circulation.  However, the circulating plasmin is rapidly inactivated by binding with a2-antiplas-  min, a circulating protease inhibitor. Clot-bound plasmin is not readily inactivated  by a-antiplasmin. Thus, plasminogen binding to fibrin facilitates its activation to  plasmin, protects it from blood serpins, and localizes it on the fibrin substrate for  subsequent efficient proteolytic attack. This mechanism allows for dissolution of  fibrin in pathologic thrombi or oversized hemostatic plugs, and at the same time  prevents degradation of fibrinogen in the circulating blood.   Two endogenous plasminogen activators are most important; both are synthe-  sized in a variety of cells. Tissue plasminogen activator (t-PA) is chiefly produced  by the vascular endothelial cells, has a high binding affinity for fibrin, and plays a  major role in fibrinolysis. Single-chain urokinase (scu-PA), is synthesized in most  cells and tissues and has a moderate affinity for fibrin. Streptokinase, the bacterial  exogenous plasminogen activator from (3 -hemolytic streptococci, is not an enzyme  but an allosteric modifier of human plasminogen that allows an autocatalytic reac-  tion such that plasmin is formed. In vivo, physical stress, hypoxia, and large num-  bers of low-molecular- weight organic compounds promote increased synthesis and  release of t-PA and scu-PA from tissues into the blood. The balance between release  of plasminogen activators, the availability of fibrin, and inhibitors of the activators  and plasmin determines regulation of the fibrinolytic response, as indicated in  Figure 45.7.   Anti-activators regulate interaction of plasminogen in blood with plasminogen acti-  vators in a dynamic equilibrium. Even if minute amounts of plasmin are generated  (e.g., after release of vascular plasminogen activator after stress), the enzyme is  probably inactivated by antiplasmin. On activation of the blood coagulation system,  a fibrin clot is formed, which not only strongly binds t-PA and plasminogen from  blood but also accelerates the rate of plasmin activation. The clot-bound plasmin is  protected from inhibitors while attached to fibrin. The enzyme is inactivated by  a2-antiplasmin and a2-macroglobulin after proteolytic dissolution of fibrin and its  liberation into the liquid phase of blood. Thus, the fibrin network catalyzes both ini-  tiation and regulation of fibrinolysis.   Sloe Klotter has hemophilia A, the most frequently encountered serious  disorder of blood coagulation in humans, occurring in 1 in every 10,000  males. The disease is transmitted with an X-linked pattern of inheritance.  The most common manifestations of hemophilia A are those caused by bleeding  into soft tissues (hematomas) such as muscle or into body spaces such as the peri-  toneal cavity or the lumen of the gastrointestinal tract. When bleeding occurs  repeatedly into joints (hemarthrosis), the joint may eventually become deformed  and immobile.   In the past, bleeding episodes have been managed primarily by administration of  Factor VIII, sometimes referred to as antihemophilia cofactor. Unfortunately, the  concentration of Factor VIII in plasma is quite low (0.3 nM compared with 8,800  nM for fibrinogen), requiring that it be prepared from multiple human donors.  Before donor screening and virus inactivation procedures during preparation   essentially eliminated transmission with blood transfusions, more than 50% of  hemophiliac patients treated with Factor VIII during the 1980s in Western Europe  or North America became infected with HIV. Recombinant Factor VIII is now avail-  able for clinical use.   A number of drugs have been developed that inhibit the blood coagulation  cascade. Such drugs are useful in cases in which patients develop sponta-  neous thrombi, which, if left untreated, would result in a fatal pulmonary  embolism. There are three major classes of such drugs; the heparins, vitamin K  antagonists, and specific inhibitors of thrombin.   Heparin will bind to and activate ATIII, which leads to thrombin inactivation.  ATIII also blocks the activity of Factors Villa, IXa, Xa, and XIa. Heparin can be  administered in either of two forms: unfractionated, or high-molecular- weight  (HMW) heparin, and fractionated, or low-molecular- weight (LMW) heparin. HMW  heparin is a heterogenous mixture of glycosaminoglycans, with an average chain  length of 45 monosaccharides with an average molecular weight of 15 kDa (the  range is 3-30 kDa). LMW heparins are fragments of HMW heparin, containing  fewer than 18 monosaccharides with an average molecular weight of 4 to 5 kDa.   HMW heparin will bind to plasma proteins and cell surfaces in addition to its  prime target, ATIII. Because different individuals synthesize different levels of  plasma proteins, the use of this form of heparin as an anticoagulant requires con-  stant monitoring of the patient to ensure that the correct dosage has been given such  that spontaneous thrombi do not develop, but not so much that spontaneous bleed-  ing occurs. LMW heparin has fewer nonspecific interactions than HMW heparin,  and its effects are easier to predict on patients, so that constant monitoring is not  required.   A major complication of heparin therapy is heparin-induced thrombocytopenia  (HIT, excessive blood clotting with a reduction in the number of circulating  platelets). This unexpected result of heparin treatment is caused by heparin bind-  ing to a platelet protein, platelet factor 4 (PF4), which induces a conformational  change in PF4 such that the immune system believes the complex is foreign. Thus,  antibodies are developed against the heparin-PF4 complex. When the antibodies  bind to the platelets, the platelets become activated, and thrombi develop. Treat-  ment consists of removing the heparin and using a different form of anti-throm-  botic agent.   The classic vitamin K antagonist is warfarin. Warfarin acts by blocking the vita-  min K reductase enzymes required to regenerate active vitamin K (see Fig. 45.5).  This results in reduced 7-carboxylation of Factors II, VII, IX, and X. In the absence  of 7-carboxylation, the factors cannot bind calcium nor form the complexes neces-  sary for the coagulation cascade to be initiated. However, warfarin also blocks the  activity of proteins S and C, so both blood clotting and the regulation of clotting are  impaired by warfarin administration.   Both heparin and warfarin therapy suffer from their lack of specificity, so drugs  specific for single steps in the blood coagulation pathway have been sought and  identified. Analysis of heparin potentiation of Factor Xa binding to ATIII showed  that a unique pentasaccharide sequence was required. An appropriate pentasaccha-  ride, named fondaparinux, was developed that would specifically enhance ATIII  interactions with Factor Xa (Fig. 45.8). Fondaparinux stimulates the binding of  ATIII to Factor Xa by 300-fold and is specific for Factor Xa inhibition. Fonda-  parinux does not affect thrombin or platelet activity, and it is not an activating agent  of platelets. Because fondaparinux does not bind to PF4, HIT is not a complication  with this therapy.   Another X-linked bleeding disorder  is hemophilia B, which is caused by  mutations in the gene for Factor IX.  Lack of Factor IX activity leads to an inability  to convert prothrombin to thrombin, and  impaired clotting.    Fig. 45.8. A. Structure of fondaparinux. B. Mechanism of fondaparinux action. The drug (shown in blue) binds to ATIII, which induces a con-  formational change such that Factor Xa can now bind to ATIII. Once Factor Xa binds, and is inactivated, the drug is released and can activate  another molecule of ATIII.   Direct thrombin inhibitors are based on the hirudin molecules, which were ini-  tially discovered in leeches and other blood- sucking organisms. These organisms  would not be able to feed if the blood clotted at the site of the puncture wound, so  the organisms secrete thrombin inhibitors to prevent clotting from occurring.  Hirudin treatment itself is dangerous in that formation of the hirudin-thrombin  complex is irreversible, and use of the drug requires constant monitoring of the  patient. Thus, to overcome this problem, rational drug design based on the hirudin  structure was used, and a synthetic 20-amino acid peptide known as bivalirudin was  synthesized. This agent has a high binding affinity and specificity for thrombin  although its effects on thrombin are transient (not irreversible), making this a safer  agent for long-term use.   Bauer KA. Selective inhibition of coagulation factors: advances in antithrombotic therapy. Seminars in   Thrombosis and Hemostasis 2002;28:15-24.  Robetorye RS, Rodgers GM. Update on selected inherited venous thrombotic disorders. Am J Hematol    1 . The edema observed in patients with non-calorie protein malnutrition is due to which of the following?   2. A recent surgery patient receiving warfarin therapy was found to be bleeding internally. The clotting process is impaired in  this patient primarily because of which of the following?   3. An inactivating mutation in which of the following proenzymes would be expected to lead to thrombosis, uncontrolled blood  clotting?   4. Classical hemophilia A results in an inability to directly activate which of the following factors?   5. Hemophilia B results in an inability to directly activate which of the following factors?   The liver is strategically interposed between the general circulation and the diges-  tive tract. It receives 20 to 25% of the volume of blood leaving the heart each  minute (the cardiac output) through the portal vein (which delivers absorbed  nutrients and other substances from the gastrointestinal tract to the liver) and  through the hepatic artery (which delivers blood from the general circulation back  to the liver). Potentially toxic agents absorbed from the gut or delivered to the  liver by the hepatic artery must pass through this metabolically active organ  before they can reach the other organs of the body. The liver s relatively large size  (approximately 3% of total body weight) allows extended residence time within  the liver for nutrients to be properly metabolized as well as for potentially harm-  ful substances to be detoxified and prepared for excretion into the urine or feces.  Among other functions , therefore, the liver, along with the kidney and gut, is an  excretory organ, equipped with a broad spectrum of detoxifying mechanisms. It  has the capacity, for example, to carry out metabolic conversion pathways as well  as secretory systems that allow the excretion of potentially toxic compounds. Con-  currently, the liver contains highly specific and selective transport mechanisms  for essential nutrients that are required not only to sustain its own energy but to  provide physiologically important substrates for the systemic needs of the organ-  ism. In addition to the myriad of transport processes within the sinusoidal and  canalicular plasma membrane sheets (see below), intracellular hepatocytic trans-  port systems exist in organelles such as endosomes, mitochondria, lysosomes, as  well as the nucleus. The sequential transport steps carried out by these organelles  include (I) uptake, (2) intracellular binding and sequestration, (3) metabolism,  (4) sinusoidal secretion, and (5) biliary excretion. The rate of hepatobiliary  transport is determined, in part, by the rate of activity of each of these steps. The  overall transport rate is also determined by such factors as hepatic blood flow,  plasma protein binding, and the rate of canalicular reabsorption. The various  aspects of the major metabolic processes performed by the liver have been dis-  cussed in greater detail elsewhere in this text. These sources are referred to as the  broad spectrum of the liver's contributions to overall health and disease are  described.   Jean Ann Tonich's family difficulties continued, and, in spite of a period  of sobriety lasting 6 months, she eventually started drinking increasing  amounts of gin again in an effort to deal with her many anxieties. Her  appetite for food declined slowly as well. She gradually withdrew from much of the   social support system that her doctors and friends had attempted to build during her  efforts for rehabilitation. Upper mid-abdominal pain became almost constant, and  she noted an increasing girth and distention of her abdomen. Early one morning, she  was awakened in excruciating pain in her upper abdomen. She vomited dark-brown  "coffee ground" material followed by copious amounts of bright red blood. She  called a friend, who rushed her to the hospital emergency room.   Amy Biasis, a 23 -year-old missionary, was brought to the hospital emer-  gency room complaining of the abrupt onset of fever, chills, and severe  pain in the right upper quadrant of her abdomen. The pain was constant in  nature and radiated to her right shoulder top. She vomited undigested food twice in  the hour before arriving at the emergency room. This did not relieve her pain.   Her medical history indicated that, while serving as a missionary in western  Belise, Central America, 2 months earlier, she had a 3 -day illness that included fever,  chills, and mild but persistent diarrhea. A friend of Amy's there, a medical mission-  ary, had given her an unidentified medication for 7 days. Amy's diarrhea slowly  resolved, and she felt well again until her current abdominal symptoms began.   On physical examination, she appeared toxic and had a temperature of 101°F.  She was sweating profusely. Her inferior anterior liver margin was palpable three  fingerbreadths below the right rib cage, suggestive of an enlarged liver. The liver  edge was rounded and tender. Gentle first percussion of the lower posterior right rib  cage caused severe pain. Routine laboratory studies were ordered, and a computed  tomogram (CT) of the upper abdomen was scheduled to be done immediately.   