NAD + (nicotinamide adenine dinucleotide) and FAD  (flavin adenine dinucleotide) are two crucial team  players in cellular metabolism. Their structures are given  in Fig. 1.16. They usually function as redox partners in  substrate oxidation reactions and act as cofactors for the  enzymes mediating these reactions.   Both NAD + and FAD function as 'electron carriers',  since they readily accept and donate electrons (associated  with FI atoms) during interaction with other molecules.  They participate in catabolic oxidation reactions (as the  oxidant, where they are reduced). Once reduced (as 're-  duced intermediates'), they each transfer an electron pair  (in association with H atoms) to electron transport chain  complexes within the mitochondria. This fuels oxidative  phosphorylation, in which they act as reductants and  are re-oxidized, reforming NAD + and FAD. Their redox  behavior is illustrated in Fig. 1.17, where 'X' represents a  substrate molecule undergoing oxidation in any catabolic  pathway (such as glycolysis).   Some scientists prefer to write 'NADH 2 ' rather than  'NADH + H + ' for simplicity. This can cause confusion  as it implies that the second hydrogen atom is covalently  associated with NADFI. The second 'atom' is in fact a  hydrogen ion, and since it 'disappears' into solution in  cellular media some scientists prefer to completely omit  the H + ion from equations. This also causes confusion as  the equation then appears unbalanced. Understand that    Fig. 1.17 Redox reactions of NAD + and FAD. Note in both  reactions that X is oxidized, whilst NAD + or FAD are reduced, as  seen in the half-equations. The two H atoms are removed from  X-H 2 in the form of a hydride ion (H _ ) and a proton (H + ion).    whenever you see 'NADH' written alone, the writer has  assumed you appreciate that a free H + ion was also  produced. Also, when you see 'NADH 2 ', mentally recog-  nize that this is being used interchangeably with  'NADH + H+'.   NAD + and FAD integrate catabolism of all the major en-  ergy substrates (carbohydrates, lipids and proteins). En-  ergy released from oxidation of these molecules is used  to reduce NAD + and FAD (by addition of a hydrogen  ion (II + ) and a hydride ion (ID)). This forms the  reduced intermediates NADFI + H + and FADH 2 .  NADH + H + and FADH 2 are then re-oxidized when they  later transfer their two hydrogen atoms (and associated  electrons) to the complexes of the electron transport  chain.   NADP + (nicotinamide adenine dinucleotide phosphate)  shares a structure with NAD + but has an additional  phosphate group at C2 of the ribose moiety. The  structure is shown in Fig. 1.18. The reduced form of  NADP + is NADPH + H + , and this is produced from  NADP + in the pentose phosphate pathway (Chapter 4).  NAD PH + H + functions as a redox partner in a number  of reductive biosynthesis reactions, including nucleotide,  fatty acid and cholesterol synthesis (Fig. 1. 19). The redox  behaviour of NADP+ is shown in Fig. 1 .20.    Fig. 1.19 Metabolic pathways requiring NAD + /NADH + H +  and FAD + /FADH 2 .   The structure of acetyl CoA consists of an acetyl group  (Cl fiCOO ) covalently linked to coenzyme A (CoA).  The functional group of CoA is a thiol group (— SH),  and to highlight this CoA is sometimes written as  CoA-SH. The structure is shown in Fig. 1.21.    Fig. 1.20 Redox reaction of NADP + . Note in this reaction that  X is oxidized, and NADP + reduced. The two H atoms are  removed from X-H 2 in the form of a hydride ion (H _ ) and a  proton (H + ion).    This molecule is central to metabolism (Fig. 1.22).  Most cellular catabolic pathways (including carbohy-  drate, fat and protein) eventually lead to acetyl  CoA. Oxidation of the acetyl residue of acetyl CoA  in the TCA cycle (Chapter 2) generates ATP directly    (substrate-level phosphorylation) and indirectly (via  oxidative phosphorylation of TCA cycle-generated  FADH 2 and NADPI + H + ). It is also a substrate for nu-  merous synthetic pathways, including fats, steroids  and ketones.    The TCA cycle (aka the 'Krebs cycle' or the 'citric acid'  cycle) is a cyclical reaction sequence (Fig. 2.1). Sequen-  tial oxidation reactions generate metabolic energy.   • The cycle occurs in the mitochondrial matrix of all  mitochondria-containing cells   • The cycle 'kicks off by accepting an acetyl CoA  molecule; this combines with an oxaloacetate (gener-  ated by a previous 'turn' of the cycle) to form citrate   • The TCA cycle generates a molecule of GTP directly  by substrate-level phosphorylation during reaction  5. This is turn generates further ATP   • The TCA cycle generates ATP indirectly via produc-  tion of the high energy intermediates FADH 2 and  NADH + H in reactions 3, 4, 6 and 8.    Fig. 2.1 The TCA cycle. 1 =citrate  synthase, 2 = aconitase, 3 = isocitrate  dehydrogenase, 4 = a-ketoglutarate  dehydrogenase, 5 = succinyl CoA  synthetase, 6 = succinate  dehydrogenase, 7 = fumarase,    Reactions 1, 3 and 4 are irreversible, rate-limiting reac-  tions. They form the main regulation points for the  cycle.   Since acetyl CoA is produced from catabolism of carbo-  hydrates, fatty acids and amino acids (the three main di-  etary sources of energy), the TCA cycle is pivotal in  metabolism. It functions as a common pathway for en-  ergy generation. Cycle intermediates also function as  'raw materials' for numerous anabolic (synthetic) path-  ways. As the TCA cycle possesses both catabolic (break-  down of energy-rich molecules to release energy) and  anabolic (synthetic) elements, it is known as an 'amphi-  bolic' pathway.   GTP is directly generated by substrate-level phosphory-  lation (reaction 5). ATP, however, is generated indi-  rectly, via production of the reducing equivalents  FADH 2 and NADH + H+.   One 'turn' of the TCA cycle generates one molecule of  FADII 2 and three NADH + H+. FADFI 2 and NADH + H+  equate to approximately 1.5 and 2. 5 ATP respectively  (Chapter 3). The single GTP generated in reaction 5 equates  to 1 ATP. Thus, 10 ATP are generated (per acetyl CoA mol-  ecule) by one complete 'turn' of the cycle:   Acetyl CoA + 2 H 2 0 + 3 NAD+ + FAD + GDP + P;   -+ 2 C0 2 + 3 (NADH + H+) + FADH 2 + GTP + CoA    Fig. 2.2 TCA cycle regulation. Note that the figure only  illustrates the key regulatory enzymes.   High intracellular [Ca 2 1 ] correlates with ATP-  demanding cellular activities. This is because Ca 2 + ions  are chemical 'signals' initiating a vast number of key  biochemical processes. Examples include muscle  contraction, cell division and neurotransmitter release  (exocytosis). This explains why Ca 2 1 has such a  powerful influence on cellular energy homeostasis.   The three irreversible reactions (1,3 and 4) are catalysed  by the enzymes citrate synthase, isocitrate dehydroge-  nase and a-ketoglutarate dehydrogenase. Since their  reactions are rate-limiting, modulating the activity of  these enzymes controls cycle activity (Chapter 1).   These enzymes are all allosterically activated by  calcium ions. Intracellular [Ca 2 1 ] is elevated when  energy-demanding processes are active. The three rate-  limiting enzymes of the cycle operate more rapidly  when [Ca 2+ ] is high. Cycle activity is enhanced, gener-  ating more metabolic energy (Fig. 2.2).   Conversely, cycle products NADH + H + and ATP (an  indirect product) allosterically inhibit these three  enzymes. Abundance of these molecules reflects high  cellular energy level, i.e. contexts in which enhanced  TCA cycle activity is not required.   The TCA cycle, like all metabolic pathways, is limited by  substrate availability. A supply of NAD + and FAD is  required to sustain the cycle. Thus NAD + and FAD  renewal (from NADH + H + and FADH 2 ) controls pat-  hway activity. These molecules are regenerated during  oxidative phosphorylation (Chapter 3), meaning that:   • An increased rate of oxidative phosphorylation  (respiration) allows greater cycle activity.   Availability of acetyl CoA, the major substrate required  for TCA cycle operation, also influences the rate at  which the cycle can function.   Many important synthetic pathways use TCA cycle  molecules as precursors, or 'raw materials'. This is the  synthetic (anabolic) aspect of the cycle, and is illustrated  in Fig. 2.3. Key examples include:   • Fatty acids and cholesterol are synthesized using  acetyl CoA, which may be derived from citrate  (Chapter 5)   • Amino acid synthesis uses both oxaloacetate and  a-ketoglutarate (Chapter 6)   When TCA cycle molecules are recruited for use in syn-  thetic pathways, they must be replenished so the cycle  can continue to operate (Fig. 2.4). Pathways and reac-  tions that replenish pathway molecules are known as  'anaplerotic'. For example, carboxylation of pyruvate  forming oxaloacetate replenishes oxaloacetate with-  drawn from the cycle to participate in nucleotide syn-  thesis or gluconeogenesis.   The key role of the TCA cycle is that it generates reducing  equivalents (FADH 2 and NADH + II + ) which undergo  oxidative phosphorylation. Oxidative phosphorylation    (rather than substrate-level phosphorylation) is respon-  sible for the vast majority of ATP generation. Oxidative  phosphorylation occurs at the inner mitochondrial  membrane, which is studded with an array of proteins  known as the electron transport system, or the 'respira-  tory chain' (Chapter 3).   • Understand how electron transfer provides energy for generating the proton gradient   • Understand how discharge of the proton gradient provides energy for ATP synthesis    ATP molecules are all created by phosphorylation of  ADP. This occurs by either 'substrate-level' phosphory-  lation or 'oxidative' phosphorylation.    This describes the reaction where ATP (or GTP) is synthe-  sized from ADP (or GDP) by transfer of a phosphoryl  group (P0 3 2 ~). The phosphoryl group is derived from a  substrate and is transferred to ADP or GDP (Fig. 3.1).  Substrate-level phosphorylation is an endergonic reac-  tion, and is therefore always accompanied by an exergonic  reaction, which provides the energy required to drive the  reaction forward.   Substrate-level phosphorylation does not require ox-  ygen, and thus is vital for energy generation in anaerobic  environments, such as rapidly contracting skeletal mus-  cle. This form of ATP generation is seen during glycoly-  sis (reactions 7 and 10), the TCA cycle (reaction 5) and  creatine kinase-mediated hydrolysis of phosphocreatine  in muscle cells.    This type of ATP production does require oxygen, and  occurs only at the inner mitochondrial membrane  (IMM) . The energy required to perform the phosphory-  lation reaction is derived from the electron pairs    associated with NADH+H + and FADIT 2 , which are  in turn generated during catabolism of high-energy  molecules such as carbohydrates, fatty acids and  amino acids. The electron pairs are transferred from  NADH+H + and FADFI 2 , along with pairs of FI + ions,  to the acceptor 'complexes' of the electron transport  chain (ETC). The electrons then transfer between the  ETC complexes.   • The protein complex that donates the electrons be-  ing oxidized   • The protein complex that receives the electrons be-  ing reduced.   Electron movement in an electronegative direction re-  leases energy. This is used to generate a chemical gradi-  ent of hydrogen ions (protons) across the IMM, with  higher [H + ] in the intermembrane space than the mito-  chondrial matrix. This sequential oxidation of ETC  complexes is the 'oxidative' component of 'oxidative  phosphorylation'.   The exergonic (energy-releasing) discharge of pro-  tons back into the mitochondrial matrix through the  ATP synthase pore (also located in the IMM) provides  the energy required for formation of the phosphoan-  hydride bond between P, and ADP, forming ATP.  This is the 'phosphorylation' part of the 'oxidative  phosphorylation'.   The ETC consists of four protein structures embedded in  the IMM. Each contains structural features that allow  complexes to readily accept and release electrons. Each   Fig. 3.1 Substrate-level phosphorylation. No oxygen is involved in this reaction. Note the two high-energy phosphoanhydride  bonds in ATP are illustrated with arrows.    structure or 'complex' is numbered in order of increas-  ing electron affinity and redox potential.   Two mobile transfer proteins also participate in oxi-  dative phosphorylation. Coenzyme Q (aka ubiqui-  none) ferries two e~ and two H + between complexes I  and III and between complexes II and III. Cytochrome  c transfers the electron and proton pair from complex  III to complex IV (Fig. 3.2).   Electron pairs arrive at the ETC incorporated within  NADH+H+ and FADII 2 . NADH+H+ transfers two e  (and two H + ) to complex I and FADH 2 transfers an  eT pair (and a H + pair) to complex II. NADH+H +  and FADH 2 are thus converted back to NAD + and  FAD. In receiving the e and 1 1 + ion pairs, each complex  is itself reduced.   Having accepted an e pair (and a 1 1 + pair), complexes  then switch function, acting as eT donors to the fol-  lowing unit of the ETC. Complex III receives electron  and proton pairs from either complex I or II via coen-  zyme Q, and complex IV receives electron and proton  pairs from complex III via cytochrome c. The final    transfer occurs when complex TV transfers both the elec-  tron pair and the proton pair to molecular oxygen (0 2 ).  This requirement for oxygen as the terminal electron  pair acceptor explains why the process of oxidative  phosphorylation requires oxygen (Fig. 3.3).   The significance of electron transfer between com-  plexes of the ETC is that it is highly exergonic. Electron  transfer releases energy. This energy is harnessed  by complexes I, III and IV and utilized to transfer  ('pump') protons from the mitochondrial matrix into  the intermembrane space (across the IMM). This trans-  fer is endergonic (requires energy), as this direction is  against a H + (proton) concentration gradient. In this  way, receipt of the e pairs is like an 'energy delivery',  providing complexes with the energy needed to trans-  port protons across the IMM against their concentra-  tion gradient.   Note that electron pairs originating from FADH 2 arrive  at complex II, bypassing complex I. Oxidation of FADI I 2  leads to proton pumping at complexes III and IV,  compared to NADII+H + oxidation, which leads to  proton pumping at complexes I, III and IV. This    Fig. 3.3 Pathway of electron and hydrogen ion transfer. Note  that the dark circle represents the transferred electron and  proton pair. C = cytochrome C, Q = coenzyme Q.    a proton gradient is formed by the action of complexes  I, III and IV, the intrinsic chemical energy contained  within the gradient (the 'proton-motive force') can be  utilized by ATP synthase.   Exploiting a chemical gradient as a source of chemical  energy to power an energy-demanding biological  process, is conceptually similar to secondary active  transport (Chapter 1).    Fig. 3.2 Schematic of oxidative phosphorylation. Note the  direction of the proton concentration gradient. C = cytochrome C,  Q = coenzyme Q.    accounts for why 1 FADIT 2 leads to generation of less  ATP per molecule than 1 NADH+H + (~ 1.5 ATP and  ~ 2 . 5 ATP respectively) .   Formation of the second phosphoanhydride bond of  ATP (from ADP and Pi) is highly endergonic. Once   ATP synthase, also located at the IMM, binds ADP and P,  and catalyses the bond formation between the two spe-  cies, generating ATP. The enzyme contains an intrinsic  pore, connecting the mitochondrial matrix with the  intermembrane space. Protons travel down their con-  centration gradient; however in doing so they impose  a transient structural alteration in the enzyme protein.  This results in the ADP and P, substrates being forced  into close contact by ATP synthase, so that the form-  ation of the phosphoanhydride bond becomes energet-  ically favourable.   ATP synthesis occurring in this manner is intimately  associated with discharge of the proton gradient. Gener-  ation of which is powered by electron transfer between  ETC complexes. This association is termed 'coupling';  ATP synthesis is coupled with proton gradient discharge.  This is often referred to as 'chemiosmotic coupling'.   Catabolism of carbohydrates, fatty acids and the carbon  skeletons of amino acids, all produce NADII+H + and  FADH 2 from their redox partners NAD + and FAD.   ETC complexes: why do they  readily accept and then transfer  onward incoming electron pairs?   For a protein to function as an electron acceptor and do-  nor, it must contain structural features that allow it to  do so. Specific features present in the proteins of the  ETC are shown in Fig. 3.4.   Both (l-oxidation of fatty acids and the TCA cycle occur  in the mitochondrial matrix. NADH+H + produced by  these pathways is therefore already in the appropriate  location for accessing the ETC and participate in oxida-  tive phosphorylation. However, NADH+II + is also gen-  erated in cell cytoplasm by glycolysis. The mitochondria  are impermeable to NADH+H + . So how does  NADH+H + gain access to the mitochondrial interior?  There are two ways, described below.   This mechanism recruits cytoplasmic NADH+H + into  a redox reaction with dihydroxyacetone-phosphate  (DHAP). NADH+H+ is oxidized to NAD+ whilst DIIAP  is reduced to glycerol-3-phosphate ( G3 P) . G3P can diffuse  across the outer mitochondrial membrane (OMM) and  into the intermembrane space. Here, G3P is re-oxidized    back to DHAP. This is mediated by glycerol-3-phosphate  dehydrogenase, an enzyme spanning the IMM. The rele-  vance of this second redox reaction is that the redox part-  ner for the second oxidation is FAD, located in the  mitochondrial matrix, on the other side of the IMM. Re-  duced FAD (FADH 2 ) is then able to participate in oxida-  tive phosphorylation by donating the electron pair to  complex II of the ETC. Whilst this is not a scenario iden-  tical to an NADH+H + itself travelling into the matrix,  there is no longer an NADH+H + in the cytoplasm and  there is a reduced equivalent in a site where it may partic-  ipate in oxidative phosphorylation.   This system uses cytoplasmic NADH+H + as the redox  partner in the reduction of oxaloacetate to malate.  This shuttle exploits the fact that malate is able to cross  mitochondrial membranes. It is represented in Fig. 3.5  and described here:   • Cytoplasmic malate dehydrogenase catalyses the ox-  idation of NADH+H+ to NAD+   • The malate then travels across both mitochondrial  membranes into the matrix via an antiport in the  inner mitochondrial membrane; in exchange,  a-ketoglutarate from the matrix is extruded into  the cytoplasm   • Once in the matrix, the reaction reverses, re-forming ox-  aloacetate and reducing matrix NAD + to NADH+H + .  Thus the reducing equivalent (NADH+H + ) 'appears'  in the matrix to participate in oxidative phosphory-  lation   • Regenerated oxaloacetate is then converted to aspar-  tate, which is extruded from the mitochondria by an  antiport in exchange for glutamate   • Once in the cytoplasm, the aspartate is converted to  oxaloacetate   • The matrix glutamate is converted to a-ketoglutarate,  completing the cycle.   Activity of malate-aspartate or glycerol-3-phosphate  shuttles ensures that cytoplasmic NAD + is continu-  ously available. Shuttle activity is driven by oxidative    Fig. 3.4 Structural features of proteins of the electron transport chain.   Iron ions are complexed with cysteine residue, sulphur atoms or inorganic sulphide groups. Iron in  this configuration can undergo oxidation and reduction by cycling between the ferric and ferrous  states   These also contain an iron ion associated with four nitrogen atoms. The iron ion likewise can  undergo oxidation and reduction by cycling between the ferric and ferrous states   Fig. 3.5 The glycerol-3-phosphate and malate-aspartate shuttles. DHAP = dihydroxyacetone phosphate, G3P = glycerol  phosphate. Note that there are both mitochondrial and cytoplasmic isoforms of the enzymes aspartate aminotransferase (3) and  glycerol-3-phosphate dehydrogenase (5).    phosphorylation, since this is the process that consumes  the reducing equivalents in the mitochondrial matrix.  Thus sustained oxidative phosphorylation ensures the  maintenance of an available pool of NAD + in the  cytoplasm.   Under anaerobic conditions, when oxidative phos-  phorylation cannot occur, NAD + is regenerated from  NADH+f I + by a different mechanism. It acts as a redox  partner in the reduction reaction pyruvate — > lactate.   Recall that 'coupling' describes the simultaneous dischar-  ging of the 1 1 + gradient with ATP synthesis. 'Uncoupling'  describes the scenario where the permeability of the IMM  to II + ions is increased. H + ions are then able to dis-  charge back into the matrix without travelling through  the ATP synthase pore. This route of return cannot gen-  erate ATP; instead, the energy is dissipated as heat.    This uncouples ATP synthesis from discharge of the  I I + gradient. Any molecule that increases permeability  of the IMM to H + ions is capable of uncoupling. 2,4-  Ninitrophenol (2,4-DNP) and FCCP (carbonyl cyanide  p-(trifluoromethoxy) -phenyl hydrazone) uncouple mi-  tochondria, short-circuiting the IT + gradient accumu-  lated by the ETC and blockading the main source of  ATP production.   Uncoupling is only physiologically advantag-  eous if heat is required, for example, in hairless  newborn mammals. Newborn babies possess spe-  cialized heat-generating cells, termed 'brown fat'  cells. These contain large numbers of uncoupled  mitochondria, which are devoted to heat product-  ion. The mitochondria are uncoupled by the presence  of proteins in the IMM that contain a proton pore,  allowing the accumulated H + gradient to discharge.  These proteins are known as 'uncoupling proteins'  or UCPs.   • Recognize the reactions of glycolysis and describe the regulation mechanisms   • Understand the influence of anaerobic and aerobic conditions on the fate of pyruvate   • Describe synthesis and degradation of glycogen, and regulation of these pathways   • Briefly outline the metabolism of ethanol, fructose, galactose and sorbitol    A carbohydrate (aka 'saccharide') is a molecule contain-  ing only carbon, hydrogen and oxygen. The ratio of these  atoms is always C: II : O = 1 :2 : 1 . The basic example of a car-  bohydrate 'unit' is the 6-carbon 'monosaccharide' such as  glucose, fructose (Fig. 4.1) or galactose. Disaccharides    comprise two linked monosaccharides. Sucrose (glucose+  fructose) and lactose (glucose + galactose) are shown  in Fig. 4.1. The more complex 'polysaccharides' consist  of numerous monosaccharide units linked by glycosidic  bonds. A physiological example is glycogen (Fig. 4.2).   In biochemistry, metabolism of carbohydrates in-  cludes glycolysis, glycogen synthesis and degradation,    Fig. 4.1 Monosaccharides; formula C x (H 2 0) y . Glucose and fructose are shown. The disaccharides lactose and sucrose are also shown.   Fig. 4.2 Macroscopic structure of glycogen. Hexagons represent glucose monomers. Note that both (1-4) and (1-6) carbon-to-  carbon bonds are present (examples shown within the dotted boxes). These bonds are detailed in Fig. 4.12.    gluconeogenesis and the pentose phosphate pathway.  These will be discussed in turn.   Six-carbon carbohydrates are also known as 'hexose'  sugars. 'Pentose' sugars are five-carbon carbohydrates.  ‘Triose’ sugars are three-carbon carbohydrates.   Glucose (or its derivatives, such as glucose-6-phosphate)  participates in all the carbohydrate pathways of metab-  olism. As phospholipid bilayers are impermeable to  polar molecules, glucose cannot directly diffuse across  plasma cell membranes. To allow glucose to move into  and out of cells, specialized transporter structures span  the membranes. Regulating transporter function there-  fore allows integrated regulation of glucose traffic across  the cell membrane.   In certain environments, glucose is a greater extracellu-  larly than intracellularly. The concentration gradient  is thus favourable for glucose to passively enter the  cell. However, a route is required to traverse the  phospholipid bilayer. This is provided by the GLUT  facilitated diffusion transporters. The different charac-  teristics of the most important subtypes are shown in  Fig. 4.3.   When the extracellular glucose is lower than the  intracellular glucose, glucose entry is coupled to sodium  transport, via the sodium-glucose symport (Chapter 1).  This allows the Na + gradient to 'power' the energy-  demanding import of glucose against its concentration  gradient. Such a system operates, for instance, in the  gastrointestinal tract, allowing the absorption of  glucose.    Fig. 4.3 Glucose transporters. Note that 'high-affinity' transporters allow more rapid glucose traffic across membranes   Erythrocytes (adult)  Blood-brain barrier  endothelia (adult)  Astrocyte glia (adult)  Widespread (fetus)   Responsible for the basal uptake of  glucose that is necessary to sustain  cellular viability  Delivers glucose from the  circulation into the brain   Allows absorption of digested  saccharides from gut lumen to  intestinal cells   The low-affinity high-capacity  characteristics allow the  intracellular glucose of pancreatic  beta 'sensor' cells to closely  resemble plasma glucose, allowing  for regulation of pancreatic  glucose-stimulated insulin secretion  This is also the main transporter  for hepatic glucose absorption   Expression of GLUT  4 is proportional to  insulin levels. This  accounts for  increased uptake of  glucose from plasma  in the presence of  insulin   Mediates blood glucose  regulation by allowing insulin to  control the extent of glucose  uptake from the circulation   Glycolysis is catabolism of glucose and the equation is  as follows (CH3COCOOH is the formula of pyruvate):   C 6 H 12 0 6 + 2NAD+ + 2ADP + HP0 4 2 ~   Glycolysis occurs in the cytoplasm of all cells. It can  occur in both aerobic and anaerobic environments.  In ten reactions, one glucose molecule is sequentially  oxidized, ultimately forming two molecules of pyruvate  (Fig. 4.4A).   During glycolysis, two ATP are generated via sub-  strate-level phosphorylation (in fact, four are generated,  but two are consumed). Two NADH + H + are also    generated, each representing ~2.5 ATP. Thus the ATP  yield of glycolysis is 7 ATP per glucose molecule  oxidized:   Much of the pyruvate generated in glycolysis is decarboxy-  lated, forming acetyl CoA. Recall that acetyl CoA  may enter the TCA cycle for further oxidation (Chapter 2),  generating further ATP and NADH + H + . Alternatively  it may participate in a number of synthetic pathways.   • Reaction 1: Glucose is phosphorylated, forming  glucose- 6-phosphate (Glc-6-P). ATP donates the  phosphoryl group   Fructose-6-phosphate + ATP — > Fructose-  1 ,6-bisphosphate + ADP + H 1   Glyceraldehyde-3-phosphate + NAD 1  + HP0 4 2 ' ->■ 1,3-  bisphosphoglycerate + NADH + H 1   • Reaction 3: Fru-6-P is phosphorylated, generating  fructose- 1,6-bisphosphate (Fru-l,6-BP). Again, ATP  is the phosphoryl donor   • Reaction 4: Fru-l,6-BP is split into two three-carbon  molecules, glyceraldehyde-3-phosphate (GAP) and  dihydroxyacetone phosphate (DI1AP)   It is important to understand that the following  glycolysis reactions occur in duplicate, since the original  six-carbon glucose molecule is split into two three-  carbon molecules, each of which progresses through  reactions 6-10.    • Reaction 6: The two three-carbon GAP molecules  undergo dehydrogenation and phosphorylation to    form 1,3-bisphosphoglycerate (1,3-BPG). NAD + is  reduced to NADH + 1 1 + . Note that two NADH + H +  are actually produced, one per GAP molecule   • Reaction 7: 1,3-BPG donates a phosphate group to  ADP, forming 3-phosphoglycerate (3-PG) and  ATP. This is a substrate-level phosphorylation   • Reaction 8: 3-PG is isomerized; the phosphate group  is transferred from the 3 rd to the 2 nd carbon atom,  forming 2-phosphoglycerate (2-PG)   • Reaction 10: The final step of glycolysis is transfer of  the phosphoryl group from PEP to ADP. This gener-  ates pyruvate and ATP (the second substrate-level  phosphorylation) (Fig. 4.4B).   The pathway also acts as an essential source of interme-  diates for other pathways, which therefore rely on gly-  colysis for substrate provision. These include:   The enzymes catalysing reactions 1, 3 and 10 of the  pathway function as glycolysis regulation points, since  these reactions are all highly exergonic and as such  are essentially irreversible.   Reaction 1 of glycolysis is catalysed by hexokinase (HK).  This enzyme is allosterically inhibited by the reaction  product Glc-6-P. Insulin up-regulates I IK transcription,  whilst glucagon down-regulates HK transcription. Insu-  lin and glucagon thus comprise the main hormonal reg-  ulation of this reaction.   Note that glucokinase (GK; the isoform of HK pre-  sent in liver, pancreatic beta cells and hypothalamic  cells) is insensitive to product-mediated inhibition by  Glc-6-P. Glucose phosphorylation will persist in these  locations even when the remainder of the pathway is  less active and Glc-6-P accumulates. This isoform also  differs from HK by affinity; GK requires 100 times    greater concentration of glucose than I IK to function.  This means that GK will only catalyse glucose phosphor-  ylation at high intracellular glucose, unlike HK, which  will operate at low concentrations.   The enzyme catalysing this reaction (phosphofructoki-  nase) is an important site of glycolysis regulation, since  reaction 3 is the rate-limiting step of the pathway.  Accelerating this pathway accelerates glycolysis. Various  factors influence (PFK-1) activity, including:   • ATP. ATP allosterically inhibits PFK-1. When ATP is  abundant, reflecting high cellular energy status, PFK-1  operates at a lower rate   • Citrate. This is not a direct product of reaction 3; the  final product, pyruvate, is decarboxylated to acetyl  CoA which combines with oxaloacetate to form cit-  rate. Citrate allosterically inhibits PFK-1. When gly-  colysis is highly active, citrate is abundant and has  an inhibitory effect on the pathway   • AMP and ADP. These allosterically activate PFK-1.  They are abundant in cells when energy status is  low, reflecting the need for more ATP to be generated   • Fructose-6-phosphate. Fru-6-P (a reaction 3 sub-  strate) allosterically activates PFK-1; accumulation   • Fructose-2, 6-bisphosphate (Fru-2,6-BP). This allo-  sterically activates PFK-1. Note that Fru-2,6-BP is  synthesized by phosphofructokinase-2 (PFK-2),  not PFK-1. When [Fru-2,6-BP] is high, glycolysis pre-  dominates over gluconeogenesis (see 'Gluconeogen-  esis' section later in this chapter)   • Insulin. Insulin regulates glycolysis by increasing  synthesis of PFK- 1 . More enzyme means that glycol-  ysis can function at a greater rate under the influence  of insulin   • Glucagon. Glucagon regulates glycolysis, by decreas-  ing synthesis of PFK-1. This means that glycolysis  operates at a lower rate under the influence of  glucagon.   • ATP. This allosterically inhibits PK by decreasing the  affinity of the enzyme for its substrate PEP. When cel-  lular energy status is high and ATP is abundant, PK is  less active    • Acetyl CoA allosterically inhibits PK. Acetyl CoA re-  flects high cellular energy status; in this context PK is  less active   • Fructose- 1-6-bisphosphate. This allosterically acti-  vates PK in a feed-forward manner, reducing the  probability of disadvantageous accumulation of  substrate   • Insulin. Insulin brings about intracellular dephos-  phorylation of PK. Dephosphorylation activates PK   Phosphofructokinase-1 is an enzyme of the glycolysis  pathway. In contrast, phosphofructokinase-2 does not  participate directly in glycolysis, but produces fructose-  2, 6, -bisphosphate (Fru-2,6-BP), which powerfully  influences glycolysis via its effect on PFK-1.   Importantly, however, Fru-2,6-BP also inhibits one of  the enzymes of the gluconeogenesis pathway. This  ensures that glucose is not degraded and synthesized  simultaneously. Figure 4.6 elaborates this concept.   Insulin is secreted when plasma glucose is high, i.e. in  the 'fed' state. When glucose is abundant, glucose oxida-  tion to provide ATP (via glycolysis) is advantageous. In-  sulin promotes glycolysis by various effects on key  regulatory enzymes. Glucagon, on the other hand, is re-  leased in the bloodstream when plasma glucose is low,    Fig. 4.6 Dual role of fructose-2, 6-bisphosphate (F2.6BP)  as a regulator of both glycolysis and gluconeogenesis.    i.e. in the 'fasting' state. It has the converse effect on the  regulatory enzymes of glycolysis; it dampens down  (rather than promotes) pathway activity.   Physiologically, insulin and glucagon are usually re-  leased by the pancreas reciprocally. This means that if  one is high, the other should be low. The relationship  between the two is reflected in the insuliniglucagon ra-  tio. If insulin is high and glucagon low, this ratio is high,  and likewise vice-versa. In health, the response of the  pancreas to changes in blood glucose is swiff. Thus  the insuliniglucagon ratio both reflects the nutritional  state of the organism in terms of carbohydrate intake  and determines the metabolic fate of glucose.   Regarding glycolysis, when the insuliniglucagon ra-  tio is high ('fed' state), pathway activity is also high.  When the ratio is low ('fasting' state), glycolysis activity  is low.   Glycolysis occurs whether or not oxygen is present. It is  the only pathway that produces ATP in the absence of  oxygen, and therefore is of paramount importance in  cells lacking mitochondria, such as erythrocytes, and  in hypoxic (low-oxygen) environments such as ischae-  mic tissue or rapidly contracting skeletal muscle.   The key difference between aerobic and anaerobic  glycolysis is that NADH + FI + generated during glycol-  ysis undergoes oxidative phosphorylation in the pres-  ence of oxygen (Chapter 3), producing— 2.5 ATP per  NADH + II + molecule. However, when oxygen is not  present to act as a terminal electron acceptor, oxidative  phosphorylation cannot occur. Instead, NADI I + H + is  oxidized to NAD + as a redox partner for the reaction  pyruvate— lactate, catalysed by lactate dehydrogenase.  This reaction yields no ATP. This lowers the ATP yield  of glycolysis in anaerobic conditions to 2 ATP per glu-  cose molecule. To compound the situation, since  pyruvate is converted to lactate it cannot enter the  TCA cycle. This would yield 10 further ATP per pyru-  vate molecule, but does not happen in anaerobic  conditions.   Cells and tissues cannot perform oxidative phosphory-  lation if they are not supplied with oxygen. Pathophy-  siologically, the state of insufficient oxygen is known  as 'hypoxia', and is seen in many disease states, for  example, ischaemia (restriction in blood supply). In this  anaerobic environment, pyruvate is converted to lactate.  When lactate rises above approximately 2.5 mmol  (hyperlactataemia), it is suggestive of tissue hypoxia,   If the patient is at rest and has elevated lactate levels  and blood pH <7.35, i.e. is also 'acidotic', this is patho-  logical, and the condition is misleadingly termed  'lactic acidosis'. It is important to appreciate that lactate  does not cause the acidosis that often accompanies  elevated lactate (although it serves as its clinical marker)  (Fig. 4.7).   Any scenario imposing anaerobic conditions will cause  lactate elevation, as pyruvate is oxidized to lactate in  order to regenerate NAD 1 . For example; compromised  oxygen delivery to respiring tissues resulting from  haemorrhage, insufficient perfusion pressure,  insufficient oxygen levels in the blood (‘hypoxaemia’)  or excessive tissue demand. All of these scenarios will  generate FI 1 ions and can cause acidosis, but this  acidosis is not due to lactate production.   Mutations in the gene coding for one of the glycolysis en-  zymes are known as inherited red cell enzymopathies.  These conditions are extremely rare; however, amongst  these glycolytic enzyme deficiencies, pymvate kinase defi-  ciency is the most common. Inheritance is autosomal re-  cessive for all but enolase (autosomal dominant) and  phosphoglycerate kinase (X-linked recessive) deficiencies.   Deficiencies in glycolytic pathway enzymes damage  red blood cells, which, lacking mitochondria, rely solely  on glycolysis for ATP generation. Unable to produce  sufficient ATP to maintain viability, their lifespan is dra-  matically shortened. This manifests clinically as chronic  haemolytic anaemia.    Acetyl CoA is formed from pyruvate. This reaction  is shown in Fig. 4.8. It is an irreversible oxidative de-  carboxylation, catalysed by pyruvate dehydrogenase    (PDII). This reaction is important, since it allows the  final common product of carbohydrate catabolism  (pyruvate) access to the TCA cycle. Note that this reac-  tion also reduces NAD + to NADI I + H + , which can gen-  erate further ATP via oxidative phosphorylation in the  presence of oxygen (Chapter 3).   PDH is in fact a trio of enzymes (El, E2 and E3) that are  all physically and spatially associated. It is located in  the mitochondrial matrix; thus the reaction occurs  in this location. There are in fact five steps in the pyru-  vate — > acetyl CoA reaction, which are not essential  to learn for medicine (however it does explain  why so many coenzymes are necessary!). These coen-  zymes are:   TPP is deficient in thiamine-deficient states, resulting in  failure of the pyruvate — > acetyl CoA reaction and thus  accumulation of pyruvate. This in part contributes to  the clinical pathology of beriberi and Wernicke's  syndrome, both manifestations of thiamine deficiency.  The excess pyruvate is converted to lactate, resulting in  hyperlactataemia.   The reaction generates NADH + H + and acetyl CoA,  which both allosterically inhibit the complex by ne-  gative feedback. PDII activity is also regulated by  phosphorylation.   