The human liver consists of two lobes, each containing multiple lobules and sinu-  soids. The liver receives 75% of its blood supply from the portal vein, which carries  blood returning to the heart from the small intestine, stomach, pancreas and spleen.  The remaining 25% of the liver's blood supply is arterial, carried to the liver by the  hepatic artery.   Blood from both the portal vein and hepatic artery empty into a common con-  duit, mixing their contents as they enter the liver sinusoids (Fig. 46.1). The sinu-  soids are expandable vascular channels that run through the hepatic lobules. They  are lined with endothelial cells that have been described as "leaky" because, as  blood flows through the sinusoids, the contents of the plasma have relatively free  access to the hepatocytes, which are located on the other side of the endothelial  cells.   The liver is also an exocrine organ, secreting bile into the biliary drainage sys-  tem. The hepatocytes secrete bile into the bile canniculus, whose contents flow par-  allel to that in the sinusoids but in the opposite direction. The canniculi empty into  the bile ducts. The lumina of the bile ducts then fuse, forming the common bile duct.  The common duct then releases bile into the duodenum. Some of the liver's efflu-  ent is stored in the gallbladder and discharged into the duodenum postprandially to  aid in digestion.   The entire liver surface is covered by a capsule of connective tissue that branches  and extends throughout the liver. This capsule provides support for the blood ves-  sels, lymphatic vessels, and bile ducts that permeate the liver. In addition, this con-  nective tissue sheet subdivides the liver lobes into the smaller lobules.   The primary cell type of the liver is the hepatocyte. Hepatocytes, also known as the  hepatic parenchymal cells, form the liver lobules. Eighty percent of the liver volume  is composed of hepatocytes, but only 60% of the total number of cells in the liver   The reports of Amy Biasis' initial  laboratory studies showed an ele-  vation in her serum hepatic  transaminases, her serum alkaline phos-  phatases, as well as her serum total bilirubin  level.    are hepatocytes. The other 40% of the cells are the nonparenchymal cells, which  constitute the lining cells of the walls of the sinusoids. The lining cells comprise the  endothelial cells, Kupffer cells, and hepatic stellate cells. In addition, intrahepatic  lymphocytes, which include pit cells (liver- specific natural killer cells) are also  present in the sinusoidal lining.   The hepatocyte is the cell that carries out the many functions of the liver. Almost all  pathways of metabolism are represented in the hepatocyte and these pathways are  controlled through the actions of hormones that bind to receptors located on the  plasma membrane of their cells. Although normally quiescent cells with low  turnover and a long life span, hepatocytes can be stimulated to grow if damage  occurs to other cells in the liver. The liver mass has a relatively constant relation-  ship to the total body mass of adult individuals. Deviation from the normal or opti-  mal ratio (caused, for example, by a partial hepatectomy or significant hepatic cell  death or injury) is rapidly corrected by hepatic growth caused by a proportional  increase in hepatocyte replication.   The sinusoidal endothelial cells constitute the lining cells of the sinusoid. Unlike  endothelial cells in other body tissues, these cells contain fenestrations with a mean  diameter of 100 nm. They do not, therefore, form a tight basement membrane bar-  rier between themselves and the hepatocytes. In this way, they allow for free diffu-  sion of small molecules to the hepatocytes but not of particles the size of chylomi-  crons (chylomicron remnants, however, which are smaller than chylomicrons, do  have free passage to the hepatocyte). The endothelial cells are capable of endocy-  tosing many ligands and also may secrete cytokines when appropriately stimulated.  Because of their positioning, lack of tight junctions, and absence of a tight basement  membrane, the liver endothelial cells do not present a significant barrier against the  movement of the contents of the sinusoids into hepatocytes. Their fenestrations or  pores further promote the free passage of blood components through this membrane  into the liver parenchymal cells.   These cells are located within the sinusoidal lining. They contain almost one quar-  ter of all the lysosomes of the liver. The Kupffer cells are tissue macrophages with  both endocytotic and phagocytic capacity. They phagocytose many substances such  as denatured albumin, bacteria, and immune complexes. They protect the liver from  gut-derived particulate materials and bacterial products. On stimulation by  immunomodulators, these cells secrete potent mediators of the inflammatory  response and play a role in liver immune defense through the release of cytokines  that lead to the inactivation of substances considered foreign to the organism. The  Kupffer cells also remove damaged erythrocytes from the circulation.   The stellate cells are also called perisinusoidal or Ito cells. There are approximately  5 to 20 of these cells per 100 hepatocytes. The stellate cells are lipid-filled cells (the  primary storage site for vitamin A). They also control the turnover of hepatic con-  nective tissue and extracellular matrix and regulate the contractility of the sinusoids.  When cirrhosis of the liver is present, the stellate cells are stimulated by various sig-  nals to increase their synthesis of extracellular matrix material. This, in turn, dif-  fusely infiltrates the liver, eventually interfering with the function of the hepatocytes.   The hepatic pit cells, also known as liver-associated lymphocytes, are natural killer  cells, which are a defense mechanism against the invasion of the liver by potentially  toxic agents, such as tumor cells or viruses.   A. The Liver Is a Central Receiving and Recycling Center  for the Body   The liver can carry out a multitude of biochemical reactions. This is necessary  because of its role in constantly monitoring, recycling, modifying, and distributing  all of the various compounds absorbed from the digestive tract and delivered to the  liver. If any portion of an ingested compound is potentially useful to that organism,  the liver will retrieve this portion and convert it to a substrate that can be used by  hepatic and nonhepatic cells. At the same time, the liver removes many of the toxic  compounds that are ingested or produced in the body and targets them for excretion  in the urine or in the bile.   As mentioned previously, the liver receives nutrient-rich blood from the enteric  circulation through the portal vein; thus, all of the compounds that enter the blood  from the digestive tract pass through the liver on their way to other tissues. The  enterohepatic circulation allows the liver first access to nutrients to fulfill specific  functions (such as the synthesis of blood coagulation proteins, heme, purines, and  pyrimidines) and first access to ingested toxic compounds (such as ethanol) and to  such potentially harmful metabolic products (such as NH 4 + produced from bacter-  ial metabolism in the gut).   In addition to the blood supply from the portal vein, the liver receives oxygen-  rich blood through the hepatic artery; this arterial blood mixes with the blood from  the portal vein in the sinusoids. This unusual mixing process gives the liver access  to various metabolites produced in the periphery and secreted into the peripheral  circulation, such as glucose, individual amino acids, certain proteins, iron-trans-  ferrin complexes, and waste metabolites as well as potential toxins produced dur-  ing substrate metabolism. As mentioned, fenestrations in the endothelial cells,  combined with gaps between the cells, the lack of a basement membrane between  the endothelial cells and the hepatocytes, and low portal blood pressure (which  results in slow blood flow) contribute to the efficient exchange of compounds  between sinusoidal blood and the hepatocyte and clearance of unwanted com-  pounds from the blood. Thus, large molecules targeted for processing, such as  serum proteins and chylomicron remnants, can be removed by hepatocytes,  degraded, and their components recycled. Similarly, newly synthesized molecules,  such as very-low-density lipoprotein (VLDL) and serum proteins, can be easily  secreted into the blood. In addition, the liver can convert all of the amino acids  found in proteins into glucose, fatty acids, or ketone bodies. The secretion of  VLDL by the liver not only delivers excess calories to adipose tissue for storage of  fatty acids in triacylglycerol, but it also delivers phospholipids and cholesterol to  tissues that are in need of these compounds for synthesis of cell walls as well as  other functions. The secretion of glycoproteins by the liver is accomplished  through the liver's gluconeogenic capacity as well as its access to a variety of  dietary sugars to form the oligosaccharide chains, as well as its access to dietary  amino acids with which is synthesizes proteins. Thus, the liver has the capacity to  carry out a large number of biosynthetic reactions. It has the biochemical where-  withal to synthesize a myriad of compounds from a broad spectrum of precursors.  At the same time, the liver metabolizes compounds into biochemically useful prod-  ucts. Alternatively, it has the ability to degrade and excrete those compounds  presented to it that cannot be further used by the body.   The CT scan of Amy Biasis' upper  abdomen showed an elevated right  hemidiaphragm as well as several  cystic masses in her liver, the largest of  which was located in the superior portion of  the right lobe. Her clinical history as well as  her history of possible exposure to various  parasites while working in a part of Belize,  Central America, that is known to practice  substandard sanitation, prompted her physi-  cians to order a titer of serum antibodies  against the parasite Entamoeba histolytica  in addition to measuring serum antibodies  against other invasive parasites.   A knowledge of functional charac-  teristics of liver cells has been used  to design diagnostic agents that  can be used to determine the normalcy of  specific biochemical pathways of the hepa-  tocytes. These "tailor-made" pharmaceuti-  cals can be designed to be taken up by one  or more of the available transport mecha-  nisms available to the liver. For example,  receptor-related endocytic processes can be  used as targets to probe specific receptor-  mediated transport functions of the liver  cells. The asialoglycoprotein receptor, also  known as the hepatic-binding protein, has  been used in this diagnostic approach. The  substrate "T c m -galactosyl-neoglycoalbumin  (NGA) was developed as a specific ligand for  selective uptake via this specific hepatic  receptor. The timing and extent of the assim-  ilation of this probe into the hepatocytes, as  determined by imaging the liver at precise  intervals after the administration of this iso-  tope, yields an estimate of hepatic blood  flow as well as the transport capacity of this  specific hepatic transporter protein.   Antibody titers against Entamoeba  histolytica by use of an enzyme  immunoassay were strongly reac-  tive in Amy Biasis' blood. A diagnosis of  amoebiasis was made. Her physicians  started nitroimidazole amoebicides intra-  venously in a dose of 500 mg every 6 hours  for 10 days. By the third day of treatment,  Amy began to feel noticeably better. Her  physicians told her that they expected a full  clinical response in 95% of patients with  amoebic liver abscesses treated in this way,  although her multiple hepatic abscesses  adversely affected her prognosis to a  degree.   Table 46.1. Examples of Enzymes  Used in Biotransformation of  Xenobiotic Compounds   Flavin-containing mono-oxygenase  Glutathione-S-transferase  Methyl transferase  Mixed-function oxidase  Reductase  Sulfotransferase  UDP-glucosyltransf erase  UDP-glucuronosyltransf erase   Each of the liver cells described contains specialized transport and uptake mech-  anisms for enzymes, infectious agents, drugs, and other xenobiotics that specifically  target these substances to certain liver cell types. These are accomplished by link-  ing these agents covalently by way of biodegradable bonds to their specific carrier.  The latter then determines the particular fate of the drug by using specific cell  recognition, uptake, transport, and biodegradation pathways.   Xenobiotics are compounds that have no nutrient value (cannot be used by the body  for energy requirements) and are potentially toxic. They are present as natural compo-  nents of foods or they may be introduced into foods as additives or through processing.  Pharmacologic and recreational drugs are also xenobiotic compounds. The liver is the  principal site in the body for the degradation of these compounds. Because many of  these substances are lipophilic, they are oxidized, hydroxylated, or hydrolyzed by  enzymes in phase I reactions. Phase I reactions introduce or expose hydroxyl groups or  other reactive sites that can be used for conjugation reactions (the phase II reactions).  The conjugation reactions add a negatively charged group such as glycine or sulfate to  the molecule. Many xenobiotic compounds will be transformed through several differ-  ent pathways. A general scheme of inactivation is shown in Figure 46.2.   The conjugation and inactivation pathways are similar to those used by the liver  to inactivate many of its own metabolic waste products. These pathways are inti-  mately related to the biosynthetic cascades that exist in the liver. The liver can syn-  thesize the precursors that are required for conjugation and inactivation reactions  from other compounds. For example, sulfation is used by the liver to clear steroid  hormones from the circulation. The sulfate used for this purpose can be obtained  from the degradation of cysteine or methionine.   The liver, kidney, and intestine are the major sites in the body for biotransfor-  mation of xenobiotic compounds. Many xenobiotic compounds contain aromatic  rings (such as benzopyrene in tobacco smoke) or heterocyclic ring structures (such  as the nitrogen-containing rings of nicotine or pyridoxine) that we are unable to  degrade or recycle into useful components. These structures are hydrophobic, caus-  ing the molecules to be retained in adipose tissue unless they are sequestered by the  liver, kidney, or intestine for biotransformation reactions. Sometimes, however, the  phase I and II reactions backfire, and harmless hydrophobic molecules are con-  verted to toxins or potent chemical carcinogens.   The toxification/detoxification of xenobiotics is accomplished through the activ-  ity of a group of enzymes with a broad spectrum of biologic activity. Some exam-  ples of enzymes involved in xenobiotic transformation are described in  Table 46.1. Of the wide variety of enzymes that are involved in xenobiotic metab-  olism, only the cytochrome P450-dependent monooxygenase system is discussed  here. The cytochrome P450-dependent monooxygenase enzymes are determi-  nants in oxidative, peroxidative, and reductive degradation of exogenous (chemi-  cals, carcinogens, and pollutants, etc.) and endogenous (steroids, prostaglandins,   retinoids, etc.) substances. The key enzymatic constituents of this system are the flavo  protein NADPH-cytochrome P450 oxidoreductase and cytochrome P450 (Fig. 46.3).  The latter is the terminal electron acceptor and substrate-binding site of the microso-  mal mixed-function oxidase complex, a very versatile catalytic system. The system  got its name in 1962, when Omura and Sato found a pigment with unique spectral  characteristics derived from liver microsome of rabbits. When reduced and complexed  with carbon monoxide, it exhibited a spectral absorbance maximum at 450 nm.   The major role of the cytochrome P450 enzymes (see Chapter 25) is to oxidize  substrates and introduce oxygen to the structure. Similar reactions can be carried  out by other flavin monoxygenases that do not contain cytochrome P450.   The cytochrome P450 enzyme family contains at least 100 to 150 different  isozymes with at least 40% sequence homology. These isozymes have different but  overlapping specificities. The human enzymes are generally divided into six major  subfamilies, and each of these is further subdivided. For example, in the naming of  the principal enzyme involved in the oxidation of ethanol to acetaldehyde, CYP2E1,  the CYP denotes the cytochrome P450 family, the 2 denotes the subfamily, the E  denotes ethanol, and the 1 denotes the specific isozyme.   The CYP3A4 isoform accounts for 60% of CYP450 enzymes in the liver and  70% of cytochrome enzymes in gut wall enterocytes. It metabolizes the greatest  number of drugs in humans. Specific drugs are substrates for CYP3A4. The con-  comitant ingestion of two CYP3A4 substrates could potentially induce competition  for the binding site, which, in turn, could alter the blood levels of these two agents.  The drug with the highest affinity for the enzyme would be preferentially metabo-  lized, whereas the metabolism (and degradation) of the other drug would be  reduced. The latter drug's concentration in the blood would then rise.   Moreover, many substances or drugs impair or inhibit the activity of the CYP3A4  enzyme, thereby impairing the body's ability to metabolize a drug. The lipid-lowering  agents known as the statins (HMGCoA reductase inhibitors) require CYP3A4 for  degradation. Appropriate drug treatment and dosing takes into account the normal  degradative pathway of the drug. However, grapefruit juice is a potent inhibitor of  CYP3A4-mediated drug metabolism. Evidence suggests that if a statin is regularly  taken with grapefruit juice, its level in the blood may increase as much as 15 -fold. This  marked increase in plasma concentration could increase the muscle and liver toxicity  of the statin in question, because side effects of the statins appear to be dose-related.   1. They all contain cytochrome P450, oxidize the substrate, and reduce oxygen.   2. They all have a flavin-containing reductase subunit that uses NADPH, and not  NADH, as a substrate.   3. They are all found in the smooth endoplasmic reticulum and are referred to as  microsomal enzymes (for example, CYP2E1 is also referred to as the microso-  mal ethanol oxidizing system, MEOS).   4. They are all bound to the lipid portion of the membrane, probably to phos-  phatidylcholine.   5. They are all inducible by the presence of their own best substrate and somewhat  less inducible by the substrates for other P450 isozymes.   6. They all generate a reactive free radical compound as an intermediate in the reaction.   Fig. 46.3. General structure of the P450  enzymes. 0 2 binds to the P450 Fe-heme in the  active site and is activated to a reactive form by  accepting electrons. The electrons are donated  by the cytochrome P450 reductase, which con-  tains an FAD plus an FMN or Fe-S center to  facilitate the transfer of single electrons from  NADPH to 0 2 . The P450 enzymes involved in  steroidogenesis have a somewhat different  structure. For CYP2E1, RH is ethanol  (CH 3 CH 2 OH), and ROH is acetaldehyde  (CH 3 COH).   The detoxification of vinyl chloride provides an example of effective detoxification  by a P450 isozyme (ethanol detoxification was previously discussed in Chapter 25).  Vinyl chloride is used in the synthesis of plastics and can cause angiosarcoma in the  liver of exposed workers. It is activated in a phase I reaction to a reactive epoxide by   a hepatic P450 isozyme (CYP2E1), which can react with guanine in DNA or other  cellular molecules. However, it also can be converted to chloroacetate, conjugated  with reduced glutathione, and excreted in a series of phase II reactions (Fig. 46.4).   Aflatoxin B 1 is an example of a compound that is made more toxic by a cytochrome  P450 reaction (CYP2A1). Current research suggests that ingested aflatoxin Bl in  contaminated food (it is produced by a fungus [Aspergillus flavus] that grows on  peanuts that may have been stored in damp conditions) is directly involved in hepa-  tocarcinogenesis in humans by introducing a G>T mutation into the p53 gene.  Aflatoxin is metabolically activated to its 8,9 epoxide by two different isozymes of  cytochrome P450. The epoxide modifies DNA by forming covalent adducts with  guanine residues. In addition, the epoxide can combine with lysine residues within  proteins and thus is also a hepatotoxin.   Acetaminophen (Tylenol) is an example of a xenobiotic that is metabolized by the  liver for safe excretion; however, it can be toxic if ingested in high doses. The  pathways for acetaminophen metabolism are shown in Fig. 46.5 As shown in the    Fig. 46.5. Pathways of acetaminophen detoxification. N-acetyl cysteine stimulates the pro-  duction of glutathione, thereby reducing the levels of NAPQI, which can damage cellular  proteins. Ethanol upregulates CYP2E1 activity (the MEOS).   figure, acetaminophen can be glucuronylated or sulfated for safe excretion by the  kidney. However, a cytochrome P450 enzyme produces the toxic intermediate N-  acetyl-p-benzoquinoneimine (NAPQI), which can be excreted safely in the urine  after conjugation with glutathione.   NAPQI is a dangerous and unstable metabolite that can damage cellular proteins  and lead to death of the hepatocyte. Under normal conditions, when acetaminophen  is taken in the correct therapeutic amounts, less than 10% of the drug forms NAPQI,  an amount that can be readily handled by the glutathione detoxifying system (phase  II reactions). However, when taken at doses that are potentially toxic, the sulfo-  transferase and glucuronyl transferase systems are overwhelmed, and more aceta-  minophen is metabolized through the NAPQI route. When this occurs, the levels of  glutathione in the hepatocyte are insufficient to detoxify NAPQI, and hepatocyte  death can result.   The enzyme that produces NAPQI, CYP2E1, is induced by alcohol (see Chapter  25, MEOS). Thus, individuals who chronically abuse alcohol have an increased sen-  sitivity to acetaminophen toxicity, because a higher percentage of acetaminophen  metabolism is directed toward NAPQI, as compared with an individual with low  levels of CYP2E1. Therefore, even recommended therapeutic doses of acetamino-  phen can be toxic to these individuals.   An effective treatment for acetaminophen poisoning involves the use of N-acetyl  cysteine. This compound supplies cysteine as a precursor for increased glutathione  production, which, in turn, enhances the phase II reactions, which reduces the lev-  els of the toxic intermediate.   One of the primary functions of the liver is to maintain blood glucose concentra-  tions within the normal range. The manner in which the liver accomplishes this  has been the subject of previous chapters (26, 31, and 36). In brief, the pancreas  monitors blood glucose levels and secretes insulin when blood glucose levels rise  and glucagon when such levels decrease. These hormones initiate regulatory cas-  cades that affect liver glycogenolysis, glycogen synthesis, glycolysis, and gluco-  neogenesis. In addition, sustained physiologic increases in growth hormone, Cor-  tisol, and catecholamine secretion help to sustain normal blood glucose levels  during fasting.   When blood glucose levels drop, glycolysis and glycogen synthesis are inhibited,  and gluconeogenesis and glycogenolysis are activated. Concurrently, fatty acid oxi-  dation is activated to provide energy for glucose synthesis. During an overnight fast,  blood glucose levels are primarily maintained by glycogenolysis and, if gluconeo-  genesis is required, the energy (6 ATP are required to produce one molecule of glu-  cose from two molecules of pyruvate) is obtained by fatty acid oxidation. On insulin  release, the opposing pathways are activated such that excess fuels can be stored  either as glycogen or fatty acids. The pathways are regulated by the activation or  inhibition of two key kinases, the cyclic adenosine monophosphate (cAMP)-  dependent protein kinase, and the AMP-activated protein kinase (see Fig. 36.1 1 for  a review of these pathways). Recall that the liver can export glucose because it is  one of only two tissues that express glucose-6-phosphatase.   When food supplies are plentiful, hormonal activation leads to fatty acid, triacylglyc-  erol, and cholesterol synthesis. A high dietary intake and intestinal absorption of cho-  lesterol will compensatorily reduce the rate of hepatic cholesterol synthesis, in which  case the liver acts as a recycling depot for sending excess dietary cholesterol to the  peripheral tissue when needed as well as accepting cholesterol from these tissues when  required. The pathways of cholesterol metabolism were discussed in Chapter 34.   Numerous other factors, beside  insulin and glucagon, can affect  liver glucose metabolism, as has  been described in Chapter 43.   The liver is the primary organ for synthesizing urea and, as such, is the central depot  for the disposition of ammonia in the body. Ammonia groups travel to the liver on  glutamine and alanine, and the liver converts these ammonia nitrogens to urea for  excretion in the urine. The reactions of the urea cycle were discussed in Chapter 38.   Table 46.2 lists some of the important nitrogen-containing compounds that are  primarily synthesized or metabolized by the liver.   The liver is the only organ that can produce ketone bodies, yet it is one of the few that  cannot use these molecules for energy production. Ketone bodies are produced when  the rate of glucose synthesis is limited (i.e., substrates for gluconeogenesis are lim-  ited), and fatty acid oxidation is occurring rapidly. Ketone bodies can cross the blood-  brain barrier and become a major fuel for the nervous system under conditions of star-  vation. Ketone body synthesis and metabolism have been described in Chapter 23.   The liver can synthesize and salvage all ribonucleotides and deoxyribonucleotides  for other cells to use. Certain cells have lost the capacity to produce nucleotides de   Liver, small amounts in brain  and cells of the immune  system   Liver, small amounts in brain  and cells of the immune  system   Forms creatine phosphate in muscle for  energy storage. Excreted as creatinine.   Protection against free radical injury by  reduction of hydrogen peroxide and lipid per-  oxides. In liver and kidney forms  mercapturic acids.   Adenine and guanine nucleosides and  nucleotides. DNA, RNA, and coenzymes, and  energy-transferring nucleotides.   In the liver, synthesis of oligosaccharide  chains on secreted proteins. Most cells, gly-  coproteins, proteoglycans, and glycolipids.   Many cells use sulfate in blood for formation  of PAPS, which transfers sulfate to proteogly-  cans, drugs, and xenobiotics   Conjugated bile salts are excreted into the bile  and assist in the absorption of lipids and  fat-soluble vitamins through the formation of  micelles   Heme from liver is incorporated into  cytochromes. Heme from bone marrow is  incorporated into hemoglobin.   Choline, phosphatidylcholine, purine and  pyrimidine synthesis, inactivation of waste  metabolites and xenobiotics through  methylation.   novo but can use the salvage pathways to convert free bases to nucleotides. The liver  can secrete free bases into the circulation for these cells to use for this purpose.  Nucleotide synthesis and degradation are discussed in Chapter 41.   The liver is the primary site of the synthesis of circulating proteins such as albumin  and the clotting factors. When liver protein synthesis is compromised, the protein  levels in the blood are reduced. Hypoproteinemia may lead to edema because of a  decrease in the protein-mediated osmotic pressure in the blood. This, in turn, causes  plasma water to leave the circulation and enter (and expand) the interstitial space,  causing edema.   