Phosphorylation inactivates PDFI, whilst dephos-  phorylation activates the enzyme. Associated with the  PDH complex are a kinase and a phosphatase enzyme.  These act on the enzyme itself. The kinase (inactivator)  is itself allosterically activated by PDH reaction products  (NADH + H + and acetyl CoA) . The phosphatase (activa-  tor) is activated by insulin (Fig. 4.9).    Fig. 4.9 Regulation of pyruvate dehydrogenase (PDH)  activity. 1 =PDH kinase, 2 = PDH phosphatase.    Gluconeogenesis is the production of glucose from  non-carbohydrate molecules. It mainly occurs in hepa-  tocytes. Most gluconeogenesis reactions occur in the  cytoplasm, but two reactions occur in mitochondria.  The substrate molecules are derived from breakdown  of lipids, carbohydrates and protein.   Recall that glycolysis includes a number of highly  exergonic reactions that are essentially irreversible.  Different enzymes take over at these steps, so the actual  reaction is not simple reversal of glycolysis. Gluconeo-  genesis is an energy-consuming pathway: 6 ATP are con-  sumed per molecule of glucose produced. This energetic  'expense' is justified by the physiological importance of  glucose (Chapter 8).   Even when fasting, blood glucose levels must be main-  tained within the range 3-6 mmol. This is achieved  through ingestion of exogenous glucose (eating), en-  dogenous release of stored glucose (glycogenolysis) or  endogenous synthesis of glucose (gluconeogenesis).   Straying outside the range 3-6 mmol may have serious  consequences; both severe hypoglycaemia and  hyperglycaemia are dangerous (Chapter 8).   Glycogen breakdown (glycogenolysis) is the 'first-line'  mechanism preventing glucose levels falling below nor-  mal. However, glycogen reserves are quickly exhausted  during fasting or prolonged exercise. Metabolism must  therefore be able to compensate by producing glucose  from other sources.   • Proteins. Muscle protein is degraded to form amino  acids, some of which ('glucogenic' amino acids) par-  ticipate in gluconeogenesis   • Lipids. Mobilized fat stores or ingested fats ('triacyl-  glycerols') are hydrolyzed, releasing glycerol and  fatty acids (Chapter 5). Glycerol enters gluconeogen-  esis. Propionyl CoA (a product of (f-oxidation of  odd-numbered fatty acids) also enters gluconeogen-  esis. However, fatty acids themselves do not give rise  to glucose   • Carbohydrates. Glycolysis under anaerobic condi-  tions ultimately generates lactate, which can be con-  verted back to pyruvate by lactate dehydrogenase.  Pyruvate in turn is a gluconeogenic substrate.   • The conversion of pyruvate to PEP in gluconeogen-  esis is a two-step reaction, rather than the single PEP  — > pyruvate reaction (glycolysis reaction 10). Also,  it requires both ATP and GTP. The responsible  enzymes are mitochondrial pyruvate carboxylase and  cytoplasmic phosphoenolpyruvate carboxykinase   • Between these first two reactions, oxaloacetate  'leaves' the mitochondria and 'enters' the cytoplasm  via the malate-aspartate shuttle (Chapter 3)   • Fru-l,6-BP is converted to Fru-6-P via a hydrolytic  reaction, with no involvement of ADP or ATP (com-  pare with glycolysis reaction 3)   • The final reaction of gluconeogenesis is dephos-  phorylation of Glc-6-P, catalysed by glucose-6-  phosphatase in a hydrolytic reaction, with no  involvement of ADP or ATP (compare with glycol-  ysis reaction 1)    2 (pyruvate) + 2C0 2 + 2ATP + 2H 2 0    • Gluconeogenesis occurs only in cells possessing the  appropriate enzymes, i.e. in hepatocytes and to a cer-  tain extent in cells of the renal cortex   • Gluconeogenesis occurs in both the cytoplasm and  the mitochondria, as opposed to purely cytoplasmic  glycolysis   This pathway is appropriately active in contexts which  require endogenous glucose production, e.g. fasting,  starvation, prolonged exercise and low-carbohydrate di-  ets. It is also inappropriately active in insulin deficiency.   Bearing in mind the reciprocity between glycolysis  and gluconeogenesis, it follows that intracellular condi-  tions that inhibit one pathway would activate the other.   The reactions of gluconeogenesis (reactions 1, 2, 9  and 11) that differ from glycolysis are also highly exer-  gonic and essentially irreversible. The enzymes catalys-  ing these reactions function as points of regulation for  gluconeogenesis.   Pyruvate carboxylase is allosterically activated by acetyl  CoA. Remember that acetyl CoA inhibits pyruvate ki-  nase (glycolysis reaction 10). High [acetyl CoA] pro-  motes gluconeogenesis but inhibits glycolysis. Acetyl  CoA is generated by fl-oxidation of fatty acids  (Chapter 5), which is maximal during fasting, and  therefore is an appropriate activator of gluconeogenesis.   Phosphoenolpyruvate carboxykinase expression is en-  hanced by glucagon and inhibited by insulin. A low in-  sulimglucagon ratio, therefore inhibits glycolysis but  promotes gluconeogenesis.    Fig. 4.10 Gluconeogenesis. The reactions that are not simply a  reversal of glycolysis are shown in bold, annotated with the  responsible enzyme. Glycolysis enzymes not shown. Please note  that two 3 -carbon pyruvate molecules enter the pathway to  form one 6-carbon molecule of glucose.   AMP promotes glycolysis (AMP activates PFK) but in-  hibits gluconeogenesis, by virtue of allosterically inhi-  biting Fru-l,6-BP. Similarly, Fru-2,6-BP promotes  glycolysis (Fru-2,6-BP activates PFK) but is inhibitory  to gluconeogenesis, since it functions as an allosteric in-  hibitor of fructose-1, 6-bisphosphatase.   Glucose-6-phosphatase is allosterically activated in a  feed-forward fashion by the reaction substrate Glc-6-P.  Remember that Glc-6-P conversely inhibits hexokinase.  High Glc-6-P promotes gluconeogenesis and inhibits  glycolysis (Fig. 4.11).    Glycogen is a polysaccharide: a polymer of glucose mol-  ecules (monomers). It is stored as intracellular granules  in liver and muscle cell cytoplasm. Glycogen is synthe-  sized from excess dietary glucose. Glycogen breakdown  ('glycogenolysis') allows rapid mobilization of glucose.  This can stave off acute hypoglycaemia until gluconeo-  genesis can pick up its pace (or until the next meal takes  place), or provide intracellular fuel for muscle contrac-  tion. Adrenaline (epinephrine) and glucagon stimulate  glycogenolysis and insulin stimulates glycogen synthe-  sis (glycogenogenesis).   Liver glycogen accounts for 10% of the liver's mass. It  functions as a systemic reserve for maintenance of blood  glucose levels during fasting. Once released into the    blood, glucose liberated from hepatic glycogen can be  utilized by any tissue of the body.   Muscle, however, lacks the necessary enzyme  (glucose-6-phosphatase) to convert Glc-6-P (generated  by glycogenolysis) to free glucose. Since Glc-6-P cannot  cross cell membranes, it remains intracellularly and  is exclusively used by the cell it originates from. The role  of muscle glycogen is to provide glucose for oxidation in  glycolysis when glucose demand exceeds the maximum  that can be absorbed from the circulation, for example  during rapid muscle contraction.   Glycogen consists of chains of glucose molecules,  linked by glycosidic bonds between the Cl of one glu-  cose and the C4 of the next. The polymer is branched; a  branch point occurs every 8 - 1 2 glucose units (residues) .  Branch bonds are between the C6 of the residue on the  main strand and the Cl of the terminal glucose of the  incoming branch (Fig. 4.12).   The branched structure allows for a large number of  'ends' or termini. Enzymes that degrade glycogen can  only operate on these termini; thus the more termini  available, the more rapidly glycogen can be degraded.  This allows for rapid mobilization of glucose, e.g. the  adrenaline-fuelled 'fight- or-flight' response.   Fig. 4.12 Glycogen structure. The (1-4) and (1-6) linkages are highlighted. Ringed numbers illustrate the numbering of carbon  atoms within each glucose monomer.   • UDP-glucose, the glucose donor. UDP is uridine di-  phosphate (a nucleotide). It is required to 'activate'  glucose molecules so they are recognizable by the  glycogenetic enzymes.   • Four enzymes: phosphoglucomutase, glycogen  synthase, branching enzyme (amylo (1,4— >1,6)  transglycosylase) and uridyl transferase.   • A pre-existing glycogen chain to link additional glu-  cose molecules to; in absence of this glycogenin  (a molecular 'primer') is required.   UDP-glucose is synthesized from Glc- 1 -P and UTP by uri-  dyl transferase (aka 'UDP-glucose pyrophosphorylase').    Glucose in this form is now 'eligible' to join the growing  glycogen chain.   Glycogen synthase now transfers the glucose from UDP-  glucose to the C4 of the terminal glucose in an existing  glycogen strand (Fig. 4.13). It is linked via a (1-4)  glycosidic bond. Elongation requires a strand of at least  four glucose residues to exist before it can occur; either  an existing 4-plus residue strand or the protein glyco-  genin ('primers') must be present.   Glycogen synthase can only lengthen strands: it cannot  create branches. For this, branching enzyme is required.  This enzyme cleaves off a length from a separately grow-  ing glycogen strand, usually around seven residues long,  and transfers this to another strand (Fig. 4.14). This  generates a branch point. A (1-4) bond is broken, but  a (1-6) bond is formed (between the Cl of the incom-  ing fragment and the C6 of the 'branch' residue on the  main strand).   Fig. 4.13 Glycogen polymer  synthesis: chain elongation. For  simplification, only the main carbon  skeleton structure is shown for  glucose. Please refer to Fig. 4.1 for the  precise structure. UTP = uridine  triphosphate, UDP = uridine  diphosphate, PP, = pyrophosphate.   4 = glycogen synthase. Note that  glycogenin can replace an existing  strand of residues as the primer.   This occurs in the cytoplasm. It is stimulated by gluca-  gon and adrenaline. There are two elements to glycogen-  olysis; strand-shortening and branch removal. The  enzymes involved are glycogen phosphorylase (which  requires pyridoxal phosphate (PLP) as a cofactor), deb-  ranching enzyme (aka (1,4)-^(1,4) glucan transferase)  and amylo-a-l,6-glucosidase.   Glucose units are cleaved off strand termini, one by one.  The cleavage of the (1— >4) glycosidic bond linking  terminal and penultimate units is performed by    glycogen phosphorylase; 'phosphorolysis', and within  intracellular organelles called lysosomes by lysosomal  a-l,4-glucosidase. Glucose- 1 -phosphate (Glc-l-P) is  released.   Unhelpfully, glycogen phosphorylase will only re-  move units if the chain is four residues or longer. It also  cannot process branch-point residues. Thus the enzyme  stops short when the strand/branch diminishes to four  residues in length (not including the 'branch' unit). This  leaves a number of branch-remnant 'stubs' (Fig. 4.15).   Glc-l-P released by glycogenolysis may be converted  by phosphoglucomutase to Glc-6-P, which can enter  glycolysis. In hepatocytes, Glc-6-P may be converted  to glucose itself by hepatocyte glucose-6-phosphatase.    Fig. 4.14 Glycogen polymer  synthesis: branch formation. Each  hexagon represents a glucose residue.  Stage 1 = excision of terminal six  residues of a strand via hydrolysis of a  (1— >4) bond, stage 2 = transfer to a  proximal residue and formation of a  (1— >6) glycosidic bond. To facilitate  understanding, the transferred  segment residues are shaded.   Once a branch has been shortened to the final four units  of a branch (stemming from a 'branch' unit), the distal  three units of this branch are trimmed off by debranch-  ing enzyme, which hydrolyzes the (1— > 4) glycosidic  bond. These three units are then attached to another  strand elsewhere in the polymer. This leaves the 'branch'  unit of the main strand with just one remaining unit  (step 1, Fig. 4.16). This protruding unit requires  amylo-a-l,6-glucosidase to hydrolyse the (1— > 6) glyco-  sidic bond, releasing free glucose (step 2, Fig. 4.16).   The activity of the key synthesis and degradation en-  zymes (glycogen synthase and glycogen phosphoryiase,  respectively) is modulated via hormonal and allosteric  mechanisms.    Fig. 4.16 Glycogenolysis: branch removal. The 'branch' unit is  shaded heavily, whilst the terminal three residues on the 'stub'  are shaded lightly to facilitate visualization.   Glycogen synthase is deactivated by phosphorylation. Re-  fer to Fig 1.7.   • The same kinase enzyme (protein kinase A; PKA)  phosphorylates both enzymes   • Intracellular cAMP is elevated by binding of adrena-  line or glucagon to membrane receptors associated  with adenylate cyclase (AC). This enzyme synthe-  sizes cAMP from AMP   • Glucagon and adrenaline therefore elevate intracel-  lular cAMP, activating PKA, which phosphorylates  (deactivates) glycogen synthase and (activates)  glycogen phosphorylase   • Insulin has the opposite effect, since insulin binding  to its intracellular receptors results in up-regulation  of intracellular protein phosphatase- 1 (PP-1). This  dephosphorylates the two enzymes, with the oppos-  ing outcome: glycogen synthesis is promoted and  glycogenolysis is inhibited.   The scenario differs according to the location of glyco-  gen: hepatocytes or muscle cells.   Glucose allosterically inhibits glycogen phosphorylase,  inhibiting glycogenolysis. Glucose binds to glycogen  phosphorylase, causing a conformational change that  exposes its phosphate group. This phosphate group is  the target for hydrolytic cleavage by PP-1 . Thus, glucose  binding increases the probability of dephosphorylation  and consequent deactivation of glycogen phosphorylase,  and inhibition of glycogenolysis. Conversely, glucose allo-  sterically activates glycogen synthase, promoting glycogen  synthesis. Glc-6-P too, allosterically inactivates phos-  phorylase and activates synthase.   Ca 2+ ions, intracell ularly elevated during skeletal mus-  cle contraction, allosterically activate glycogen phos-  phorylase. Thus glycogenolysis is promoted in a  context where glucose mobilization is desirable. Simi-  larly, AMP, plentiful in active cells undergoing high  levels of ATP hydrolysis, allosterically activates glycogen  phosphorylase.   This term describes genetic diseases arising from defi-  ciencies of one of the enzymes of either glycogen synthe-  sis or degradation. They result in clinical manifestations  reflecting the resulting abnormalities of glycogen syn-  thesis or degradation (Fig. 4.17).    The PPP, aka the 'hexose monophosphate shunt', is a pri-  marily anabolic pathway that uses glucose-6-phosphate  (Glc-6-P) as an initial subsUate. The PPP generates  NADPIT + PI + , pentose sugars and other intermediates.  NADPH + H + is both vital for fatty acid and cholesterol  synthesis (Chapter 5) and also regeneration of glutathi-  one. Pentose sugars are required for nucleotide and nucleic  acid synthesis. Other pathway intermediates are impor-  tant raw materials for numerous synthetic reactions.   Whilst not the pathway's primary role, the PPP allows  conversion of dietary pentose sugars into hexose and th-  ose intermediates that are then able to enter glycolysis,  conserving energy substrates.   • An initial oxidative phase. Three exergonic reactions  ultimately generate ribulose-5-phosphate, C0 2 and  two molecules of NADPFI + H + per Glc-6-P oxidized   • A reversible non-oxidative phase, consisting of a  series of reactions converting ribulose-5-phosphate  into intermediates with varying carbon numbers.   The main regulatory influence is exerted at the glucose-  6-phosphate — > 6-phosphogluconolactone reaction.  The substrate:product ratio drives the reaction forward;  the higher the [NADP + ] (substrate) relative to the  [NADPH + H + ] (product), the greater the pathway ac-  tivity. Higher activity results in more Glc-6-P entering  the PPP (and less participating in glycolysis).   Demand for intermediates (generated during the sec-  ond, non-oxidative phase) determines which products  are predominantly generated. For example, if ribose-5-  phosphate is withdrawn from the PPP to enter nucleic  acid synthesis, it will not be able to combine with  xylulose-5-phosphate and progress along the PPP to  produce downstream intermediates (Fig. 4.18).   Glucose-6-phosphate from  gluconeogenesis or glycogenolysis  cannot be converted to glucose and thus  released from liver cells. Glycogen  accumulates within liver and renal cells  (gluconeogenesis sites)   Hepatomegaly (enlarged liver)  and enlarged kidneys; both due  to accumulated unmobilized  glycogen. Fasting  hypoglycaemia from inability to  release glucose from glycogen  or gluconeogenesis (glucose-6-  phosphatase deficiency) or from  glycogenoloysis (debranching  enzyme)   Glycogen strands cannot be shortened  lysosomally. Glycogen accumulates  intralysosomally within heart muscle,  skeletal muscles, liver and CNS   Hepatomegaly, cardiomegaly  (enlarged heart), muscular and  neurological symptoms.  Restrictive cardiomyopathy can  develop from glycogen  accumulation in the  myocardium. Inheritance is  autosomal recessive   The final four residues of shortened  branches cannot be removed. Glycogen  has abnormal structure, with large  numbers of short 'branchlets'   Hepatomegaly, hypoglycaemia,  late-onset muscle weakness and  cardiomyopathy. Inheritance is  autosomal recessive   Glycogen synthesis is abnormal; long  unbranched strands accumulate. This  abnormal structure is less soluble than  normal glycogen   Accumulation is most  pronounced in the heart muscle  and the liver. Progressive causes  death, usually before the age of  5. A number of genes can cause  this syndrome; inheritance  varies according to the  particular gene   Muscles cannot perform glycogenolysis,  therefore must rely on glucose absorbed  from the bloodstream   Decreased exercise tolerance,  (increased fatigue and muscle  cramps) on sustained exercise  since local glycogen  mobilisation is impossible. No  hypoglycaemia, since hepatic  glycogenolysis is unimpaired   Impaired glycogenolysis results in  normal-structured glycogen  accumulation in liver and muscle   Hepatomegaly. Hypoglycaemia  on fasting. 75% cases are X-  linked recessive, remaining  cases are autosomal recessive   Complex. Essentially, glycogen synthase  is abnormally activated, resulting in  glycogen accumulation in muscles   Glutathione is a 'tripeptide'; a trio of amino acids (glutathi-  one, cysteine and glycine). Glutathione is the primary in-  tracellular antioxidant, neutralizing harmful intracellular    reactive oxygen species (ROS) (Chapter 1) and limiting  oxidative damage. This function is particularly important  in immune cells. Glutathione also plays fundamental roles  in many vital metabolic processes, including enzyme acti-  vation, protein synthesis, DNA synthesis and DNA repair.    Fig. 4.18 Pentose phosphate pathway. 1 =glucose-6-  phosphate dehydrogenase, 2 = gluconolactonase, 3 = 6-  phosphogluconate dehydrogenase, 4 = ribulose-5-phosphate-  3-epimerase, 5 = ribulose-5-phosphate isomerase,   Glutathione operates to neutralize ROS by donating H +  and e~ (from the thiol group of the cysteine residue) to  the unstable ROS. In donating reducing equivalents, gluta-  thione is itself oxidized, and becomes unstable. It then rap-  idly reacts with another (unoxidized) glutathione molecule,  forming glutathione disulphide (GSSG) . GSSG is inactive in    terms of its antioxidant role. To return glutathione to its ac-  tive, unoxidized form, NADPH+ H + is required (Fig. 4.19).   Glutathione is of key importance in hepatocytes. They re-  quire a constant supply of active (unoxidized) glutathione  for participation in conjugation (excretion) of numerous  foreign compounds - including dietary molecules, drugs  and toxins. If active glutathione becomes unavailable, these  substances accumulate to toxic levels. As a constant supply  of NAD PH + H + is required in order to maintain a constant  supply of active glutathione, the PPP assumes partial re-  sponsibility for maintaining the liver's excretion role.   When high levels of ROS are present in a context of cel-  lular oxidative stress, the cytochrome b 5 reductase system  (which normally maintains haemoglobin in the reduced  state (Fe 2+ )) becomes overwhelmed. Active glutathione  protects against oxidation of haemoglobin (to a non-  functional methaemoglobin (Fe 3+ )) and allows erythro-  cytes to continue performing their oxygen-carrying role.  In this way, the PPP is also very important in the context  of oxidative stress in erythrocytes, since PPP-mediated  NADPH + H + production ensures that sufficient gluta-  thione is available to sustain Hb function.   The y-glutamyl cycle is a mechanism for importing  various amino acids into cells (Chapter 6). Glutathione  is required for this import process to operate.   Glucose-6-phosphate dehydrogenase deficiency is an  X-linked recessive condition, affecting over 400 million  persons worldwide, making it the most common enzymo-  pathy. Since inheritance is X-linked and recessive, females  are rarely seriously affected.    Absence of functioning glucose-6-phosphate dehy-  drogenase results in failure of the PPP. The mitochon-  drial citrate shuttle (Chapter 5) is the only other  mechanism generating NADPH + H + . Erythrocytes,  however, lack mitochondria, and so cannot counteract  the NADPH + H + deficit. Absence of active glutathione  leaves erythrocytes unable to survive oxidative assaults,  dramatically shortening their lifespans. This manifests  clinically as haemolysis in situations of oxidative stress.   There are a number of factors that enhance cellular ox-  idative stress. For example:   • Broad beans (fava beans) contain high levels of vicine  compounds, which are potent cellular oxidants   • Various drugs, in particular sulphonamides, certain  antibiotics, aspirin and antimalarials   Fructose is a hexose (C 6 ITi20 6 ). This formula is the  same as glucose; however fructose is altered in structure.  Its main dietary source is the disaccharide sucrose. Gut  sucrase hydrolyzes sucrose to component monosaccha-  rides glucose and fructose. Absorption of fructose into  intestinal cells (enterocytes) is via the GFUT-5 trans-  porter. Fructose then leaves the enterocytes into the  bloodstream (this time via GLUT-2) (Fig. 4.20).   Fructose is phosphorylated by hepatocyte fructoki-  nase, forming fructose- 1 -phosphate. This is further me-  tabolized to glyceraldehyde-3 -phosphate (GAP) by  aldolase B, which enters glycolysis or gluconeogenesis  according to cellular energy status (Fig. 4. 1 1 ) . In muscle  cells, fructose is phosphorylated to Fru-6-P by HK (like  glucose) and enters glycolysis.   The two main enzymopathies of fructose metabolism are  fructokinase deficiency and fructose- 1 -phosphate aldolase  deficiency. Both are autosomal recessive conditions.   Deficiency of fructokinase results in failure of hepatic  fructose catabolism. Fructose therefore can only be  degraded by muscle hexokinase. This leads to increased   Fig. 4.20 Fructose metabolism in a liver cell. In a muscle cell,  hexokinase would perform the first phosphorylation.   plasma fructose, which leads to elevated urinary  fructose. This is clinically asymptomatic.   This is also called 'hereditary fructose intolerance'. Lack of  aldolase B leads to accumulation of fructose- 1-phosphate  and failure of dietary fructose to be diverted towards gly-  colysis or gluconeogenesis. Fructose- 1 -phosphate eleva-  tion is toxic, because high intracellular concentration  sequesters intracellular phosphate. Enzymes reliant on  phosphorylation for their activation thus fail. Glycogen  phosphorylase (required for glycogen mobilization)  and aldolase A (required for gluconeogenesis) are inacti-  vated. Therefore the two mechanisms for maintaining  plasma glucose on fasting fail, and fasting hypoglycaemia  soon occurs following fructose exposure in deficient indi-  viduals. Treatment is by complete dietary exclusion of  anything containing fructose, including sucrose.   Recall that Fru-6-P participates in glycolysis, when PFK-  1 mediates addition of a second phosphate, generating  Fru-l,6-BP. In the liver, Fru-6-P can also participate in  gluconeogenesis or the PPP, after conversion to Glc-6-P  by phosphoglucoisomerase.    In erythrocytes, fructose-derived Glc-6-P enters the  PPP or glycolysis (gluconeogenesis does not occur in  erythrocytes), allowing NADPPI + H + production.   Galactose is also a hexose, and shares the formula  C 6 H 12 0 6 with fructose and glucose. The main dietary  source is the disaccharide lactose, found in milk products.  Gut lactase hydrolyzes lactose to its components glucose  and galactose. Absorption into enterocytes is via the  sodium/glucose symport. Like glucose, diffusion into  the bloodstream from the enterocytes is via the GLUT-2  transporter. Galactose is converted into Glc-6-P, which  can then enter gluconeogenesis (in the liver), glycolysis  or the PPP (Fig. 4.21).   • Galactose- 1 -phosphate then reacts with UDP-glucose,  forming glucose- 1 -phosphate and UDP-galactose. This  is catalysed by galactose- 1 -phosphate uridyl transferase   • Glucose- 1 -phosphate is then converted to glucose- 6-  phosphate by phosphoglucomutase   This is a rare, autosomal recessive condition, arising  from deficiency of either galactose- 1 -phosphate uridyl  transferase, galactokinase or galactose-6-phosphate  epimerase. Failure of galactose catabolism results in  toxic accumulation of galactose or galactose- 1-  phosphate in various tissues, particularly the liver, kid-  ney, lens and CNS. These serious and irreversible events  occur soon after feeding commences postpartum; even  breastfeeding galactosaemic infants exposes them to ga-  lactose. Treatment is complete exclusion of all galactose-  containing foods from the diet.   Sorbitol (aka glucitol) is a sugar-alcohol, produced en-  dogenously from excess glucose via the polyol pathway.  It can also be obtained from the diet. Tissues containing  sorbitol dehydrogenase (such as the liver or sperm cells)  oxidize sorbitol to fructose. This reaction uses NAD + as  a redox partner, thus generating NADI I + H + . This can  then undergo oxidative phosphorylation. The fructose  may ultimately feed into glycolysis or the PPP (or gluco-  neogenesis in the liver) (Fig. 4.22).   At high plasma glucose, such as is seen in uncon-  trolled diabetes for example, aldose reductase up-  regulates. As this enzyme is part of the sorbitol synthesis  (polyol) pathway, this results in enhanced sorbitol  production. This is significant for two reasons:   • The polyol pathway utilizes NADPFI + H + as a redox  partner in the glucose — > sorbitol reduction, result-  ing in lower availability of NAD PH + H + for regen-  eration of glutathione. This renders cells less able  to survive oxidative assaults   • Certain tissues lack sorbitol dehydrogenase and can-  not divert sorbitol into glycolysis via fructose. Sorbi-  tol accumulates and exerts a pathological osmotic  effect, leading to structural damage and impaired  function. This is seen in Schwann cells and retinal  cells in particular.   Ethanol (C 2 H 5 OFI) is a component of alcoholic bever-  ages. It binds to many CNS receptors, including acetyl-  choline, serotonin, GABA and glutamate, with an  overall depressant effect on neuronal activity.   Ethanol detoxification occurs in liver cells via two  sequential oxidations, culminating in the less toxic  ethanoic acid (aka 'acetic acid'). There are three differ-  ent systems that mediate the first oxidation, which    Fig. 4.22 Polyol pathway. 1 = aldose  reductase, 2 = sorbitol dehydrogenase.   • Microsomal ethanol oxidizing system, which uses  the P450 enzyme system   Ethanal (produced by one of the mechanisms above)  enters the mitochondria, where it is further oxidized  to ethanoate (acetate) by mitochondrial aldehyde de-  hydrogenase. A cytoplasmic isoform of this enzyme also  exists. The acetate produced can then be coupled with  coenzyme A by the enzyme acetyl CoA synthetase, form-  ing acetyl CoA.   Aldehydes are highly reactive and react promiscuously  with numerous biological molecules. The ethanal aris-  ing from biological ethanol processing is responsible  for the unpleasant effects of ethanol consumption.  These are usually seen when individuals consume exces-  sively and exceed their own personal capacity for main-  taining ethanal at an asymptomatic concentration.  Nausea, vomiting, flushing, dizziness, elevated heart  rate, headache and shortness of breath are seen.   These symptoms are exploited by Disulfiram  (Antabuse), a drug used to deter recovering alcoho-  lics from consuming alcohol. Disulfiram inhibits  aldehyde dehydrogenase (mitochondrial and cyto-  plasmic), resulting in rapid ethanal accumulation  following ethanol consumption. The unpleasant  symptoms of ethanal toxicity act as a deterrent against  repeated consumption.   Approximately 50% of Oriental individuals lack the  mitochondrial isoform of aldehyde dehydrogenase.  With only the cytoplasmic isoform to deal with an  ethanal load following ethanol consumption,  degradation is retarded, leading to accumulation of  ethanal. These individuals manifest early signs of  ethanal toxicity such as flushing, nausea and vomiting.   (Fig. 4.23) . This depletes the NAD + available to act as a re-  dox partner in numerous metabolic reactions, including:   • Glycolysis. Less carbohydrate catabolism occurs,  with the consequence of less ATP production   • The TCA cycle. Less oxidation of TCA cycle substrates  occurs, with the consequence of less ATP generation   • Gluconeogenesis. Inhibition of this pathway causes  hypoglycaemia once glycogen reserves are exhausted.   Lactate and urate compete for the same cellular  excretion apparatus in the renal tubules. Thus excess  lactate results in proportional retention of urate. This  may crystallize in connective tissue, leading to an  inflammatory reaction. This causes the substantial pain  of gout, and explains its association with excessive  ethanol intake (Chapter 7).   Ethanol consumption causes an up-regulation in syn-  thesis of alcohol-detoxifying enzymes. This includes  the cytochrome P450 system. However, these enzymes    are also solely responsible for metabolism and clearance  of many individual therapeutic drugs. An alcohol-  induced increase in enzyme presence results in in-  creased clearance of such drugs. This can result in drug  levels falling to sub-therapeutic, i.e. ineffective levels.   Warfarin, for example, is metabolized and cleared from  the body via the cytochrome P450 system. Alcohol-  induced up-regulation of the P450 system means that  the usual dose of warfarin will be degraded too rapidly  reach a sufficient level to exert therapeutic  anticoagulative effects. This can be catastrophic for  those relying on warfarin anticoagulation to prevent  thrombotic events such as stroke, pulmonary embolism  and myocardial infarction.   • Appreciate the mechanism of the citrate shuttle in fatty acid transport   • Describe mobilization of fatty acids from triacylglycerols, and conversely triacylglycerol synthesis from  fatty acid substrates   • Understand the physiological role of cholesterol and recognize the structure   • Recognize the different lipoprotein particles and their various functions and characteristics   • Describe ketone synthesis and degradation, appreciating the role in metabolism of these pathways   Lipids are a large group of diverse molecules ranging  from waxes to sterols. In this chapter the term 'lipids'  is used synonymously with 'fats', i.e. referring to fatty  acids and their derivatives. Lipid molecules all have  the general structure shown in Fig. 5.1.   Lipids have varied physiological roles; both structural  and within metabolism. In anabolism, triacylglycerol  (TAG, aka triglyceride) molecules are the major form    of energy storage. TAGs consist of three fatty acids linked  to a glycerol backbone by ester bonds. Figure 5.2 shows  an example. In catabolism, lipid stores are mobilized, re-  leasing fatty acids and glycerol, which enter the blood-  stream and act as substrates for oxidation by distant  tissues, generating energy.   Fatty acids (FA) are excellent examples for illustrating  lipid structure: a carboxylate ('head') linked to a long  unbranched hydrocarbon chain ('tail') (Fig. 5.3).    Fig. 5.1 General lipid structure; a hydrophobic 'tail' composed  of a hydrocarbon chain (C and H) and a hydrophilic 'head'  consisting of C, H and O.    Fatty acids are required as substrates for lipogenesis, i.e.,  synthesis of triacylglycerols; energy-dense storage mole-  cules. FA are synthesized de novo from acetyl CoA.  When acetyl CoA is abundant, i.e. the fed state, the en-  ergy intrinsic in acetyl CoA can be 'stored' by synthesis  of triaclyglycerols.   Conversely, FA can be released into the bloodstream  from triaclyglycerols (stored in adipose tissue) by lipol-  ysis; once released they can be catabolized to release en-  ergy. The long hydrocarbon tails of FA molecules are  incredibly efficient in terms of energy storage, because  they contain fully reduced carbon and release maximal  energy on oxidation (catabolism). FA catabolism is via  (3-oxidation, which both generates FADH 2 and  NADH+H + and releases acetyl CoA. Acetyl CoA can  enter the TCA cycle for oxidation, and reducing equiva-  lents can undergo oxidative phosphorylation, releasing  energy.    bonds within the hydrocarbon chain, and so no opportu-  nity for any new bonds to be formed; the molecule is 'sat-  urated' with hydrogen atoms. 'Polyunsaturated' FA have  more than one double bond present, whereas monoun-  saturated FA have a single double bond (Fig. 5.4).    Note that fatty acids all have molecular formula  'R-COOIT', i.e. 'something' carrying a carboxylic acid  group. Therefore, fatty acids are weak acids:   Due to the average pKa value (for FA) being ~4.5, at  physiological pH the equilibrium position lies to the  right of the equation. FA exist in their anionic form un-  der physiological conditions. This has the suffix '-ate'  rather than '-ic acid', e.g. stearate rather than stearic acid.  These terms are often incorrectly used synonymously. In  this text the physiological anionic versions will be dis-  cussed, i.e. the suffix '-ate' is to be used.   Double bonds within an unsaturated FA may adopt one  of two stereoisomeric configurations: cis or trans. The  nature of a C=C double bond can have significant con-  sequences for molecular properties, for example struc-  ture and chemical characteristics, cis configurations  impose a sharp bend in the hydrocarbon chain, whereas  trans configurations do not. Most naturally occurring  unsaturated FA possess C=C bonds in the cis configura-  tion (Fig. 5.5).   Fatty acids are described as 'saturated' or 'unsaturated'.  Unsaturated FA contain a double bond at some location  within the hydrocarbon tail, whilst saturated FA contain  no double bonds. In saturated FA, there are no double   With unsaturated FA, the number of carbons as well as  the number, position and nature of the double bonds  must be communicated by the notation. This is illus-  trated using the example of palmitoleate (16:lcA9)  and arachidonate (20:4cA5,8,ll,14) in Fig. 5.6.   Fig. 5.4 Saturated and unsaturated fatty acids: stearate vs.  elaidate. Note the ringed numbers illustrate the carbon number.  Appreciate that stearate is the same molecule as in Fig. 5.3,  however for brevity not every single carbon and bond has been  drawn.    Fig. 5.6 Fatty acid nomenclature: palmitoleate (16:1cA9) and  arachidonate (20:4cA5,8,1 1 ,14). Ringed numbers indicate  the carbon numbering.    The number before the colon describes the total num-  ber of carbons in the molecule (16), whilst the number  directly after the colon describes the number of double  bonds (1). The V indicates that the configuration of  that double bond is cis (as opposed to trans). The 'A'  symbol is followed by a number representing the car-  bon atom at which the double bond starts.    Fig. 5.5 Stereoisomerism: cis (oleate) and trans (elaidate)  configuration of CH 3 (CH 2 ) 7 CH = CH(CH 2 ) 7 COCr.   Carbon counting in chemistry begins from the carbon  attached to the main functional group of the molecule  (in FA this is the carboxyl group). The carbon bearing  this functional group is carbon-1 (Cl). Cl is also  sometimes called the 'alpha' carbon, and C2 the 'beta'  carbon. This explains why Greek letters are part of the  names of biochemical molecules; for example  oc-ketoglutarate (this tells us that the keto (C=0)  functional group originates from the alpha carbon of  glutarate).   Omega-3 fatty acids are a family of FA molecules with  a common structural feature: a double bond between  the 3 rd and 4 th carbon counted from the end of the  hydrocarbon tail. The final carbon in the hydrocarbon  tail is the "omega" carbon, irrespective of whether it is  C5 or C25. This final carbon is at the opposite end of  the molecule to the carboxyl group. Whilst confusing  that this naming system counts from the other end  to the functional group, it is used because the proximity  of the first unsaturation (C=C double bond) to the  terminal (omega) carbon has more influence upon  molecular properties of the fatty acid than its proximity  to the carboxylate group at the other end of the  molecule.   Although fatty adds (FA) are accessible from dietary fats  (hydrolysis of ingested triacylglycerols (TAG) releases FA)  the bulk of human energy intake is via carbohydrates.  Carbohydrate storage (as glycogen) is limited; thus a pro-  cess for conversion of carbohydrate to fat is required. This  process is fatty acid synthesis; acetyl CoA derived from  pyruvate (a glycolysis product) is incorporated into  new fatty acid molecules. These may then be esterified,  forming TAG for storage in adipose tissue.   