Most circulating plasma proteins are synthesized by the liver. Therefore, the  hepatocyte has a well-developed endoplasmic reticulum, Golgi system, and cellular  cytoskeleton, all of which function in the synthesis, processing, and secretion of  proteins. The most abundant plasma protein produced by the liver is albumin, which  represents 55 to 60% of the total plasma protein pool. Albumin serves as a carrier  for a large number of hydrophobic compounds, such as fatty acids, steroids,  hydrophobic amino acids, vitamins, and pharmacologic agents. It is also an impor-  tant osmotic regulator in the maintenance of normal plasma osmotic pressure. The  other proteins synthesized by the liver are, for the most part, glycoproteins. They  function in hemostasis, transport, protease inhibition, and ligand binding, as well as  secretogogues for hormone release. The acute phase proteins that are part of the  immune response and the body's response to many forms of "injury" are also syn-  thesized in the liver. Table 46.3 lists some of the proteins and their functions.   The liver, because it is the site of synthesis of most of the blood proteins (including  the glycoproteins), has a high requirement for the sugars that go into the oligosac-  charide portion of glycoproteins (The synthesis of glycoproteins is discussed in  Chapter 30.). These include mannose, fructose, galactose, and amino sugars.   One of the intriguing aspects of the hepatic biosynthetic pathways that use car-  bohydrate in the synthesis of these compounds is that the liver is not dependent on  either dietary glucose or hepatic glucose to generate the precursor intermediates for  these pathways. This is because the liver can generate carbohydrates from dietary  amino acids (which enter gluconeogenesis generally as pyruvate or an intermediate  of the TCA cycle), lactate (generated from anaerobic glycolysis in other tissues),  and glycerol (generated by the release of free fatty acids from the adipocyte). Of  course, if dietary carbohydrate is available, the liver can use that source as well.   Most of the sugars secreted by the liver are O-linked, that is, the carbohydrate is  attached to the protein at its anomeric carbon through a glucosidic link to the -OH  of a serine or a threonine residue. This is in contrast to the N-linked arrangement in  which there is an N-glycosyl link to the amide nitrogen of an asparagine residue  (Fig. 46.6). A particularly important O-linked sugar is N-acetylneuraminic acid  (NANA or sialic acid), a nine-carbon sugar that is synthesized from fructose-  6-phosphate and phosphoenolpyruvate (see Fig. 30.8). As circulating proteins age,   0 © Cirrhosis of the liver results in por-  ( /~oA ) tal hypertension, which because of  ™ increasing back pressure into the  esophageal veins promotes the develop-  ment of dilated thin-walled esophageal  veins (varices). At the same time, synthesis  of blood coagulation proteins by the liver  and required vitamin K-dependent reactions  are greatly diminished (resulting in a pro-  longed prothrombin time; which, in turn,  increases clotting time). When the  esophageal varices rupture, massive bleed-  ing into the thoracic or abdominal cavity as  well as the stomach may occur. Much of the  protein content of the blood entering the  gastrointestinal tract is metabolized by intes-  tinal bacteria, releasing ammonium ion,  which enters the portal vein. Because hepa-  tocellular function has been compromised,  the urea cycle capacity is inadequate, and  the ammonium ion enters the peripheral cir-  culation, thereby contributing to hepatic  encephalopathy (brain toxicity due to ele-  vated ammonia levels).   Table 46.3. A Partial List of Proteins Synthesized in the Liver   Blood coagulation factors: fibrinogen, prothrombin,  Factors V, VII, IX and X. Also a-2 macroglobulin.   NANA (sialic acid) residues are lost from the serum proteins. This change signals  their removal from the circulation and their eventual degradation. An asialoglyco-  protein receptor on the liver cell surface binds such proteins, and the receptor—  ligand complex is endocytosed and transported to the lysosomes. The amino acids  from the degraded protein are then recycled within the liver.   The major functions of the pentose phosphate pathway (see Chapter 29) are the gen-  eration of NADPH and five-carbon sugars. All cell types, including the red blood  cell, can carry out this pathway because they need to generate NADPH so that the  activity of glutathione reductase, the enzyme that catalyzes the conversion of oxi-  dized glutathione (GSSG) back to reduced glutathione (GSH) can be maintained.  Without the activity of this enzyme, the protection against free radical injury is lost.  All cells also need this pathway for the generation of ribose, especially those cells  that are dividing rapidly or have high rates of protein synthesis.   The liver has a much greater demand for NADPH than do most other organs. It  uses NADPH for the biosynthesis of fatty acids and cholesterol, which the liver  must make to produce phospholipids, and for the synthesis of VLDL and bile salts.  It also uses NADPH for other biosynthetic reactions, such as that of proline syn-  thesis. NADPH is also used by mixed-function oxidases such as cytochrome P450  that are involved in the metabolism of xenobiotics and of a variety of pharmaceuti-  cals. Because the liver participates in so many reactions capable of generating free  radicals, the liver uses more glutathione and NADPH to maintain glutathione reduc-  tase and catalase activity than any other tissue. Consequently, the concentration of  glucose-6-phosphate dehydrogenase (the rate-limiting and regulated enzyme in the  pentose phosphate pathway) is high in the liver, and the rate of flux through this  pathway may be as high as 30% of the rate of flux through glycolysis.   The reactions used to modify and inactivate dietary toxins and waste metabolites are  energy requiring, as are the reactions used by anabolic (biosynthetic) pathways such  as gluconeogenesis and fatty acid synthesis. Thus, the liver has a high energy  requirement and consumes approximately 20% of the total oxygen used by the  body. The principle forms in which energy is supplied to these reactions is the high-  energy phosphate bonds of adenosine triphosphate (ATP), uridine triphosphate  (UTP), and guanosine triphosphate (GTP), reduced NADPH, and acyl-CoA  thioesters. The energy for the formation of these compounds is obtained directly   • Followed by oxidation to carbon dioxide in the TCA cycle   • Precursors for the synthesis of glycerol-3-phosphate (the backbone of triacylglycerols  and other glyceolipids), sialic acid, and serine   • Entry into the TCA cycle and exit as citrate, followed by conversion to acetyl CoA, mal-  onyl CoA, and entry into fatty acid synthesis and secretion as VLDL   • Conversion to mannose, sialic acid, and other sugars necessary for the synthesis of  oligosaccharides for glycoproteins, including those secreted into blood   • Synthesis of acid sugars for proteoglycan synthesis and formation of glucuronides   • Oxidation in the pentose phosphate pathway for the formation of NADPH (necessary for  biosynthetic reactions such as fatty acid synthesis, glutathione reduction, and other  NADPH-utilizing detoxification reactions)   from oxidative metabolism, the TCA cycle, or the electron transport chain and  oxidative phosphorylation. After a mixed meal containing carbohydrate, the major  fuels used by the liver are glucose, galactose, and fructose. If ethanol is consumed,  the liver is the major site of ethanol oxidation, yielding principally acetate and then  acetyl CoA. During an overnight fast, fatty acids become the major fuel for the liver.  They are oxidized to carbon dioxide or ketone bodies. The liver also can use all of  the amino acids as fuels, converting many of them to glucose. The urea cycle dis-  poses of the ammonia that is generated from amino acid oxidation.   After a carbohydrate-containing meal, glucose, galactose, and fructose enter the  portal circulation and flow to the liver. This organ serves as the major site in the  body for the utilization of dietary galactose and fructose. It metabolizes these com-  pounds by converting them to glucose and intermediates of glycolysis. Their fate is  essentially the same as that of glucose (Table 46.4).   Why would you expect fructose 1-  phosphate levels to promote the  dissociation of glucokinase from  regulatory protein (RP)?   The entry of glucose into the liver is dependent on a high concentration of glucose  in the portal vein after a high-carbohydrate meal. Because the K m for both the glu-  cose transporter (GLUT2) and glucokinase is so high (approximately 10 mM), glu-  cose will enter the liver principally after its concentration rises to 10 to 40 mM in  the portal blood and not at the lower 5-mM concentration in the hepatic artery. The  increase in insulin secretion that follows a high-carbohydrate meal will promote the  conversion of glucose to glycogen. In addition, the rate of glycolysis will  be increased (PFK-2 is active; thus, PFK-1 is activated by fructose-2, 6 bisphos-  phate) such that acetyl CoA can be produced for fatty acid synthesis (acetyl CoA  carboxylase will be activated by citrate; see Chapter 33). Thus, after a high-carbo-  hydrate meal, the liver uses glucose as its major fuel, while activating the pathways  for glycogen and fatty acid synthesis.   The rate of glucose utilization by the liver is determined, in part, by the level of  activity of glucokinase. Glucokinase activity is regulated by a glucokinase regula-  tory protein (RP, Fig. 46.7), which is located in the nucleus. In the absence of glu-  cose, glucokinase is partially sequestered within the nucleus, bound to RP, in an  inactive form. High concentrations of fructose 6-phosphate promote the interaction  of glucokinase with RP, whereas high levels of either glucose or fructose  1 -phosphate block glucokinase from binding to RP and promote the dissociation of  the complex. Thus, as glucose levels rise in the cytoplasm and nucleus (because of  increased blood glucose levels after a meal, for example), there is a significant  enhancement of glucose phosphorylation as glucokinase is released from the  nucleus, travels to the cytoplasm, and phosphorylates glucose.   The role of glucokinase RP is very  \\ j complex. Mice that have been  *r genetically engineered to no  longer express the RP (a knockout mouse)  display reduced levels of total glucokinase  activity in the liver. This is attributable to the  finding that RP is important in the post-tran-  scriptional processing of the mRNA for glu-  cokinase. In the absence of RP, less glucoki-  nase is produced. These mice, therefore,  have no glucokinase in the nucleus, a  reduced cytoplasmic glucokinase content,  and inefficient glucose phosphorylation in  the liver when glucose levels rise.    Fructose-1-phosphate is produced  from fructose metabolism. The  major dietary source of fructose,   the ingestion of which would lead to  increased fructose 1-phosphate levels, is  sucrose. Sucrose is a disaccharide of glu-  cose and fructose. Thus, an elevation of fruc-  tose 1-phosphate usually indicates an eleva-  tion of glucose levels as well.   Fig. 46.7. Regulation of glucokinase by regulatory protein (RP). RP is localized to the  nucleus, and in the absence of glucose or presence of fructose 6-phosphate, most glucokinase  is translocated to the nucleus and binds RP. This leads to the formation of the inactive form  of glucokinase. When glucose or fructose- 1-phosphate levels rise, glucokinase is released  from RP. It then translocates to the cytoplasm and actively converts glucose to glucose  6-phosphate.   The major regulatory step for liver glycolysis is the PFK-1 step. Even under fast-  ing conditions, the ATP concentration in the liver (approximately 2.5 mM) is suffi-  ciently high to inhibit PFK-1 activity. Thus, liver glycolysis is basically controlled  by modulating the levels of fructose 2,6-bisphosphate, the product of the PFK-2  reaction. As fructose 2,6-bisphosphate levels increase (which would occur in the  presence of insulin) the rate of glycolysis increases; when glucagon levels increase  and protein kinase A is activated such that PFK-2 is phosphorylated and inactive,  glycolysis will slow down, and gluconeogenesis will be enhanced (see Chapters 22  and 31).   Long-chain fatty acids are a major fuel for the liver during periods of fasting, when  they are released from adipose tissue triacylglycerols and travel to the liver as fatty  acids bound to albumin.   Within the liver, they bind to fatty acid-binding proteins and are then activated  on the outer mitochondrial membrane, the peroxisomal membrane, and the smooth  endoplasmic reticulum by fatty acyl Co A synthetases. The fatty acyl group is trans-  ferred from CoA to carnitine for transport through the inner mitochondrial mem-  brane, where it is reconverted back into fatty acyl CoA and oxidized to acetyl CoA  in the p-oxidation spiral (see Chapter 23).   The enzymes in the pathways of fatty acid activation and p-oxidation (the syn-  thetases, the carnitine acyltransf erases, and the dehydrogenases of p-oxidation) are  somewhat specific for the length of the fatty acid carbon chain. The chain length  specificity is divided into enzymes for long-chain fatty acids (C20 to approximately  CI 2), medium-chain (approximately C12 to C4), and short-chain (C4-C2). The  major lipids oxidized in the liver as fuels are the long-chain fatty acids (palmitic,  stearic, and oleic acids), because these are the lipids that are synthesized in the liver,  are the major lipids ingested from meat or dairy sources, and are the major form of  fatty acids present in adipose tissue triacylglycerols. The liver, as well as many other  tissues, uses fatty acids as fuels when the concentration of the fatty acid-albumin  complex is increased in the blood.   The liver and certain cells in the kidney are the major sites for the oxidation of  medium-chain-length fatty acids. These fatty acids usually enter the diet of infants   in maternal milk as medium-chain-length triacylglycerols (MCT). In the intestine,  the MCT are hydrolyzed by gastric lipase, bile salt-dependent lipases, and pancre-  atic lipase more readily than long-chain triacylglycerols. Within the enterocytes,  they are neither reconverted to triacylglycerols nor incorporated into chylomicrons.  Instead, they are directly released into the portal circulation (fatty acids of approx-  imately 8-carbon chain lengths or less are water-soluble). In the liver, they diffuse  through the inner mitochondrial membrane and are activated to acyl CoA deriva-  tives by medium-chain-length fatty acid activating enzyme (MMFAE), a family of  similar isozymes present only in liver and kidney. The medium-chain fatty acyl-  CoA is then oxidized by the normal route, beginning with medium-chain-length  acyl CoA dehydrogenase (MCAD; see Chapter 23).   Peroxisomes are present in greater number in the liver than in other tissues. Liver  peroxisomes contain the enzymes for the oxidation of very-long-chain fatty acids  such as C24:0 and phytanic acid, for the cleavage of the cholesterol side chain nec-  essary for the synthesis of bile salts, for a step in the biosynthesis of ether lipids,  and for several steps in arachidonic acid metabolism. Peroxisomes also contain  catalase and are capable of detoxifying hydrogen peroxide.   Very-long-chain fatty acids of C20 to C26 or greater are activated to CoA deriv-  atives by very-long-chain acyl CoA synthetase present in the peroxisomal mem-  brane. The very-long-chain acyl CoA derivatives are then oxidized in liver peroxi-  somes to the 8-carbon octanoyl CoA level. In contrast to mitochondrial p -oxidation,  the first enzyme in peroxisomal p -oxidation introduces a double bond and gener-  ates hydrogen peroxide instead of FAD(2H). The remainder of the cycle, however,  remains the same, releasing NADH and acetyl CoA. Peroxisomal catalase inacti-  vates the hydrogen peroxide, and the acetyl CoA can be used in biosynthetic path-  ways such as those of cholesterol and dolichol synthesis.   The octanoyl CoA that is the end-product of peroxisomal oxidation leaves the  peroxisomes and the octanoyl group is transferred through the inner mitochondrial  membrane by medium-chain-length acylcarnitine transferase. In the mitochondria,  it enters the regular (3-oxidation pathway, beginning with medium-chain-length acyl  CoA dehydrogenase (MCAD).   The peroxisome proliferator activated receptors (PPAR) play an important role in  liver metabolism. These receptors obtained their name from the finding that certain  agonists were able to induce the proliferation of peroxisomes in liver. These ago-  nists included hypolipidemic agents, nonsteroidal anti-inflammatory agents, and  environmental toxins. The receptors that bind these agents, the PPAR, are members  of a nuclear receptor family and, when activated, stimulate new gene transcription.  Within the liver, the major form of PPAR directly regulates the activity of genes that  are involved in fatty acid uptake and p- and co-oxidation of fatty acids.   There are three major PPAR isoforms, a, 8/(3, and 7,. The major form found in  the liver is the a form. Fatty acids are an endogenous ligand for PPAR a, such that  when the level of fatty acids in the circulation is increased (with a concurrent  increase in the fatty acid content of hepatocytes), there is increased gene transcrip-  tion for those proteins involved in regulating fatty acid metabolism (Table 46.5).  Genetically altered mice have been generated that lack PPAR a. These "knockout"  mice exhibit no abnormal phenotype when fed a normal diet. When fasted, however,  or when fed a high-fat diet, these mice develop severe fatty infiltration of the liver.  The inability to increase the rate of fatty acid oxidation in this organ leads to exces-  sive fatty acid buildup in the hepatocytes. It also leads to an insufficient energy sup-  ply with which to make glucose (leading to hypoglycemia) as well as an inability to    Medium-chain triglycerides (MCT)  are important components of nutri-  tional supplements used in patients  with digestive disorders. They therefore can  be employed as an easily absorbed source of  calories in patients who have a gastrointesti-  nal (Gl) disorder that may result in malab-  sorption of nutrients. These diseases include  pancreatic insufficiency, intraluminal bile salt  deficiency due to cholestatic liver disease,  biliary obstruction, ileal disease or resection,  and disease causing obstruction of intestinal  lymphatics. Remember, however, that MCT  do not contain polyunsaturated fatty acids  that can be used for synthesis of eicosanoids  (see Chapter 35).    Zellweger's (cerebrohepatorenal)  syndrome occurs in individuals  with a rare inherited absence of  peroxisomes in all tissues. Patients accumu-  late C26-C38 polyenoic acids in brain tissue  owing to defective peroxisomal oxidation of  the very-long-chain fatty acids synthesized  in the brain for myelin formation. In liver,  bile acid and ether lipid synthesis are  affected, as is the oxidation of very-long-  chain fatty acids.   Fatty acid transport proteins  The mitochondrial and peroxisomal enzymes  of fatty acid oxidation  Carnitine palmitoyl transferase I  HMG-CoA synthase  Apoprotein Clll (suppression)   The fibrates (e.g., clofibrate) are a  class of drugs that bind to PPARs to  elicit changes in lipid metabolism.  They are typically prescribed for individuals  with elevated triglyceride levels because they  increase the rate of triglyceride oxidation.  This, in turn, leads to a reduction in serum tri-  acylglycerol levels. Fibrates, through PPAR-a  stimulation, also suppress apoprotein 01 1 1  synthesis and stimulate LPL activity. Apo 0 1 1 1  normally inhibits LPL activity, so by reducing  Clll synthesis overall, LPL activity is  increased. Apo Clll also blocks apoprotein E  on IDL particles, causing the IDL particles to  accumulate because they cannot be taken up  by the apo E receptor in the liver. The sup-  pression of apo Clll levels allows more IDL to  be endocytosed, thereby also reducing circu-  lating triacylglycerol levels.   Reye's syndrome is characterized  clinically by vomiting with signs of  progressive central nervous system  damage. In addition, there are signs of  hepatic injury and hypoglycemia. There is  mitochondrial dysfunction with decreased  activity of hepatic mitochondrial enzymes.  Hepatic coma may occur as serum ammonia  levels rise. It is epidemiologically associated  with the consumption of aspirin by children  during a viral illness, but it may occur in the  absence of exposure to salicylates. The inci-  dence in the United States has decreased  dramatically since the 1980s, when parents  were made aware of the dangers of giving  aspirin to children to reduce fever. Reye's  syndrome is not necessarily confined to chil-  dren. In patients who die of this disease, the  liver at autopsy shows swollen and disrupted  mitochondria and extensive accumulation of  lipid droplets with fatty vacuolization of cells  in both the liver and the renal tubules.   produce ketone bodies. In normal fasted mice or mice fed a high-fat diet, fatty acids  would eventually stimulate their own oxidation, via peroxisome proliferation and by  induction of other enzymes needed for their oxidation. The knockout mice cannot  make these compensations.   The liver uses the pathways of fatty acid metabolism to detoxify very hydrophobic  and lipid-soluble xenobiotics that, like fatty acids, either have carboxylic acid  groups or can be metabolized to compounds that contain carboxylic acids. Benzoate  and salicylate are examples of xenobiotics that are metabolized in this way. Ben-  zoate is naturally present in plant foods and is added to foods such as sodas as a pre-  servative. Its structure is similar to salicylic acid (which is derived from the degra-  dation of aspirin). Salicylic acid and benzoate are similar in size to  medium-chain-length fatty acids and are activated to an acyl CoA derivative by  MMFAE (Fig. 46.8). The acyl group is then conjugated with glycine, which targets  the compound for urinary excretion. The glycine derivatives of salicylate and ben-  zoate are called salicylurate and hippurate, respectively. Salicylurate is the major  urinary metabolite of aspirin in humans. Benzoate has been administered to treat  hyperammonemia associated with congenital defects, because urinary hippurate  excretion tends to lower the free ammonia pool. Aspirin cannot be used for this pur-  pose because it is toxic in the large doses required   Chronic parenchymal liver disease is associated with relatively predictable changes  in plasma lipids and lipoproteins. Some of these changes are related to a reduction  in the activity of lethicin cholesterol acyltransf erase (LCAT). This plasma enzyme  is synthesized and glycosylated in the liver; then enters the blood, where it catalyzes  the transfer of a fatty acid from the 2-position of lecithin to the 3p-OH group of free  cholesterol to produce cholesterol ester and lysolecithin. As expected, in severe  parenchymal liver disease, in which LCAT activity is decreased, plasma levels of  cholesterol ester are reduced and free cholesterol levels normal or increased.   Plasma triacylglycerols are normally cleared by peripheral lipases (lipoprotein  lipase or LPL and hepatic triglyceride lipase or HTGL). Because the activities of  both LPL and HTGL are reduced in patients with hepatacellular disease, a relatively  high level of plasma triacylglycerols may be found in both acute and chronic hepa-  titis, in patients with cirrhosis of the liver, and in patients with other diffuse hepa-  tocellular disorders.   With low LCAT activity and the elevated triacylglycerol level described, low-  density lipoprotein (LDL) particles have an abnormal composition. They are rela-  tively triacylglycerol rich and cholesterol ester poor.   High-density lipoprotein (HDL) metabolism may be abnormal in chronic liver  disease as well. For example, because the conversion of HDL 3 (less antiatheroscle-  rotic) to HDL 2 (more antiatherosclerotic) is catalyzed by LCAT, the reduced activ-  ity of LCAT in patients with cirrhosis leads to a decrease in the HDL 2 :HDL 3 ratio.  Conversely, the conversion of HDL 2 to HDL 3 requires hepatic lipases. If the activ-  ity of this lipase is reduced, one would expect an elevation in the HDL 2 :HDL 3 ratio.  Because the HDL 2 :HDL 3 ratio is usually elevated in cirrhosis, the lipase deficiency  appears to be the more dominant of the two mechanisms. These changes may result  in an overall increase in serum total HDL levels. How this affects the efficiency of  the reverse cholesterol transport mechanism and the predisposition to atherosclero-  sis is not fully understood.   With regard to triacylglycerol levels in patients with severe parenchymal liver  disease, the hepatic production of the triacylglycerol-rich, very-low-density lipopro-  tein (VLDL) particle is impaired. Yet the total level of plasma triacylglycerols  remains relatively normal because the LDL particle in such patients is triacylglyc-  erol-rich, for reasons that have not been fully elucidated.   Non-esterified fatty acid (NEFA) levels are elevated in patients with cirrhosis.  This change might be expected because basal hepatic glucose output is low in these  patients. As a result, more NEFA are presumably required (via increased lipolysis)  to meet the fasting energy requirements of peripheral tissues.   The liver is the principle site of amino acid metabolism in humans. It essentially bal-  ances the free amino acid pool in the blood through the metabolism of amino acids  supplied by the diet after a protein-containing meal and through metabolism of amino  acids supplied principally by skeletal muscles during an overnight fast. In an adult  who is no longer growing linearly, the total protein content of the body on a daily basis  is approximately constant, such that the net degradation of amino acids (either to other  compounds or used for energy) is approximately equal to the amount consumed. The  key points concerning hepatic amino acid metabolism are the following:   1 . The liver contains all the pathways for catabolism of all of the amino acids and  can oxidize most of the carbon skeletons to carbon dioxide. A small proportion  of the carbon skeletons are converted to ketone bodies. The liver also contains  the pathways for converting amino acid carbon skeletons to glucose (gluconeo-  genesis) that can be released into the blood.   2. Because the liver is the principle site of amino acid catabolism, it also contains  the urea cycle, the pathway that converts toxic ammonium ion to nontoxic urea.  The urea is then excreted in the urine.   3. After a mixed or high-protein meal, the gut uses dietary aspartate, glutamate, and  glutamine as a fuel (during fasting the gut uses glutamine from the blood as a  major fuel). Thus, the ingested acidic amino acids do not enter the general cir-  culation. The nitrogen from gut metabolism of these amino acids is passed to the  liver as citrulline or ammonium ion via the portal vein.   4. The branched-chain amino acids (valine, leucine, and isoleucine) can be used by  most cell types as a fuel, including cells of the gut and skeletal muscle. After a   Unlike Amy Biasis, whose hepatic  amoebic disorder was more local-  ized (abscesses), Jean Anne Tonich  had a diffuse hepatic disease, known as alco-  hol-induced cirrhosis (historically referred to  as "Laennec's cirrhosis"). The latter is charac-  terized by diffuse fine scarring, a fairly uni-  form loss of hepatic cells, and the formation  of small regenerative nodules (sometimes  referred to as "micronodular cirrhosis"). With  continued alcohol intake, fibroblasts and acti-  vated stellate cells deposit collagen at the site  of persistent injury. This leads to the forma-  tion of weblike septa of connective tissue in  periportal and pericentral zones. These even-  tually connect portal triads and central veins.  