FA synthesis occurs in cell cytoplasm. It occurs  mainly in specialised fat cells; adipocytes, but also  in liver, kidney and of course lactating mammary  glands. It requires various substrates but the most  important ones to remember are acetyl CoA and  NADPH+HT   1 . Transport of acetyl CoA to the cytoplasm, where the  synthetic enzymes are located   2. 'Activation'; synthesis of malonyl CoA and localising  at fatty acid synthase   3 . A sequence of condensation, reduction, dehydration  and a second reduction   4. Addition of a 2-carbon unit (derived from another  malonyl CoA)    The number of 'repeats' determines the length of the hy-  drocarbon chain. In the example ofpalmitate (16:0), af-  ter the initial process ([1] through [4]), this would  repeat a further six times. Note that this process gener-  ates a saturated FA, with no double bonds (hence the '0'  in 16:0). Synthesis of unsaturated fatty acids requires a  saturated FA to first be synthesized and then enzymati-  cally modified.   This is via a mechanism known as the 'citrate shuttle',  aka the 'pyruvate-malate' cycle (Fig. 5.7).   • Acetyl CoA condenses with oxaloacetate (in the  mitochondrial matrix) forming citrate, as in the  TCA cycle. This is catalysed by citrate synthase   • Citrate is then exported from the matrix into the cell  cytoplasm in exchange for malate   • Here it reacts with ATP and CoA to form oxaloacetate  and acetyl CoA. This is catalysed by citrate lyase. The  acetyl CoA is now in the cytoplasm and able to par-  ticipate in fatty acid synthesis   • The oxaloacetate is reduced to malate by malate  dehydrogenase, using NADH+FI + as a redox  partner   • Malate is decarboxylated to pyruvate by malic en-  zyme, using NADP + as a redox partner. This gener-  ates NADPH+FI + , which is also required for FA    Fig. 5.7 The citrate shuttle  mechanism for mitochondrial import  of acetyl CoA. 1 = citrate synthase,  2 = citrate lyase, 3 = malate  dehydrogenase, 4 = malic enzyme,    synthesis, or is returned to the mitochondrial matrix  in exchange for citrate   • Pyruvate then re-enters the mitochondria, where it  may either be used to regenerate oxaloacetate or ace-  tyl CoA, completing the circuit. Figure 5.7 illustrates  the entire process   This is the irreversible rate-limiting step of FA synthesis.  Carboxylation of acetyl CoA is mediated by acetyl CoA  carboxylase (it requires biotin as a cofactor). The reac-  tion is shown in Fig. 5.8.   Getting it together: assembly of malonyl and acetyl  groups on ACP   ACP is 'acyl carrier protein', and is a component of  fatty acid synthase (FAS). ACP accepts acyl groups,  such as the acetyl and malonyl groups of acetyl CoA  and malonyl CoA, thus anchoring them to the enzyme  in preparation for FA synthesis. The CoA components  are released and the acyl components bind to the  terminal sulphur atom of the phosphopantetheine  moiety (Fig. 5.9). This reaction is mediated by acetyl  transacylase and malonyl transacylase. Note that    two ACP are present; this is because FAS exists as a  homodimer.   This enzyme is a homodimer, consisting of two copies  (a 'dimer') of identical ('homo') enzymatic units. The  units themselves are also composed of a number of sub-  units, each fulfilling various enzymatic roles in the reac-  tions of FA synthesis.   The ACP-anchored acetyl group is cleaved off and trans-  ferred to the protruding end of the malonyl group  (bound to the other ACP molecule). This transfer dis-  places the carboxyl group, liberating C0 2 . This conden-  sation reaction is catalysed by (f-ketoacyl-ACP synthase  and results in a saturated four-carbon chain, still at-  tached to the ACP. This forms the basic skeleton of a  fatty acid (Fig. 5.10).   The nascent fatty acid has a C=0 double bond at the C3  position, which is now removed. Two new bonds  are created for C3; one with -H and one with -OH.  NADPIT+H + is oxidized as the redox partner for  this reduction. This reduction reaction is mediated by  (3-ketoacyl-ACP reductase.   A double bond is then introduced between C2 and  C3. This releases H 2 0 ('dehydration'), and is catalysed  by 3-hydroxyacyl-ACP dehydrase.   Finally this double bond is removed, and C2 and C3  are fully saturated with H atoms. This second reduction  reaction is mediated by enoyl-ACP reductase, and the re-  dox partner again is NADPH+H + . This has generated a  four-carbon acyl-group, which remains attached to its  ACP anchor (Fig. 5.11).    Fig. 5.9 Synthesis of acetyl-ACP  and malonyl-ACP. FAS = fatty acid  synthase.   P-ketoacyl-ACP synthase now transfers the four-carbon  acyl chain from ACP onto one of its own cysteine resi-  dues (cysteine contains sulphur in its side-chain). This  leaves the ACP free to receive further incoming malonyl  CoA (Fig 5.12).   Further malonyl CoA (produced by conversion of acetyl  CoA) arrives, sheds its CoA and binds to the available  ACP in a condensation reaction, forming malonyl-  ACP (Fig. 5.9). Malonyl-ACP is now ready to receive  the growing acyl chain, which is currently attached to  a Cys residue within P-ketoacyl-ACP synthase.    Fig. 5.10 Condensation: acetyl transfer to malonyl-ACP. The  dotted lines indicate where p-ketoacyl synthase breaks bonds  prior to transfer of the acetyl group.    Fig. 5.11 The condensation, reduction, dehydration and reduction  cycle. 1 = p-ketoacyl-ACP-synthase, 2 = p-ketoacyl reductase,   The enzyme P-ketoacyl-ACP synthase catalyses the  transfer (condensation) of the nascent acyl chain from  itself onto the malonyl-ACP. This marks the start of a  new cycle of fatty acid synthesis (Fig. 5.13).   At this point, the developing fatty acid undergoes six  further cyles of condensation/reduction/dehydration/  reduction/site switch/transfer (seven times in total).  This produces an acyl chain 16 carbon atoms in  length. A thioesterase enzyme then cleaves off the acyl  chain from where it is bound to the ACP, releasing a  16-carbon saturated fatty acid (palmitate). This may  later then be further elongated (via a different    Fig. 5.12 Transfer of the growing chain to p-ketoacyl  synthase. The chain binds to the S atom in the cysteine residue.    mechanism) or undergo further reactions to introduce  double bonds, generating an unsaturated fatty acid  (Figs 5.14A and 5.14B).   Fatty acid synthase can only manufacture fatty acids  up to 16 carbons in length. Additional enzymes are re-  quired to lengthen 16-carbon FA. These are located  within the mitochondria and the endoplasmic reticu-  lum. The process is complex and is not addressed  in this text; however it is important to be aware  that further elongation occurs in the ER and in  mitochondria.   This occurs in the smooth endoplasmic reticulum. A sat-  urated fatty acyl CoA, oxygen and NADH+II + are re-  quired. A mono-unsaturated equivalent fatty acyl CoA  is produced, along with NAD + and two H 2 0  (Fig. 5.15). Three enzymes are required:   In mammals, double bounds can only be introduced at  positions A4, A5, A6 and A9 due to enzyme availability:  there are four subtypes of desaturase enzymes, each re-  sponsible for introduction of a double bond at these    positions. However, FA with double bonds at the co-6  and co-3 positions, are also physiologically required,  for example, for the synthesis of eicosanoids (such as  prostaglandins). As these cannot be synthesised, they  must be obtained in the diet and are termed 'essential  fatty acids'. Linoleate (18:2 cA9,12 - double bond at  the co-6 carbon) and a-linolenate (18:3 cA9,12,15 -  double bonds at co-6 and co-3) are the two unsaturated  FA that cannot be endogenously synthesized in  humans. Some plants possess desaturase enzymes capa-  ble of introducing the double bond at the co-6 and co-3  locations, hence the presence of co-6 and co-3 oils in  plant seed oil.   Malonyl CoA availability is directly related to acetyl  CoA availability. Acetyl CoA is abundant following  carbohydrate intake (high glycolysis activity — ► high  [pyruvate] — > high [acetyl CoA]). Initially, elevated  acetyl CoA fuels sustained high activity of the TCA  cycle. Once TCA cycle products accumulate, they in-  hibit cycle activity. At this point acetyl CoA becomes  available for FA synthesis. Citrate (a TCA cycle inter-  mediate; Chapter 2) also allosterically activates the  rate-limiting enzyme of FA synthesis, acetyl CoA  carboxylase.    Fig. 5.15 Conversion of saturated fatty acids to unsaturated  Cdesaturation'). This example illustrates introduction of a c/s  double bond at the C9 position. 1 = NADH-cytochrome b 5  reductase, 2 = Cytochrome b 5 , 3 = fatty acyl CoA desaturase.  Ringed numbers indicate carbon numbering.    When FA synthesis occurs, NADPH+H + is oxidized to  NADP + , lifting the inhibition of glucose-6-phosphate  dehydrogenase and increasing PPP activity. Thus FA  synthesis activity promotes further NADPH+H + syn-  thesis. Additionally, the citrate shuttle (which is highly  active when [acetyl CoA] is high) also generates more  NADITP+H + when acetyl CoA (and therefore malonyl  CoA) is abundant.   Acetyl CoA carboxylase is also subject to hormonal reg-  ulation. Insulin (a fed-state hormone) binding to its in-  tracellular receptors activates a signalling cascade,  including activation of a phosphatase enzyme (which  dephosphorylates acetyl CoA carboxylase, activating  it). Acetyl CoA carboxylase is re-phosphorylated (inacti-  vated) in cells exposed to glucagon or adrenaline (epi-  nephrine) (fasting-state hormones). The enzyme is of  key regulatory importance because it mediates conver-  sion of acetyl CoA to malonyl CoA, thereby controlling  substrate provision for FA synthesis. Fatty acid synthesis  is promoted by insulin, but inhibited by glucagon. Addi-  tionally, insulin promotes dephosphorylation (activa-  tion) of pyruvate dehydrogenase, which converts  pyruvate to acetyl CoA (Fig. 5.16).    Fig. 5.14B Synthesis of palmitic acid. 1 = p-ketoacyl-ACP-  synthase, 2 = p-ketoacyl reducatase, 3 = 3-hydroxyacyl-ACP  dehydrase, 4 = enoyl ACP reductase, 5 = thioesterase.   Recall that NADPH+H+ inhibits glucose-6-phosphate  dehydrogenase, and in doing so slows flux through  the pentose phosphate pathway (PPP) (Chapter 4).   Triacylglycerol (TAG) molecules, in medicine more  commonly called 'triglycerides', consist of three FA  linked to glycerol by ester bonds. TAG synthesis allows  fatty acid storage in adipocytes (specialized fat storage  cells). Synthesis from glycerol and FA occurs in three  stages, and is illustrated in Fig. 5.17.    Fig. 5.16 Adipocyte: regulation of  fatty acid synthesis. For simplicity, no  shuttle mechanisms are shown, but  recall that acetyl CoA leaves the  mitochondria via the citrate shuttle  (Fig. 5.7). PPP = pentose phosphate  pathway, PDH = pyruvate  dehyrogenase.    • Formation of glycerol-3-phosphate via one of two  mechanisms. Either glycerol is phosphorylated at  C3 by glycerol kinase using ATP as the phosphate  donor, or the glycolytic intermediate dihydroxyace-  tone phosphate (DHAP, Chapter 4) is reduced by  glycerol-3-phosphate dehydrogenase   • Fatty acid activation. FA must be linked to CoA in or-  der to undergo lipogenesis. Fatty acyl CoA synthe-  tase performs this reaction   • The three activated FA are esterified to glycerol-  3-phosphate in stages.    The major physiological identity of lipids is as energy-  dense molecules, which can be rapidly mobilized. Lipid  degradation releases FA for energy provision during pro-  longed exercise or when energy utilization exceeds  dietary energy intake. Lipid breakdown includes the  following stages:   2. FA activation (molecular modification of liberated  FA necessary to obtain access to the mitochondria)   This occurs in the cytoplasm, where TAG are stored in  droplets. A hormone-sensitive lipase hydrolyzes the  ester bonds at Cl or C3, liberating one FA, leaving a  di-acyl glycerol (DAG) comprising the glycerol back-  bone with two remaining FA attached. Next, di-acyl  glycerol lipase removes a second FA, leaving mono-acyl  glycerol (MAG). Mono-acyl glycerol lipase then cleaves  off the remaining FA, freeing glycerol (Fig. 5.18).   The newly freed fatty acids are released into the blood-  stream, where they bind to proteins. They are taken up  by muscle or liver cells and oxidized. They may also    Fig. 5.17 Triacylglycerol synthesis.  The 'x' is to emphasize that the  fatty acid varies in tail length and  saturation. Also note that DHAP forms  glycerol-3-phosphate in adipocytes  since they lack glycerol kinase.   DHAP = dihydroxyacetone  phosphate. 1 = glycerol-3-phosphate  dehydrogenase, 2 = glycerol kinase,    be taken up again by adipocytes and re-esterified to participate in glycolysis or gluconeogenesis, depending  triacylglycerols. on cellular energy status.   Intracellular glycerol is phosphorylated and oxidized to  DHAP, which is then isomerized to glyceraldehyde-3-  phosphate (GAP, Chapter 4). This may then enter gly-  colysis. Alternatively, glycerol may be released into the  bloodstream and enter hepatocytes, where it may   Thiokinase enzymes convert FA to fatty acyl-CoA. This  reaction requires ATP for generation of an adenylyl in-  termediate (Fig. 5.19). The second high-energy phos-  phoanhydride bond is also hydrolyzed. Thus the  equivalent of ttw ATP is consumed during fatty acid    Fig. 5.18 Lipolysis of triaclyglycerols. Please note that 'R'  represents any acyl chain, for example -(CH 2 )x-CH 3 . DAG  lipase = diacylglycerol lipase, MAG lipase = monoacylglycerol  lipase, Gly-3-PDH = glycerol-3-phosphate dehydrogenase.    activation. Now activated, fatty acyl CoA is then ready to  enter the mitochondrial matrix via the carnitine shuttle.   Due to mitochondrial membrane impermeability to  fatty acyl CoA, a specialized mechanism exists to allow  these molecules to access their site of catabolism (the  matrix) . This is the carnitine shuttle, which is best illus-  trated diagrammatically (Fig. 5.19).   This occurs in the mitochondrial matrix and is a four-  stage process which repeats itself until the fatty acid  molecule is completely consumed.   The fatty acyl CoA is oxidized by fatty acyl-CoA dehy-  drogenase. This enzyme exists in various isoforms, each  specific for different length FA (long, medium and  short). Oxidation converts the single bond between  C2 (the '(V carbon) and C3 to a double bond. The H  atoms are accepted by the redox partner FAD, which re-  duces to FADH 2 . This can enter the ETC, generating  ~ 1.5 ATP.   Enoyl-CoA hydratase introduces an O atom and two H  atoms to the newly formed double bond between C2  and C3.   (3-Hydroxyacyl CoA dehydrogenase mediates oxidation  of the -OH group at C3, converting it to a C=0 (ke-  tone) functional group. This molecule is 3-ketoacyl-  CoA. The two H atoms removed are transferred to  NAD + (the redox partner) generating NADH+H + . This  may enter the electron transport chain (ETC), generat-  ing ~2.5 ATP.   Thiolase enzyme cleaves off Cl and C2 from 3-ketoacyl  CoA, releasing acetyl CoA. This shortens the fatty acyl  chain by two carbons. Another CoA is required to  'cap' the newly shortened molecule. This molecule is  a fatty acyl CoA, but with two fewer carbons than the  molecule in step 1 .    Fig. 5.19 Fatty acid activation and  entry to the mitochondria: the  carnitine shuttle. OMM = outer  mitochondrial membrane,   IMM = inner mitochondrial  membrane, CAT = carnitine  acyltransferase - note the two  isoforms - CAT I at the OMM and  CAT II at the IMM.   Steps 1 to 4 are repeated until the fatty acid is almost  completely degraded. The penultimate repeat leaves a  four-carbon fatty acyl CoA, which is then undergoes  steps 1— >4 as previously; however this final 'round' of  P-oxidation differs in that produces two acetyl CoA  rather than one. Note that this final step is only the case  for FA with an even number of carbons (Fig. 5.20).   This final step differs for FA with an odd number of car-  bons; the penultimate repeat leaves a five-carbon fatty  acyl CoA. This undergoes a final round of steps 1— >4,  but the final two products are one acetyl CoA molecule  and one three-carbon propionyl CoA molecule (instead  of two acetyl CoA) (Fig. 5.21).   This molecule may be converted to succinyl CoA in  three steps. This requires 1 ATP and a bicarbonate ion  (HCOs ). One of the requisite enzymes requires a    vitamin B12-derived cofactor and another uses biotin  as a cofactor. Succinyl CoA oxidation generates one  NADH+H + and one FADH 2 , together representing  4 ATP.   Every round of P-oxidation generates 1 FADH 2 , 1  NADIT+F1 + , and 1 acetyl CoA. Recall that full oxidation  of one molecule of acetyl CoA generates 10 ATP  (Chapter 2), and that FADIT 2 and NADH-f IT + generate  approximately 1.5 and 2.5 ATP per oxidized molecule,  respectively. Therefore, each round of P-oxidation repre-  sents 14 ATP.   To calculate the number of rounds of p-oxidation an  even-numbered fatty acid must undertake, divide the  number of carbons by two, and then minus 1 . In the  example of the sixteen -carbon palmitate: (1 6 -f 2)- 1 = 7,  so palmitate undergoes seven cycles of p-oxidation.    Fig. 5.20 (3-Oxidation of fatty acids.  Palmitate is used for illustration.   Palmitate (16C) undergoes seven rounds of P-  oxidation. Therefore, 14x7 = 98 ATP are produced from  these seven rounds. However, remember that the final    round generates an extra acetyl CoA, representing an ad-  ditional 10 ATP; 98+10 = 108 ATP. Also, remember  that 2 ATP are consumed during fatty acid activation.  The net generation of ATP from the complete oxidation  of palmitate is 108 - 2 = 106 ATP.   The final round of p-oxidation for an odd-numbered  FA generates a propionyl CoA rather than an acetyl  CoA. Propionyl CoA is metabolized to succinyl  CoA, a TCA cycle intermediate (Chapter 4). Entry to  the TCA cycle and complete oxidation to oxaloacetate  yields one each of GTP, FADH 2 and NADIT+H + . This  equates to 5 ATP; however, 1 ATP is consumed during  conversion of propionyl CoA to succinyl CoA, so the  energy yield represented by propionyl CoA is 4 ATP.   To calculate the number of rounds of p-oxidation an  odd-numbered fatty acid must undertake, first minus 1  from the number of carbons and then divide by two  and then minus 1 as with even-numbered FA. In the  example of the seventeen-carbon margarate: 17 - 1 =  16. (16 = 2) - 1 = 7, so margarate undergoes seven  cycles of p-oxidation.    Bearing this in mind, using margarate (C 1 7 ) as an ex-  ample, seven rounds of p-oxidation generate 14x7 = 98  ATP. The final round generates propionyl CoA, repre-  senting 4 ATP: 98+4 = 102. Also, activation consumes  2 ATP: 102 - 2 = 100. Therefore, total generation of ATP  from complete oxidation of margarate is 100 ATP  (Fig. 5.22).   This process is slower than oxidation of saturated FA,  since the carnitine shuttle is retarded by a cargo of unsat-  urated FA. The process of catabolism is similar to that of  saturated FA, however two extra enzymes are required:   • Enoyl CoA isomerase converts cis C = C bonds to the  trans configuration, allowing the FA to be recognised  by the enzymes of P-oxidation. cis configurations are  present in naturally occurring unsaturated FA, but    the double bond introduced in P-oxidation is of  the trans configuration   • NADP-dependent 2,4-dienoyl reductase participates  in metabolism of unsaturated FA with double bonds  at both even and odd-numbered carbon positions.  For example, during the metabolism of the essential  fatty acid linoleate (18:2 cA9,12) (note the double  bonds at 9 and 12), the intermediate 2,4-dienoyl  CoA is produced.   P-Oxidation occurs in peroxisomes as well as mitochon-  dria. Peroxisomes are intracellular membrane-bound  structures. The relative contributions of mitochondrial  and peroxisomal P-oxidation are unclear and appear  to be influenced by numerous factors in health and dis-  ease. However, catabolism of all very-long-chain (24 or  more carbons) fatty acids and branched chain FA occurs  in mammalian peroxisomes. This is because these FA  are unable to use the carnitine shuttle and so cannot ac-  cess the mitochondria. The actual process of P-oxidation  is the same; however be aware that:   • The enzymes differ, for example a single peroxi-  somal enzyme performs hydration and oxidation   • FADH 2 produced in the first oxidation cannot enter  the ETC; FADH 2 's pairs of H + ions and electrons are  instead transferred to molecular 0 2 , forming H 2 0 2  and generating heat, rather than ATP   • Acetyl CoA produced by each round of P-oxidation  must transfer into the mitochondria for TCA cycle  oxidation    Fig. 5.22 Comparison of ATP yields of six-carbon glucose and the six-carbon fatty acid caproiate   2 (Pyruvate ^acetyl CoA conversion yield = 1 NADPI+H + ),  and since 1 NADH+H + ~ 2.5 ATP:    • Once the fatty acyl CoA is shortened to medium  length (6-12 carbons), it is esterified with carnitine,  and then diffuses out from the peroxisome to the cy-  toplasm. It must enter the mitochondria via the car-  nitine shuttle for further (1-oxidation.   Control of lipid degradation operates at three levels.  These are described below.   Lipolysis controls oxidation of fatty acids by virtue of  regulating FA availability. Hormone-sensitive lipase  (ITSL) is a major control point: remember, this enzyme  catalyses the first step of TAG degradation, where the  first FA is cleaved off the TAG. It is activated by phos-  phorylation, mediated by a cAMP-dependent protein  kinase. Increase in intracellular cAMP increases phos-  phorylation of HSL. cAMP is synthesized from AMP  by adenylate cyclase. This enzyme is activated by hor-  mones adrenaline and glucagon - released in context  requiring mobilization of energy reserves. Insulin  lowers cAMP levels by activation of a phosphodiesterase  that degrades cAMP. Therefore, insulin ultimately  inhibits phosphorylation (activation) of HSL, inhibit-  ing lipolysis (Chapter 8).   Oxidation of fatty acids is limited by the rate they can  access their oxidation site, i.e. the mitochondria, where  (S-oxidation of all short- and medium-chain FA occurs.  Malonyl CoA, which increases when fatty acid synthesis  is active, inhibits carnitine acyl transferase I and there-  fore FA import. This prevents simultaneous synthesis  and oxidation occurring (a 'futile cycle').   Both NAD + and FAD are required to function as redox  partners in (3-oxidation reactions. When these are scarce,  due to cellular energy status being high (i.e. all the  NAD + and FAD have been converted to NADIT+H +  and FADH 2 ), FA catabolism is inhibited due to lack of  substrate availability. Conversely, when NAD + is plen-  tiful, indicating low cellular energy status, FA catabo-  lism is promoted.   Deficiencies of the enzymes catalysing the first step of (S-  oxidation result in inability to successfully oxidize fatty  acids. This imposes reliance upon catabolism of    glucose, and can cause life-threatening hypoglycaemia  when glycogen reserves and ultimately gluconeogenic  substrates become exhausted. Individuals recovering  from such metabolic crises may still suffer developmen-  tal delay. Clinical severity varies depending on how se-  verely |3-oxidation is impaired. Severe phenotypes can  cause sudden unexplained death in infants, leading to  this being a valid clinical differential for sudden infant  death syndrome ('cot death').   Medium-chain acyl-CoA dehydrogenase deficiency is  the most common disorder of |3-oxidation and with an  incidence of 1/14 600 is one of the most common in-  born errors of metabolism. Newborn screening aims  to identify individuals prior to clinical symptom  development.    Cholesterol is a 27-carbon molecule. It is an integral  structural component of all cell membranes, conferring  permeability and regulating fluidity, as well as being a  precursor of a wide range of hormones and other signal-  ling molecules. There is continuous demand for this  molecule, which is synthesized endogenously, but is  also obtained in the diet from animal fat.   Cholesterol is transported in the vascular system as a  component of lipoprotein particles. Long-term elevation  of plasma cholesterol above a certain level has potentially  serious pathological consequences for cardiovascular  health. Excessive consumption of high-cholesterol foods  contributes to development of high cholesterol, but, inter-  estingly, ingested saturated fats contribute more to blood  cholesterol levels than actual cholesterol intake.   Cholesterol is classed as a steroid because it contains a  17-carbon sterane core, a feature of all steroid mole-  cules. Cholesterol contains 27 carbon atoms in total,  all of which are derived from acetyl CoA. It contains four  fused ring structures, two cyclohexane and one cyclo-  pentane (Fig. 5.23). Different steroids vary by virtue  of different functional groups attached to these rings,  and also via the oxidation state of the rings themselves.   Cholesterol is a precursor of various important physio-  logical molecules, including:   • All steroid hormones, e.g. glucocorticoids, mineral-  ocorticoids and sex hormones    Fig. 5.23 Structure of cholesterol.  Note that the hydrogen atoms are not  detailed; appreciate that each C atom  is fully saturated. If it is bonded to three  other C atoms, one hydrogen atom  fulfils the valence requirement. If  bonded to two C atoms, two hydrogen  atoms likewise fulfil the valence  requirement, and so on.   • Vitamin D metabolites, which are mandatory for  bone health and intact immune system function  amongst their numerous diverse roles.   This occurs in the cytoplasm, particularly the liver. The  process comprises three stages:   • Stage II: progressive assimilation of isoprene units to  form squalene   • Stage III: conversion of squalene to lanosterol, and  then lanosterol to cholesterol.   • Condensation of a pair of acetyl CoA molecules,  generating the 4 C acetoacetyl CoA   • HMG-CoA synthase catalyses incorporation of a  third acetyl CoA, generating HMG-CoA. This mole-  cule is also an intermediate in ketogenesis, but the  enzymes underpinning ketogenesis are mitochon-  drial, so cytoplasmic HMG-CoA cannot be diverted  to ketogenesis.   HMG-CoA is then converted to isopentenyl pyrophos-  phate (IPP) in a two-step process:   • HMG-CoA is reduced to mevalonate by HMG-CoA  reductase. NADPH+H + is the redox partner, under-  going oxidation to NADP + . This is the irreversible,  rate-limiting step of cholesterol synthesis   • Mevalonate is then phosphorylated and decarboxy-  lated, forming IPP (the basic 5 C isoprene unit).   • IPP units are first isomerized to dimethylallyl pyro-  phosphate (DMAP)   • DMAP combines with another IPP, forming geranyl  pyrophosphate (GPP), a 10-carbon molecule   • GPP combines with another IPP unit, forming farne-  syl pyrophosphate (FPP), a 15-carbon unit   • Two FFP now unite in a condensation reaction cata-  lysed by squalene synthase. This generates squalene,  a 30-carbon molecule. This reaction requires  NADPH+H+ as a redox partner.   Ergosterol, a vital component of fungal cell  membranes, is a useful antifungal drug target,  particularly since it is absent in animal (including  human) cell membranes. For example, allylamines  such as terbinafine inhibit squalene epoxidase,  blocking synthesis of ergosterol (which is derived  from lanosterol). Imidazole antifungals such as  clotrimazole inhibit fungal synthesis of ergosterol  from lanosterol, whilst amphotericin B physically  associates with ergosterol forming a monovalent ion  channel.   • Squalene epoxidase recruits molecular oxygen and  NADPH+H + , oxidizing squalene to squalene  epoxide   • Finally, lanosterol is converted to cholesterol. This  conversion involves a complex series of reactions,  many of have yet to be characterized. In essence,  three methyl (— CH 3 ) functional groups are re-  moved, the double bond between C8 and C9 of  lanosterol is found (in cholesterol) between C5  and C6 and the double bond between C24 and  C25 is opened (Fig. 5.24).   Cholesterol, the pathway product, allosterically inhibits  the synthesis pathway's rate-limiting enzyme, HMG-  CoA reductase. Furthermore, IlMG-CoA reductase  synthesis is inversely proportional to intracellular  cholesterol. Abundant intracellular cholesterol inhibits  cholesterol synthesis by decreasing availability of the  pathway's rate-limiting enzyme.   The insulimglucagon ratio regulates cholesterol synthe-  sis activity; the greater this ratio the greater pathway  activity. To understand the mechanism, it is important  to recognise that HMG-CoA reductase is activated by  dephosphorylation and inactivated by phosphoryla-  tion. Insulin and glucagon influence phosphorylation  in the following ways:   • Insulin lowers intracellular cAMP (by up-regulation  of a cAMP-degrading phosphodiesterase). Recall  that cAMP activates a cAMP-dependent protein  kinase, which is responsible for phosphorylation  of a wide range of intracellular enzymes. Insulin  also upregulates FlMG-CoA reductase expression.  Similarly, it down-regulates expression of HMG-  CoA reductase kinase, which would otherwise  phosphorylate and inactivate the enzyme. These fac-  tors contribute to insulin stimulation promoting  endogenous cholesterol synthesis   • Glucagon and adrenaline activate adenylyl cyclase, in-  creasing intracellular cAMP and consequently activity  of the cAMP-dependent protein kinase (Chapter 8).  This phosphorylates (inactivates) HMG-CoA  reductase, inhibiting endogenous cholesterol synthe-  sis in situations where these hormones are elevated,  such as fasting. Glucagon also increases expression  of HMG-CoA reductase kinase, thus promoting  phosphorylation (inactivation) of HMG-CoA  reductase.   IPP = isopentylpyrophosphate, DMAP = dimethylallyl  pyrophosphate, GPP = geranyl pyrophosphate, FPP = farnesyl  pyrophosphate.   Statins are important drugs in lowering of pathological  cardiovascular event risk. Their mechanism of action is via  inhibition of HMG-CoA reductase (the rate-limiting  enzyme of cholesterol synthesis). This results in down-  regulated endogenous cholesterol synthesis and reduced  intracellular cholesterol. In response, cellular LDL-  receptor expression up-regulates. This results in increased  extraction of cholesterol from the blood, and lowered  plasma cholesterol. This in turn reduces the impact of  raised cholesterol on atherogenesis (formation of  atherosclerotic plaques) and thus cardiovascular health.    Although lipids are technically amphipathic (both hy-  drophobic and hydrophilic), they are predominantly  hydrophobic. This is because whilst the carboxyl group  is hydrophilic, the hydrophobic tail represents the bulk  of the molecule. The consequence of this is that lipids  are insoluble in water. Therefore, they require special-  ized transport vehicles to travel in the blood, since  blood is largely water, a polar solvent.   Lipid transport vehicles are known as lipoproteins.  A typical lipoprotein (Fig. 5.25) consists of:   Fig. 5.25 Basic lipoprotein structure. Note phospholipid  orientation; hydrophilic head groups orientated to the exterior  of the structure, hydrophobic tail groups to the interior.    • A hydrophilic surface shell, typically composed of a  phospholipid monolayer (hydrophilic head groups  orientated outwards)   • Cholesterol. This is present in both the surface shell  as free cholesterol and the lipoprotein interior as  cholesterol esters   • Apoproteins; specialized proteins of lipoproteins  (Fig. 5.26). They function as structural components,  enzyme cofactors, and receptor binding sites for the  lipoproteins   • Hydrophobic interior, consisting of the lipid cargo.  This includes triacylglycerol and esterified cholesterol.   The different classes of lipoprotein are characterized by  density, size, origin of lipid cargo, specific array of apo-  protein and physiological role. The main properties are  illustrated in Fig. 5.27.   There are two main routes by which lipids are processed  and access peripheral tissues, and these are differenti-  ated by the origin of the lipid; exogenous (from the diet)  or endogenous (synthesized physiologically).   This describes the physiological digestion, absorption  and transport of ingested lipids to tissue destination.   Al, All, B48, Cl,  CII (mature CM),  CII, E (mature  CM)   Transport of lipids absorbed from  ingested fats to the liver and rest of  body   Distribution of lipids from the liver to  the rest of the body   IDL are a consequence of VLDLs  progressively offloading lipid cargo and  thus increasing in density. They  continue in the same fashion,  distributing lipids peripherally   LDL are a consequence of IDL  progressively offloading lipid cargo and  thus increasing in density. They  continue in the same fashion,  distributing lipids peripherally   Transport peripheral cholesterol and  other lipids to the liver for biliary  excretion   Lipases in the gastrointestinal (GI) tract hydrolyse  dietary triacylglycerol, generating free fatty acids and  2-monoacylglycerol. These are absorbed across the  lumenal face of enterocytes (epithelial cells lining the  gut). Once in the enterocyte cell cytoplasm, products  of lipid hydrolysis are assembled, along with apopro-  tein B-48, into chylomicrons (CM) (refer to Fig. 5.27).   CM are extruded from enterocytes into lacteals by exo-  cytosis. Lacteals are blind-ending projections of the lym-  phatic system that protrude into intestinal villi. CM  travel in the lymphatic system to the main vascular cir-  culation via the thoracic duct. Once in the bloodstream,  CM 'steal' apoproteins CII and E from LIDL particles  they encounter, and integrate them into the stmcture  of their own surface shells.   Once chylomicrons (CM) have acquired surface CII  apoproteins, their role can now switch from transport  mode to delivery mode. This is because the newly ac-  quired surface CII apoproteins allow CM to react with  the enzyme lipoprotein lipase (LPL), since CII functions  as a cofactor for this enzyme. LPL is sited at the surface  of endothelial cells throughout the entire vascular    system. On contact with CII (CM), LPL is activated  and becomes able to perform its hydrolytic function  on triacylglycerols in the CM interior. These are hydro-  lyzed to their fatty acid and glycerol components.   FA and glycerol diffuse across the vascular lumenal  surface into endothelial cells. From here they are able  to access the body tissues, such as adipocytes or muscle  cells. They may be re-assembled into TAG for storage or  catabolized to produce energy.   Now the triacylglycerol cargo has been delivered, the  chylomicron is greatly reduced in size. The CM now  thoughtfully returns CII apoproteins to any passing  LIDL it encounters. It does not, however, return the 'sto-  len' E apoproteins. These smaller structures, with E but  not CII apoproteins, are known as chylomicron rem-  nants. Note that at no point have the CM lost their  B-48 apoproteins; these have been present throughout  and maintain the CM remnant structure. When the rem-  nants next traverse the hepatic circulation, they are  endocytosed and degraded by hepatocytes (i.e. removed  from the circulation) (Fig. 5.28).   This describes transport of hepatically synthesized tria-  cylglycerols from the liver to peripheral tissues.    Fig. 5.28 Chylomicron life cycle:  distribution of exogenous lipids. LPL =  lipoprotein lipase, HDL = high-density  lipoprotein. Endothelial LPL hydrolyzes  CM TAG cargo to FA and glycerol  which enters the endothelial cells.  Please note this diagram is schematic  not anatomically precise.   Very low-density lipoproteins (VLDL) are assembled  in hepatocytes from triacylglycerol, cholesterol and  apoprotein B100, and released into the circulation.  Like chylomicrons, VLDL 'steal' apoproteins CII and  E from HDL particles they encounter in the blood-  stream. Once these are acquired, VLDL are termed 'ma-  ture'; prior to acquisition of CII and E, they are termed  'nascent'.   Just as with chylomicrons, CII functions as a cofactor for  endothelial LPL, allowing hydrolysis of VLDL triacylgly-  cerol cargo and release of fatty acids and glycerol. The  only difference is that the triacylglycerol cargo of the  VLDL is endogenously synthesized rather than acquired  from the diet (as in chylomicrons).   Progressive offloading of cargo leaves the VLDL reduced  in size. IDL now return CII (but not apoprotein E)  to HDL they encounter in the circulation, just like  triacylglycerol-depleted chylomicrons. During this en-  counter, LIDLs also donate cholesterol esters (mediated  by plasma cholesterol ester transfer protein), and in  exchange receive phospholipids and triacylglycerols  from the VLDL. The resulting lipoprotein is now known  as VLDL remnants (a term pretty much equivalent to  intermediate-density lipoprotein (IDL)).   'Intermediate density’ in this context refers to density  intermediate to 'very low density' and ‘low density',  rather than intermediate between ‘high density' and  ‘low density'.    Some remmants are endocytosed and dismanded by  the liver, but those remaining in the circulation are  further depleted of their TAG cargo, progressively re-  ducing in size (and increasing in density). Further  encounters with HDL results in further depletion of  TAG, further loss of CII and increase in cholesterol  ester load. This results in remnants (IDL) progressively  transforming into LDL.   LDL apoprotein E binds to endothelial LDL-receptors,  mediating internalization of the entire particle by endo-  cytosis. Internalized LDL fuse with lysosomes, lysosomal  enzymes dismantling the LDL and releasing cholesterol  intracellularly. This may enter various synthetic path-  ways, or may be esterified with fatty acids to form choles-  terol esters. This esterification is mediated by acyl-CoA:  cholesterol acyl transferase (ACAT) (Fig. 5.29).   I IDL (the smallest, densest lipid transport vehicle) are  assembled in the liver and intestine from apoproteins  AI, All, E and phospholipids. LIDL donate apoproteins  to chylomicrons and VLDL particles. HDL also play a  very important role in cholesterol metabolism; by   Fig. 5.29 Transport of endogenously synthesized lipids via  VLDL, IDL and LDL particles. VLDL CII acts as a cofactor for  endothelial LPL, allowing hydrolysis of the TAG cargo and  offloading the products FA and glycerol. ACAT = acyl-CoA:  cholesterol acyltransferase.    