With further exposure to alcohol, the liver  shrinks and then becomes nodular and firm  as "end-stage" cirrhosis develops. Unless  successfully weaned from alcohol, these  patients eventually die of liver failure. Amy  Biasis, however, can probably look forward to  enjoying normal liver function after success-  ful amoebicidal therapy without evidence of  residual hepatic scarring.    high-protein meal, most of the branched-chain amino acids are not oxidized by  the liver (because of very low activity of the branched-chain amino acid transam-  inase) and instead enter the peripheral circulation to be used as a fuel by other  tissues or for protein synthesis (these amino acids are essential amino acids). The  liver does, however, take up whatever amino acids it needs to carry out its own  protein synthesis.   5. Most tissues transfer the amino acid nitrogen to the liver to dispose of as urea.  They, therefore, produce either alanine (from the pyruvate-glucose-alanine  cycle, in skeletal muscle, kidney, and intestinal mucosa) or glutamine (skeletal  muscle, lungs, neural tissues) or serine (kidney), which are released into the  blood and taken up by the liver.   6. The liver uses amino acids for the synthesis of proteins that it requires as well as  for the synthesis of proteins to be used elsewhere. For example, the liver uses the  carbon skeletons and nitrogens of amino acids for the synthesis of nitrogen-con-  taining compounds such as heme, purines, and pyrimidines. The amino acid pre-  cursors for these compounds are all nonessential, because they can be synthe-  sized in the liver.   The concentration of amino acids in the blood of patients with liver disease is  often elevated. This change is, in part, attributable to a significantly increased  rate of protein turnover (general catabolic effect seen in severely ill patients) as  well as to impaired amino acid uptake by the diseased liver. It is unlikely that the  increased levels are due to degradation of liver protein and the subsequent  release of amino acids from the failing hepatocyte into the blood. This is true  because the total protein content of the liver is only approximately 300 g. To  account for the elevated amino acid levels in the blood, the entire protein content  of the liver would have to be degraded within 6 to 8 hours to account for the  increased protein turnover rates found. Because 18 to 20 times more protein is  present in skeletal muscle (greater mass), the muscle is probably the major  source of the elevated plasma levels of amino acids seen in catabolic states such  as cirrhosis of the liver.   In cirrhotic patients, such as Jean Ann Tonich, the fasting blood a-amino nitro-  gen level is elevated as a result of reduced clearance. Urea synthesis is reduced as  well.   The plasma profile of amino acids in cirrhosis characteristically shows an ele-  vation in aromatic amino acids, phenylalanine and tyrosine, and in free tryptophan  and methionine. The latter changes may be caused by impaired hepatic utilization  of these amino acids as well as to portosystemic shunting. Although the mecha-  nism is not known, a reduction in fasting plasma levels of the branched-chain  amino acids (BCAA) is also seen in cirrhotic patients. These findings, however,  must be interpreted with caution because most of the free amino acid pool in  humans is found in the intracellular space. Therefore, changes seen in their plasma  concentrations do not necessarily reflect their general metabolic fate. Yet the ele-  vation in aromatic amino acids and the suppression of the level of BCAAs in the  blood of cirrhotics have been implicated in the pathogenesis of hepatic  encephalopathy.   Diseases of the liver can be clinically and biochemically devastating, because no  other organ can compensate for the loss of the multitude of functions that the liver  normally performs. Alcohol-induced liver disease has been discussed in Chapter  25. A number of diseases can lead to hepatic fibrosis (see Biochemical Comments)   and cirrhosis. When this occurs to a great enough extent, liver function becomes  inadequate for life. Signs and symptoms of liver disease include elevated levels of  the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase  (AST) in the plasma (due to hepatocyte injury or death with a consequent release  of these enzymes into the blood), jaundice (an accumulation of bilirubin in the  blood caused by inefficient bilirubin glucuronidation by the liver; see Chapter 45),  increased clotting times (the liver has difficulty producing clotting factors for  secretion), edema (reduced albumin synthesis by the liver leads to a reduction in  osmotic pressure in the blood), and hepatic encephalopathy (reduced urea cycle  activity leading to excessive levels of ammonia and other toxic compounds in the  central nervous system).   Alanine aminotransferase and  \\ ) aspartate aminotransferase are the  *S modern versions of older names.  ALT was formally known as serum glutamic  pyruvate transaminase (SPGT), and AST  was formally known as serum glutamic  oxaloacetic transaminase (SGOT).   Patients with cirrhosis of the liver who have no known genetic propen-  sity to glucose intolerance, such as Jean Ann Tonich, tend to have higher  blood glucose levels than do normal subjects in both fasting and fed  states. The mechanisms that may increase glucose levels in the fasting state  include a reduction in the metabolic clearance rate of glucose by 25 to 40% com-  pared with normal subjects. This reduction in glucose clearance results, in part,  from increased oxidation of fatty acids and ketone bodies and the consequent  decrease in glucose oxidation by peripheral tissues in cirrhosis patients. This is  suggested by the discovery that plasma non-esterified fatty acid (NEFA) levels are  high in many patients with hepatocellular dysfunction, in part because of  decreased hepatic clearance of NEFA and in part because of increased adipose  tissue lipolysis. Another possible explanation for the reduction in whole body glu-  cose utilization in cirrhotic patients relates to the finding that ketone body pro-  duction is increased in some patients with cirrhosis. This could lead to enhanced  utilization of ketone bodies for fuel by the central nervous system in such  patients, thereby reducing the need for glucose oxidation by the highly metaboli-  cally active brain.   After glucose ingestion (fed state), many patients with liver disease have abnor-  mally elevated blood glucose levels ("hepatogenous diabetes"). Using World Health  Organization (WHO) criteria, 60 to 80% of cirrhotic patients have varying degrees  of glucose intolerance, and overt diabetes mellitus occurs 2 to 4 times as often in cir-  rhotics than it does in subjects without liver disease. The proposed mechanisms  include a degree of insulin resistance in peripheral tissues; however, as the cirrhotic  process progresses, they develop a marked impairment of insulin secretion as well.  Although the mechanisms are not well understood, this decrease in insulin secretion  leads to increased hepatic glucose output (leading to fasting hyperglycemia) and  reduced suppression of hepatic glucose output after meals, leading to postprandial  hyperglycemia as well. If the patient has an underlying genetic predisposition to dia-  betes mellitus, the superimposition of the mechanisms outlined above will lead to an  earlier and more significant breakdown in glucose tolerance in these specific  patients.   Extensive and progressive fibrosis of the hepatic parenchyma leads to cir-  rhosis of the liver, a process that has many causes. The development of  fibrosis requires the activities of hepatic stellate cells, cytokines, proteases,  and protease inhibitors.   A major change that occurs when fibrosis is initiated is that the normally  "sparse" or "leaky" basement membrane between the endothelial cells and the  hepatocyte is replaced with a high-density membrane containing fibrillar collagen.  This occurs because of both an increased synthesis of a different type of collagen  than is normally produced and a reduction in the turnover rate of existing extracel-  lular matrix components.   The supportive tissues of the normal liver contain an extracellular matrix that,  among other proteins, includes type IV collagen (which does not form fibers), gly-  coproteins, and proteoglycans. After a sustained insult to the liver, a threefold to  eightfold increase occurs in extracellular matrix components, some of which con-  tain fibril-producing collagen (types I and III), glycoproteins, and proteoglycans.  The accumulation of these fibril-producing compounds leads to a loss of endothe-  lial cell fenestrations and, therefore, a loss of the normal sieve-like function of the  basement membranes. These changes interfere with normal transmembrane meta-  bolic exchanges between the blood and hepatocytes.   The hepatic stellate cell is the source of the increased and abnormal collagen  production. These cells are activated by growth factors whose secretion is induced  by injury to the hepatocytes or endothelial cells. Growth factors involved in cellu-  lar activation include TGF-(31 (which is derived from the endothelial cells, Kupffer  cells, and platelets) and platelet-derived growth factor (PDGF) and epidermal  growth factor (EGF) from platelets. The release of PDGF stimulates stellate cell  proliferation and, in the process, increases their synthesis and release of extracellu-  lar matrix materials and remodeling enzymes. These enzymes include matrix  metalloproteinases (MMPs) and tissue inhibitors of MMPs, as well as converting  (activating) enzymes. This cascade leads to the degradation of the normal extracel-  lular matrix and replacement with a much denser and more rigid type of matrix  material. These changes are, in part, the result of an increase in the activity of tis-  sue inhibitors of MMP's for the new collagen relative to the original collagen in the  extracellular matrix.   One consequence of the increasing stiffness of the hepatic vascular channels  through which hepatic blood must flow is a greater resistance to the free flow of  blood through the liver as a whole. Resistance to intrahepatic blood flow is also  increased by a loss of vascular endothelial cell fenestrations, loss of free space  between the endothelial cells and the hepatocytes (space of Disse), and even loss of  vascular channels per se. This increased vascular resistance leads to an elevation in  intrasinusoidal fluid pressure. When this intrahepatic (portal) hypertension reaches  a critical threshold, the shunting of portal blood away from the liver (portosystemic  shunting) further contributes to hepatic dysfunction. If the portal hypertension can-  not be reduced, portal blood will continue to bypass the liver and return to the heart  through the normally low-pressure esophageal veins. When this increasing intrae-  sophageal venous pressure becomes severe enough, the walls of these veins thin  dramatically and expand to form varices, which may suddenly burst, causing life-  threatening esophageal variceal hemorrhage. This is a potentially fatal complication  of cirrhosis of the liver.   Berger J, Moller DE. The mechanism of action of PPARs. Annu Rev Med 2002;53:409-435.  Gibson DM, Harris RA. Liver metabolism, in Metabolic Regulation in Mammals. New York: Francis,  Inc, 2002:177-210.   Sherlock S, Dooley J. Hepatic cirrhosis, in Diseases of the Liver and Biliary System, Chapter 21.  Maiden, MA: Blackwell Science, 2002:365-380.   Meijer DKF, Jansen PLM, Groothuis GMM. Hepatobiliary disposition and targeting of drugs and  genes, in Oxford Textbook of Clinical Hepatology, vol 1, 2nd Ed. New York: Oxford Medical Pub-  lications, 1999.   1. Drinking grapefruit juice while taking statins can lead to potentially devastating side effects. This is due to a component of  grapefruit juice doing which of the following?   (B) Accelerating the conversion of the statin to a more toxic form   2. Which one of the following characteristics of cytochrome P450 enzymes is correct?   (A) They are all found in the Golgi apparatus and are referred to as microsomal enzymes.   (B) They all contain a flavin-containing reductase unit that uses NADH and not NADPH as a source of electrons.   (C) They are all inducible by oxygen, which binds to the iron of the cytochrome.   (E) They all generate a free radical compound as final products of the reaction.   3. Fairly predictable changes occur in the various metabolic pathways of lipid metabolism in patients with moderately advanced  hepatocellular disease. Which one of the following changes would you expect to see under these conditions?   (A) The activity of plasma lecithin cholesterol acyltransferase (LCAT) is increased.   4. After a 2-week alcoholic binge, Jean Ann Tonich ingested some Tylenol to help her with a severe headache. She took three  times the suggested dose because of the severity of the pain. However, within 24 hours Jean Ann became very lethargic, vom-  ited frequently, and developed severe abdominal pain. The symptoms Jean Ann is experiencing are attributable to a reaction  to the Tylenol due to which of the following?   5. An individual displays impaired glucose tolerance; blood glucose levels remain elevated after a meal for a longer time than is  normal, although they do eventually go down to fasting levels. The patient has a normal release of insulin from the pancreas  in response to elevated blood glucose levels. Fibroblasts obtained from the patient display normal levels of insulin binding to  its receptor, and normal activation of the intrinsic tyrosine kinase activity associated with the insulin receptor. Analysis of glu-  cose 6-phosphate formation within the fibroblasts, however, indicated a much slower rate of formation than in fibroblasts  obtained from a normal control. A possible mutation that could lead to these results is which of the following?   There are three types of muscle cells: smooth, skeletal, and cardiac. In all types  of muscle, contraction occurs via an actinl myosin sliding filament system, which  is regulated by oscillations in intracellular calcium levels.   Muscle cells use stored glycogen and circulating glucose, fatty acids, and  amino acids as energy sources. Muscle glycolysis is regulated differently from the  liver, with the key difference being the regulation of phosphofructokinase-2  (PFK-2). Muscle PFK-2 is not inhibited by phosphorylation; cardiac PFK-2 is  actually activated by an insulin- stimulated protein kinase. Thus, under conditions  in which liver PFK-2 is inactive, and glycolysis is running slowly, muscle glycoly-  sis is either unaffected, or even stimulated, depending on the isoform of PFK-2  being expressed.   Although muscle cells do not synthesize fatty acids, they do contain an isozyme  of acetyl Co A carboxylase (ACC-2) to regulate the rate of fatty acid oxidation.  ACC-2 produces malonyl Co A, which inhibits carnitine palmitoyl transferase I,  thereby blocking fatty acid entry into the mitochondria. Muscle also contains mal-  onyl Co A decarboxylase, which catalyzes the conversion of malonyl Co A to acetyl  Co A and carbon dioxide. Thus, both the synthesis and degradation of malonyl  Co A is carefully regulated in muscle cells to balance glucose and fatty acid oxida-  tion. Both allosteric and covalent means of regulation are employed. Citrate acti-  vates ACC-2, and phosphorylation of ACC-2 by the adenosine monophosphate  (AMP)-activated protein kinase inhibits ACC-2 activity. Phosphorylation of mal-  onyl CoA decarboxylase by the AMP -activated protein kinase activates the  enzyme , further enhancing fatty acid oxidation when energy levels are low.   Muscles use creatine phosphate to store high-energy bonds. Creatine is  derived from arginine and glycine in the kidney, and the guanidino acetate formed  is methylated (using S-adenosyl methionine) in the liver to form creatine. The  enzyme creatine phosphokinase (CPK) then catalyzes the reversible transfer of a  high-energy phosphate from adenosine triphosphate (ATP) to creatine, forming  creatine -phosphate and adenosine diphosphate (ADP). Creatine phosphate is  unstable and spontaneously cyclizes to form creatinine, which is excreted in the  urine. The spontaneous production of creatinine occurs at a constant rate and is  proportional to body muscle mass. Thus, the amount of creatinine excreted each  day (the creatinine clearance rate) is constant and can be used as an indicator of  the normalcy of the excretory function of the kidneys.   Skeletal muscle cells can be subdivided into type I and type II fibers. Type I  fibers are slow-twitch fibers that use primarily oxidative metabolism for energy,  whereas the type II fibers (fast-twitch) use glycolysis as their primary energy-gen-  erating pathway.   Glucose transport into muscle cells can be stimulated during exercise because  of the activity of the AMP -activated protein kinase. Fatty acid uptake into exer-  cising muscle is dependent on the levels of circulating fatty acids, which are  increased by epinephrine release.   CHAPTER 47 / METABOLISM OF MUSCLE AT REST AND DURING EXERCISE   Rena Felya, a 9-year-old girl, complained of a severe pain in her throat  and difficulty in swallowing. She had chills, sweats, headache, and a fever  of 102.4 Q F. When her symptoms persisted for several days, her mother  took her to the pediatrician, who found diffuse erythema (redness) in her posterior  pharynx (throat) with yellow exudates (patches) on her tonsils. Large, tender lymph  nodes were present under her jaw on both sides of her neck. A throat culture was  taken, and therapy with penicillin was begun.   Although the sore throat and fever improved, 8 days after the onset of the origi-  nal infection, Rena's eyes and legs became swollen and her urine suddenly turned  the color of "Coca-Cola." Her blood pressure was elevated. Protein and red blood  cells were found in her urine. Her serum creatinine level was elevated at 1.8 mg/dL  (reference range = 0.3-0.7 for a child). Because the throat culture grew out group  A ^-hemolytic streptococci, the doctor ordered a Streptozyme test. This test was  positive for antibodies to streptolysin O and several other streptococcal antigens. As  a result, a diagnosis of acute poststreptococcal glomerulonephritis was made. Sup-  portive therapy, including bed rest and treatment for hypertension, was initiated.   Muscle consists of three different types: skeletal, smooth, and cardiac (Fig. 47.1). The  metabolism of each is similar, but the functions of the muscle are quite different.   Skeletal muscles are those muscles that are attached to bone and facilitate the move-  ment of the skeleton. Skeletal muscles are found in pairs, which are responsible for  opposing, coordinated directions of motion on the skeleton. The muscles appear  striated under the microscope, and are controlled voluntarily (you think about mov-  ing a specific muscle group, and then it happens).   Skeletal muscle cells are long, cylindrical fibers that run the length of the mus-  cle. The fibers are multinucleated because of cell fusion during embryogenesis. The  cell membrane surrounding the fibers is called the sarcolemma, and the sarcoplasm  is the intracellular milieu, which contains the proteins, organelles, and contractile  apparatus of the cell. The sarcoplasmic reticulum is analogous to the endoplasmic  reticulum in other cell types and is an internal membrane system that runs through-  out the length of the muscle fiber. Another membrane structure, the transverse  tubules (T-tubules), are thousands of invaginations of the sarcolemma that tunnel  from the surface toward the center of the muscle fiber to make contact with the ter-  minal cisterns of the sarcoplasmic reticulum. Because the T tubules are open to the  outside of the muscle fiber and filled with extracellular fluid, the muscle action  potential that propagates along the surface of the muscle fiber's sarcolemma travels  into the T tubules and to the sarcoplasmic reticulum.   The striations in skeletal muscle are attributable to the presence and organization  of myofibrils in the cells. Myofibrils are thread-like structures consisting of thin and  thick filaments. The contractile proteins actin and myosin are contained within the  filaments; myosin within the thick filaments, actin within the thin filaments. The  sliding of these filaments relative to each other, using myosin-catalyzed ATP  hydrolysis as an energy source, allows for the contraction and relaxation of the mus-  cle (see Fig. 19.4).   Fig. 47.1. Structures of the three different  muscle types. Smooth Cardiac. Adapted from  Basic Histology, Text and Atlas, Junqueira and  Carneiro, 10th Ed. Lange, McGraw-Hill, 2002.   Duchenne's muscular dystrophy is  caused by the absence of the pro-  tein dystrophin, which is a struc-  tural protein located in the sarcolemma.  Dystrophin is required to maintain the  integrity of the sarcolemma, and when  absent leads to a loss of muscle function,  caused by breakdown of the sarcolemma.  The gene is X-linked, and mutations that  lead to Duchenne's muscular dystrophy gen-  erally result from large deletions of the gene,  such that dystrophin is absent from the  membrane. Becker's muscular dystrophy, a  milder form of disease, is caused by point  mutations in the dystrophin gene. In  Becker's muscular dystrophy, dystrophin is  present in the sarcolemma, but in a mutated  form.   Muscle fibers can be classified as either fast-twitch or slow-twitch. The slow-  twitch fibers, or type I fibers (also called slow-oxidative), contain large amounts of  mitochondria and myoglobin (giving them a red color), utilize respiration and  oxidative phosphorylation for energy, and are relatively resistant to fatigue. Com-  pared with fast-twitch fibers, their glycogen content is low. The slow-twitch fibers  develop force slowly but maintain contractions longer than fast-twitch muscle.   The fast-twitch fibers, or type II, can be subdivided as type Ha or lib. Type lib  fibers (also called fast-glycolytic) have few mitochondria and low levels of myo-  globin (hence, they appear white). They are rich in glycogen and use glycogenoly-  sis and glycolysis as their primary energy source. These muscles are prone to  fatigue, because a continued reliance on glycolysis to produce ATP leads to an  increase in lactic acid levels, resulting in a drop in the intracellular pH. As the pH  drops, the ability of the muscle to produce ATP also diminishes. However, fast-  twitch muscle can develop greater forces than slow-twitch muscle, such that con-  tractions occur more rapidly. Type Ha fibers (also called fast- oxidative glycolytic)  have properties of both type I and lib fibers and thus display functional character-  istics of both fiber types. The properties of types I, Ha, and lib fibers are summa-  rized in Table 47.1.   Muscles are a mixture of the different fiber types, but depending on the function  a muscle could have a preponderance of one fiber type over another. Type I fibers  are found in postural muscles such as the psoas in the back musculature or the  soleus in the leg. The percentage of type I to type II will vary with the muscle. The  triceps, which functions phasically, has 32.6% type I, whereas the soleus, which  functions tonically, has 87.7% type I. Type II fibers are more prevalent in the large  muscles of the limbs that are responsible for sudden, powerful movements. Extraoc-  ular muscles would also have more of these fibers than type I.   Smooth muscle cells are found in the digestive system, blood vessels, bladder, air-  ways, and uterus. The cells have a spindle shape with a central nucleus (see Fig.  47. IB). The designation of smooth refers to the fact that these cells, which contain a  single nucleus, display no striations under the microscope. The contraction of smooth  muscle is controlled involuntarily (the cells contract and relax without any conscious  attempt to have them do so; examples of smooth muscle activity include moving food   CHAPTER 47 / METABOLISM OF MUSCLE AT REST AND DURING EXERCISE   along the digestive tract, altering the diameter of the blood vessels, and expelling urine  from the bladder). In contrast to skeletal muscle, these cells have the ability to main-  tain tension for extended periods, and do so efficiently, with a low use of energy.   The cardiac cells are similar to skeletal muscle in that they are striated (contain fibers),  but like smooth muscle cells they are regulated involuntarily (we do not have to think  about making our heart beat). The cells are quadrangular in shape (see Fig. 47. 1C) and  form a network with multiple other cells through tight membrane junctions and gap  junctions. The multicellular contacts allow the cells to act as a common unit and to con-  tract and relax synchronously. Cardiac muscle cells are designed for endurance and  consistency. They depend on aerobic metabolism for their energy needs because they  contain many mitochondria and very little glycogen. These cells thus generate only a  small amount of their energy from glycolysis using glucose derived from glycogen.   For an extensive review of how muscle contracts or a detailed view of the signaling  to allow muscle contraction, consult a medical physiology book. Only a brief  overview is presented here.   The nerve-muscle cell junction is called the neuromuscular junction (Fig. 47.2).  When appropriately stimulated, the nerve cell releases acetylcholine at the junction,  which binds to acetylcholine receptors on the muscle membrane. This binding stim-  ulates the opening of sodium channels on the sarcolemma. The massive influx of  sodium ions results in the generation of an action potential in the sarcolemma at the  edges of the motor end plate of the neuromuscular junction. The action potential  sweeps across the surface of the muscle fiber and down the transverse tubules to the  sarcoplasmic reticulum, where it initiates the release of calcium from its lumen, via  the ryanodine receptor (Fig. 47.3). The calcium ion binds to troponin, resulting in a  conformational change in the troponin-tropomyosin complexes such that they move  away from the myosin-binding sites on the actin. When the binding site becomes  available, the myosin head attaches to the myosin-binding site on the actin. The bind-  ing is followed by a conformational change (pivoting) in the myosin head, which  shortens the sarcomere. After the pivoting, ATP binds the myosin head, which  detaches from the actin and is available to bind another myosin-binding site on the   Fig. 47.2. The neuromuscular junction. When appropriately stimulated, the synaptic vesi-  cles, containing acetylcholine, fuse with the axonal membrane and release acetylcholine into  the synaptic cleft. The acetylcholine binds to its receptors on the muscle cells, which will ini-  tiate signaling for muscle contraction.    A reduced flow of oxygen-rich  blood to the heart muscle may lead  to a myocardial infarction (heart   attack). The amount of ATP that can be gen-  erated by glycolysis alone is not sufficient to  meet the energy requirements of the con-  tracting heart.   The ryanodine receptors are cal-  cium release channels found in the  endoplasmic reticulum and sar-  coplasmic reticulum of muscle cells. One  type of receptor can be activated by a depo-  larization signal (depolarization-induced cal-  cium release). Another receptor type is acti-  vated by calcium ions (calcium-induced  calcium release). The proteins received their  name because they bind ryanodine, a toxin  obtained from the stem and roots of the  plant Ryania speciosa. Ryanodine inhibits  sarcoplasmic reticulum calcium release, and  acts as a paralytic agent. It was first used  commercially in insecticides.   