transporting excess cellular cholesterol through the  bloodstream to the liver for excretion in bile.   Remember that intracellular cholesterol inhibits HMG-  CoA reductase, inhibiting endogenous synthesis and  reducing intracellular cholesterol. Statins also inhibit  this enzyme, with the same effect. Note also that  reduced intracellular cholesterol enhances transcription  of the LDL-receptor, increasing the amount of  cholesterol the cell can assimilate via LDL. This up-  regulation of LDL-receptor expression is the main  physiological mechanism for lowering plasma  cholesterol, and is also the mechanism by which statins  exert their therapeutic effects.   Prior to integration into HDL, cholesterol undergoes es-  terification. The polar hydroxyl group at C3 (see  Fig. 5.23) is hydrophilic, and cholesterol tends to locate  itself superficially in lipid phases, such as the phospho-  lipid monolayer. However, molecular 'shielding' of this  hydroxyl group allows cholesterol to be packaged in  the hydrophobic interior of lipoproteins, allowing for  increased transport capacity.   'Shielding' involves covering the -OH group with a  fatty acid acyl group. This is an esterification reaction  and the reaction product is a cholesterol ester. This  may occur intracellularly, in which case it is catalysed  by acyl CoA: cholesterol acyl transferase (ACAT). It  may also be performed by LIDL, in which case it is  catalysed by plasma lecithinxholesterol acyl transferase  (LCAT). In the case of HDL, the fatty acid used to mod-  ify cholesterol is sourced from the phospholipid phos-  phatidylcholine, which is present within the LIDL shell.   LIDL apoprotein AI interacts with the ATP-binding cas-  sette transporter 1 (ABCA1), aka cholesterol efflux reg-  ulatory protein. ABCA1 is a cellular cholesterol efflux  pump, ejecting excess cholesterol from cells into blood  plasma. This free cholesterol is then esterified by plasma  LCAT, and internalized to LIDL interior.   LIDL interacts with (and removes cholesterol from) pe-  ripheral cells such as macrophages, which may contrib-  ute to the well-established association between higher  LIDL levels and reduced atheromatous disease.   Atheromata (atherosclerotic plaques) are arterial wall  deposits, which may be fibrous or lipid-rich. Lipid-rich  atheromata consist of cells, connective tissue elements,  lipids and debris. They are medically significant since  they narrow arterial lumens and, if they rupture,  promote thrombus formation and rapid occlusion. In  the coronary arteries, complete occlusion results in  ischaemia and necrosis of downstream heart muscle; a  myocardial infarction.   Dyslipidaemia is elevation of plasma triglycerides or to-  tal cholesterol (or both) resulting from an abnormality  of lipid metabolism or uptake. This can be due to a de-  ficiency in either:   The term 'triglycerides' is used synonymously with  'triacylglycerol', however as ‘triglycerides' is used in  clinical biochemistry and medicine, this term will be  used in this section.    Inappropriately high cholesterol or triglycerides  result in equally inappropriate deposition of lipid in  multiple locations, including the skin, connective tis-  sue, the corneal limbus and arterial walls. This results    in development of palmar and tendon xanthomata,  periorbital xanthelasma, corneal arcus and atheromata  respectively. These features comprise the clinical pheno-  type of dyslipidaemia. Severe hypertriglyceridaemia  may also cause acute pancreatitis.   Two clinically important examples of genetic dyslipi-  daemia are discussed below, but be aware that there are  many; consult a reference textbook for more detail.   Familial hypercholesterolaemia is the most common  inherited dyslipidaemia. This condition results from an  absent or dysfunctional LDL-receptor (95%) or defective  B 100 apoproteins (remaining 5%) . This results in reduced  (heterozygotes) or absent (homozygotes) cellular uptake  of circulating LDL, resulting in increased plasma LDL.   Homozygotes have extreme hypercholesterolaemia,  exhibit all the clinical features of the hyperlipidaemias  phenotype and experience childhood onset of symp-  tomatic coronary atherosclerosis. In heterozygotes the  hypercholesterolaemia is less pronounced, since half  the receptor population is functional, but individuals  are still at increased susceptibility of cardiovascular  disease.   This is a very rare, autosomal recessive condition. It is  defined by gross elevation of plasma chylomicrons. This  occurs because of deficiency or absence of either the en-  zyme LPL or its cofactor, apoprotein CII. This results in  hypertriglyceridaemia from the extremely high plasma  chylomicron. This causes frequent pancreatitis, and  strict avoidance of dietary fat and alcohol is mandatory  to manage symptoms.   Potentiate the hepatocyte PPAR-a receptor signaling cascade. Amongst  the many effects, hepatic (S-oxidation of FA is enhanced and increased  synthesis of HDL particles ensues   These bind and 'sequester' bile acids in the gut lumen, preventing their  reabsorption. Their subsequent loss reduces the total capacity for lipid  absorption. Now rarely used   Localizes to enterocyte apical membranes (within the gut lumen). Here it  inhibits absorption of dietary cholesterol. Decreased cholesterol absorption  results in up-regulation of LDL receptors, increasing LDL clearance from  the blood   This is raised total plasma cholesterol (>5.0 mmol) in  the absence of an inherited disorder of lipid metabo-  lism. It is typically accompanied by raised LDL  (> 3.0 mmol). It is at least partially attributable to a  high intake of saturated fat, and is partially correctable  by diet and lifestyle modification.   However, since elevated plasma cholesterol has such  serious implications for cardiovascular health, a num-  ber of therapeutic drugs have been developed to manage  this risk (Fig. 5.30). Statins are by far the most com-  monly used preparations.    In biochemistry, the term 'ketones' (also called 'ketone  bodies') refers to three particular molecules. They are  all acetyl CoA-derived, and function as substrates  for ATP generation. The three biologically significant  ketones are:   They are continuously synthesized in the liver at low  levels, but when intracellular glucose is low, their syn-  thesis is up-regulated.   Ketone synthesis, or 'ketogenesis', occurs in the mito-  chondrial matrix of hepatocytes. Acetyl CoA is the sub-  strate of the synthesis pathway. It may derive from:   • Catabolism of the ketogenic amino acids leucine,  tryptophan and isoleucine   Three acetyl CoA molecules condense to form HMG-  CoA, just as in the cholesterol synthesis pathway  (Fig. 5.24). However, cholesterol synthesis occurs in  the cytoplasm, and ketogenesis in the mitochondria,  so HMG-CoA can only participate in one of these path-  ways, determined by its location. HMG-CoA then re-  leases acetyl CoA (mediated by HMG-CoA lyase),  forming acetoacetate.    Fig. 5.31 Ketone synthesis. HMG-CoA = 3-hydroxy-2-  methylglutaryl-CoA. 1 = p-ketothiolase, 2 = HMG-CoA  synthase, 3 = HMG-CoA lyase, 4 = p-hydroxybutyrate-  dehydrogenase.   Note that catabolism of the amino acids tyrosine,  phenylalanine, leucine and lysine generates acetoacetate,  which may integrate into the ketogenesis pathway at this  point. Acetoacetate may spontaneously decarboxylate,  forming acetone, or may be reduced by 3-hydroxybutyrate  dehydrogenase to 3-hydroxybutyrate. This latter reaction  requires NADH+H + as a redox partner and represents  the main fate of acetoacetate.   Regulation of ketone synthesis is primarily by substrate  (acetyl CoA) availability. There are two main meta-  bolic contexts in which ketogenesis is particularly ac-  tive; high lipid catabolism activity and high rate of  gluconeogenesis.   When gluconeogenesis is active, oxaloacetate is diverted  to gluconeogenesis, rather than participating in the TCA  cycle. Reduced oxaloacetate availability has the conse-  quence of less acetyl CoA consumption by the TCA  cycle. This increases acetyl CoA availability for ketogen-  esis. A high rate of gluconeogenesis results in increased  ketogenesis.   In the context of lipid catabolism, plasma FA is elevated.  This provides abundant substrate for f5-oxidalion by nu-  merous tissues. In the liver, increased (3-oxidation in-  creases hepatocyte mitochondrial acetyl CoA. This  increases acetyl CoA availability for ketogenesis.   During prolonged fasting, once glycogen reserves have  been exhausted, hepatic gluconeogenesis is up-regulated,  since plasma glucose levels must be maintained.  Additionally, lipid catabolism becomes predominant.  Therefore, in starvation, both metabolic scenarios  promoting ketone synthesis are present. This explains  the observed rise in plasma ketones (Chapter 8).   Untreated or inadequately treated (insufficient insulin  replacement) type 1 diabetic patients are insulin-  deficient. This insulin insufficiency is accompanied by  elevation of hormones such as glucagon and adrenaline  (epinephrine). These promote widespread lipid mobili-  zation, leading to an increase in plasma FA level and  ultimately increased ketogenesis via the mechanisms  discussed previously. In addition, hepatic gluconeogen-  esis is highly active during insulin insufficiency (even  though blood glucose is elevated), due in part to  the low insulimglucagon ratio (Chapter 4). Insulin  insufficiency promotes ketogenesis (leading to increased  plasma ketones) by both mechanisms. Metabolic acido-  sis and elevation of plasma ketones secondary to insulin  insufficiency is known as diabetic ketoacidosis (DKA).  DKA is uncommon in type 2 diabetes.   Ketoacidosis results from ketone accumulation. Acetoa-  cetate and 3-hydroxybutyrate are chemically carboxylic  acids; at physiological pH they are in the anionic form.    dissociated from their FI + ions. If ketones production is  excessive and prolonged, these I l + ions cause acidosis  (plasma pFl < 7.35, or [H+] > 10 7 3S ) once normal  physiological buffering systems are overwhelmed. This  scenario is known as ketoacidosis; acidosis due to path-  ological ketone elevation.   It is important to understand the difference between  the terms 'ketosis' and 'ketoacidosis'. 'Ketosis' describes  an elevated level of ketones in the blood, and is a nor-  mal consequence of ketone synthesis. 'Ketoacidosis' is a  pathological state occurring when ketones accumulate  to the extent they cause significant metabolic acidosis.  Ketoacidosis is a feature of decompensated diabetes  mellitus (particularly type 1; see Chapter 8).   Ketones are oxidized in mitochondria, generating acetyl  CoA. In this way, ketones may be thought of as 'alterna-  tives' to glucose, since their oxidation generates acetyl  CoA to fuel TCA cycle oxidation, ultimately generating  ATP to meet the cell's energy requirements.   In starvation, once glycogen reserves are exhausted,  plasma glucose is maintained at the expense of muscle  protein breakdown. This provides amino acids to sus-  tain hepatic gluconeogenesis, but with the unhelpful  side-effect of further weakening the starving individual.    Fig. 5.32 Ketone catabolism. 1 = p-hydroxybutyrate  dehydrogenase, 2 = p-ketoacyl-CoA transferase, 3 =  P-ketothiolase.    Existence of ketones as 'back-up' oxidation substrate  reduces the demand placed on gluconeogenesis to pro-  duce glucose for cellular oxidation in the various  tissues.   Ketone catabolism can occur in all mitochondria-  containing cells (except hepatocytes, since the enzyme  3-ketoacyl-CoA transferase is absent in the liver).  The pathway is shown in Fig. 5.32. NAD + is required  as a redox partner for oxidation of 3-hydroxybutyrate  to acetoacetate, producing NADH+H + ; however since  the reverse of this reaction occurs during original  synthesis of 3-hydroxybutyrate from acetoacetate, there  is no net generation of NADH+H + .   In hypoglycaemic states, the CNS is forced to par-  tially switch from glycolysis to ketone oxidation for  acetyl CoA production. Fatty acids cannot cross the    blood-brain barrier and so are of no use to the CNS  for acetyl CoA generation; thus the CNS relies on  glucose and ketones to meet its metabolic demands.  The CNS is able to gradually increase ketone utilization  relative to glycolysis during sustained hypoglycaemia,  but unable to make a complete transition. This persis-  tent reliance on glucose accounts for why blood glucose  levels must be always maintained.   Surprisingly, cardiac myocytes preferentially catabolize  fatty acids in all metabolic contexts except starvation.  This means that these cells utilize (3-oxidation of fatty  acids (rather than glycolysis) to generate acetyl CoA  for TCA cycle oxidation and energy generation. In star-  vation, when plasma FA gradually diminishes, cardiac  myocytes are able to switch to ketone catabolism to  meet their energy requirements.   • Appreciate the need for and outline the mechanism of the urea cycle    Amino acids link with each other via peptide bonds, form-  ing a polymer ('polypeptide'). The name 'protein' is re-  served for polypeptides containing 50 or more amino  acid residues. A protein may also consist of a single poly-  peptide, or may be a number of closely associated poly-  peptides. The linear order of the amino-acid sequence  in a polypeptide (protein) is termed the primary structure.   Amino acids also associate with each other via a sec-  ond type of bond. They form hydrogen bonds between  the oxygen atom of the C = 0 component of the car-  boxyl group of one amino acid and the amino N atom  of another (see Fig. 6.1). These hydrogen bonds are  responsible for the spontaneous formation of protein  'secondary structure'. These are ubiquitous architectural  features, such as a-helices and (3-pleated sheets.   Protein 'tertiary structure' describes the three-  dimensional structure that a polypeptide adopts, whilst  retaining the secondary structure features. Finally,  'quaternary structure' describes the interaction between  different polypeptide chains - how they associate with  each other to form the complete protein.    Proteins are composed of amino acids. Amino acids all  share a common structural 'skeleton', which includes an  amino group (the N-terminus of the protein molecule)  and a carboxyl group (the C-terminus) (Fig. 6.1). Individ-  ual amino acids have different side-chains ('R-groups'),  which confer them with different properties (Fig. 6.2).  Amino acids form bonds with each other via peptide  bonds (Fig. 6.3).    There are two types of reaction that one must appreciate  before understanding amino acid metabolism. These  are transamination and oxidative deamination. Both  perform the same function; removal of the amino group  (— NH 2 ) from the carbon skeleton of the amino acid.    Transamination: conversion of any  amino acid into alanine, glutamate  or aspartate   The amino group of any amino acid is transferred  to a-ketoglutarate, converting it to glutamate. The  deaminated molecule is now structurally a keto-acid.  This is catalysed by the aminotransferase enzymes  (Fig. 6.4), all of which use pyridoxal phosphate (PLP)  as a co-factor. The second reaction converts the newly  formed glutamate into alanine, aspartate or glutamate,  depending on whether the glutamate reacts with pyruvate,   Precursor for nitric oxide. Role in the urea  cycle. Basic R-group   Hydrophobic R-group; hence normally  located in the interior of protein structures   Like isoleucine, leucine has a  hydrophobic R-group; hence  normally located in the interior  of protein structures   -OH group in the R-group is a site for  post-translational modification such as  O-linked glycosylation   Precursor to catecholamine hormones  noradrenaline, adrenaline and dopamine, and  also the pigment melanin. Tyrosine precursor   Hydrophobic R-group; hence normally located  in the interior of protein structures   In muscle, via the glucose-alanine cycle,  alanine fulfils the role glutamine performs in  all other tissues, i.e. transport of nitrogen to  the liver for urea synthesis   Amino group donor of the urea cycle. Role in  purine and pyrimidine synthesis. Acidic  R-group. Precursor of threonine   Sulphur-containing R-group allows important  structural role in proteins (disulphide bridges).  Component of glutathione   Able to cross the blood-brain barrier. Functions  to transport nitrogen from most tissues to the  liver for urea synthesis   'Imino' amino acid. Central neurotransmitter.  Important structural component of collagen.  Role in synthesis of purines, porphyrins,  creatine and glutathione. Participation in drug  metabolism. Component of glutathione   Also an 'imino' amino acid. This is the only  amino acid where the side chain incorporates  the a-amino group of the amino acid skeleton   Contribution of activated one-carbon units  required for tetrahydrofolate conenzymes.  Required for biosynthesis of cysteine and  phospholipids    Fig. 6.3 Formation of a peptide bond between two amino acids.   In these reactions, the amino group of an amino acid is  removed (resulting in the formation of a-ketoglutarate)  and released as ammonia (NH 3 ) . This enters the urea cy-  cle for excretion. The redox partner for this oxidation  is NAD + (Fig. 6.5). The main substrate for oxidative  deamination is glutamate, since most amino acids  are degraded (as described previously) by the transam-  ination generating glutamate.    oxaloacetate or a-ketoglutarate respectively. The transam-  inase enzyme catalysing this second reaction is named  according to the end product; e.g. alanine transaminase  (ALT) or aspartate transaminase (AST).   Since amino acids are present at much higher concentra-  tions intracell ularly than extracellularly, amino acid im-  port requires energy. Transporters exist that couple ATP   Hartnup's disease: autosomal recessive absence of this carrier  system. These amino acids cannot be absorbed across the gut  or reabsorbed from renal ultrafiltrate, leading to deficiency of  these particular amino acids   Cystinuria: autosomal recessive condition resulting from  absence of this carrier system. This causes inability to reabsorb  cysteine from renal ultrafiltrate, leading to crystallisation of  cysteine and stone formation in the renal tract   Iminoglycinuria: autosomal recessive illness resulting from the  carrier deficiency, causing failure of both absorption of dietary  proline and glycine and reabsorption from renal ultrafiltrate.  Important components of collagen    hydrolysis (directly or indirectly) to amino acid import.  Transporters are grouped by specificity for R-group  properties (Fig. 6.6).   This process imports a wide range of amino acids into  cells. It is active, requiring three ATP molecules per  amino acid imported, but rapid and high-capacity.  It utilizes the peptide glutathione   • Glutathione donates y-glutamyl residue to the trans-  membrane enzyme y-glutamyl transpeptidase  (GGT), which transfers y-glutamyl to the exterior  face of the membrane. The glutathione Cys-Gly  'remnant' remains intracellular    • At the exterior surface of the cell membrane, the  y-glutamyl is attached to the awaiting amino acid,  also catalysed by GGT. This modification allows  the y-glutamyl-amino acid to be imported by GGT   • Once imported, the y-glutamyl component releases  the amino acid intracellularly. The y-glutamate is con-  verted to 5'oxoproline. Both these functions are per-  formed by intracellular y-glutamyl cyclo-transferase   • 5'oxoproline is then converted to glutamate by oxo-  prolinase, utilizing 1 ATP   • Glutamate recombines with Cys forming y-Glu-Cys,  again utilizing 1 ATP. This is catalysed by y-Glu-Cys  synthetase   • y-Glu-Cys then reacts with Gly, re-forming glutathi-  one. This is catalysed by glutathione synthetase,  again utilizing 1 ATP (Fig. 6.7).   Fig. 6.7 General amino acid import: the y-glutamyl cycle.  GLU = glutamate, CYS = cysteine, GLY = glycine.   Essential amino acids are mainly those that humans  cannot endogenously synthesize de novo and thus re-  quired in the diet. However, arginine, phenylalanine  and methionine are considered essential although  they can be synthesized endogenously. This is for the  following reasons:   • Arginine is not synthesized at a fast enough rate to  meet physiological demand, mainly because the  majority of arginine synthesis occurs in the context  of nitrogen excretion during the urea cycle   • Phenylalanine likewise is not synthesized suffi-  ciently rapidly for normal requirements, since a large  amount is diverted towards tyrosine synthesis   • Methionine, similarly, is required in high quantities  to synthesize cysteine, and is considered 'essential'  since maximal endogenous synthesis still requires  dietary supplementation.    The non-essential amino acids are those that can be syn-  thesized endogenously. Their synthesis is described  below.   These are grouped together since glutamate is used as a  substrate for synthesis of the other three.   a-Ketoglutarate is converted to glutamate by transami-  nation. This is, as described earlier, mediated by gluta-  mate dehydrogenase (Fig. 6.4).   • Reduction of glutamate to glutamate y-semialdehyde  (redox partner is NADPH + I I + )   • Glutamate y-semialdehyde spontaneously undergoes  molecular rearrangement to lower-energy pyrroline-  5-carboxylate   • Pyrroline-5-carboxylate is reduced to proline (reac-  tion redox partner is NADPH + H + ).   Like proline synthesis, glutamate is first reduced to  glutamate y-semialdehyde (redox partner NADPH + H + ) .  This is then transaminated, forming ornithine. Ornithine  enters the urea cycle, ultimately forming arginine.   • Aspartate is formed by transamination of oxaloacetate.  This is catalysed by aspartate-aminotransferase, and  the amino group is helpfully provided by glutamate  (which is converted to a-ketoglutarate)   • Aspartate is then converted to asparagine by as-  paragine synthetase. The amide group is donated  by glutamine. This reaction consumes an ATP  (Fig. 6.9).   The majority of serine synthesis occurs in cell cyto-  plasm. This utilizes 3-phosphoglycerate (a glycolytic  intermediate; Chapter 4). The pathway involves three  steps: oxidation, transamination and hydrolysis   This occurs mitochondrially, via two routes. One is the  generation of glycine from serine (serine hydroxy-  methyl transferase). The other (not illustrated) is cata-  lysed by glycine synthase, uses NADH + I I + as a redox  partner and requires the ammonium ion (NH 4 + ),  C0 2 , and N 5 N 10 -methylene-THF as substrates.    Fig. 6.10 Serine and glycine synthesis. As many of the  enzymes in serine synthesis have not yet been conclusively  identified, some names are omitted.   Cysteine is formed from methionine and serine in the  cytoplasm. The synthesis pathway is not yet fully iden-  tified; however, the main steps are shown in Fig. 6.11.  Deficiency of the enzyme cystathionine- (3-synthase  results in accumulation of the intermediate homo-  cysteine. This causes homocystinuria; a multisystem  disorder characterized by elevated serum and urinary  homocysteine.   Tyrosine synthesis is simply hydroxylation of phenylala-  nine (Fig. 6. 12). The reaction is highly exergonic, explain-  ing why phenylalanine cannot be synthesized from  tyrosine. The reaction is catalysed by phenylalanine hy-  droxylase and requires tetrahydrobiopterin as a cofactor.   The autosomal recessive disease phenylketonuria  (PKU) arises from deficiency or absence of  phenylalanine hydroxylase. Tyrosine cannot be  synthesized endogenously, making it an essential  amino acid in individuals with PKU. Phenylalanine  accumulates, saturating the large-neutral R-  group/aromatic R-group amino acid transporter  at the blood-brain barrier. This denies other  molecules relying on this transporter access to the  developing CNS. This manifests with progressive brain  damage and seizures. Treatment is by strict dietary  exclusion and tyrosine supplementation.   Tyrosine is oxidized by the enzyme tyrosinase to  produce the pigment melanin. Tyrosinase deficiency  results in failure of melanogenesis, leading to  oculocutaneous albinism type 1 . Ocular complication  arising from iris translucency and reduced retinal  pigmentation are common.   Alanine is synthesized by transamination of pyruvate to  alanine (Fig. 6.13). This is catalysed by alanine amino-  transferase (cofactor PLP), and requires glutamate to  donate the amino group.    Amino acids are essential as the building blocks of pro-  teins; however they also fulfil important functions as in-  dividual molecules; for example, the majority of central  neurotransmitters are derived from amino acids.   Serotonin (5-hydroxytryptamine, 5-HT) is synthesized  from tryptophan. This is a two-step pathway (Fig. 6.14):  tryptophan is hydroxylated to 5-hydroxytryptophan, by  tryptophan hydroxylase (cofactor tetrahydrobiopterin).  5-OH-tryptophan is then decarboxylated by amino acid  decarboxylase (cofactor PLP) to form 5-HT.   These three neurotransmitters ('catecholamines') are all  derived from tyrosine. The pathway is best shown as a  diagram (Fig. 6.15).   Two tyrosine residues in the same polypeptide (the  glycoprotein thyroglobulin) are iodinated at their R-  groups, forming mono-iodotyrosine or di-iodotyrosine  depending on whether iodination occurs at one or two  sites. These two iodinated tyrosine residues react with  each other. This forms T3 (tri-iodothyronine) when  one di-iodotyrosine combined with another mono-  iodotyrosine, and T4 (thyroxine) if two di-iodotyrosines  combined with each other. Generation of T3 and T4  (both termed 'thyroid hormones') is catalysed by thyroid  peroxidase. The newly formed thyroid hormones are  excised from their thyroglobulin scaffold by lysosomal  enzymes and are then released in a regulated fashion  by the thyroid gland. T3 is much more active than T4;  T4 can also be peripherally converted to T3.    As nitrogen breakdown yields toxic products, and biolog-  ical availability of nitrogen is limited, the balance be-  tween synthesis and breakdown of nitrogen-containing    ('nitrogenous') molecules is closely regulated. Nitroge-  nous molecules include amino acids and their  derivatives, nucleotides, nucleic acids and, of course, pro-  teins. Nitrogen presence in the body is largely accounted  for by proteins, and therefore nitrogen balance is primar-  ily determined by the balance between synthesis and  degradation of proteins.   Positive nitrogen balance describes the scenario  where N intake exceeds N excretion. This is seen during  growth, pregnancy, and wound healing; all situations  where new body tissue (protein) is being generated.  Negative nitrogen balance describes the converse sce-  nario; a net loss of N occurs due to excretion exceeding  intake. This can be due to:   • Combination of both: cachexia (emaciation associ-  ated with severe illness).   In order to excrete nitrogen, proteins are degraded  to their constituent amino acids, which are then  processed so that the nitrogen they contain can be off-  loaded. This offloading can be conceptually divided  into two stages; removal of the amino group and  formation of urea.   This may occur via either transdeamination or transam-  ination. Each route generates different products, but  both are able to enter the urea cycle.   'Transdeamination' is where amino acids are converted  to their corresponding keto acid (which is further  metabolized) and NH 3 is released, entering the urea cy-  cle. It involves sequential transamination and oxidative  deamination:   • Cytoplasmic transamination, where aminotransfer-  ases 'convert' the amino acid to glutamate; in reality  the amino group is transferred to a-ketoglutarate,  generating glutamate, whilst the amino acid skeleton  is released as the corresponding keto acid   • This is followed by mitochondrial oxidative  deamination of glutamate (Fig. 6.5) releasing  a-ketoglutarate and NH 3 (Fig. 6.16).   Transamination was described earlier in the chapter  (Fig. 6.4). In the context of nitrogen excretion, the sec-  ond transaminase reaction generates aspartate and is  mediated by AST. This aspartate is then able to enter  the urea cycle (Fig. 6.19).   Once the amino group has been removed (from an  amino acid), the carbon 'skeleton' remains. This skele-  ton (the corresponding keto-acid) will vary in structure  according to the individual amino acid. Each structure  is further catabolized to form intermediates of the  TCA cycle or ketogenesis pathways. Figure 6.17 provides  further detail.   Amino acids whose skeletons are ultimately processed  into TCA cycle intermediates are termed 'glucogenic'.  This term is used since all TCA cycle intermediates ulti-  mately form oxaloacetate, which can be converted to  glucose via gluconeogenesis. Some amino acid catabo-  lism products, however, do not correspond to any of  the TCA cycle intermediates, instead becoming interme-  diates of the ketogenesis pathway; acetoacetate and ace-  tyl CoA (Chapter 5 ) . These are termed 'ketogenic' amino  acids. Some amino acids are able, depending on their  route of catabolism, to generate intermediates of both  ketogenesis and the TCA cycle. Others are only able  to ultimately produce intermediates of one or other  pathways:   • Isoleucine, phenylalanine and tyrosine are both ke-  togenic and glucogenic   The BCAA are isoleucine, leucine and valine. They  are degraded primarily in muscle tissue, since the liver  (the usual site for amino acid catabolism) lacks the  specific aminotransferase enzyme required to cleave  off the amino group from BCAA. Their catabolism is  as follows: BCAA aminotransferase transfers the amino  group from BCAA to a-ketoglutarate. This forms gluta-  mate and a branched-chain a-ketoacid. This a-ketoacid  then undergoes oxidative decarboxylation, mediated  by mitochondrial branched-chain a-ketoacid dehy-  drogenase complex (cofactor PLP). This produces  branched-chain acyl-CoA derivatives, which are then  dehydrogenated by two separate dehydrogenase  enzymes (Fig. 6.18). Each separate BCAA derivative  undergoes oxidation reactions reminiscent of (1-  oxidation, ultimately producing:   Deficiency of mitochondrial branched-chain a-  ketoacid dehydrogenase results in a failure to  catabolise all three branched-chain amino acids.  Plasma levels of lie, Leu and Val accumulate, leading to  severe neurological dysfunction. The BCAA are also  seen at high concentration in the urine as well as the  blood, ‘branched-chain ketoaciduria’. This gave rise to  the name ‘maple-syrup urine disease', as the odour of  affected individuals' urine is reminiscent of maple  syrup.    The urea cycle, aka the ‘ornithine cycle', is the process  via which ammonia (NIT 3 ) is converted to urea  ((NH 2 ) 2 C = 0) in the liver. The cycle also accepts  nitrogen in the form of aspartate and integrates it  into the urea molecule. The urea cycle operates within  hepatocytes across two cellular compartments; the  cytoplasm and the mitochondrial matrix. The cycle  and relevant enzymes is detailed in (Fig. 6.19).  In brief:   • Ammonia combines with a bicarbonate ion and  2 ATP molecules, forming carbamoyl phosphate  (catalysed by carbamoyl synthetase-1)   • Citrulline then combines with aspartate, forming  arginosuccinate. ATP is hydrolyzed   • Arginine remains in the urea cycle for the final reac-  tion; hydration of arginine generates urea and re-  generates ornithine   Fumarate produced by cleavage of arginosuccinate is  converted by cytoplasmic fumarase to malate. This ma-  late may follow one of two routes:   • Further oxidize to oxaloacetate (catalysed by malate  dehydrogenase). This is then transaminated, gener-  ating aspartate, by AST. Aspartate can re-enter the  urea cycle, as shown in Fig. 6.19.   Undergoes oxidative deamination (Fig. 6.5), forming  a-ketoglutarate, NH 3 and NADH + H 1   Cleaved to form ornithine and urea in the urea cycle.  Ornithine is transaminated to form glutamate y-  semialdehyde, which converts to glutamate   Histidine catabolism involves a complex, seven-enzyme  pathway culminating in formation of glutamate. Initial  de-amination is mediated by histidase: deficiency of this  enzyme causes histidinaemia   Glycine is converted to serine (which is catabolized  ultimately to pyruvate) by serine hydroxymethyltransferase   Methionine donates its methyl group to various acceptors  via the intermediate S-adenosyl-methionine (SAM). This  generates propionyl CoA, which is metabolised to succinyl  CoA by the odd-chain fatty acid oxidation pathway   Serine-threonine dehydratase coverts threonine to a-  ketobutyrate, which is converted to propionyl CoA and  C0 2 . Propionyl CoA is metabolized to succinyl CoA by the  odd-chain fatty add oxidation pathway   Tyrosine aminotransferase catalyses the first step in this  5-step pathway, culminating in fumarate and acetoacetate.  Deficiency of homogentisic acid dioxygenase, one of the  tyrosine catabolism enzymes, causes alkaptonuria  (accumulation of the intermediate homogentisate)   Branched chain amino acid catabolism: see following  section. Catabolism yields acetyl CoA and succinyl CoA   The complex catabolism of tryptophan has not yet been  fully elucidated; however the TRY catabolism pathway  culminates with acetyl CoA    Fig. 6.18 Branched-chain amino acid (BCAA) catabolism. 'B'  indicates a branched-chain ' R' group.   Aspartate may be derived from urea cycle fumarate  (as described previously). Alternatively, it may origi-  nate from transaminated oxaloacetate. Any amino acid  can be transaminated (first via the relevant aminotrans-  ferase, and then via AST) to generate aspartate.   The urea cycle is the main physiological mechanism  for nitrogen elimination. Produced in the liver, urea  is excreted from the body via specialized mechanisms  in the kidney. The urea is nearly 50% nitrogen by mo-  lecular weight and is an efficient nitrogen transport  molecule. It diffuses freely across cell membranes  and is highly soluble in plasma; therefore, it is easily  transported from the liver to the kidneys via the  bloodstream. It is electrically neutral and does not af-  fect blood pH.   Sustained increases in nitrogen intake result in in-  creased expression of urea cycle enzymes. Therefore,  the cycle can operate at a faster rate, processing more  NH 3 and aspartate and thus eliminating more nitrogen.    Sustained high-protein intake is the most common con-  text in which the urea cycle is up-regulated, since more  ingested protein results in more nitrogen requiring  processing for elimination.   Short-term regulation of the urea cycle is mainly via  modulation of mitochondrial carbamoyl-phosphate  synthase-I (CPS-I) activity, the enzyme that uses ATP,  Nll 3 and I IC0 3 to form carbamoyl phosphate. This  enzyme is allosterically activated by N-acetyl gluta-  mate, itself formed by the combination of acetyl  CoA and glutamate. This is appropriate, since intra-  cellular glutamate will rise following catabolism of  ingested protein. Remember that the amino group  of amino acids undergoing catabolism is transferred  to a-ketoglutarate, forming glutamate - this is why this  amino acid in particular is a sensitive indicator of  amino acid catabolism.   Deficiencies of each urea cycle enzyme and carriers  have been identified. These are rare, and inheritance  varies according to enzyme. For example, the most  common inborn error of metabolism of the urea cycle,  ornithine trancarbamoylase deficiency, follows an  X-linked recessive pattern of inheritance. However,  all disorders of the urea cycle result in a failure  to synthesize urea and accumulation of NII 3 :  'hyperammonaemia'.   Ammonia toxicity manifests initially with  decreased level of cognition, tremors and blurred  vision, progressing with increasing [NH 3 ] to  seizures, brain damage, coma and death. This occurs  soon after birth with inborn enzyme deficiencies,  or develops gradually with progressive failure  of a previously functioning urea cycle, e.g. in  liver failure. In this scenario progressive loss of  functioning hepatocytes results in failure of  the urea cycle to function at a rate sufficient to  prevent accumulation of NH 3 , resulting in  hyperammonaemia.   Proteins, both structural and functional, undergo con-  tinuous synthesis and degradation. If these processes    Fig. 6.19 The urea cycle. Note the cytoplasmic/mitochondrial location of the cycle enzymes. Appreciate that the fumarate  malate — > oxaloacetate stages are part of the TCA cycle; for simplicity the enzymes are not shown in this illustration.    occur at equal rates, the total protein quantity remains  constant. Since proteins are comprised of amino acids  (nitrogen-containing molecules), protein intake and  excretion broadly approximates to nitrogen balance  (see above).    This section describes enzymatic breakdown of proteins  to their constituent amino acids. Enzymes that  hydrolyse peptide bonds (between amino acids) are  called proteases. There are two major routes of   This is an ATP-dependent cytoplasmic mechanism of  protein destruction. Ubiquitin, a small protein present  in all cells, is attached to proteins by ubiquitin ligase.  This is called 'ubiquitination'. It functions as a molecu-  lar label, identifying proteins destined for destruction  by the 25S proteosome. The proteosome is a large  cylindrical protease complex with a central access pore.  Ubiquitinated proteins enter the pore, possibly due  to ubiquitin acting as a recognition element, and are  proteolytically degraded to their component amino  acids. Ubiquitination is a four-step process (Fig. 6.20)  that includes:   • Production of an active adenylated ubiquitin inter-  mediate and covalent binding of activated ubiquitin  to enzyme El (ubiquitin-activating enzyme)   • Association of E2-ubiquitin with substrate-bound  E3 (ubiquitin ligase). E3 'reads' the N terminus of  a protein    • If the N-terminus is unstable, a peptide bond is  generated between ubiquitin and the E3-bound  substrate. E2 and E3 then dissociate.   Lysosomes are intracellular organelles. Intralysosomal  protease enzymes ('cathepsins') mediate lysosomal pro-  teolysis. Proteins gain access to lysosomal interior by ei-  ther endocytosis (extracellular proteins) or intracellular  autophagy. Autophagy is when endoplasmic reticulum  wraps around intracellular organelles (e.g. mitochon-  dria), forming an 'autophagosome'.   There are various structural features that decrease the  lifespan of proteins by conferring susceptibility to vari-  ous cellular degradation processes. Appreciate that ex-  posure of such features during conformational change  also renders a protein vulnerable to degradation.   High density of the amino acids Met, Gly, Ala and Ser  promotes N-terminus stability, since these are not read-  ily ubiquitinated. Thus dense presence of these amino  acids at the N-terminus of a protein renders it relatively  insensitive to ubiquitin-mediated destruction. Con-  versely, Phe, Try, Asp, Asn and Lys destabilize the  N-terminus. These amino acids are an N-terminal fea-  ture of short-lived proteins, since they are readily  ubiquitinated by E3.   These are features of a protein's primary structure; re-  gions with high density of Pro, Glu, Ser and Thr (P, E,  S and T according to the one-letter amino acid code) res-  idues are common in proteins with short lifespans.  PEST regions are examples of a 'degradation motif; their  presence condemns a protein to a rapid turnover. The  mechanism of PEST-mediated susceptibility is not yet  clarified but may be due to caspase or proteasome  action.   Proteins are synthesized in the cytoplasm by ribosomes.  Ribosomes assemble amino acids in the correct order to  form individual polypeptides. This order is determined,  or 'coded for' by the ribonucleotide sequence of the  particular messenger RNA (mRNA). Ribosomes are  structures composed of protein and ribosomal RNA  (rRNA). Ribosomes associate with mRNA strands in    the cytoplasm and function as 'assembly stations' for in-  coming amino acids.   These amino acids arrive bound to individual trans-  fer RNA molecules (tRNA). Amino acids are integrated  into growing polypeptides by ribosomes if, and only if,  the anticodon on their tRNA is complementary to the  next codon in the ribonucleotide sequence of the mRNA  strand. So, if the mRNA strand sequence is A-U-G, only  a tRNA with anticodon U-A-C can 'dock' at the ribo-  some for incorporation into the growing protein.   A codon is three sequential nucleotides representing  (‘coding for’) a particular amino acid. For example; the  mRNA sequence 'A-U-G' (adenine, uracil and  guanine) codes for methionine. The nuclear DNA  sequence encoding methionine is however 'A-T-G',  since RNA contains uracil (U) in place of thymine (T).   mRNA is 'messenger' RNA. This is an RNA sequence  reflecting the DNA sequence of a gene, except that  RNA cannot incorporate thymine, so utilizes uracil in  its place. The DNA sequence 'A-C-T-T-T-C-G' would  be represented in mRNA by 'A-C-U-U-U-C-G'. Every  three ribonucleotides (a 'codon') represents an amino acid.   The mRNA sequence codes for the gene from which  it was originally transcribed. This gene lies within  chromosomal DNA, in the nucleus. A base sequence  on one strand (the 'sense' strand) of the double-  stranded DNA helix codes for a gene. The complemen-  tary strand (the 'antisense' strand) acts as a template  for mRNA. RNA polymerase catalyses the assembly  of a ribonucleotide polymer (mRNA) complementary  to the antisense strand. This means the new mRNA  strand has a base sequence identical to the gene  sequence, except that 'U' is substituted for T in the  mRNA sequence.   • Discuss the roles of one-carbon units in amino acid synthesis   Single carbon units exist in a number of oxidation  states; for example, methane, formaldehyde and meth-  anol. They are used in the synthesis and elongation of  many organic compounds. To do this, carbon units re-  quire a carrier to activate them and to enable their trans-  fer to the molecule being synthesized. The main carriers  used are folate and S-adenosyl methionine. The term  'one-carbon pool' refers to single-carbon units attached  to these carriers.   S-adenosyl methionine (SAM) is a high-energy com-  pound formed by the condensation of the amino acid  methionine with ATP. It contains an activated methyl  group, which can be transferred easily to a variety of  molecules. SAM is the major donor of methyl groups  for biosynthetic reactions; for example, the methylation  of norepinephrine to epinephrine.   The active form of folate is 5,6,7,8-tetrahydrofolate  (THF). THF is a carrier of one-carbon units, which bind  to its nitrogen atoms at positions N 5 , N 10 or both, to  form the compounds shown in Fig. 7.1. THF receives  these one-carbon fragments from donor molecules such  as serine, glycine or histidine, and transfers them to  intermediates in the synthesis of other amino acids,  purines and thymidine. These THF compounds are all  interconvertible except the N 5 -methyl group.   The one carbon pool concept might be confusing;  simply remember that THF and SAM are carriers of  one-carbon groups which are used in the synthesis of a  range of molecules, mainly amino acids, purines and  pyrimidines.   THF is formed by the two-step reduction of folate by  dihydrofolate reductase (DIIFR) (Fig. 7.2). DHRF is  competitively inhibited by methotrexate, a folic acid an-  alogue used in the treatment of certain cancers. Metho-  trexate decreases THF synthesis and its availability for  purine and pyrimidine formation, which decreases  DNA and RNA synthesis in cells.   Use: As a chemotherapy treatment for cancer and for  autoimmune diseases.   Action: The affinity of methotrexate for DHFR is  about 1 000-fold higher than that of folate. It also has a  greater negative effect on rapidly dividing cells (such as  malignant and myeloid cells), which rapidly replicate  their DNA, thus inhibiting growth and proliferation of  these cancer cells.   Adverse effects: Toxicity to the rapidly dividing cells  of bone marrow and gastrointestinal mucosa, causing  anaemia, neutropenia and nausea.    Fig. 7.1 Compounds formed by the binding of THF to  various one-carbon compounds.    Fig. 7.2 Formation of tetrahydrofolate (THF). THF is formed  by the two-step reduction of folate by dihydrofolate (DHF)  reductase.   Reactions involving transfer of methyl groups result in  the formation of N 5 -methyl THF. Unlike other THF  compounds, N 5 -methyl THF is not interconvertible fur-  ther, therefore THF cannot be released and remains  trapped as N 5 -methyl THF. However, the methionine  salvage pathway is present. Methionine is formed by  methylation of homocysteine using N 5 -methyl THF as  the methyl group donor, therefore releasing THF. This  reaction is catalysed by homocysteine methyltransferase    and requires vitamin B 12 as an essential co factor  (methylcobalamin).    In vitamin B 12 deficiency, the methionine salvage  pathway is inhibited and THF remains as N 5 -methyl  THF. Eventually, all the body's folate becomes trapped,  resulting in folate deficiency secondary to E>i 2  deficiency (see Chapter 8). This results in decreased  nucleotide synthesis and DNA and RNA formation.   As blood cells require high levels of nucleotides for their  turnover, they are particularly sensitive to folate  deficiency, leading to megaloblastic anaemia.   The synthesis and breakdown of certain amino acids  produce THF carriers that can be used in the synthesis  of other amino acids and nucleotides. The following  reactions demonstrate the use of the one-carbon pool.   2. SAM contains an activated methyl group that can be  donated to a number of acceptor molecules, while  SAM transforms into S-adenosyl homocysteine   4. Homocysteine can be used either for the synthesis of  the amino acid cysteine, or for point 5 below   5. Regeneration of methionine (and THF) in the me-  thionine salvage pathway.   Purines are the nitrogenous bases adenine, guanine and  hypoxanthine. They have a double-ring structure, con-  sisting of a six-carbon ring and a five-carbon ring. They  can either exist as free bases or with a pentose sugar  (5 C), usually ribose or deoxyribose, attached at the  N9 position to form a nucleoside (i.e. adenosine). Phos-  phorylation of the sugar at the C5 position leads to the  formation of mono-, di- and tri-nucleotides as shown in  Fig. 7.4. The phosphate groups cause these molecules to  be negatively charged. The main functions of purines  are listed in Fig. 7.5.    Fig. 7.3 Formation of S-adenosyl methionine (SAM) and the methionine salvage pathway. (Numbers refer to the text.)   adenine (A) when base is free  7 adenosine when it is attached to sugar   Diet provides negligible amounts of purines because  they are broken down in the gut to form uric acid.  Two pathways are concerned with the formation of  purine nucleotides (Fig. 7.6).   The purine ring is assembled on a molecule of ribose-5-  phosphate; therefore, the purines are synthesized as  mononucleotides instead of as free bases. This process  occurs in the cytosol of hepatocytes and there are two  stages:   • Formation of inosine monophosphate. Eleven reac-  tions are necessary to form inosine monophosphate  (IMP), the nucleotide of hypoxanthine. In the  first reaction, ribose phosphate pyrophosphokinase  catalyses the phosphorylation of ribose-5-phosphate  at the Cl position, forming 5 -phosphoribosyl -  1 -pyrophosphate. In the second reaction, PRPP  amidotransferase catalyses the synthesis of 5-  phosphoribosylamine, which is an irreversible  rate-limiting step of the pathway. The rest of the re-  actions are concerned with the construction of the  purine ring of the inosine monophosphate by the  addition of five carbon and four nitrogen atoms  from amino acids (aspartate, glycine and gluta-  mine), C0 2 and THF derivatives   • Conversion of IMP to AMP (adenosine monopho-  sphate) and guanosine monophosphate (GMP)  (Fig. 7.7).   When nucleic acids and nucleotides are broken down,  free bases are released. The salvage pathway recycles  these free bases by re-attaching ribose-5-phosphate to    them (Fig. 7.8). It is a one-step pathway where the  ribose-5-phosphate is transferred to the free bases from  PRPP. The release of pyrophosphate makes the reactions  irreversible. Only two enzymes are necessary: adenine  phosphoribosyl transferase (APRT) and hypoxanthine  guanine phosphoribosyl transferase (HGPRT). The  pathway is simple and requires much less ATP than  de novo synthesis because the bases do not have to be  made first.   Purine synthesis is controlled allosterically by feedback  inhibition at four major control sites:   • PRPP synthetase. This is inhibited by the end prod-  ucts GMP and AMP. As PRPP is also an intermediate  in both the salvage pathway and pyrimidine synthe-  sis (discussed later in this chapter), this is not the  major control site   • PRPP amidotransferase. This irreversible, rate-  limiting reaction is unique to purine synthesis. It is  allosterically inhibited by the end products IMP,  AMP and GMP   • Adenylsuccinate synthase. This is inhibited by the  end product AMP   • IMP dehydrogenase. This is inhibited by the end  product GMP.   If regulation is lost because of a defect in one of these  four regulatory enzymes, this may lead to the overpro-  duction of AMP and GMP, in excess of the requirements  for nucleic acid synthesis and other functions. The  excess purines are broken down to uric acid, which  may become deposited in tissues, leading to symptoms  of gout.    used for DNA or RNA  synthesis, energy  transfer, etc. (Fig. 6.5)    Fig. 7.6 Overview of purine metabolism. Two pathways are concerned with the formation of purine nucleotides:   1 . De novo synthesis of purines, where the purine ring is assembled on a molecule of ribose-5-phosphate. The pathway consists of  two stages:   a. Formation of IMP, which occurs in 1 1 reactions. The purine ring is constructed by addition of C and N atoms from a number of  sources: amino acids, C0 2 and THF derivatives   2. Salvage pathways 'recycle' free purines released during nucleic acid turnover by re-attaching a sugar phosphate unit to them.  There is only one pathway for purine breakdown, which converts the purines to the free bases hypoxanthine and xanthine; these are  then oxidized to uric acid for excretion by the kidney.   Breakdown of purines occurs in two stages: the break-  down of the nucleotide to a free base hypoxanthine  or xanthine, and the formation of uric acid (Fig. 7.9).   1. Breakdown of the nucleotide to a free base:  hypoxanthine or xanthine   Three reactions are necessary (numbers and letters refer  to Fig. 7.9):   AMP and GMP are degraded by the same three reac-  tions; only the order differs (see Fig. 7.11). AMP and  IMP form hypoxanthine and GMP forms xanthine.   1 . Oxidation at the C2 position by IMP dehydrogenase, forming  xanthosine monophosphate   2. The amino group of glutamine insertion at the C2  position by GMP synthase to form GMP. This reaction  requires ATP.   1 . Addition of aspartate at the C6 position to form  adenylsuccinate. This reaction requires GTP   2. Adenylsuccinate lyase then eliminates the C-skeleton of  aspartate as fumarate, leaving behind the amino group at  C6 to form AMP.   Uric acid formation requires two steps, which are both  catalysed by the enzyme xanthine oxidase (see Fig. 7.9):   Xanthine oxidase is the key enzyme involved in purine  degradation. It is unusual because it is a molybdenum-  and iron-containing flavoprotein that uses molecular  oxygen as an oxidizing agent.   In humans, the uric acid formed is excreted in the  urine. Uric acid is insoluble. The acidic pH of urine al-  lows it to precipitate out at high concentrations as so-  dium urate. Hyperuricaemia, that is, high serum levels  of uric acid, may lead to gout (see below).    Fig. 7.8 Salvage pathways. When nucleic acids and  nucleotides are broken down, free bases are released.  The salvage pathway recycles these free bases by  re-attaching ribose-5-phosphate to them by transfer  from PRPP.   Xanthine oxidase is the key enzyme involved in control-  ling the amount of uric acid produced. Treatment with  xanthine oxidase inhibitors decreases the amount of  uric acid formed and increases the amounts of the sol-  uble precursors hypoxanthine and xanthine, which are  easily excreted in the urine. Allopurinol, an analogue of  hypoxanthine, is the most commonly used xanthine ox-  idase inhibitor. It has a number of actions:   • The salvage enzyme can catalyse addition of ribose-  5 -phosphate to allopurinol, forming allopurinol  ribonucleotide. This can inhibit the rate-limiting en-  zyme of de novo purine synthesis, namely by PRPP  amidotransferase, leading to a decrease in the level  of purines and also of the PRPP pool   • Allopurinol can be metabolized by xanthine oxidase  to oxypurinol, an even stronger inhibitor of xan-  thine oxidase.    Fig. 7.9 Breakdown of purines.  Breakdown of purines occurs in two  stages:   1. The breakdown of the nucleotide  to a free base, hypoxanthine or  xanthine. For both AMP and GMP,  three reactions are necessary,  although the order differs:   2. Formation of uric acid by the  oxidation of hypoxanthine and  xanthine by xanthine oxidase.   The prevalence of gout varies from about 0. 1-0.2% in Eu-  rope to as high as 10% in the Maori population of New  Zealand. It is caused by an abnormality of uric acid me-  tabolism, resulting in hyperuricaemia and the deposition  of sodium urate crystals in joints (first metatarsal phalan-  geal (MTP)), soft tissues and the kidney (Fig. 7.10).   Gout predominantly affects men in middle life. It  does not occur before puberty (unless it is part of  Lesch-Nyhan syndrome). In women, it only occurs after  menopause (the male to female ratio is 8:1). It is an  inherited condition in some families.   Gout intitially presents as recurrent, acute attacks of ar-  thritis, usually affecting only one joint (monoarthropa-  thy) . Presenting complaint: A warm swollen and very  tender joint, (usually the first meta-tarsophalangeal  joint of the big toe). The most common differential di-  agnoses for the acute symptoms of gout are trauma and  aseptic arthritis (Fig. 7.11). After repeated attacks occur    and resolve, symptoms may start to persist due to per-  manent urate crystal deposition, leading to chronic  tophaceous gout. Further complications can develop in-  cluding calcium oxalate kidney stones causing dysuria  and renal colic.   • Decreased HGPRT levels to 2-5% of normal. Similar  to Lesch-Nyhan syndrome but not as severe   • Overactive PRPP synthetase, involved in the regula-  tion of purine biosynthesis. Overactivity causes re-  lease from normal control, leading to increased  rates of de novo synthesis of purines   • Insensitive PRPP amidotransferase, the rate-controlling  enzyme of purine synthesis. A mutant form has full ac-  tivity but no regulatory sites, therefore feedback control  is lost, causing overproduction of purines   • The excess purines produced in these conditions are  broken down to uric acid, leading to hyperuricaemia  and gout.   Recurrent attacks of acute arthritis caused by  deposition of sodium urate crystals in joints; usually  only one joint is affected (big toe >90%)   Synovial fluid examination: affected joint is  aspirated and fluid examined under polarized light  microscopy for long, needle shaped, negatively  birefringent crystals (detected in 85% of cases of gout)  Flyperuricaemia does not necessarily cause gout   • Increased purine turnover, for example in leukae-  mia, myeloproliferative disorders, and due to  the use of cytotoxic drugs in the treatment of cancers   • Decreased excretion of uric acid, for example drug  therapy (thiazides, low-dose aspirin), lead toxicity,  excess alcohol.   Aspirin is contraindicated in gout because it impairs the  excretion of uric acid by the renal tubules, thus  aggravating hyperuricaemia.   Acute attacks are treated with anti-inflammatory drugs:  colchicine or non-steroidal anti-inflammatory drugs  (e.g. indometacin) provide relief within 24-48 hours.   • Simple measures are weight reduction (reduce  energy intake especially of saturated fats), decreased  alcohol intake and withdrawal of drugs such as  salicylates and thiazides   • Allopurinol, a xanthine oxidase inhibitor, is the main  drug used for the prevention of gout not to be initi-  ated as a treatment during an acute attack of gout    • Probenecid, a uricosuric drug, is an alternative to al-  lopurinol. It has a direct action on the renal tubule,  preventing the reabsorption of uric acid in the kid-  ney, causing it to be excreted.   Lesch-Nyhan syndrome is a very rare, X-linked dis-  order caused by an almost complete absence of the sal-  vage enzyme hypoxanthine guanine phosphoribosyl  transferase (HGPRT). HGPRT catalyses the addition  of 5-phosphoribosyl-l-pyrophosphate (PRPP) to the pu-  rine bases, guanine and hypoxanthine, in the salvage  pathway (see Fig. 7.12). In Lesch-Nyhan syndrome, a  decreased level of HGPRT results in:   • Excess guanine and hypoxanthine, which are broken  down to form large amounts of uric acid, leading to  severe hyperuricaemia (causing kidney stones, ar-  thritis) and gout.   • Increased PRPP, which is therefore used for the de  novo synthesis of purines, causing purine overpro-  duction and severe neurological disturbances (spas-  ticity and mental retardation) .    These symptoms begin at 3 months, which can lead to  diagnosis, or this may be detected by an orange nappy,  or HGPRT activity. Treatment is also with allopurinol to  control gout and arthritis. The prognosis is very poor;  with irreversible neurological impairment, and death  by the age of 5 years; usually from kidney failure (espe-  cially in boys).   There are three main pyrimidines: thymine, cytosine  and uracil. Like purines, the pyrimidines are mostly  found associated with a five-carbon sugar attached at  N1 to form the nucleosides thymidine, cytidine and  uridine (Fig. 7.13). The sugar may be mono-, di- or  tri-phosphorylated to form the corresponding  nucleotides.   • Allopurinol lowers uric acid levels and helps to control gout and arthritis   • With time, high purine levels result in worsening of neurological   • Boys usually die from kidney failure because of high sodium urate  deposits causing kidney stones    Fig. 7.13 Structure of pyrimidines.  Like purines, the pyrimidines are  mostly found associated with a  five-carbon sugar attached at N1  NH 2 to form nucleosides.   Pyrimidines are the building blocks of DNA and RNA:  thymine and cytosine are present in DNA and cytosine  and uracil are present in RNA.   Pyrimidine nucleotide derivatives are activated inter-  mediates in a number of synthetic reactions; for exam-  ple, UDP-glucose, the precursor of glycogen (see  Chapter 4).   There are three main stages in the biosynthesis of pyrim-  idines (see Fig. 7.14 and 7.16). All of the three stages  take place in the cell cytosol.    a. Construction of the pyrimidine ring to form uri-  dine monophosphate (UMP). Unlike purine syn-  thesis, the pyrimidine ring is synthesized before  attachment to ribose-5-phosphate, that is, it is  formed as a free base. The ring is derived from glu-  tamine, aspartate and C0 2 (Fig. 7.15). There are six  steps in the reaction sequence (Fig. 7.14). In the  first three steps, the enzymes involved are present  as a single polypeptide chain, forming a multifunc-  tional enzyme (CAD), which consists of carbamoyl  phosphate synthase II, aspartate trans carbamoy-  lase and dihydroorotase. In a way similar to fatty  acid synthase, the enzymes are linked together to  minimize side reactions and loss of substrate   1. Synthesis of carbamoyl phosphate by carbamoyl phosphate synthase II (CPSII). This is the rate-limiting step. Carbamoyl  phosphate is also the precursor of urea; however, urea is formed by the mitochondrial enzyme, carbamoyl phosphate synthase I   5. Conversion of the free pyrimidine to a nucleotide by the addition of ribose-5-phosphate from PRPP. This is catalysed by orotate  phosphoribosyl transferase (OPRT) and is driven by the hydrolysis of pyrophosphate to two free molecules of inorganic  phosphate. PRPP is thus required for the synthesis of both purines and pyrimidines   6. Decarboxylation of orotidine monophosphate (OMP) to UMP by orotidylate decarboxylase. Both OPRT and orotidylate  decarboxylase are also found together as a single polypeptide.    Fig. 7.15 The pyrimidine ring. The ring is derived from  glutamine, aspartate and C0 2 .   b. Conversion of UMP to uridine triphosphate (UTP)  and cytidine triphosphate (CTP), the ribonucleotides  found in RNA. UMP is phosphorylated to UDP and  UTP as shown in Fig. 7.14. CTP is formed from UTP  by amination, that is, the addition of an NH 2 group  from glutamine to position 4 of the pyrimidine ring.  Both UTP and CTP are used for RNA synthesis   c. Formation of the deoxyribonucleotides dCTP and  dTTP found in DNA (Fig. 7.16).   The rate-limiting step is the formation of carbamoyl  phosphate by carbamoyl phosphate synthase II. CPSII  is inhibited by the end products of pyrimidine    synthesis, namely UDP and UTP. The reaction is acti-  vated by ATP and PRPP.   The regulatory enzyme of deoxyribonudeotide synthe-  sis is ribonucleotide reductase, which catalyses the irre-  versible reduction of all four nucleoside diphosphates  (ADP, GDP, CDP and UDP) to their corresponding de-  oxy forms. The enzyme has four subunits (two B1 and  two B2). Each B1 subunit has two allosteric sites distinct  from the active site: an activity site and a substrate spec-  ificity site. The binding of the product dATP to the activ-  ity site inhibits the enzyme. The binding of the  substrate, a ribonucleotide, e.g. ATP, to the substrate  specificity site, activates the enzyme.   The salvage pathways for pyrimidines are similar to  those for purines (Fig. 7.17). The breakdown of nucle-  otides releases free pyrimidines, thymine and uracil.  These pyrimidines are salvaged by the enzyme uracil/  thymine phosphoribosyl transferase (UTPRT), which  transfers a ribose-5-phosphate from PRPP to the free py-  rimidines to re-form the mononucleotides. However,  the enzyme UTPRT cannot salvage cytosine. Therefore  cytidine (nucleoside) is deaminated to uridine; this is  then converted to uracil, which can be salvaged.    Fig. 7.16 Third stage of pyrimidine synthesis: the formation of deoxyribonucleotides.   1 . Ribonucleotide reductase reduces CDP todeoxyCDP by removal of the C2 hydroxyl group on ribose, converting it to deoxyribose.  The dCDP formed is phosphorylated to dCTP   2. dTTP is formed by the methylation of dUMP. Thymidylate synthetase transfers a methyl group from N 5 ,N 10 -methylene THF to  position 5 of the pyrimidine ring, forming dTMP, which can be phosphorylated to dTTP (numbers refer to text here). DHF,  dihydrofolate; THF, tetrahydrofolate.   Purines are excreted with their ring still intact as  uric acid. The pyrimidine ring, however, can be  split and broken down to soluble structures.  Uracil and cytosine are broken down to P-alanine,  which forms acetyl CoA. Thymine is degraded to  P-amino-isobutyrate, which forms succinyl CoA. The  carbon skeletons of the pyrimidines, namely acetyl  CoA and succinyl CoA, can be oxidized by the  TCA cycle.   These drugs inhibit the formation of nucleotides, lead-  ing to a decrease in DNA synthesis and cell growth. Can-  cer cells divide rapidly and have an increased demand    for DNA synthesis. These drugs help to slow down  the growth of cancer cells. However, they also affect  normal cell replication, leading to serious side effects  (Fig. 7.18).   Most anti-cancer drugs affect normal cell  replication and proliferation, especially cells of the bone  marrow, gastrointestinal tract, gonads, skin and hair  follicles. This results in severe side effects such as  anaemia, neutropenia (making patients susceptibile to  infection), hair loss, vomiting, infertility, impaired  wound healing and stunting of growth.   Analogues of glutamine: competitively inhibit  enzymes that use glutamine as substrate   J, pyrimidine synthesis: inhibits CPS II  l purine synthesis: inhibits PRPP  amidotransferase and reaction 5  (Fig. 7.13)   Inhibits DFIF reductase leading to decreased  available TFHF for transfer of one-carbon units   Inhibits methylation of dUMP to  dTMP causing  i dTMP synthesis  l pyrimidine synthesis (Fig. 7.15)   Haem is a complex structure containing an iron atom  (as Fe 2+ ) placed in the centre of a tetrapyrrole ring of  ferroprotoporphyrin (formerly protoporphyrin IX)  (Fig. 7.19).   • The basic structure is a four-ringed cyclic structure  called a porphyrin   • Each ring is called a pyrrole ring and the rings are  linked together via methenyl bridges   • Three types of side chains can be attached to the  pyrrole ring - methyl, vinyl or propionyl - and the  arrangement of these is important to the activity   Haem is the prosthetic group found in a number of pro-  teins. The function of haem in each group can vary  (Fig. 7.20). The Fe 2+ atom (ferrous form) at the centre  of the haem structure can undergo oxidation to Fe 3 +  (ferric form); this is important for its function in cyto-  chromes and enzymes, enabling it to act as a recyclable  electron carrier. However, in haemoglobin and myoglo-  bin, Fe 3+ cannot bind oxygen, and its function as an ox-  ygen transporter is impaired (it forms methaemoglobin,  see Chapter 3).   Haemoglobin and myoglobin are oxygen-binding  proteins with different mechanisms of action.    I Iaemoglobin-oxygen binding is described as being co-  operative, and follows a sigmoidal curve; compared to  the hyperbolic curve which myoglobin exhibits. The sig-  moidal curve means that haemoglobin becomes highly  saturated at high oxygen partial pressures, and releases a  significant amount of oxygen at pressures which are  fairly low, but not extremely so. This cooperative bind-  ing mechanism is more efficient at collecting oxygen  where it is in high concentration, and supplying it where  it is needed. In contrast, low partial pressures of oxygen  can almost totally saturate myoglobin. As a result, hae-  moglobin is described as having a lower affinity for ox-  ygen than myoglobin.   • Bone marrow erythroid cells, where haem is used to  form haemoglobin   • Hepatocytes, where haem is used for cytochrome  synthesis, particularly cytochrome P450 which is in-  volved in drug metabolism.    Fig. 7.19 Structure of haem. Haem  consists of an Fe 2 + atom placed in the  centre of protoporphyrin IX.    The human body makes 40-50 mg/ day of haem, about  80-85% of which is used for haemoglobin synthesis.  Mature erythrocytes lack mitochondria and therefore  cannot make haem.   Haem biosynthesis is partitioned between mitochon-  dria and the cytosol (Fig. 7.21).   • There are eight reactions; the first and last three occur  in the mitochondria, the rest are in the cytosol   • The substrates for haem synthesis are glycine and  succinyl CoA   • Eight moles of each are required to form eight moles  of 8-aminolevulinic acid (ALA), which condenses to  form four moles of porphobilinogen (PBG). These,  in turn, condense to form one mole of uroporphyr-  inogen I (UROgen I)    Protoporphyrinogen IX is a colourless, unstable and  easily oxidized precursor of porphyrin. Porphyrins are  highly coloured (red), stable compounds that charac-  teristically absorb ultraviolet light at a wavelength of  400 nm.   The rate-limiting enzyme of haem synthesis is ALA  synthase. It is a good control point because the enzyme  undergoes rapid turnover (has a half-life of 60-70 mi-  nutes). ALA synthase is inhibited by high levels of the  end product haem (fe 2+ ) and also haemin (ferriproto-  porphyrin; fe 3+ ), formed by the oxidation of haem.   In the liver, control of ALA synthase by haem is con-  sidered at three levels (numbers refer to fig. 7.22):   1 . Allosteric inhibition of the enzyme by haem. How-  ever, high concentrations of haem are necessary  (1(T 5 M) and, therefore, this is not an important  control mechanism   2. Haem also inhibits the transport of newly synthe-  sized enzyme from cytosol into mitochondria   1 . The synthesis of ALA. ALA synthase  catalyses the condensation of  glycine and succinyl CoA in the  mitochondria. The reaction requires  pyridoxal phosphate (PLP) as a  cofactor. This is the irreversible, rate-  limiting step of haem synthesis   2. The formation of porphobilinogen  (PBG). ALA dehydrase catalyses the  dehydration of two molecules of  ALA to form PBG. The enzyme is  inhibited by heavy metals such   I (UROgen I). UROgen I synthase  catalyses the condensation of four  molecules of PBG to form UROgen  I (inactive)   4. UROgen III cosynthase produces  the asymmetrical active  uroporphyrinogen III (UROgen III).  The rest of the reactions alter the  side chains and the degree of  unsaturation of the porphyrin ring   5. The first decarboxylation results in  the formation of  coproporphyrinogen III   8. Ferrochelatase inserts the Fe 2+ ion  into the ring to form haem.    3 . Repression of transcription of the ALA synthase gene  by haem. This is probably the most effective regula-  tion because it works at low concentrations (1CT 7 M).   In erythroid tissue, the same regulatory mechanisms apply  as in the liver but additionally, under certain conditions  such as chronic hypoxia or anaemia, erythropoietin pro-  duction is stimulated, leading to an increase in red cell  production and, therefore, an increase in haem.   A number of drugs, such as steroids and barbiturates,  cause an increase in the amount of hepatic ALA  synthase. The mechanism proceeds as follows:   • Drugs are metabolized by microsomal cytochrome  P450 enzymes, which are haem-containing proteins  themselves   • Certain drugs induce the synthesis of cytochrome  P450, leading to an increase in the consumption  and breakdown of haem   • This leads to an overall decrease in the concentration  of haem in the liver cells, which in turn stimulates or    induces the transcription of ALA synthase and haem  synthesis (Fig. 7.22)   About 80-85% of haem that is broken down comes  from old erythrocytes; the rest comes from cytochrome  turnover (Fig. 7.23).   Kupffer cells and macrophages of the reticuloendothe-  lial system (mainly liver, spleen and bone marrow).   The two steps in the pathway are (steps refer to  Fig. 7.24):   1. Cleavage of the porphyrin ring to form biliverdin.  Haem oxygenase found in microsomes splits the por-  phyrin ring by breaking one of the methenyl bridges  between two pyrrole rings. This produces biliverdin,    Fe 3+ and carbon monoxide (this is the only reaction  in vivo that produces carbon monoxide)   Bilirubin is non-polar and lipophilic and is only spar-  ingly soluble in aqueous solutions. Within the blood,  it is bound to albumin and very little remains free in  solution. Bilirubin bound to albumin is taken up by  the liver. Bilirubin is conjugated within the hepatic en-  doplasmic reticulum by a UDP glucuronyl transferase  to produce a water-soluble bilimbin diglucuronide  (Fig 7.24).   The conjugated bilirubin moves into the bile can-  aliculi of the liver and is then stored in the gall  bladder. When stimulated by eating, bile (including  the conjugated bilirubin) is secreted into the small  intestine. Within the large intestine, bilirubin is further  metabolized by bacteria present in the gut into    urobilinogen. Some of this is absorbed from the intestine  and enters the blood. Much of this in turn is taken up by  the liver from the portal vein, but a small proportion en-  ters the general circulation, is filtered through the glomer-  ulus, and enters urine (giving its characteristic yellow  colour).   The liver has a large capacity to conjugate bilirubin and  can normally cope with moderately elevated levels.  Flowever, in patients with haemolytic anaemia, such as  during a sickle cell crisis, there is a very large increase in  haem breakdown, resulting in high bilirubin levels,  which exceed the conjugating capacity of the liver.   This results in elevated plasma levels of unconjugated  bilirubin, causing jaundice. In jaundice, the deposition  of bilirubin leads to a yellow colouring of the skin,  mucosal membranes and the sclerae of eyes (Fig. 12. 10  describes this clinical sign in the clinical assessment)   Haem breakdown occurs at sites of minor trauma  underneath the skin. The changing colours of  a bruise represent the different pigments  produced.    haem synthesis and thus the formation of excessive  quantities of either porphyrin precursors, for example  5-aminolevulinic acid (ALA) or porphobilinogen  (PBG), or porphyrins themselves, depending upon  which enzyme is deficient.   PBG is readily measured in urine, and usually is crucial in  the diagnosis of porphyrias.    This is a group of rare, inherited disorders in which  there is a partial deficiency of one of the enzymes  of haem synthesis. This results in the inhibition of    The key, rate-limiting enzyme of haem synthesis is  ALA synthase which is normally inhibited by haem    (see Fig. 7.22). In porphyrias, the absence of haem re-  leases the inhibition (and the control) of ALA synthase,  resulting in the increased formation of intermediates  preceding the defective enzyme in each porphyria.   When porphyrin precursors (ALA and PBG) are pro-  duced in excess, they cause mainly neuropsychiatric  symptoms and abdominal pain. When porphyrins  themselves are produced in excess, they cause skin pho-  tosensitivity (i.e., the skin burns and itches on exposure  to light). This is because porphyrins absorb light, which  excites them and induces the formation of oxygen free  radicals. These can attack membranes, particularly lyso-  somal membranes, leading to the release of enzymes  which damage underlying layers of skin, rendering it  susceptible to the light.   Porphyrias are diagnosed on the basis of symptoms  and the pattern of porphyrins and their precursors pre-  sent in the blood and urine.   Porphyrias are classified as either hepatic or erythro-  poeitic and also as acute or chronic (Fig. 7.25). They  are all rare; the most common in the UK is acute inter-  mittent porphyria which is discussed below. The fea-  tures of other types of porphyrias are summarized in  Fig. 7.25.   Acute intermittent porphyria is an autosomal dominant  disease with prevalence in the UK of 1:100 000. The de-  fect is a deficiency of uroporphyrinogen 1 synthase.  Characteristically, acute attacks are separated by long  periods of remission. The attacks are precipitated by  various factors, including alcohol, barbiturates, oral   Increased levels of PBG and ALA can be found in the urine  of these patients. The urine darkens to a port-wine colour  on exposure to air, due to the presence of PBG. The classic  bedside test for excess PBG is to add Ehrlich's reagent (an  aldehyde) to urine, which causes it to go pink. The colour  persists when excess chloroform is added.   The treatment is with fluids, pain relief and a high-  carbohydrate diet, which inhibits the pathway. Avoiding  precipitants is important. It is important to ask about  inherited disorders when pre-assessing patients for  surgery.   The effects of all porphyrias can be decreased by intra-  venous haemin which inhibits ALA synthase, the rate-  controlling enzyme, regaining the control of haem syn-  thesis. An increased dietary intake of antioxidant  vitamins A, C and E also helps to protect against free  radical damage. Intravenous haematin can be given.   Porphyrias are very rare. You will seldom see or be  asked aboutthem. Fig. 7.25 summarizes all you need to  know.   