Acetylcholine levels in the neuro-  muscular junction are rapidly  reduced by the enzyme acetyl-  cholinesterase. A number of nerve gas poi-  sons act to inhibit acetylcholinesterase (such  as sarin and VX), such that muscles are con-  tinuously stimulated to contract. This leads  to blurred vision, bronchoconstriction,  seizures, respiratory arrest, and death. The  poisons are covalent modifiers of acetyl-  cholinesterase; therefore, recovery from  exposure to such poisons requires the syn-  thesis of new enzyme. A new generation of  acetylcholinesterase inhibitors, which act  reversibly (i.e., they do not form covalent  bonds with the enzyme), are now being used  to treat dementia, in particular dementia as  brought about by Alzheimer's disease.   Fig. 47.3. Events leading to sarcoplasmic reticulum calcium release in skeletal muscle.  1. Acetylcholine, released at the synaptic cleft, binds to acetylcholine receptors on the sar-  colemma, leading to a change of conformation of the receptors such that they now act as an  ion pore. This allows sodium to enter the cell and potassium to leave. 2. The membrane polar-  ization that results from these ion movements is transmitted throughout the muscle fiber by  the T-tubule system. 3. A receptor in the T-tubules (the dihydropyridine receptor, DHPR) is  activated by membrane polarization (a voltage-gated activation) such that activated DHPR  physically binds to and activates the ryanodine receptor in the sarcoplasmic reticulum (depo-  larization-induced calcium release). 4. The activation of the ryanodine receptor, which is a  calcium channel, leads to calcium release from the SR into the sarcoplasm. In cardiac mus-  cle, activation of DHPR leads to calcium release from the T-tubules, and this small calcium  release is responsible for the activation of the cardiac ryanodine receptor (calcium-induced  calcium release) to release large amounts of calcium into the sarcoplasm.   actin. As long as calcium ion and ATP remain available, the myosin heads will repeat  this cycle of attachment, pivoting, and detachment (Fig. 47.4). This movement  requires ATP, and when ATP levels are low (such as occurs during ischemia), the  ability of the muscle to relax or contract is compromised. As the calcium release  channel closes, the calcium is pumped back into the sarcoplasmic reticulum against  its concentration gradient by use of the energy-requiring protein SERCA (sarcoplas-  mic reticulum Ca 2+ ATPase), and contraction stops. This basic process occurs in all  muscle cell types, with some slight variations between cell types.   The pathways of glycolysis and fatty acid oxidation in muscle are the same as has  been previously described (see Chapters 22 and 23). The difference between mus-  cles and other tissues is how these pathways are regulated.    Fig. 47.4. An overview of muscle contraction. A. Muscle contraction. During muscle contraction, the myosin head binds to the actin thin fila-  ment. A pivoting of the myosin head toward the center of the sarcomere pulls the Z-lines closer together, with subsequent shortening of the sar-  comere. B. A closer look at myosin-actin interactions. 1. A resting sarcomere. The troponin-tropomyosin complex is blocking the myosin-bind-  ing sites on the actin. The myosin head is already energized to power a contraction. 2. Exposure of the active site. After calcium binding to  troponin, a conformational change in the troponin molecule pulls the troponin away from the binding site. 3. Cross-bridge attachment. Once the  binding sites on the actin are exposed, the myosin head binds to it. 4. Myosin head pivoting. After cross-bridge attachment, the energy stored in  the myosin head is released, and the myosin head pivots toward the center of the sarcomere (power stroke). Now the ADP and phosphate bound  to the myosin head are released. 5. Detachment of the cross-bridge. Now a molecule of ATP binds to the myosin head with simultaneous detach-  ment of the myosin head from the binding site on the actin molecule. 6. Reactivation of the myosin head. The ATPase activity of the myosin head  hydrolyzes the ATP into ADP and phosphate. The energy released from the hydrolysis of this high-energy bond is used to re-energize the myosin  head, and the entire cycle can be repeated as long as calcium is present and there are sufficient ATP reserves.   ACC-1 is the isozyme of acetyl-coA  \\ ) carboxylase, which is used to syn-  *S thesize malonyl-CoA for fatty acid  synthesis. Muscle cells do not synthesize  fatty acids; however, they do carefully regu-  late the oxidation of fatty acids through the  synthesis and destruction of malonyl-CoA.  ACC-1 is a cytosolic protein, whereas ACC-2  is mitochondrial, closely linked to CPT-I in  the outer mitochondrial membrane. Mice  that have been bred to lack ACC-2 have a  50% reduction of fat stores as compared  with "normal" mice. This was shown to be  attributable to a 30% increase in skeletal  muscle fatty acid oxidation because of the  dysregulation of CPT-I, because malonyl  CoA could not be produced to regulate the  rate at which fatty acid oxidation occurred.   Phosphofructokinase 2 (PFK-2) is negatively regulated by phosphorylation in  the liver (the enzyme that catalyzes the phosphorylation is the cyclic adenosine  monophosphate [c AMP] -dependent protein kinase). However, in skeletal muscle,  PFK-2 is not regulated by phosphorylation. This is because the skeletal muscle  isozyme of PFK-2 lacks the regulatory serine residue, which is phosphorylated in  the liver. However, the cardiac isozyme of PFK-2 is phosphorylated and activated  by a kinase cascade initiated by insulin. This allows the heart to activate glycolysis  and to use blood glucose when blood glucose levels are elevated.   Fatty acid uptake by muscle requires the participation of fatty acid-binding pro-  teins and the usual enzymes of fatty acid oxidation. Fatty acyl-CoA uptake into the  mitochondria is controlled by malonyl-CoA, which is produced by an isozyme of  acetyl-coA carboxylase (ACC-2; the ACC-1 isozyme is found in liver and adipose  tissue and is used for fatty acid biosynthesis). ACC-2 is inhibited by phosphoryla-  tion by the AMP- activated protein kinase (AMP-PK) such that when energy levels  are low the levels of malonyl CoA will drop, allowing fatty acid oxidation by the  mitochondria. In addition, muscle cells also contain the enzyme malonyl CoA  decarboxylase, which is activated by phosphorylation by the AMP-PK. Malonyl  CoA decarboxylase converts malonyl CoA to acetyl CoA, thereby relieving the  inhibition of carnitine palmitoyl transferase I (CPT-I) and stimulating fatty acid oxi-  dation (Fig. 47.5). Muscle cells do not synthesize fatty acids; the presence of acetyl  CoA carboxylase in muscle is exclusively for regulatory purposes.   Under conditions in which ketone  \\ ) bodies are produced, fatty acid lev-   els in the plasma are also elevated.  Because the heart preferentially burns fatty  acids as a fuel rather than the ketone bodies  produced by the liver, the ketone bodies are  spared for use by the nervous system.   The heart primarily uses fatty acids (60-80%), lactate, and glucose (20-40%) as its  energy sources. Ninety-eight percent of cardiac ATP is generated by oxidative  means; 2% is derived from glycolysis. The lactate used by the heart is taken up by  a monocarboxylate transporter in the cell membrane that is also used for the trans-  port of ketone bodies. However, ketone bodies are not a preferred fuel for the heart,  because the heart prefers to use fatty acids.   Lactate is generated by red blood cells and working skeletal muscle. When the  lactate is used by the heart, it is oxidized to carbon dioxide and water, following the    Fig. 47.5. Regulation of fatty acyl CoA entry into muscle mitochondria. 1 . Acetyl CoA car-  boxylase^ (ACC-2) converts acetyl CoA to malonyl CoA, which inhibits carnitine palmitoyl  transferase I (CPT-I), thereby blocking fatty acyl CoA entry into the mitochondria. 2. How-  ever, as energy levels drop, AMP levels rise because of the activity of the adenylate kinase  reaction. 3. The increase in AMP levels activates the AMP-activated protein kinase (AMP-  PK), which phosphorylates and inactivates ACC-2, and also phosphorylates and activates  malonyl CoA decarboxylase (MCoADC). The decarboxylase converts malonyl CoA to acetyl  CoA, thereby relieving the inhibition of CPT-1, and allowing fatty acyl CoA entry into the  mitochondria. This allows the muscle to generate ATP via the oxidation of fatty acids.   CHAPTER 47 / METABOLISM OF MUSCLE AT REST AND DURING EXERCISE   pathway lactate to pyruvate, pyruvate to acetyl-coA, acetyl CoA oxidation in the  TCA cycle, and ATP synthesis through oxidative phosphorylation. An alternative  fate for lactate is its utilization in the reactions of the Cori cycle in the liver.   Glucose transport into the cardiocyte occurs via both GLUT1 and GLUT4 trans-  porters, although approximately 90% of the transporters are GLUT4. Insulin stim-  ulates an increase in the number of GLUT4 transporters in the cardiac cell mem-  brane, as does myocardial ischemia. This ischemia-induced increase in GLUT4  transporter number is additive to the effect of insulin on the translocation of GLUT4  transporters to the plasma membrane.   Fatty acid uptake into cardiac muscle is similar to that for other muscle cell types  and requires fatty acid-binding proteins and carnitine palmitoyl transferase I for  transfer into the mitochondria. Fatty acid oxidation in cardiac muscle cells is regu-  lated by altering the activities of ACC-2 and malonyl CoA decarboxylase.   When blood flow to the heart is interrupted, the heart switches to anaerobic metab-  olism. The rate of glycolysis increases, but the accumulation of protons (via lactate  formation) is detrimental to the heart. Ischemia also increases the levels of free fatty  acids in the blood and, surprisingly, when oxygen is reintroduced to the heart, the  high rate of fatty acid oxidation in the heart is detrimental to the recovery of the  damaged heart cells. Fatty acid oxidation occurs so rapidly that NADH accumulates  in the mitochondria, leading to a reduced rate of NADH shuttle activity, an  increased cytoplasmic NADH level, and lactate formation, which generates more  protons. In addition, fatty acid oxidation increases the levels of mitochondrial acetyl  CoA, which inhibits pyruvate dehydrogenase, leading to cytoplasmic pyruvate  accumulation and lactate production. As lactate production increases and the intra-  cellular pH of the heart drops, it is more difficult to maintain ion gradients across  the sarcolemma. ATP hydrolysis is required to repair these gradients, which are  essential for heart function. However, the use of ATP for gradient repair reduces the  amount of ATP available for the heart to use in contraction, which, in turn, com-  promises the ability of the heart to recover from the ischemic event.   Skeletal muscles use many fuels to generate ATP. The most abundant immediate  source of ATP is creatine phosphate. ATP also can be generated from glycogen  stores either anaerobically (generating lactate) or aerobically, in which case pyru-  vate is converted to acetyl CoA for oxidation via the TCA cycle. All human skele-  tal muscles have some mitochondria and thus are capable of fatty acid and ketone  body oxidation. Skeletal muscles are also capable of completely oxidizing the car-  bon skeletons of alanine, aspartate, glutamate, valine, leucine, and isoleucine, but  not other amino acids. Each of these fuel oxidation pathways plays a somewhat  unique role in skeletal muscle metabolism.   ATP is not a good choice as a molecule to store in quantity for energy reserves.  Many reactions are allosterically activated or inhibited by ATP levels, especially  those that generate energy. Muscle cells solve this problem by storing high-energy  phosphate bonds in the form of creatine phosphate. When energy is required, crea-  tine phosphate will donate a phosphate to ADP, to regenerate ATP for muscle con-  traction (Fig. 47.6).   Creatine synthesis begins in the kidney and is completed in the liver. In the kid-  ney, glycine combines with arginine to form guanidinoacetate. In this reaction, the  guanidinium group of arginine (the group that also forms urea), is transferred to   A new class of drugs, known as  partial fatty acid oxidation (pFOX)  inhibitors, are being developed to  reduce the extensive fatty acid oxidation in  heart after an ischemic episode (the proto-  type drug is ranolazine). The reduction in  fatty acid oxidation induced by the drug will  allow glucose oxidation to occur and reduce  lactate buildup in the damaged heart mus-  cle. Other possible targets of such drugs,  which have yet to be developed, include  ACC-2, malonyl CoA decarboxylase, and car-  nitine palmitoyl transferase I.   Fig. 47.6. The creatine phosphokinase reaction. The high-energy bond is the unusual nitro-  gen-phosphate bond, as indicated by the blue squiggle.   glycine, and the remainder of the arginine molecule is released as ornithine. Guani-  dinoacetate then travels to the liver, where it is methylated by S-adenosyl methionine  to form creatine (Fig 47.7).   The creatine formed is released from the liver and travels through the blood-  stream to other tissues, particularly skeletal muscle, heart, and brain, where it  reacts with ATP to form the high-energy compound creatine phosphate (see  Fig. 47.6). This reaction, catalyzed by creatine phosphokinase (CK, also abbrevi-  ated as CPK), is reversible. Therefore, cells can use creatine phosphate to regen-  erate ATP.   Creatine phosphate serves as a small reservoir of high-energy phosphate that  can readily regenerate ATP from ADP. As a result, it plays a particularly important  role in muscle during exercise. It also carries high-energy phosphate from 
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