The human body contains about 120 mg of lead. Exces-  sive ingestion or inhalation can result from contami-  nated food, water or air. In the UK, the common  sources were old lead piping and petrol. Lead inhibits  three key enzymes of haem synthesis, resulting in the  accumulation of intermediates:   • ALA dehydrase: this leads to the accumulation of  ALA, which can be measured in urine   • Coproporphyrinogen III oxidase: this leads to the  accumulation of coproporphyrinogen III   • Ferrochelatase: this leads to the accumulation of  protoporphyrin IX in erythrocytes.   Overall, this results in the inhibition of haem synthesis  and microcytic anaemia. Lead also binds to bone.  The main clinical features and diagnostic criteria are  discussed in Fig. 7.26.   • Causes staining of teeth  and bones, myopathy,  peripheral neuropathy,  renal damage and  sideroblastic anaemia   Blood film: anaemia with  basophil stippling; red  cells may contain small,  blue deposits   Treatment is with lead chelators such as desferrioxa-  mine mesilate, sodium calcium edetate or penicilla-  mine. They all bind lead, forming a complex, which  can be excreted in the urine.   • Describe the main effects of insulin and glucagon on carbohydrate, protein and lipid metabolism   • Discuss the main metabolic effects of diabetes and its long-term complications    Glucose homeostasis can be conveniently discussed by  looking at three basic states: the fed, fasted (post-  absorptive) and starved state (Fig. 8.1). The starved state  can be further subdivided into early and late, since dif-  ferent metabolic fuels are used depending on the length  of starvation (Fig. 8.2).   It is important to realize that glucose homeostasis is a  dynamic process. There are no well-defined boundaries    between the different states; instead, there is some de-  gree of overlap between them.   This is the period 0-4 hours after a meal and is summa-  rized in Fig. 8.3. During the fed state (numbers refer  to Fig. 8.3):   1 . An increase in plasma glucose results in the release of  insulin from the (1 cells in the pancreas. The availabil-  ity of substrate and the increase in insulin stimulates   II Fasted state most glucose is provided by the breakdown of liver   breakdown of protein and fat provides amino acids  and glycerol, substrates for gluconeogenesis   T insulin results in:  f glucose uptake by peripheral tissues  f glycogen, TG, and protein synthesis   Brain: glucose and some ketone bodies  liver: fatty acids muscle: mainly fatty acids  and some ketone bodies   Brain: uses more ketone bodies and less  glucose to preserve body protein. Muscle:  only fatty acids   Fig. 8.3 Summary of fuel metabolism in the fed state (numbers 1-3 refer to the text).  108    glycogen, triacylglycerol (triglyceride), and protein  synthesis by tissues; this is an anabolic state   2. Glucose is the sole fuel for the brain; its uptake there  is insulin-independent   3. Muscle and adipose tissue also use glucose; however  here its uptake is insulin dependent.   An increase in glucose and insulin activates glucokinase in  the liver. Glucokinase, unlike hexokinase, is not inhibited  by glucose-6-phosphate, enabling the liver to respond to  the high blood glucose levels that occur after a meal. Glu-  cokinase phosphorylates glucose, enabling its further me-  tabolism, including glycogen synthesis (see Chapter 4).   Hexokinase, present in most cells, is also active when  the concentration of glucose in the blood is low.   This is the period 4-12 hours after a meal, also called  the post-absorptive state (Fig. 8.4). During the fasted  state (numbers refer to Fig. 8.4):    1 . The breakdown of liver glycogen stores provides glu-  cose for oxidation by the brain. These stores are suf-  ficient to last only between 12 and 24 hours   2. The hydrolysis of triacylglycerols from stores re-  leases fatty acids, which are used preferentially as a  fuel by muscle and liver   3. Muscle can also use its own glycogen as a fuel.   All these processes are activated by the increase in the  ratio of glucagon to insulin. This activates (by phosphor-  ylation) glycogen phosphorylase and hormone-sensitive  lipase, leading respectively to glycogen breakdown and  lipolysis.   Once the liver glycogen begins to be depleted, an alter-  native substrate is required to provide glucose (Fig. 8.5).  In short-term starvation (numbers refer to Fig. 8.5):   Fig. 8.4 Summary of fuel metabolism in the fasted state; 4-12 hours after a meal. A high glucagon to insulin ratio activates the  breakdown of liver glycogen, which provides glucose for the brain. Both the fall in insulin concentration and the increase in  norepinephrine promote hydrolysis of triacylglycerol stores, releasing fatty acids which can be used as a fuel by muscle and liver.  Muscle uses its own glycogen as fuel (numbers refer to text above).   Fig. 8.5 Summary of fuel metabolism in early starvation. Norepinephrine and cortisol activate the breakdown of muscle protein to  release amino acids, particularly alanine and glutamine. Norepinephrine also activates hydrolysis of triacylglycerols to release  glycerol. Glycerol, alanine and glutamine are taken to the liver, where they enter gluconeogenesis and are oxidized to glucose.  Glucose is used mainly by the brain. Fatty acids released from hydrolysis of triacylglycerols can be taken to the liver and used to  generate ketone bodies, which can be used by brain and other tissues (numbers refer to text below).    1 . Glucagon and later cortisol activate protein break-  down in muscle, which releases amino acids (partic-  ularly alanine and glutamine)   2. Hydrolysis of triacylglycerol stores (adipose tissue)  releases glycerol. Both the amino acids and glycerol  are used by the liver for gluconeogenesis   4. The fatty acids released from triacylglycerols are also  used by the liver to make ketone bodies which can  be used as an alternative fuel by peripheral tissues  as well as the brain.   This is the period of starvation of longer than 1 6 days. In  prolonged starvation the breakdown of muscle protein  slows down. This is because there is less need for glucose  to be supplied via gluconeogenesis, because the brain  adapts to using more ketone bodies. This is further helped  by muscle using, almost exclusively, fatty acids as fuel.   A comparison of the fed and the fasted state is a  commonly examined ‘metabolic’ question, because it  requires overall knowledge of protein, fat and  carbohydrate metabolism and its regulation. The way  to answer this for each state is to think about time-  course, hormonal influences, main active pathways,  substrate availability and any special tissue  requirements.   Gluconeogenesis is the major source of glucose once  glycogen stores are depleted. The main role of gluconeo-  genesis is the maintenance of blood glucose and the  provision of glucose for the brain and erythrocytes dur-  ing fasting. An increased glucagominsulin ratio activates    gluconeogenesis and causes the reciprocal inhibition of  glycolysis (see Chapter 4). In muscle, cortisol activates  protein breakdown, releasing, in particular, alanine  and glutamine (alanine is one of the main substrates  for gluconeogenesis).   Ketone body synthesis begins during the first few days of  starvation and increases as the brain adapts to using  ketone bodies as its major fuel, therefore reducing the  need for glucose. Once significant ketone body synthe-  sis occurs, a decrease in the level of gluconeogenesis  from amino acids is seen. This results in a reduction  in muscle proteolysis, therefore sparing protein.   After 2-3 weeks of starvation, muscle reduces its use  of ketone bodies and uses fatty acids almost exclusively;  this leads to an increase in ketone bodies available for  the brain.   Both ketogenesis and gluconeogenesis are balanced to  ensure efficient use of metabolic fuels during starvation.  Gluconeogenesis activates ketogenesis by depleting    oxaloacetate, which ensures that the concentration of  acetyl CoA exceeds the oxidative capacity of the TCA  cycle; acetyl CoA can therefore be used for ketone body  synthesis.    Insulin is an anabolic hormone that increases the glu-  cose uptake and synthesis of glycogen, triacylglycerol  and protein. Glucagon, norepinephrine, epinephrine  and cortisol are catabolic hormones. The main effects  of glucagon are summarized in Fig. 8.6. Norepine-  phrine and epinephrine (stress, or fight-and-flight  hormones) have some similar effects to glucagon  where they:   Increases glycogen breakdown in liver only (NA and  epinephrine increase breakdown in muscle) decreases glycogen  synthesis   • In the muscle during intense activity, there is only  time for anaerobic glycolysis, resulting in the  build-up of lactate   • Lactate diffuses out of muscle and is taken to the liver  where it is oxidized to pyruvate, which can then be  converted back to glucose via gluconeogenesis   • The formed glucose diffuses out of the liver and  returns to the muscle to be further used as fuel.   This series of reactions, which 'shifts the metabolic bur-  den from the muscle to the liver', is known as the Cori  cycle (Fig. 8.7). (Compare it with the glucose-  alanine cycle.)   The body does not store enough glycogen to provide  the energy necessary to run long distances. If the respi-  ratory quotient (RQ: the ratio of the amount of 0 2 con-  sumed to the amount of C0 2 released) is measured  during a run, initially it is about 1.0, indicating that   Fig. 8.7 The Cori cycle distributes the metabolic burden  between the muscle and the liver. Lactate, which builds up in  muscle during intense activity, is taken to the liver to be  converted back to glucose via gluconeogenesis. This replenishes  fuel for the muscle and prevents lactic acidosis.    mainly carbohydrate is being used. However, the RQ  falls during running to a value of about 0.77 after about  1 hour, indicating that mainly fats are being oxidized.   The type and amount of substrate used varies with  the intensity and duration of exercise, in a similar way  to starvation. As glycogen stores are depleted, an in-  crease in glucagon, norepinephrine and epinephrine  stimulates lipolysis, releasing fatty acids for muscle to  use to conserve glucose. An increase in these hormones,  along with an increase in cortisol, leads to stimulation  of gluconeogenesis and protein degradation in muscle.  These changes are similar to those of the fasting state;  the difference is that the level of ketone bodies in the  blood is low. It is not clear whether this is because they  are not being synthesized or if they are being oxidized as  soon as they are formed.   Insulin is an anabolic hormone, and the understanding  of its actions has recently greatly improved. It promotes  the synthesis and storage of carbohydrates, lipids and  proteins and inhibits their degradation and release back  into the circulation; these actions involve multiple sig-  naling pathways.   Basically, the binding of insulin to its tyrosine kinase  receptor on the outside surface of the cells induces the  receptor to undergo autophosphorylation at several ty-  rosine kinase residues located inside the cell. This  autophosphorylation facilitates binding and phosphor-  ylation of cytosolic substrate proteins, such as insulin  receptor substrate-1 (IRS-1) and Cbl proteins. LIpon  phosphorylation, these proteins interact with other sig-  naling molecules through their SH2 (Src-homology-2)  domains, which then activate several diverse pathways.   Such pathways include activation of PI 3 kinase and  TC10 (a small GTP binding protein). PI-3 kinase acti-  vates protein kinase B (PKB) and PKB in turn phosphor-  ylates a range of target enzymes. The net result of these  diverse pathways is regulation of glucose, lipid and  protein metabolism as well as cell growth and differen-  tiation (Fig. 8.8).   Diabetes mellitus is a syndrome caused by the lack, or  diminished effectiveness, of insulin. It results in a raised  blood glucose known as hyperglycaemia and can lead  to the development of diverse vascular complications  over long periods of time. There are two main types  of diabetes:    Fig. 8.8 The insulin signaling pathway. Insulin binds to its  receptor, resulting in autophosphorylation. This then  phosphorylates the protein Cbl which is in complex with the  adaptor protein CAP. Cbl/CAP complex then interacts with the  adaptor protein Crk which is constitutively associated with C3G,  a GTP/GDP exchange protein. C3G activates TC10, which  themselves promote GLUT4 translocation to the plasma  membrane. Autophosphorylation of the insulin receptor also  phosphorylates insulin receptor substrate 1 (IRS-1). IRS-1  attracts p85, which binds to pi 10, which then activates  phophatidylinositol-3 kinase (PI-3 kinase). PI-3 kinase activates  protein kinase B (PKB; Akt) which act on further pathways,  resulting in glucose, lipid and protein metabolism, and specific  gene expression. Akt also promotes GLUT-4 translocation to  plasma membrane, resulting in increased glucose uptake.    identical twins, which implies a mixture of both ge-  netic and environmental factors. Secondly, there is a  positive family history in approximately 10% of pa-  tients. Thirdly, more than 90% of patients with Type  1 diabetes carry HLA DR3 and DR4 antigens, com-  pared with 40% of the general population  • A viral cause, for example mumps or Coxsackie B,  has also been considered. However, it is likely that  viral infections provide the stimulus for auto-  immune destruction rather than actually initiating  diabetes.   Therefore, the cause is probably a mixture of all three -  auto-immune destruction of the P cells in genetically  susceptible patients, which may be precipitated by a vi-  ral infection.   The presentation of the disease is usually of rapid on-  set, weeks or days, with the characteristic symptoms of  polyuria, polydipsia and weight loss.   This was also known as maturity-onset diabetes, because  it typically presents after the age of 35 years. The inci-  dence is more common, and it accounts for 80-90%  of the total number of people with diabetes.    • Type 1 : formerly known as insulin-dependent diabe-  tes mellitus (IDDM) in which there is an absolute  failure of the pancreas to produce insulin   • Type 2: formerly known as non-insulin-dependent  diabetes mellitus (NIDDM) in which there is a fail-  ure of the tissues to respond normally to insulin,  together with a compensatory rise in plasma insulin  concentration at early stages. As the disease pro-  gresses, insulin secretion deteriorates.   Type 1 was often referred to as juvenile-onset diabetes  because it typically presents in childhood or puberty.  It accounts for only 10-20% of the total number of  people with diabetes and has an incidence rate of about  1 in 3000.   The aetiology of the disease is a complete deficiency  of insulin that can only be corrected by life-long insulin  treatment. There are three theories as to its cause:   • Auto-immune destruction of the p cells in the islets  of Langerhans in the pancreas by islet cell auto-  antibodies, resulting in insulin deficiency   • Genetic factors. The evidence for a genetic cause is  that, firstly, there is a 50% concordance between    Insulin resistance (metabolic) syndrome is a new  concept, which helps to identify individuals with risk  factors for cardiovascular disease and type 2 diabetes  (obesity, abnormal glucose tolerance). Defining criteria  vary, but according to the International Diabetes  Federation (2006) there needs to be:   Central obesity (defined as waist circumference with  ethnicity specific values, but if BMI is >30 kg/m 2 ,  central obesity can be assumed)   • Reduced FIDL cholesterol: <1.03 mmol/L in males,  < 1 .29 mmol/L in females   • Raised blood pressure: systolic BP >130 or diastolic  BP >85 mmHg   • Raised fasting plasma glucose (FPG)>(5.6 mmol/L).  Note: If FPG > 5.6 mmol/L, an oral glucose tolerance   test is strongly recommended (but is not necessary to  define the syndrome).   • Impaired insulin secretion from the (I cells; they  fail to secrete enough insulin to correct the blood  glucose level.   • Insulin resistance in the tissues; cells failing to re-  spond adequately to insulin.   Genetic factors are very important; there is almost 100%  concordance between identical twins and about 30% of  patients have a first-degree relative with Type 2 diabetes.  There is no auto-immune or viral involvement.   The presentation is of an insidious onset and more  than 80% of patients are obese.   In every medical examination, there will always  be questions on diabetes. Know the effects of an  increased glucagon/insulin ratio - the rest can be  easily worked out!   There are a number of other types of diabetes, which  usually occur secondary to a predisposing factor, for  example:   • Secondary diabetes: this may be the result of damage  to the pancreas itself, for example, in chronic pancre-  atitis or haemochromatosis, where iron may deposit  in the pancreas (see Chapter 11). Diabetes may also  occur secondary to the excessive secretion of cata-  bolic hormones, resulting in hyperglycaemia and in-  sulin resistance. For example, in acromegaly, where  there is over-secretion of growth hormone, or in  Cushing's syndrome, where there are high levels of  glucocorticoids such as cortisol; also in poorly mon-  itored long-term steroid therapy.   These other types of diabetes are covered in more detail  in textbooks of endocrinology and clinical medicine.   Insulin normally facilitates the uptake of glucose by  peripheral tissues. In its absence, glucose remains in  the blood, resulting in a decreased tissue availability of  glucose, but a high plasma concentration of glucose.  The phrase 'starvation in the midst of plenty' is fre-  quently used to describe this. As there is a low concentra-  tion of insulin, the metabolic effects of glucagon and the    other catabolic hormones are unopposed (see Fig. 8.6).  This results in the predominance of catabolic processes;  the breakdown of carbohydrate, protein and fat (see  Fig. 8.9). This aggravates hyperglycaemia, and leads  to ketoacidosis, hypertriglyceridaemia and importantly,  dehydration (because of osmotic diuresis, causing  large amounts of glucose to enter urine). Ketoacidosis  is life-threatening. In addition, because cells cannot  obtain glucose from the diet they have to obtain it by  the breakdown of body stores or by synthesizing it from  non-carbohydrate precursors (gluconeogenesis).  Hyperglycaemia is caused by:   • Glucagon-stimulated increase in the breakdown of  liver glycogen and gluconeogenesis, leading to an  increased hepatic output of glucose.   • An increase in triacylglycerol hydrolysis in adipose  tissue that releases fatty acids   The release of fatty acids is much greater than in starva-  tion; therefore, the rate of formation of ketone bodies is  much greater than the rate of use, leading to ketonaemia  (see Chapter 5).   Hypertriglyceridaemia is an increase in the concen-  tration of triacylglycerols in plasma. (Note that in clin-  ical medicine triacylglycerols are usually referred to as  triglycerides.) Some of the fatty acids released from tria-  cylglycerols are packaged in the liver into very-low-  density lipoproteins (VLDLs). Dietary triacylglycerols  are assembled into chylomicrons. In the absence of  insulin, the activity of lipoprotein lipase decreases,  and the VLDLs and chylomicrons remain in the plasma,  and are responsible for hypertriglyceridaemia  (see Fig. 8.9).   A lot of people liken diabetes to starvation but  there are some very important differences that can  lead to fatal consequences for a diabetic patient  (Fig. 8.10).   The metabolic effects are essentially the same as for Type  1 but usually they are milder because insulin is present.  However:   • The amount of insulin secreted from the pancreas may  be insufficient to cope with the blood glucose level   • Target tissues or organs fail to respond correctly to  insulin (they show insulin resistance).    Fig. 8.9 Effect of an increased glucagon to insulin ratio in diabetes.   Large increase in production of ketone bodies where rate of  formation exceeds rate of use; can lead to life-threatening  ketoacidosis   Increased concentration, but  usually rate of formation equals  rate of use    In Type 2 diabetes, insulin resistance may be due to a  number of defects. For example, an abnormal insulin  receptor or a defect in a glucose transporter. Insulin  resistance in the liver results in uncontrolled glucose  production and its decreased uptake by the peripheral  tissues. Both phenomena contribute to hyperglycaemia.  Hyperglycaemia in turn further stimulates insulin secre-  tion by the pancreas. Type 2 diabetes is typically associ-  ated with older age of onset than Type 1 diabetes and,  most importantly, with obesity.   Obesity is associated with an increase in the number  and/or size of adipocytes. These cells overproduce hor-  mones and cytokines, collectively known as adipokines,    such as leptin and tumour necrosis factor-alpha,  (TNF-a) which induce cellular resistance to insulin by  interfering with the phosphorylation of the insulin re-  ceptor and IRS-1. Adipocytes also decrease synthesis  of hormones such as adiponectin, which normally en-  hance insulin responsiveness. As a result, there is insulin  resistance in muscle and liver. Initially, the pancreas  maintains glycaemic control by overproducing insulin  but prolonged overproduction of insulin eventually re-  sults in failure of the P-cells, leading to Type 2 diabetes.  However, there is still hope for improvement since  insulin resistance has been shown to be reversible with  weight loss and increased exercise.   During pregnancy, there is a decrease in insulin  sensitivity to help provide the developing fetus with  adequate glucose. However, in 3-5% of pregnant  women, glucose intolerance develops. This is known as  gestational diabetes mellitus (GDM), which is defined  by an additional decrease in insulin sensitivity and an  inability to compensate with increased insulin  secretion. GDM is generally reversible after pregnancy,  but about 30-50% of women with GDM go on to  develop Type 2 diabetes later in life; overweight  women tend to have a higher risk of this.   The presentation of the symptoms of diabetes mellitus  may be acute or emergency or insidious in onset (types  are listed in Fig. 8.1 1).   Young people often present with a brief 2-4 week his-  tory of the classical symptoms, namely polyuria, poly-  dipsia, and weight loss, accompanied by tiredness.  These patients usually have Type 1 diabetes.   The onset of symptoms is usually over months to years.  Patients may still present with the classic symptoms al-  though, quite often, tiredness is the prominent symp-  tom, particularly Type 2 diabetes.   Glycosuria or raised blood glucose may be detected dur-  ing a routine medical examination.   If the early symptoms are not recognized, patients can  present with ketoacidosis (see Fig. 8.12), where:   • Severe hyperglycaemia causes an osmotic diuresis.  The consequent loss of fluid and electrolytes results  in dehydration. If this is severe, the patient may  be confused and be in shock. Remember to consi-  der this diagnosis in patients presenting with ab-  dominal pain   • Increased production of ketone bodies results in  metabolic acidosis and characteristic ketotic breath.  The acidosis typically causes nausea and vomiting  and further loss of fluid and electrolytes. Respiratory  compensation results in hyperventilation (Kussmaul  breathing). Diabetic ketoacidosis is a medical emer-  gency: failure to treat a patient in ketoacidosis may  result in coma and death.   Patients may also present with diabetic complications  such as retinopathy, neuropathy or nephropathy. For  example, they may present after visits to the opticians  (diabetic retinopathy), or with tingling and numbness  in the leg, or with leg or foot ulcers or impotence  (neuropathy).   The clinical features and diagnosis of Type 1 diabetes are  listed in Fig. 8.13. The treatment consists of:   • Insulin. There are three main types of insulin: short-  acting, intermediate-acting and long-acting. The du-  ration of action of insulin is increased by forming a  complex with a protamine salt and/or varying the  size of the crystals   Overall incidence, approximately 2% in Western world  Patients are usually younger than 25 years   Patients are usually older than 25 years and can often be overweight   Affects about 5% of population; these patients are more likely to develop diabetes  when they are older   Either due to pancreatic damage, e.g. chronic pancreatitis, haemochromatosis or  Wilson's disease, or due to endocrine disease, e.g. acromegaly, Cushing's disease   Note that a new category of impaired fasting glucose (>6 mmol/L) has been recently recognized   can occur in type 1+2 diabetes  (drug side effect from sulphonylurea  in type 2 diabetes)   (previously known as HONK,  is the equivalent problem related  to high sugars in type 2 diabetics)    Fig. 8.12 Diabetic ketoacidosis. In the absence of insulin, hyperglycaemia causes osmotic diuresis. The loss of fluid and electrolytes  results in dehydration. Increased ketogenesis causes metabolic acidosis. Respiratory compensation results in hyperventilation. Both  dehydration and hyperglycaemia must be corrected in parallel with insulin treatment.    Fig. 8.13 Clinical features and diagnosis of Type 1 (insulin-dependent) diabetes mellitus.   • acute onset of symptoms (2-4 weeks) polyuria, poly-  dipsia, accompanied by weight loss, abdominal pain  and tiredness   (oral glucose tolerance test is not necessary - reserved for  borderline cases; glycosuria is not diagnostic due to  variation in renal threshold for glucose)   • Diet. Ensuring the correct content and timing of  meals. The diet should be high in fibre and unre-  fined carbohydrate, low in saturated fat and refined  carbohydrate   • The detection of ketones in urine (and blood),  important for the detection of developing  ketoacidosis    • Education. It is crucial that patients understand their  disease, and the short- and long-term benefits of  treatment.    There are a number of methods for monitoring the con-  trol of diabetes and these are covered in detail in  Chapter 12. They include:   • Measuring blood glucose levels, using reagent strips  based on the glucose oxidase reaction, or portable  glucose meters   • Monitoring the level of glycated haemoglobin  (HbAi c ). This provides a measure of the average  blood glucose control over the past 4-6 weeks   Type 2 diabetes is increasing in prevalence and not only  can lead to specific long-term complications but also  has an impact on cardiovascular risk. This should be  considered and appropriately managed in patients  identified as being 'at risk'.    Fig. 8.14 Diagnosis, management and treatment of Type 2 diabetes (non-insulin dependent diabetes, NIDDM). (References:  http://guidance.nice.org.uk/CG66/Cuidance/pdf/English)   • May be asymptomatic- detection of  f blood glucose on routine check-up   4 main classes (in the usual order of use, with addition for further medications  as needed for optimal control):   1) Biguanides, e.g. metformin: j glucose uptake by peripheral tissues and  j glucose production by liver. Key point: metformin aids weight loss   2) Sulphonylureas, e.g. glibenclamide: \ insulin secretion by islet cells (inhibits  ATP-sensitive K 1 channels in [3 cell membranes). Key point: due to this  mechanism of action, this class of oral hypoglycaemics have a risk of  hypoglycaemia   4) Acarbose inhibits intestinal enzyme, glucosidase and therefore, delays the  digestion of starch   Metformin is usually the first choice, except in cases where the patient is not  overweight ora rapid response to therapy is required because of hyperglycaemic  symptoms, in which case sulphonylureas are initiated.    The diagnosis, management and treatment of Type 2  diabetes are covered in Fig. 8.14.   The aim of treatment of Type 1 diabetes with insulin is  to maintain a normal blood glucose level, which de-  creases the long-term effects of diabetes. However,  too much insulin or too infrequent 'top ups' of glucose  (i.e. insufficient intake of carbohydrate) lead to a low  blood glucose (hypoglycaemia). Hypoglycaemia causes  unpleasant autonomic symptoms, such as sweating,  nausea and palpitations, and more severe neuroglyco-  penic symptoms as a result of a decrease in glucose  supply to the brain: drowsiness, unsteadiness, confu-  sion and coma (these patients may look drunk). This  is a very serious condition and must be treated without  delay with an intravenous 50% dextrose infusion. Mild  hypoglycaemia can be treated with sugar or sweet  drinks.   In the absence of insulin, effects of glucagon are unop-  posed. Decreased uptake of glucose by tissues, coupled  with an increased hepatic glucose production, leads to  hyperglycaemia. This causes an osmotic diuresis, and  the resulting loss of fluid and electrolytes causes  dehydration. An increase in lipolysis leads to increased    ketogenesis and a metabolic acidosis (ketones will be  detected on urinalysis). Respiratory compensation re-  sults in hyperventilation.   Hyperosmolar hyperglycemic non-ketotic syndrome  (HITNS) is a diabetic emergency which occurs rarely,  mostly in elderly Type 2 diabetic patients. It is also  due to hyperglycaemia. It is defined by a serum glucose  that is usually higher than 33 mmol/1 (600 mg/dl), and  a resulting serum osmolarity that is greater than  350 mOsm. An osmotic diuresis will result in polyuria  and severe volume depletion, which causes a hemocon-  centration that further increases blood glucose level and  osmolarity.   • HHNS usually occurs in Type 2 diabetics, DKA usu-  ally in Type 1   • Ketosis; absent in HHNS. (The presence of some in-  sulin inhibits lipolysis so these acidic by-products  may not form, unlike in DKA)   • Onset; HHNS can be insidious and develop over  weeks, and in about a third of cases it is the first man-  ifestation of type 2 diabetes. (Note that HHNS is very  rare.)   • Precipitating factors; both HHNS and DKA are  usually precipitated by an illness/infection/acute  illness (myocardial infarction)   Rehydration is the first priority, followed by  management of electrolyte imbalances (potassium)  and by control of blood glucose with an insulin infusion  (sliding scale). Rehydration can be aggressive in young  patients with ketoacidosis, but, care needs to be taken  in elderly patients with HHNS who could have  cardiovascular problems.    • Both are life threatening; DKA continues to be a  prominent cause of death, particularly in young  diabetics. Cerebral oedema (swelling of the brain)  is a serious complication of DKA and carries a  high risk of permanent neurological damage  and death. Equally, reported death rates from  HHNS are very high (58%).   Be aware of warning signs: dry mouth, confusion, weak-  ness, and visual losses. And knowledgeable about the  emergency management of DKA and HHNS (Fig. 8.13).   In the long term the major cause of death in Type 1 di-  abetes are chronic complications, particularly diabetic  retinopathy, nephropathy and neuropathy. In type 2 di-  abetes, heart disease, peripheral vascular disease and  stroke are the major causes of death.   Good control of diabetes (i.e. when the blood glu-  cose is maintained close to normal) decreases the  frequency and progression of microangiopathy (but  apparently not macroangiopathy) (Fig. 8.14).    Fig. 8.16 Comparison of Type 1 and Type 2 diabetes mellitus.    The WHO criteria for diagnosis of diabetes are as fol-  lows: fasting venous plasma glucose equal or above  7 mmol/L. A glucose level of 6-7 mmol/L is defined  as impaired fasting glucose (IFG). If the patient has  no diabetic symptoms, diagnosis should not be based  on a single glucose value.   'Compare Type 1 diabetes mellitus with Type 2'. This is  a commonly asked exam question; Fig. 8.16 should  help.   • Discuss the causes of obesity, its complications, prevention and treatment   • Discuss protein requirements and the main causes and clinical features of protein deficiency diseases   • Assess nutritional status of patients and consider optimal feeding routes when necessary   Nutrients are essential dietary factors, such as vitamins,  minerals, essential amino acids and essential fatty acids  that cannot be synthesized by the body, and can only be  obtained from the food we eat.   Macronutrients are present in our diets in large  amounts, and make up the bulk of our diets. They  can be found in carbohydrates, fat, protein and water.   Micronutrients are present in our diet, but in very  small amounts. These are vitamins, minerals and trace  elements. They do not provide energy, however they  are still needed in adequate amounts to ensure that  all our body cells function properly. Despite their  presence in minute amounts their importance is  paramount.   Staple foods are the principal sources of energy in the  diet. They are specific to a particular country; for exam-  ple, in parts of Africa and Asia cereals provide more than  70% of the energy in the diet. As countries become more  prosperous, the percentage of energy derived from a sin-  gle staple food declines. In the UK, flour and flour prod-  ucts provide only about 25% of food energy.   Figure 9.1 shows the anatomy of the intestinal tract and  summarizes the main steps in the digestion of carbohy-  drates, proteins and fats.   Digestion of carbohydrates begins in the mouth and  stomach. Saliva contains an enzyme, a-amylase, which  hydrolyzes starch into maltose and other small poly-  mers of glucose. Digestion continues in the stomach  for about an hour before the activity of salivary amylase  is blocked by gastric acid.   Pancreatic secretions, similar to saliva, contain  large quantities of a-amylase. It is identical to the  a-amylase in saliva, therefore virtually all the starches  are digested by the time they enter the duodenum.  Disaccharides and small glucose polymers are hydro-  lyzed into monosaccharides by intestinal epithelial  enzymes.   Protein digestion begins in the stomach. The enzyme  pepsin breaks down collagen to allow other enzymes  to penetrate meats and digest cellular proteins. Most  protein digestion occurs through the actions of pancre-  atic proteolytic enzymes.   • Trypsin and chymotrypsin break down protein mol-  ecules into small polypeptides   • Carboxypolypeptidase cleaves amino acids from the  carboxyl ends of the polypeptides   • Proelastase gives rise to elastase, which then digests  the elastin fibres that hold meat together.   The last stage of digestion of proteins is carried out in  the intestinal lumen by enterocytes, which contain    Digestion continues for  about an hour before  gastric acid blocks the  action of salivary amylase   Pancreatic secretion contains  large amounts of a amylase  (identical to amylase in saliva)  Virtually all starches are  digested by the duodenum   Most protein digestion  occurs through the action  of pancreatic proteolytic  enzymes  (e.g. trypsin)    -(T) Emulsification by bile  breaks fat globules down  to smaller pieces to  increase surface area    (D Micelles carry these fats to the  brush border of the intestine    multiple peptidases that break down remaining tripep-  tides and dipeptides into amino acids, which then enter  the blood.   Fat digestion begins with the emulsification by bile  acids and lecithin, where fat globules are broken into  smaller pieces to increase their surface area. Pancreatic  lipase breaks down triglycerides into free fatty acids  and 2-monoglycerides which are carried to the brush  border of the intestinal epithelial cells by micelles. Mi-  celles are composed of a central fat globule (containing  monoglycerides and free fatty acids) with molecules of  bile salt projecting outward covering the surface of the  micelle.   There are three main methods for estimating an individ-  ual's dietary intake:   • Dietary recall. Ask the patient what he or she has  eaten. This is the least accurate because it relies on  the patient's recall and willingness to cooperate   • Complete chemical analysis. This is the most  expensive but the most accurate method.   The definitions in Fig. 9.2 are in keeping with the dietary  reference values (DRVs) for food energy and nutrients  for the UK.   This is the average requirement of a group of people for energy  or a nutrient. About 50% of the population will need less than  the EAR and 50% will need more.   This is the amount of nutrient that is enough or more than  enough for about 97% of people in the group (EAR + 2SD)  Lower reference nutrient intake (LRNI)   This is the amount of nutrient that is sufficient for only a few  people in a group with low needs (EAR - 2SD)   This is the amount of nutrient enough for almost everyone, but  not so much as to cause undesirable effects. This term is applied  for nutrients for which there is not enough information known  to estimate EAR, RNI or LRNI (e.g. vitamin E).   For instance, the DRVs for vitamin C are: LRNI, 10 mg/day;   EAR, 25 mg/day; RNI, 40 mg/day. Therefore below the LRNI,  symptoms of vitamin C deficiency (scurvy) are seen and above  the RNI, symptoms of excess may be seen.   The total energy content of food is the amount of energy  released when food is completely burnt in air to C0 2 and  1T 2 0, that is, the heat of combustion (Fig. 9.3). The total  energy is equal to the sum of the digestible energy and the  non-digestible energy (Fig. 9.4). From Fig. 9.3 it can be  seen that protein has a higher total energy content than  carbohydrate. However, protein is not as efficiently oxi-  dized (it forms urea and requires ATP for this; see  Chapter 6) and only about 4kcal/g are available as me-  tabolizable energy. Carbohydrate is oxidized completely  to C0 2 and H 2 0 and all the energy produced is available  for use; the metabolizable energy is also 4 kcal/g.   Lean body mass (LBM) is made up of (these are approx-  imate values):   Generally, women have a higher fat content (about 25%  fat) than men. Fat content tends to increase with age.  An average 72-kg man can survive on his energy stores  for about 50-60 days provided he is given water. This is  mostly due to fat reserves; glycogen stores last only   Fat: essential for absorption of fat-soluble vitamins (A, D, E and K)   Carbohydrate: as either starch, sugar or non-starch polysaccharide (NSP), i.e. fibre   thermic effect of food  = enegy released during  the digestion, absorption  and transport of food  (also called diet-induced  or post-prandial  thermogenesis)    25-40%  trapped as ATP  or useful energy  the body is only  25-40% efficient    • Non-digestible energy is the energy in food that we cannot  break down and is lost in faeces (e.g. cellulose)   • Digestible energy is the total energy minus energy that is  lost in faeces.   Metabolizable energy is the energy available to the body for use;   • 5-10% of energy is released during the digestion, absorption  and transport of food. This is known as either the thermic  effect of food, diet-induced thermogenesis (DIT), or  post-prandial thermogenesis (they all mean the same thing)   • Only about 25-40% of the energy is trapped as ATP; that is,  the body is only 25-40% efficient.    12-24 hours. Fig. 9.5 summarizes the methods avail-  able to measure body composition. However, most of  the methods, with the exception of anthropometry,  are rarely used in clinical practice.   Energy is used by the body for three main processes  (Fig. 9.6).   The basal metabolic rate (BMR) is the energy used to  carry out normal body functions (such as breathing); it  is the energy expended doing nothing. The units of  BMR are kj/hour/kg of body weight. To calculate the  BMR:   • The patient must be at rest, lying down but not  asleep   • The temperature of the environment must be mod-  erate and constant   • The patient must be assessed about 1 2 hours after the  last meal or any exercise.   The BMR is usually measured first thing in the morning.  It is proportional to LBM, therefore, men have a higher  BMR than women. Women have a greater percentage   Weigh in air to give fat content  (density = 0.9 mg/mL); weigh in water  to give lean body mass (density =   The patient is injected with a known  volume of tritiated water  Its concentration at equilibrium is  measured   This is a measure of lean body mass as  there is no potassium in fat   The uptake of a fat-soluble gas, e.g.  xenon or cyclopropane is measured  Biopsy to measure concentration   • skin-fold thickness (subscapular and  suprailiac) compare with normograms  for weight and height   (when organism is at  rest, all energy  expenditure is equal  to heat produced,  that is, themogenesis)   • Environmental temperature changes. This is a very  small effect unless the temperature is either ex-  tremely high or low   • Pregnancy and lactation. For the first 6 months of  pregnancy no extra energy is necessary, but for the  last 3 months, an extra 800 k) (200kcal) are needed  each day. During lactation, an extra 2000 kj  (500kcal) are required each day   • Growth. The energy requirement in the first year of  life is double that of adulthood   • Age. The BMR decreases after the age of about  20years.   The measurement of 0 2 consumption allows indirect  measurement of the metabolic rate. This is because 1 li-  tre of 0 2 consumed at rest is equal to 20 kj of energy  expenditure.   The incorporation of doubly labelled water ( 2 F1 2 18 0)  into body fluids and its loss in the urine can be mea-  sured. H is incorporated only into H 2 0 but O is incor-  porated into both H 2 0 and C0 2 . The difference  between them is equal to the C0 2 produced.    of fat which is less metabolically active. The BMR  usually accounts for 50-70% of the total energy expended.   This is the energy required for the digestion and absorp-  tion of food and accounts for 5-10% of the energy  expenditure.   The amount of energy consumed depends on the dura-  tion and intensity of exercise. The physical activity ratio  (PAR) can be measured for situations where activity is  expressed as a multiple of the BMR (i.e. BMR= 1).    The physical activity level (PAL) can also be cal-  culated. This is equal to the total energy expenditure in  1 day divided by the BMR.   A number of systems are thought to participate in the  regulation of food intake (Fig. 9.7).   Overall control is by the hypothalamus. Hormones  and signals from the stomach, pancreas, and adipose tis-  sue interact to control the balance of hunger/ satiety and  energy expenditure, through the mechanisms described  in the figure.   • Ghrelin is secreted by the stomach and stimulates  NPY/AgRP-expressing neurons, increasing appetite   • Cholecystokinin (CCK) is known to decrease appe-  tite. CCK slows gastric emptying, maintaining gastric  distension, which is thought to be an important sa-  tiety signal   • Pancreatic signals include insulin, which regulates  food intake, promotes satiety by stimulating the up-  take of glucose by peripheral tissues. Therefore,  levels of glucose and glucagon regulate intake. Orig-  inally it was thought that low plasma glucose levels  had a direct stimulatory effect on the hunger centre.  Now it is believed that it is the increased availability  of glucose to tissues that produces satiety (the gluco-  stat hypothesis)    • Adipose tissue is an active endocrine organ produc-  ing adipokines such as leptin.   - Leptin has a key role in decreasing appetite and  increasing thermogenesis, and leptin levels are  proportional to body fat content. Animal studies  on leptin found deficient cases to be hyperphagic  and obese.   If energy intake is equal to energy expenditure, there is  no change in body mass. Obesity develops when there is  an imbalance; and energy intake is greater than  expenditure.   Obesity can be categorized in terms of the body mass  index (BMI) (Fig. 9.8). The cut-off values for classifica-  tions are in the table below, but consideration of ethnic  group should be noted, as cut-offs for obesity in China  are BMI > 28 and in Japan > 25 (lower thresholds due to  the negative consequences of a higher BMI).   Obesity is rising to epidemic proportions in men,  women and children, especially in countries with West-  ern influences. This problem is beginning to replace in-  fectious diseases and under nutrition as the most  significant contributor to ill health worldwide, as it is  associated with an increased risk of various clinical dis-  orders. In developing countries, it is estimated that  more than 115 million people suffer from weight-  related problems.   Fig. 9.9 discusses some of the proposed theories for  obesity. Twin studies suggest a genetic background.  However, genetic factors are greatly influenced by  environmental and socio-economic factors. Poor educa-  tion, high alcohol intake, and less energy expenditure,  increase the incidence of obesity. This may also be  related to the type of food consumed, which is largely  governed by financial status. The most obvious cause  of obesity is an excessive intake of calories accompanied  by a decrease in energy expenditure. The reasons for  overeating are usually complex and may be psycholog-  ical in origin, related to stress or a life event. Only rarely  are there metabolic causes.   Identical twins are not always the same weight  Adopted children resemble their new family  weightwise   Likely genetic predisposition but also  modified by environmental factors (diet,  social-economic status)   In the West, low socio-economic class — > obesity  In the East, high socio-economic class — ► obesity   Adrenal hyperfunction (Cushing's syndrome),  hypothyroidism, and Type 2 diabetes mellitus are all  associated with obesity   Diet-induced thermogenesis (DIT) is greater in lean  people (N.B. basal metabolic rate is not lower in  obese people!)   The main cause of obesity is usually an excessive intake  of calories accompanied by a decrease in energy  expenditure.   • Type 2 diabetes. Obesity results in persistently high  insulin levels, leading to a down-regulation of  insulin receptors and thus insulin resistance in the  tissues   • Coronary heart disease. There is an increase in mor-  bidity and mortality caused by coronary heart disease  in obese individuals. It may be that other risk factors  are more likely to be present in obese patients   Morbidity is the incidence or prevalence of disease in a  population. Mortality is the number of deaths from  disease in a population.   • Reduction of energy intake. The main treatment of  an obese patient is an appropriate diet, with plenty  of support and encouragement from a doctor.  Lots of different weight-reducing diets have been  formulated; most do not work, particularly in the  long term. For example, on a low-carbohydrate diet,  where bread, potatoes, cakes and any starch-  containing foods are cut out of the diet, initially,  weight loss is fast (0.5kg/day) but most of the loss  is water. Protein is also broken down to maintain  the blood glucose, but is replaced as soon as the diet  is stopped. The loss of fat is the same as for a normal  mixed diet. However, low-carbohydrate diets im-  prove glucose tolerance   • Other diets such as low-fat diets and low-cholesterol  diets are also used to help treat obesity. Recent trials,  utilizing diets low in saturated fat and supplemented  with polyunsaturated fatty acids, mainly from  omega-3 fatty acids (three helpings of oily fish per  week, fish oil capsules and alpha-linoleic acid mar-  garine), have been shown to be beneficial in helping  patients to lose weight   • Most weight-reducing diets allow an intake of  lOOOkcal/day. This must be a balanced intake of  protein, carbohydrate and fat (i.e. a mixed diet).  Why is it that 80-100% of obese people regain lost  weight? During starvation, the metabolic rate falls by    15-30%. Therefore, after dieting, to remain at a  lower weight, a lower energy intake must be main-  tained otherwise the weight will be put straight back  on. The only way to lose weight is a prolonged mod-  eration of intake and then a permanent change in  eating habits to maintain the weight loss  Increase energy expenditure in a way appropriate to  age and health   Drug therapy should only be considered after dietary,  exercise and behavioural approaches have been  started and evaluated for patients who have not  reached their target weight loss or have reached a pla-  teau on dietary, activity and behavioural changes  alone. Any medications should be reviewed regularly.  There is only one key medication you need to know  about: Orlistat, which is a pancreatic lipase inhibitor  which is licensed for use together with a mildly  hypocaloric diet in those with a BMI of greater than  30 kg/m 2 . Part of its effect may be related to the reduc-  tion of fat intake necessary to avoid severe gastrointes-  tinal effects such as steatorrhoea. It should only be  continued beyond 3 months if the patient has lost at  least 5% of their initial body weight since starting  the drug treatment. Appetite suppressants such as  phentermine (a catecholaminergic drug with minor  sympathomimetic and stimulant effects) are not  presently used. Sibutramine has recently been sus-  pended and is no longer used. Currendy, research is  being done on unraveling the links between obesity  and adipokine secretion to investigate leptin and other  adipokines for use in the diagnosis and treatment of  obesity   Surgery. Examples include: jaw wiring, gastric plica-  tion (stapling the walls of the stomach together to  form a smaller stomach), bypass of the small intes-  tine, and gastric distension.   • Morbid obesity (BMI >40 kg/m 2 OR 35-40 kg/m 2 )  AND other significant disease (diabetes, high blood  pressure) that may be improved if they lose weight.   • Receiving treatment in a specialist obesity clinic  (providing full assessments, counseling and support  pre and post-surgery)   • Unable to maintain weight loss despite trying all other  appropriate non-surgical treatments   • No medical or psychological contraindications for this  type of surgery   Reference proteins contain all the amino acids in the exact  proportions needed forprotein synthesis. Albumin (found  in egg white) and casein (milk) are closest to the ideal.  Other proteins are compared with these reference proteins.   A limiting amino acid is the essential amino acid present  in a protein in the lowest amount relative to its require-  ment for protein synthesis. Examples of protein-  containing foods and their limiting amino acids are:   Combining different protein-containing foods, such as  meat and the pulses, ensures an adequate intake of all  the amino acids, that is, protein complementation  (e.g. eating baked beans on toast). This is particularly  important in vegetarian diets.   The quality of any protein can be assessed using a rating  system based on a number of variables.   The chemical score is the ratio of the amount of limiting  amino acid to its requirement, expressed as percentage  points. For example, if the amount of limiting amino acid  in a test protein is 2% and the amount of limiting amino acid  in the reference protein is 5%; the chemical score is 40%.   The biological value is the proportion of the absorbed pro-  tein which is retained by the body for protein synthesis.   The net protein utilization (NPU) is the proportion of  dietary protein which is retained by the body for protein  synthesis. For example:   Net dietary protein as a percentage of energy (NDPE%)  is the proportion of total dietary energy provided by  fully 'usable' protein. This method provides a way of  comparing different diets. For example:   Children require an NDPE% of greater than 8%, that is, at  least 8% of their diet must come from usable protein.  Adults require an NDPE% of greater than 5%. In areas  where the staple food is starch (e.g. yam, cassava), the  diet provides only low levels of protein. It would be phys-  ically impossible to consume the amount of food neces-  sary to satisfy the protein requirement, especially for  children, and this leads to protein deficiency states.  Cereal-based diets are adequate for adults but not  children.   Diet should provide the essential amino acids and  enough amino acid nitrogen to synthesize the non-  essential amino acids. These are required for:   • Formation of body proteins during periods of  growth, pregnancy, lactation, infection, and after  major trauma or illness such as cancer.   The recommended protein requirement for an adult in  the UK is 0.8g/kg/day of protein and should not be  greater than 1.5g/kg/day.   The RNI for protein is 55 g/day for men and 44 g/day  for women.   Protein-energy malnutrition (PEM) arises when the  body's need for protein or energy, or both, is not met  by the diet. It is most commonly seen in developing  countries. However, in the industrialized world, it can  be present in the elderly or chronically ill patients.   • Increased requirement; for example, in preterm in-  fants, infection (septic state increases catabolism),  major trauma or surgery   The bulk of excess protein is oxidized via  gluconeogenesis to glycogen or fat and stored by the  body. Therefore, protein is not a slimming food. One  famous diet consists of a protein-sparing modified fast  (PSMF) which is hydrolyzed gelatine and collagen, thus  it is cheap. However, during the hydrolysis process a lot  of electrolytes are lost, including potassium, which may  lead to serious problems.    In developing countries, PEM manifests as two con-  ditions in children:   • Kwashiorkor: lack of protein only - energy supply is  adequate.   In developing countries, 20-75% of children below  5 years of age have some form of malnutrition. Five mil-  lion children die every year because of malnutrition.   Marasmus is the childhood form of starvation (Figs 9.10  and 9.11). Both protein and energy are limited, leading  to a low concentration of insulin but increased levels of  glucagon and cortisol, that is, a starved state (see  Chapter 8). As no fuel is available for the body, muscle  protein and fat are broken down to provide energy,  which leads to wasting. Muscle protein is broken down  to amino acids which are used for the synthesis of albu-  min by the liver; therefore, this prevents oedema.   Translated this means the 'disease the first child gets  when the second child is born'. In kwashiorkor severe  protein deficiency occurs but energy is maintained  (Figs 9.12 and 9.13). It usually occurs when a young  child is weaned from breastfeeding because of the ar-  rival of a new baby. The first child is fed a low-protein,  high-starch diet instead. Kwashiorkor often develops af-  ter an acute infection, such as measles or gastroenteritis,  when the demand for protein is increased.   As energy is not limiting, there is a high insulin to  glucagon, and insulin to cortisol ratio. Amino acids  are taken up by muscle for protein synthesis. This diverts  amino acids from the liver, so fewer are available for al-  bumin synthesis: the resulting low albumin levels re-  duce the plasma oncotic pressure, causing oedema.  The oedema causes a deceptively fat appearance and    children are known as water or 'sugar' babies. It is pos-  sible there may also be some degree of energy loss in  kwashiorkor and therefore other factors may contribute  to, or cause, the oedema. For example:   • Excessive generation of free radicals causing mem-  brane damage and oedema   • Infection diverts protein synthesis from albumin to  the synthesis of immunoglobulins and acute-phase  proteins (e.g. C-reactive protein).   A comparison of kwashiorkor and marasmus is given in  Fig. 9.14.   It is important to restore fluid and electrolyte balance  first. Following this:   • Carefully re-feed initially, just enough to maintain a  steady state to satisfy the normal daily requirement.  Milk is often given with flour or maize, slowly and reg-  ularly. There is a risk of refeeding syndrome, which re-  sults within the first few days of starting to feed after a  period of starvation/prolonged undernutrition. The  availability of glucose causes surges in insulin levels,  which moves phosphate, potassium, and other elec-  troltytes into cells, reducing levels in the blood. These  low levels, especially hypophosphataemia, can result   • Eventually, high-energy foods are given to restore  weight and also any necessary vitamin and mineral  supplements.   Mortality rates for children with severe malnutrition are  about 50%. The rate is so high because adequate treat-  ment is usually not available.   Malnourished children are less active and more apathetic;  these behavioural abnormalities are usually reversed by  re-feeding. However, severe, prolonged malnutrition  causes much reduced brain growth and permanent dam-  age to both physical and mental development. Immunity  is impaired, leading to delayed wound healing; protein  loss from muscle may eventually include the diaphragm,  leading to death. The physiological effects of severe  prolonged malnutrition are listed in Fig. 9.15.   Prevention of childhood malnutrition is a World Health  Organization priority. The main targets are to provide:   However, drought, famine and war in affected countries  often make these targets impossible to achieve.   In Western countries, a degree of PEM maybe seen in  hospitalized patients with the following conditions:    That is, anything that causes a negative nitrogen balance  (see Chapter 6).   Malnutrition in adults in developing countries has  symptoms similar to those seen in children but the  results are not as devastating. This is because adults are  already physically and mentally mature and are  therefore more resilient.   Nutritional support is provided for all patients who  are severely malnourished or are unable to eat because  of physical illness. Whenever possible, enteral nutri-  tion is used, that is, delivery of nutrients via the gastro-  intestinal tract, including eating and drinking  normally, or a nasogastric (NG) tube or gastrostomy;  a tube placed directly in the stomach. Enteral nutrition  is more natural, cheaper and far less hazardous in  terms of the effects on fluid and electrolyte balance  than parenteral nutrition. Enteral nutrition is always  given in preference if the gastrointestinal tract is func-  tional. Parenteral nutrition may be indicated in the  following situations:   • Intestinal failure; either as the result of surgery (gut  resection) or because of a fistula or gastrointestinal  tract obstruction by tumour.   • Unconscious patients with a very high energy re-  quirement; that is, those in hypercatabolic state,  for example severe trauma or burns patients.   Administration is usually via a central venous catheter  into the superior vena cava, or sometimes into a   Decreased cell-mediated response, immunoglobulin production is maintained: this can  have harmful effects as it depletes production of other proteins   May effect Na + /K + pump and the maintenance of ion gradients across cells   Fat accumulates since its transport requires apolipoproteins that are deficient (not seen  in marasmus)    peripheral vein. It involves the intravenous infusion of a  mixture of high-concentration glucose, fat emulsion,  amino acids, vitamins, electrolytes and trace elements.   The most frequent complication of total parenteral nu-  trition (TPN) is infection of the delivery line; therefore,  a meticulous aseptic technique is essential. Also, these  patients require careful daily clinical monitoring to  avoid complications:   • Plasma electrolytes (sodium, potassium, chloride,  bicarbonate, urea, and creatinine) and glucose are  measured at least once a day to prevent the develop-  ment of refeeding syndrome   • Regular haematological measurements (full blood  count and so on) are necessary, as is the monitoring  of iron, vitamin B 12 or folate deficiencies    • In a patient with stable renal function, 24 h urinary  urea excretion provide an index of the body's protein  status. This is not used as a routine test   • Liver function tests are checked about three times a  week   Refeeding syndrome is usually within 4 days of starting  artificial refeeding (enteral or parenteral) in patients  who are severely malnourished and it encompasses  potentially fatal metabolic disturbances (a fall in serum  phosphate, potassium, magnesium, glucose, and  thiamine), which can cause confusion, coma,  convulsions, and even death.   • Describe the main causes and clinical features of vitamin deficiency diseases   Vitamin: A complex organic substance required in the  diet in small amounts, the absence of which leads to  a deficiency disease.   Vitamins can be divided into two main groups, fat-  soluble and water-soluble.   • Can be toxic in excess (particularly A and D)  (Fig. 10.1).   Animal sources are butter, whole milk, egg yolk, liver  and fish liver oils; they contain retinol.   Plant sources are most green, yellow or orange vege-  tables; they contain (1-carotene, the precursor of retinol.   Retinol is absorbed in the intestinal mucosa and ester-  ified to long-chain fatty acids, forming retinyl esters.    These are packaged in chylomicrons and transported  to the liver for storage. When required, retinol is re-  leased and transported, bound to retinol-binding pro-  tein. Retinol can be oxidized to other active forms,  namely retinoic acid and retinal. (S-carotene is absorbed  in the intestine and converted into retinal.   • Retinoic acid. This acts as a typical steroid hormone.  It binds to chromatin to increase the synthesis of  proteins controlling cell growth and differentiation  of epithelial cells. Therefore, it increases epithelial  cell turnover   • Retinal. 11-cis retinal binds to opsin to form rho-  dopsin, the visual pigment of the rod cells in the ret-  ina, involved in vision and particularly night vision  (Fig. 10.2)   • (1-carotene is an antioxidant. The role of antioxidants  in the prevention of heart disease and lung cancer is  being studied intensively, but has not yet produced  any conclusive results.   Vitamin A deficiency is rarely seen in developed coun-  tries because liver stores are sufficient to last 3-4 years.  It is commonly found in children in developing coun-  tries such as India and parts of South-East Asia, where  about 500 000 children each year are blinded as a result  of vitamin A deficiency.   Vitamin A deficiency may be caused by a decreased di-  etary intake; usually only seen in very severe malnutri-  tion. It may also occur secondary to fat malabsorption.   • Initially, deficiency causes impaired dark adaptation  and night blindness. This is reversible   Dermatitis, mucous membrane defects and hair loss  Hepatomegaly, thinning and fracture of the long bones  Increased intracranial pressure   Toxicity is very unlikely with normal sources, be wary when prescribing high doses of retinoic acid for  severe acne sufferers   The most toxic of all vitamins. As it is fat soluble, it is stored in the body. Normally, this is well tolerated  but in high doses over a period of time, hypervitaminosis D may occur. This presents with nausea,  vomiting and muscle weakness. Very high levels of vitamin D result in greatly increased rates of  calcium absorption and bone resorption, causing hypercalcaemia and calcium deposition in tissues,  particularly the arteries, heart, liver, kidneys and pancreas. This is known as metastatic calcification and  may interfere with the correct functioning of the organs, possibly causing renal stones, calcification of  other arteries and heart failure   High intake disturbs liver function, carbohydrate tolerance and urate metabolism. (>200 mg/day)  causes vasodilatation and flushing   Rare; an excess can result in sensory neuropathy. It can be used in treatment of premenstrual tension   Fig. 10.2 Role of vitamin A in vision. 11 -c/s retinal binds to  opsin, converting it to rhodopsin, the visual pigment of the rod  cells in the retina involved in vision and dark adaptation to light.  Low light intensity (scotopic vision) activates a series of  photochemical reactions that bleach rhodopsin, converting it to  a\\-trans retinal, which triggers a nerve impulse in the optic nerve  to the brain.    • Severe prolonged deficiency results in xerophthal-  mia: a dryness of the cornea and conjunctiva due  to progressive epithelial keratinization. Bitot's spots  may be seen, which are white plaques of keratinized  epithelial cells on the conjunctiva   • If untreated, keratomalacia develops, causing cor-  neal ulceration and the formation of opaque scar  tissue (cataracts); this causes irreversible blindness.   Diagnosis and treatment are usually on the basis of the  above clinical features. The following can also be  measured:   • The plasma concentration of vitamin A and retinol  binding protein   Urgent treatment with vitamin A (as retinol palmitate)  orally or intramuscularly prevents blindness. If the defi-  ciency is severe and has already caused keratomalacia,  eyesight cannot be restored. It is interesting to note that  vitamin A is also used successfully to treat a number of  skin problems, including acne (Fig. 10.3).    Fig. 10.3 Uses of vitamin Ain the treatment of skin disorders.   Pregnant women must not take more than 3.3 mg/ day  because vitamin A causes congenital defects. Therefore,  they must avoid vitamin A supplements or eating liver  because it contains about 13-40 mg of vitamin A per  100 g. Isotretinoin treatment for acne is absolutely con-  traindicated in pregnancy.   There is no RNI for vitamin D because it is synthesized  by the body.   • Endogenous synthesis: most vitamin D is made by  the body.   Vitamin D is a derivative of cholesterol and is therefore  not present in plants; vegetarians must make their own.   Vitamin D is manufactured in the skin by the action of  sunlight of wavelength 290-310 nm. No radiation of  this length is available between October and March in  the UK; therefore, the body relies on stores made during  summer.   Cholecalciferol undergoes two hydroxylation reactions,  the first in the liver and the second in the kidney, to form  the active form, 1,25-dihydroxycholecalciferol (Fig. 10.4).  Vitamin D is mostly stored as 25-hydroxycholecalciferol  in the liver.   The main role of vitamin D is in calcium homeostasis,  which it controls in three ways (Fig. 10.4):   • Increases uptake of calcium (and inorganic phos-  phate) from the intestine (main role)    Fig. 10.4 Synthesis, metabolism and functions of vitamin D.  The active form has three main effects:   1. Increases uptake of Ca 2+ (and inorganic phosphate) from  the intestine   3. Increases resorption of bone (when necessary) so that  calcium is released.   The causes of deficiency are also highlighted in blue at the levels  of synthesis affected.   • Increases resorption of bone (when necessary) so  that calcium is released.   Therefore, vitamin D increases the plasma concentra-  tion of calcium ions.   The active form, 1,25-dihydroxycholecalciferol, is a ste-  roid hormone. In intestinal cells it binds to a cytosolic  receptor. The resulting complex enters the nucleus and  binds to chromatin at a specific site (enhancer region  or response element) to increase the synthesis of a  calcium-binding protein, cal-bindin, resulting in in-  creased calcium reabsorption in the intestine.   • Children and women of Asian origin in sunlight-  poor areas   • Babies breastfed in winter because light of the correct  wavelength for production of vitamin D is not avail-  able for mothers   Vitamin D deficiency disrupts bone mineralization. In  children, this causes rickets; in adults, it causes osteoma-  lacia. These disorders are covered later in this chapter  with calcium deficiency.   A disruption of calcium homeostasis also causes  hypocalcaemia and hypophosphataemia (low plasma  calcium and phosphate). This may cause symptoms  of neuromuscular irritability, numbness, parasthesiae,  tetany and, possibly seizures.   Vitamin E consists of eight naturally occurring tocoph-  erols; a-tocopherol is the most active.   None. A diet high in polyunsaturated fatty acids (PUFA)  requires a high vitamin E intake.   Tocopherol is found 'dissolved' in dietary fat and is  absorbed with it. It is transported in the blood by lipo-  proteins, initially in chylomicrons which deliver dietary  vitamin E to the tissues. Vitamin E is transported from  the liver with very-low-density lipoproteins (VLDL)  and is stored in adipose tissue. Therefore, a defect in li-  poprotein and fat metabolism may lead to a deficiency  of vitamin E.   The functions and clinical manifestations of a deficiency  of vitamin E are listed in Fig. 10.5.   In humans, vitamin E deficiency is very rare and is only  seen in:   • Premature infants, causing haemolytic anaemia of  the newborn. Vitamin E crosses the placenta in the    Fig. 10.5 Vitamin E: function and effects of deficiency.  PUFA, polyunsaturated fatty acid; LDL, low-density  lipoprotein   Naturally occurring  antioxidant which  prevents oxidation of  cell components by  free radicals, e.g.  PUFA present in cell  membranes   Very rare except in  premature infants in  whom it can cause  haemolytic anaemia of  newborn    last trimester of pregnancy; therefore, premature  infants have very small vitamin E stores. Their eryth-  rocyte membranes are fragile and are susceptible  to free radical damage, leading to lysis of erythro-  cytes. Vitamin E supplements are given to pregnant  mothers to prevent this   • Children and adults, secondary to severe fat malab-  sorption. For example, biliary atresia, cholestatic  liver disease or a lipoprotein deficiency (e.g.  abetalipoproteinaemia) .   Vitamin E deficiency causes muscle weakness, periph-  eral neuropathy, ataxia and nystagmus. In children with  abetalipoproteinaemia, vitamin E therapy can prevent  the occurrence of severe spino-cerebellar degeneration  and gross ataxia.   • It is made mostly by the normal bacterial flora of  jejunum and ileum   The functions and clinical manifestations of a deficiency  of vitamin K are listed in Fig. 10.6.   A true deficiency is rare because most of the body's vita-  min K is synthesized by bacteria in the gut.    Vitamin K is a coenzyme  for the carboxylation of  glutamate residues of  blood clotting factors II,  VII, IX and X    True deficiency is rare  because bacteria in the  gut usually produce  enough   Long-term antibiotic  therapy leads to J, bacteria  and | vitamin K, resulting  in poor blood clotting and  bleeding disorders    HDNB is a coagulation disturbance, resulting from  vitamin K deficiency and consequently impaired  hepatic production of factors II, VII, IX and X.  Premature infants, infants exposed to perinatal  asphyxia and breastfed babies are most at risk of  developing HDNB. It classically presents in the fourth  day of life with gastrointestinal bleeding. Usually, the  bleeding is minor but can also result in major  haemorrhage and death. Therefore, every newborn  baby in the UK is given prophylactic intramuscular or  oral vitamin K.   • A decreased level of bacteria in the gut (e.g. due to  long-term antibiotic therapy)   • Newborn babies have sterile guts and cannot make  vitamin K initially   A deficiency of vitamin K results in low levels of the vi-  tamin K-dependent clotting factors II, VII, IX and X and  inhibition of the clotting cascade. Patients will have an  increased tendency to bleed and bruise.   The diagnosis and treatment of vitamin K deficiency is  covered in Fig. 10.7.   Newborn babies have sterile gut and have no bacteria to  make vitamin K. The newborn infant has virtually no  hepatic stores of vitamin K and it is present in only  low concentrations in human milk. Vitamin K defi-  ciency causes haemorrhagic disease of the newborn  (HDNB).   Thiamine pyrophosphate (TPP), which is formed by the  transfer of a pyrophosphate group from ATP to  thiamine.   The functions of thiamine are listed in Fig. 10.8, with its  mechanism of action described in Fig. 10.9.   • Beriberi. This occurs in two forms: wet beriberi,  which results in oedema, cardiovascular symptoms  and heart failure, and dry beriberi, which causes  muscle wasting and peripheral neuropathy   Beriberi (Fig. 10.10) is now seen only in the poorest  areas of South-East Asia where the staple food is   Decreased activity of pentose phosphate pathway results in low levels  of NADPH necessary for fatty acid synthesis; therefore this leads to a  decrease in synthesis of myelin, which may cause peripheral neuropathy   Fig. 10.9 Mechanism of action of thiamine. Thiamine  pyrophosphate (TPP), the active form of thiamine, acts as  coenzyme for pyruvate dehydrogenase and a-ketoglutarate  dehydrogenase reactions in the TCA cycle and for transketolase  in the pentose phosphate pathway.   polished rice, that is, the husk that contains most of the  vitamins, including thiamine, has been removed.   Diagnosis is by measurement of the transketolase  activity in erythrocytes, before and after the addition of    TPP. A greater than 30% increase in activity with TPP in-  dicates a deficiency. Thiamine can now also be mea-  sured directly in plasma.   Initially, treatment is with intramuscular injections  of thiamine for approximately 3 days (varies according  to severity) followed by daily, oral supplements of  thiamine. For wet beriberi, treatment results in a dra-  matic decrease in oedema and a quick improvement  of symptoms. For dry beriberi, there is a slower  improvement.   Diagnosis: made on  clinical grounds; condition  is reversible with  immediate thiamine  therapy   If untreated it may  develop into Korsakoffs  psychosis: a severe  irreversible syndrome  characterized by loss of  short-term memory   Major cause of dementia  in developed countries  Progression from  untreated Wernicke's  encephalopathy   As thiamine is present in most foods, a dietary deficiency  is rare in developed countries. The deficiency manifests  itself as Wernicke's encephalopathy (Fig. 10.11). In the  UK, a low thiamine intake is seen in:   • People with diseases of the upper gastrointestinal  tract (e.g. gastric cancer).    FAD and FMN are  coenzymes for a number of  oxidases and  dehydrogenases   They can accept two  hydrogens to form  FADH 2 and FMNH 2  respectively and take part  in redox reactions, e.g.  electron transport chain  or act as antioxidants   The functions and clinical manifestations of a deficiency  of riboflavin are listed in Fig. 10.12. Riboflavin is not  toxic in excess.   The synthesis of niacin from trytophan is a very ineffi-  cient process: as much as 60 mg of tryptophan is needed  to make 1 mg of niacin. Synthesis requires thiamine, ri-  boflavin and pyridoxine as cofactors, and only occurs  after the needs of protein synthesis are met. This means,  in theory, that niacin deficiency can be treated with a  high-protein diet, but lots would be needed!   The functions and clinical manifestations of a deficiency  of niacin are listed in Fig. 10.13.   Milk, eggs, liver. Riboflavin is readily destroyed by ultra-  violet light.   NAD is required for repair of UV light-  damaged DNA in areas of exposed skin  (nothing to do with redox state)   certain dyslipidaemias because it  inhibits lipolysis, leading to decreased  VLDL synthesis (see Chapter 5)    Pellagra: a disease of the skin,  gastrointestinal tract and central nervous  system  Incidence   Pellagra is rare and is found in areas where maize is the  staple food. It is now seen only in certain parts of Africa.  Maize contains niacin in a biologically unavailable  form, niacytin. Niacin can only be removed from the  maize by alkali treatment (Mexicans soak maize in lime  juice to release the niacin). Pellagra (Fig. 10. 14) can also  occur in conditions in which large amounts of trypto-  phan are metabolized; for example, carcinoid syn-  drome, which is very rare.   • Vitamin B 6 and pyridoxal phosphate deficiency (pyr-  idoxal phosphate is a cofactor for niacin synthesis  from tryptophan)   • Hartnup's disease: a failure to absorb tryptophan  from the diet (Fig. 6.6).   • Isoniazid treatment for tuberculosis inhibits vitamin  B s , causing a decrease in tryptophan synthesis.   Diagnosis is by the measurement of niacin or its metabo-  lites (N-methylnicotinamide or 2-pyridone) in the urine.   Photosensitive  symmetrical skin rash  occurs when skin is  exposed to sunlight:   Dementia occurs in  chronic disease and is  usually irreversible; may  develop tremor and  encephalopathy   Mild cases are reversible, dementia usually is not, and  may lead to death.   A high intake upsets liver function, carbohydrate toler-  ance and urate metabolism. More than 200 mg/day will  cause vasodilatation and flushing.   Vitamin B 6 exists in three forms: pyridoxine, pyridoxal  and pyridoxamine.   All three forms can be converted to the coenzyme pyri-  doxal phosphate (PLP).   The functions and clinical manifestations of a deficiency  of vitamin B 6 are listed in Fig. 10.15.    Fig. 10.15 Vitamin B 6 : functions and effects of deficiency.    The drug isoniazid binds to pyridoxal phosphate to  form an inactive hydrazone derivative, which is rapidly  excreted, causing the deficiency.   Vitamin B 6 supplements are given to all patients on iso-  niazid therapy   Toxicity is rare. Vitamin B 6 is actually used in the treat-  ment of premenstrual tension (PMT) . An excess is, how-  ever, associated with the development of a sensory  neuropathy.    (CH 2 ) 4 C _ N (CH 2 ) 4 LYS  H    Fig. 10.17 Biotin is a coenzyme for carboxylation reactions. It  binds to a lysine residue in carboxylase enzyme molecules.   As a coenzyme for carboxylation reactions, biotin binds  to a lysine residue in carboxylase enzymes (Fig. 10.17).   The functions and clinical manifestations of a deficiency  of biotin are listed in Fig. 10.18.   The functions and manifestations of a deficiency of pan-  tothenic acid are listed in Fig. 10.16. Panthothenic acid  is not toxic in excess.   Only animal sources: liver, meat, dairy foods; vegans are  at risk of deficiency.   Most foods, especially egg yolk, offal, yeast and nuts. A  significant amount is synthesized by bacteria in the  intestine.   As coenzyme A, it is  involved in the transfer of  acyl groups, e.g. acetyl  CoA, succinyl CoA, fatty  acyl CoA   It is also a component of  fatty acid synthase: acyl  carrier protein (see  Chapter 4)   N.B. a deficiency in rats  causes depigmentation of  fur, i.e. it turns grey. Not  toxic in excess   • acetyl CoA carboxylase  in fatty acid synthesis  (see Chapter 4)   • propionyl CoA carboxyl-  ase in p oxidation of  odd-numbered fatty  acids (see Fig. 10.20)   • eating lots of raw egg  whites, rich in a glyco-  protein, avidin, that binds  to biotin in the intestine  preventing its absorption   1 . Vitamin B 12 , released from food in the stomach, be-  comes bound to a glycoprotein carrier, intrinsic fac-  tor (IF), produced by gastric parietal cells   2. The complex of B 12 and intrinsic factor binds to re-  ceptors on the mucosal cells of the terminal ileum   3. B 12 is absorbed and transported to tissues, attached  to transcobalamin II. About 2-3 mg of B 12 are stored  by the body, mainly in the liver; this is relatively  large compared with its daily requirement.   Vitamin B 12 is a carrier of methyl groups. It is the coen-  zyme for two enzymes:   • Methylmalonyl CoA mutase, as deoxyadenosylcoba-  lamin, to assist in the breakdown of odd-numbered  fatty acids (Fig. 10.20)   Fig. 10.19 The absorption and transport of vitamin B 12  (numbers refer to the text).    Fig. 10.20 |3 Oxidation of odd-numbered fatty acids. B 12 is a  carrier of methyl groups. It is the coenzyme for methylmalonyl  CoA mutase, assisting in the breakdown of odd-numbered  fatty acids.    • Homocysteine methyltransferase, as methylcobala-  min, to assist in the synthesis of methionine. This re-  action also reverses the methylfolate trap,  regenerating tetrahydrofolate (THF) from methyl-  THF (discussed below with folate).   A significant amount of vitamin B 12 is stored; it takes   • The accumulation of abnormal odd-numbered  fatty acids, which may be incorporated into the cell  membranes of nerves, resulting in neurological  symptoms, inadequate myelin synthesis and nerve  degeneration   • Secondary 'artificial' folate deficiency since folate is  'trapped' as methyl TFIF. This causes a decrease in  nucleotide synthesis, resulting in megaloblastic  anaemia (see Chapter 7).   Subacute combined degeneration of the spinal cord is  the metabolic disorder due to vitamin B 12 and even  folate deficiency. It is characterized by a symmetrical  loss of posterior columns, causing an ataxic gait,  asymmetrical upper motor neurone signs in the lower  limbs, but absent reflexes, peripheral sensory  neuropathy, optic atrophy and dementia. It is treated  with intramuscular injections of hydroxycobalamin.    The most common cause of vitamin B 12 deficiency is  pernicious anaemia, an auto-immune condition where  antibodies are made by the body to intrinsic factor.   Reduced intake, for example, by vegans because vitamin  B 12 is only found in animal-derived foods. Reduced ab-  sorption caused by:   • Diseases of the terminal ileum which is the site of B 12  absorption (e.g. Crohn's disease or tuberculosis)   • Bypass of the B 12 absorption site (e.g. fistulae or sur-  gical resection of gut)   Body stores (mainly in the liver) are large relative to the  daily requirement, therefore a reduced intake alone  takes about 2-3 years to cause a deficiency.   Pernicious anaemia is the commonest cause of vitamin  B 12 deficiency.   It is more common in older women and is often associ-  ated with fair-haired and blue-eyed individuals, and  also the presence of other auto-immune disorders  (e.g. thyroid disease and Addison's disease).   Pernicious anaemia is an auto-immune disorder where  antibodies are made to either:   • Gastric parietal cells, causing atrophy or wasting of  the cells, thus preventing the production of intrinsic  factor and stomach acid (Fig. 10.21A)   • Intrinsic factor itself (Fig. 10.21B); antibodies bind  to the intrinsic factor, preventing it from either bind-  ing to vitamin B 12 (blocking antibodies) or binding  to the receptors in the terminal ileum (binding anti-  bodies). A lack of intrinsic factor leads to a decreased  uptake of vitamin B 12 . The clinical features of perni-  cious anaemia are discussed in Fig. 10.22.   Diagnosis is performed by analysis of the blood film  and bone marrow specimens and by the Schilling test,  which measures the absorption of vitamin B 12 :   • A 24 hr urine collection is performed to measure the  percentage of the dose of radioactive vitamin B 12 ex-  creted in the urine   • If the subject is vitamin B 12 deficient, less than 10%  will be excreted because the vitamin B 12 is being  used to replenish depleted stores   • If the result is abnormal, the test is repeated with the  addition of intrinsic factor   • If excretion is now normal, the diagnosis is perni-  cious anaemia.   The treatment of vitamin B 12 deficiency is intramuscular  injections of hydroxycobalamin for life. Initially, these  are more frequent to fill the stores. Pernicious anaemia  carries a slightly increased risk of carcinoma of the  stomach.   5,6,7,8-TITF, which is involved in the transfer of one-  carbon units.   Folate is absorbed in the duodenum and jejunum.  About 10 mg of folate is stored, mainly in the liver.   All one-carbon THF units are interconvertible except N 5 -  methyl THF; the THF cannot be released from it and is  trapped, forming the 'methyl- folate trap'.   The only way to re-form THF is via vitamin B 12 -  dependent synthesis of methionine: the methionine sal-  vage pathway (Fig. 10.23).   The functions and clinical manifestations of a deficiency  of folate are listed in Fig. 10.24.   The stores of folate are small relative to the daily require-  ments, therefore a deficiency state can develop in a few  months, particularly if it is associated with a period of  rapid growth.   1 . In normal individuals, vitamin B 12 released from food in the  stomach becomes bound to intrinsic factor (IF) produced by  gastric parietal cells   2. In individuals with pernicious anaemia, antibodies to the  gastric parietal cells cause wasting of the cells and therefore  prevent production of intrinsic factos by them. Vitamin B 12  is not absorbed, resulting in B 12 deficiency.   Fig. 10.21B Antibodies to intrinsic factor. Intrinsic factor  contains two binding sites; one for vitamin B 12 and a second one  for ileal receptors in the terminal ileum, its site of absorption. In  pernicious anaemia, antibodies produced may bind to either or  both of these sites.   • Secondary to B 12 deficiency: vitamin B 12 is essential  to maintain an adequate supply of the active form  of folate, that is 5,6,7,8-tetrahydrofolate. It regen-  erates THF from N 5 -methyl-THF in the methionine  salvage pathway (Fig. 7.3). Even if there are adequate  amounts of folate in the diet, in the absence of vita-  min B 12 , folate deficiency arises.   • Decreased intake: a poor diet is the most common  cause (e.g. inslimmers, elderly people and alcoholics)   • Increased requirement: during periods of rapid cell  growth, such as:   The clinical features and diagnosis of folate deficiency  are covered in Fig. 10.25.   The development of the neural tube in the fetus is de-  pendent on the presence of folic acid. Pregnant women  should take prophylactic folate supplements to reduce  the risk of neural tube defects such as spina bifida or an-  encephaly. The critical time is the first few weeks after   bone marrow:  megaloblasts (developing  red cells where nuclei  mature more slowly than  the cytoplasm)   B 12 deficiency causes  secondary folate  deficiency, which leads to  decreased production of  DNA and defective cell  division   peripheral neuropathy  affecting sensory neurons  of posterior and lateral  columns of spinal cord;  leads to subacute  combined degeneration of  spinal cord   Pathogenesis of CNS  damage unknown  impairment of CNS amino  acid and fatty acid  metabolism has been  implicated   Combination of jaundice  from red cell lysis and  pallor because of anaemia    conception: women should therefore start supplements  before conception to cover this period. A woman who  has already had a baby with a neural tube defect has  about a 1:20 risk of a second affected baby; the use of  folate supplements has been shown to reduce this risk.   A comparison of folate and vitamin B 12 deficiencies is  given in Fig. 10.26. A deficiency of either can cause a  macrocytic, megaloblastic anaemia. Patients suspected  of having either deficiency, must always be investigated  for both folate and B i2 deficiency since the administra-  tion of folic acid corrects the anaemia but masks a B 12  deficiency. Therefore, folate should never be given alone  in treatment of pernicious anaemia and other B 12 defi-  ciency states because it may precipitate an irreversible  peripheral neuropathy.   Fig. 10.23 Folate and vitamin B 12 . The only way to re-form  tetrahydrofolate is via vitamin B 12 -dependent synthesis of  methionine: the methionine salvage pathway.   The functions and clinical manifestations of a deficiency  of ascorbate are listed in Fig. 10.27.   this is identical to vitamin  B 12 deficiency (see  Fig. 10.22)   Blood film: macrocytes  (MCV > 100 fL)  megaloblasts in bone  marrow   N.B. peripheral  neuropathy and  neurological symptoms do  not occur in folate  deficiency   Red cell folate is a better  test of folate stores  normal = IBS-  750 mg/mL   Must always consider and  eliminate vitamin B 12  deficiency and  malignancy   In the past, this used to be common among sailors who  spent weeks at sea without any fresh fruit or vegetables.   Scurvy is caused by a poor dietary intake of fresh fruit  and vegetables. In the UK, it is seen in elderly people,  alcoholics and smokers. Smokers require twice the nor-  mal intake of vitamin C (80 mg/day). Humans have  about 6 months' store of vitamin C.   • reduces dietary Fe 3+ to  Fe 24 in the gut, allowing its  absorption (therefore, de-  ficiency can lead to  anaemia)   • inactivates free oxygen  radicals which damage  lipid membranes, proteins  and DNA   The treatment of vitamin C deficiency is 1 g daily of  ascorbate and lots of fresh fruit and vegetables in the diet.   Some researchers believe that large doses of vitamin C  cure many illnesses, such as the common cold and certain  immune-mediated diseases, and even help in cancer pre-  vention and promote fertility. The benefits of large doses  are unresolved and under review. It is thought that  1 -4 g/day of vitamin C can decrease the severity of symp-  toms of cold but not decrease the incidence. Vitamin C is  an antioxidant and it is thought that, along with vitamins   most symptoms are due  to a decrease in  collagen synthesis,  leading to poor  connective tissue  formation and wound  healing    A and E, it might decrease the incidence of coronary  heart disease and certain cancers by scavenging free  radicals, preventing oxidative damage to cells and their  components. This has not been confirmed.   A high intake of vitamin C may lead to the formation of  kidney stones, diarrhoea and also cause systemic condi-  tioning, that is, requirements increase as the body  adapts to metabolizing more.   The role of ascorbate in hydroxylation reactions:  Hydroxylase enzymes contain iron, which exists in  two oxidation states: Fe 3+ which is inactive, and Fe 2 +  which is reduced and active. Ascorbate is necessary to  maintain iron in its reduced and active state (Fe 2+ ).   The best way to learn this sort of information is to take a  large piece of paper and for each vitamin list only the  main points mentioned above. Examiners love to ask  about deficiency diseases.   The symptoms and signs of each individual vitamin de-  ficiency are covered in Chapter 1 1 and therefore will  only be covered briefly here (Fig. 10.29). General causes  of deficiency are:   Other causes of degenerative  eye changes, e.g. infection  such as syphilis, gonorrhoea,  chlamydia in neonates (rare)   Vitamin K: increased  bleeding produces  clotting problems (in  newborn babies,  causes haemorrhagic  disease of the  newborn)   • antibiotic therapy which  destroys vitamin K-  producing bacteria in gut    generalized malnutrition, mainly seen in developing  countries; or to poor diet, commonly seen in the  elderly and in the housebound in developed  countries, or it may occur in people on a vegan diet  • Fat malabsorption; for example, due to liver and bil-  iary tract disease or obstmction preventing bile salts  from facilitating absorption   The symptoms of a deficiency of vitamins B and C are  listed in Fig. 10.30.   Oedema, tachycardia, shortness of breath and other signs of heart  failure   Ascending peripheral neuropathy: initially weakness and numbness  of legs that ascends to involve trunk, arms and eventually brain  Confusion, ataxia, ophthalmoplegia and peripheral neuropathy   Failure of wound healing hypochromic, microcytic anaemia  Swollen, sore, spongy gums with bleeding   • Describe the main causes and clinical features of mineral deficiencies along with the treatments available    There are 103 known elements. Living organisms are  composed mainly of 11 of these. Namely carbon, hy-  drogen, oxygen, nitrogen and the seven major minerals:   • Calcium, phosphorus and magnesium, which are  used mainly in bone   • Sodium, potassium and chloride, which are the  main electrolytes present in the intracellular and ex-  tracellular fluid   The RNI is greater than 100 mg/ day for each of these  (the exception is sulphur for which no RNI has been  published).   In addition, there are at least 12 other elements that  are required in the diet in smaller quantities. These are  known as the essential trace elements, for which the RNI  is less than 100 mg/ day: iron, zinc, copper, cobalt, io-  dine, chromium, manganese, molybdenum, selenium,  vanadium, nickel and silicon.    Calcium is the most abundant mineral in the human  body. There is about 1.2 kg of calcium in the average  70-kg adult, of which 99% is in bone.   The RNI of calcium is 700 mg/day; it is higher during  periods of growth, pregnancy, lactation and after the  menopause.   Milk and milk products; a lot of foods are fortified with  calcium, for example, bread.   Absorption of calcium from the diet is variable depend-  ing on the following factors:   • Lactose and basic amino acids increase absorption  because they form complexes with calcium    • Fibre decreases absorption, therefore, vegans need a  lot more calcium.   • Parathyroid hormone, which increases plasma  calcium but decreases levels of inorganic  phosphate   • Vitamin D, which increases both plasma calcium  and inorganic phosphate levels   • Calcitonin, which decreases both plasma calcium  and inorganic phosphate levels.   • Secondary to vitamin D deficiency. Vitamin D is nec-  essary for the intestinal absorption of calcium and  phosphate (Fig. 11.2)    reduction in its normal strength, leading to soft, easily  deformed bones. The difference is that they occur at dif-  ferent stages of bone development. In rickets the  production of undermineralized bone results in a fail-  ure of adequate growth, whereas in osteomalacia, de-  mineralization of existing bones leads to an increased  risk of fractures.   • Rickets. The characteristics of rickets are shown in  Figs 11.3 and 11.4. Treatment is with calcium sup-  plements and education on a balanced diet. Vitamin  D supplements may also be required    Fig. 11.2 Synthesis, metabolism and  functions of vitamin D. Active form,   1 ,25-dihydroxycholecalciferol has  three main effects which increase the  plasma calcium concentration:   1. Increases uptake of Ca 2+ (and  inorganic phosphate) from the  intestine   3. Increases resorption of bone (when  necessary) so that calcium is  released.   • Osteomalacia. This is seen particularly in elderly  people and is usually secondary to vitamin D defi-  ciency. The characteristics of osteomalacia are  shown in Fig. 11.5   • Osteoporosis. This is the progressive reduction of to-  tal bone mass, usually due to the effects of oestrogen  deficiency, post-menopause. It is prevented by the  use of hormone replacement therapy but calcium  is also thought to have a role in its prevention. Ade-  quate calcium nutrition when young helps to  achieve a peak bone mass and this decreases the ef-  fects of loss and osteoporosis in later life. Calcium  supplements both before and after menopause, usu-  ally with vitamin D, are recommended.   Major causes of hypercalcaemia are primary hyperpara-  thyroidism and malignant disease and have nothing to  do with nutrition, and are therefore, beyond the scope  of this book. Very rarely, hypercalcaemia is associated  with the excessive ingestion of milk and antacids for  the control of indigestion. This decreases the renal  excretion of calcium: milk-alkali syndrome.   Weakness of proximal  muscles causing a proximal  myopathy with a  characteristic waddling gait   Calcium ions are normally found in cells and deposited  calcium salts are present in bones and teeth. In over-  load, calcium salts are deposited in normal tissues, lead-  ing to tissue metastatic calcification and impaired  function. This may cause renal stones, arrhythmias,  heart failure and calcification of the arteries. Muscle  weakness, tiredness, anorexia, constipation and a slug-  gish, nervous response may also be seen.   • It is required for the production of ATP and other  phosphorylated metabolic intermediates. Therefore,  it is fundamental to the maintenance of function of  cells in the body.   • Cofactor for more than 300 enzymes in the body,  that is, those enzymes that catalyse ATP-dependent  reactions. Magnesium binds to ATP, forming a mag-  nesium-ATP complex which is the substrate for en-  zymes such as kinases   • Interacts with calcium to affect the permeability of  excitable membranes and neuromuscular  transmission.   Seen in alcoholics; patients with liver cirrhosis; follow-  ing diuretic therapy; and in renal and intestinal disease.    Fig. 1 1 .6 The effects of phosphorous deficiency and excess.    Sodium, potassium and chloride function together to  regulate the osmolality of intracellular and extracellular  fluids. Sodium is the most important one in this respect.  Importantly, both high and low potassium concentra-  tion in plasma can cause severe cardiac problems. The  characteristics of sodium and potassium are listed in  Fig. 11.7.   Fluid and electrolyte balance management is an  important aspect of medicine, and rarely well  understood.   Key intravenous fluids you may prescribe for patients  include 3 main classes:   • Crystalloids (0.9% Saline, 5% Dextrose (glucose))  and on rarer occasions Hartmann's   The choice depends on whether you wish to replete  the circulatory volume, top up electrolytes or maintain  fluid balance. The fluids all have different compositions  of electrolytes and should be used with caution in cer-  tain groups of patients. Key composition of commonly  prescribed crystalloids:   It is important to note that Hartmann's solution is the  most physiological of the solutions with a composition  and osmolality most similar to plasma values (normal  plasma Osm: 275-299 milli-osmoles per kilogram). Also  note that 0.9% Saline is often referred to as 'normal sa-  line'; please remember this is not a 'normal' fluid as  the name implies, and as with all fluids, it should be only  prescribed once existing electrolyte and fluid statuses are  known.    The dietary intake of the sulphur-containing amino acid  methionine is essential for synthesis of cysteine (see  Fig. 6.11); both can then be incorporated into proteins  and enzymes.   Principal cation of ECF: plasma concentration  maintained between 135 and 145 mmol/L;  necessary for:   • Na + /K 1 -ATPase and uptake of solutes by cell   • Na + gradient provides driving force for secondary  active transport   • Na + /K ' -ATPase and uptake of molecules by cell   • disturbances common in hospitalized patients;  causes of loss include: vomiting, diarrhoea, use of  diuretics, Addison's disease, hyperglycaemia  (causing an osmotic diuresis) or renal failure   • sodium loss is usually accompanied by water loss,  leading to decrease in plasma volume and signs of  circulatory failure and collapse   • loss may be secondary to vomiting and the use  of diuretics, diarrhoea, excess steroids, hyper-  aldosteronism (e.g. Conn's syndrome), Cush-  ing's syndrome or alkalosis   • remember, intravenous insulin treatment without  supplementation of potassium leads to  hypokalaemia   Role in hypertension: sodium overload linked to  water overload can lead to oedema and to cardiac  failure   • the most common cause of potassium retention  and hyperkalaemia is renal failure   • both severe hypokalaemia and hyperkalaemia are  dangerous and require immediate treatment   Hyperkalaemia is a medical emergency and requires  immediate attention, especially when the potassium  concentration is above 6 mmol/l or there are associated  ECG changes. Refer to local and current guidelines  when managing, focus on the protection of the cardiac  membrane by giving calcium gluconate intravenously.  This buys one time to treat the hyperkalaemia by giving  infusion of both insulin and glucose to shift potassium  ions into the cell, lowering the plasma potassium  concentration.   Hypokalaemia (< 2.5 mmol./L) is also a medical  emergency that can result in cardiac arrythmias and  muscle weakness. This should be managed with  potassium replacement.   The daily loss of iron from the body is 0.5- 1.0 mg/day  and is due to:   • Desquamation of intestinal mucosal cells and biliary  excretion, about 0.3 mg/day   • Sweat and desquamation of skin cells, about 0. 1 mg/  day   Small daily losses are accounted for by the absorption of  dietary iron in the duodenum. The demand for iron in-  creases during growth, pregnancy and menstruation  (1 ml of blood loss is equal to 0.5 mg of iron). The daily  iron requirements are:    Only 10% of dietary iron is absorbed, therefore,  the amount ingested daily is equal to the daily  requirementx 10. Therefore, the RN1= 10-20 mg/day.   Liver, meat, green vegetables and cereals. Dietary iron  exists in two forms:   • Haem iron, which is derived from haemoglobin or  myoglobin in meat and is rapidly absorbed   • Non-haem iron, which is present in vegetables and  cereals and is absorbed slowly.   A summary of the absorption, transport, and functions  of iron is given in Fig. 11.8. Total body iron is about  3-5 g. About 60% is in haemoglobin and most of the  rest is stored mainly as ferritin, which is a protein-iron  complex. A small amount of iron is also stored as  haemosiderin.   Haem iron, present in meat, is readily absorbed. Inor-  ganic (non-haem) iron, present in vegetables and ce-  reals, is mostly in the oxidized (Fe 3+ ) state and must    Fig. 11.8 A summary of the absorption, transport and functions of iron. Iron is transported in the blood bound to transferrin;  each molecule of transferrin binds two Fe 2+ ions. This transports iron from sites of absorption and haemoglobin breakdown to  storage sites: mainly the reticuloendothelial cells (bone marrow, spleen), hepatocytes (liver) and muscle cells. These cells have  transferrin receptors enabling iron to be taken up by receptor-mediated endocytosis. Iron is also transported to these sites for  production of haemoglobin (bone marrow), myoglobin (muscle) or production of enzymes (liver).   Electron transport chain Cytochrome P450  (drug metabolism) Catalase (Fl 2 0 2 breakdown)  peroxidase   • Absorption is favoured in the ferrous (Fe 2 + ) as op-  posed to the ferric (Fe 3 + ) form   • Stomach hydrochloric acid and ascorbic acid both  favour absorption by reducing iron to the ferrous  form   • Increased erythropoietic activity (e.g. due to  bleeding, haemolysis or high altitude) increases  absorption   • Alcohol increases absorption. Phosphates and phy-  tates (from plants) form insoluble complexes with  iron and prevent absorption.   This is probably the most common cause of iron defi-  ciency, particularly in a vegan diet.   This occurs in premature babies as iron is trans-  ferred to the fetus during the last trimester of preg-  nancy. It also occurs during infancy, adolescence  and pregnancy, that is, during periods of increased  growth.   1 mL of blood contains 0.5 mg of iron. Therefore, a  small blood loss of 3-4 mL/day over a period of weeks  to months can cause chronic iron deficiency. Losses can  be from:   • The gut (e.g. due to peptic ulcer, hiatus hernia,  cancer of the stomach or caecum and ulcerative  colitis)   Always look for causes of chronic blood loss in a person  with iron deficiency anaemia.   Due to high levels of phytates in the diet, secondary to  vitamin C deficiency or after surgery (partial or total  gastrectomy).   Often, there is more than one cause, for example,  a poor quality diet and heavy periods in an  adolescent.   Infants, toddlers, adolescents, pregnant women, men-  struating women and elderly people are all at risk of iron  deficiency.   Highlight the importance to learning this concept of  bioavailability for exams.   Bioavailability is the efficiency  with which any  dietary nutrient is used in the body. A number of factors  can influence the absorption and use of nutrients. For  example:   The clinical features and diagnosis of iron deficiency  anaemia are covered in Fig. 11.10.   Finding the cause is essential. The treatment for iron de-  ficiency anaemia is oral iron supplements (e.g. ferrous  sulphate or gluconate). If malabsorption is suspected,  use intramuscular or intravenous iron. Iron supple-  ments should be given for long enough to correct the  haemoglobin level; when this is normal, iron must then  be continued for 3-6 months to replenish stores.   Iron overload leads to iron deposition in the tissues,  which may interfere with their function (Fig. 11.11).   There are two principal causes of iron overload. There is  an inherited form, called idiopathic primary haemochro-  matosis, which has a homozygote prevalence in the    l Production of  haemoglobin: less  oxygen reaches the  tissues especially the  brain and heart muscle  causing pallor, tiredness,  shortness of breath,  giddines, palpitations  symptoms usually occur  when haemoglobin  <8 g/dL   abnormalities: angular  stomatitis (cracked  corners of mouth)  glossitis (sore tongue)  koilonychia (spoon-  shaped nails)   • ) total iron-binding ca-  pacity (TIBC) (due to  increase in 'free' transferrin)    Cold standard for diagnosis  of iron deficiency is the  absence of iron stores in the  bone marrow   N.B. normal haemoglobin  concentration is 13-18 g/dL  in men, 11.5-16 g/dL in  women    population of 0.5%. Iron overload may also be acquired,  where it occurs secondary to an increased administration  of iron. This is called transfusional iron overload.   Idiopathic primary haemochromatosis is an autosomal  recessive disorder, characterized by the excessive absorp-  tion of iron in the small intestine. The gene defect is lo-  cated on chromosome 6. Only homozygotes manifest  clinical features; the accumulation of iron is gradual.  It usually presents in the fifth decade when levels of iron  are about 40-60 g compared with 3-5 g in a normal per-  son. The disease is clinically manifested more commonly  in men as women can compensate for the excess absorp-  tion by menstrual bleeding. The course of the disease de-  pends on the amount of dietary iron and the presence of  other dietary factors, such as vitamin C or alcohol.   Iron is deposited as insoluble haemosiderin, forming yel-  low granules in tissues (Fig. 11.12), which eventually in-  terfere with tissue function. An increase in iron levels  leads to an increase formation of free radicals, especially  the hydroxyl radical. At normal iron levels, a reactive su-  peroxide radical 0 2 * is removed effectively by the enzyme  superoxide dismutase, as shown in the reaction below:   20 2 ' + 2H+ -> H 2 0 2 + 0 2   However, in iron overload, Fe 3+ reacts with the  superoxide radical to form an extremely reactive  hydroxyl radical, OH", this hydroxyl radical is capa-  ble of damaging biological molecules, particularly  lipids, leading to lipid peroxidation and membrane  damage (especially of lysosomal membranes).   0 2 _* + Fe 3+ -> Fe 2+ + 0 2  Fe 2+ + H 2 0 2 -> Fe 3+ + OFT + OH*   Liver fibrosis and pigmentation resulting in  cirrhosis and eventually liver failure; may  progress to hepatocellular carcinoma (30%)   This results in the oxidation and destruction of cell  membranes and tissues.   The treatment for idiopathic primary haemochromato-  sis is regular venesection (i.e. removal of blood) to re-  duce the iron load. Plasma iron and ferritin levels are  used to monitor the treatment. Once the excess iron is  removed, the frequency of venesection is reduced.   Repeated blood transfusions over a long period of time  can cause iron overload. The ability of the reticuloendo-  thelial cells (spleen, liver and bone marrow) to store iron  is exceeded and iron is deposited at other sites. As with  primary iron overload, iron is deposited mainly in the  skin, heart, liver and pancreas. Patients with any condi-  tion requiring regular blood transfusions are regarded as  high risk (e.g. thalassaemia major, aplastic anaemia).   Chelation therapy with desferrioxamine is highly  effective in chelating the iron, thus enabling its  excretion.    The daily zinc requirement is 2-3 mg/ day but absorp-  tion is only about 30% effective. Therefore, the RNI is  10 mg/day. Zinc can be found in most foods. The total  body zinc is 2-3 g. It is found in all tissues but high con-  centrations are present in the liver, kidney, bone, retina,  muscle and prostate. The role of zinc in the body is  described in Fig. 11.12.   Acrodermatitis enteropathica is an extremely rare, auto-  somal recessive disorder that leads to the malabsorption  of zinc in the small intestine. It presents in infancy with  a severe symmetrical, eczematous rash around orifices  and on the hands and feet. Frequently, the lesions be-  come severely infected with Candida or bacterial infec-  tions, leading to death. Infants may also develop  growth retardation, hypogonadism and poor wound  healing.   The condition is completely cured by zinc therapy.  Zinc deficiency is also a very rare complication of par-  enteral nutrition when insufficient supplementation  is given.   The total body copper is about 75-150 mg. High copper  concentrations are found in the liver, brain, heart and  kidneys. Dietary copper is absorbed in the stomach  and duodenum and transported to the liver loosely  bound to albumin; the absorption is about 30% effec-  tive. It is incorporated into caeruloplasmin, a glycopro-  tein synthesized by the liver, which transports copper to  the tissues where it can be used for the synthesis of other  copper-containing enzymes. Normally, it is excreted in  the bile (daily loss is approximately 2-3 mg/day). In the  blood, 80-90% of the copper present is bound to  caeruloplasmin.   Copper is required for the synthesis of a number of cop-  per-containing enzymes (Fig. 11.13).   Menkes' kinky hair syndrome is a rare, X-linked disease  with an incidence of 1 in 50000-100000. It is caused by  the defective absorption of copper from the intestine,  leading to a decreased synthesis of copper-containing  enzymes (Fig. 11.14).    Fig. 11.13 The role of copper and the effects of copper deficiency.   Scavenges the superoxide radical and prevents  lipid peroxidation and membrane damage   Copper therapy has no significant effect. The life expec-  tancy is less than 2 years.   Wilson's disease is a rare, autosomal recessive disorder  (incidence of 1 in 100000). The defect has been iden-  tified on chromosome 13 and results in failure of the  liver to excrete copper in the bile. Copper incorpora-  tion into caeruloplasmin is also impaired. Copper ac-  cumulates and is deposited in the liver, basal ganglia  of the brain, kidneys and the eyes, causing damage  (Fig. 11.15).   Wilson's disease is treated by daily chelation therapy  with D-penicillamine. This is very effective at binding  copper and eliminating it in the urine. However, the  resulting liver and neurological damage is  permanent.    The human body contains about 15-20 mg of iodine,  most of which is in the thyroid gland. It is essential  for the synthesis of the thyroid hormones thyroxine  and triiodothyronine.   Endemic goitre, a generalized enlargement of the thy-  roid gland, occurs in areas where the soil and water lack  iodine, such that the daily intake is less than 70 mg  (usually mountainous areas). In the UK, it used to be  found in people in Derbyshire, causing the so-called  Derbyshire neck. The problem has now been eliminated  in most countries by the addition of iodine to table salt  and its prevalence is mostly restricted to developing  countries.   Normally iodine is used to make thyroxine and triiodo-  thyronine. Increased levels of these hormones exert a  negative feedback effect on the hypothalamus and    T 3 exerts negative  feedback on thyroid-  releasing hormone and  thyroid-stimulating  hormone release,  i.e. inhibits further  release T 3 is the main  active species   low levels of triiodothyronine (T 3 ) and thyroxine (T 4 )  no negative feedback on hypothalamus and  pituitary — ► J j thyroid-stimulating hormone    Fig.11.17 The hypothalamic-pituitary-thyroid feedback  system: dietary iodine limiting. Decreased production of thyroid  hormones releases negative feedback on hypothalamus and  anterior pituitary.    Fig. 11.16 The hypothalamic-pituitary-thyroid feedback  system: normal status. In the presence of dietary iodine, thyroid  hormones are produced which exert a negative feedback effect  on hypothalamus and pituitary, inhibiting release of TRH and  TSH.    anterior pituitary inhibiting further release of thyroid-  releasing hormone and thyroid-stimulating hormone  (Fig. 11.16), resulting in a decrease in their synthesis.  However, low levels of iodine decrease thyroxine for-  mation by the thyroid gland. This releases the negative  feedback on the hypothalamic-pituitary axis, causing  an uncontrolled increase in thyroid-stimulating hor-  mone secretion. High levels of TSH overstimulate  the thyroid gland, causing hyperplasia of the thyroid  epithelium and generalized enlargement (Fig. 11.17).  The addition of iodine to the diet should reverse  this effect.   Pregnant mothers who are deficient in iodine may give  birth to babies who are hypothyroid. Growth and    mental development in these babies are severely im-  paired and may be irreversible. The diagnosis is made  by the neonatal screening test, the Guthrie test, which  is performed on all newborn babies and looks for  raised thyroid-stimulating hormone levels. The same  sample is used to screen for phenylketonuria. Treatment  is lifelong oral replacement of thyroxine. Cretinism  can be prevented by the iodination of salt in the  maternal diet.   Hypothyroidism has an insidious onset with vague,  non-specific and diverse features. It usually presents  with fatigue, weight gain and cold intolerance. It may  also present with bradycardia, constipation, delayed  puberty, growth and mental retardation, and dry,  scaly, cold and thickened skin.    Fig. 11.18 The characteristics of some of the other trace elements.   Endemic  in parts of  China — >  cardio-  myopathy  (Keshan  disease)   Excessive dietary iodine may cause the symptoms of hy-  perthyroidism, that is, an overactive thyroid.    The characteristics of some of the other trace elements  not covered here are listed in Fig. 11.18.    The symptoms and signs of each individual mineral  deficiency are covered in this chapter. The symptoms  of the more important mineral deficiency disorders  are listed in Fig. 11.19. The differential diagnosis is bi-  ased towards metabolic causes.   Know about iron deficiency and overload, calcium  deficiency, and copper overload because they are  commonly asked about.   J absorption caused by: intestinal malabsorption such as  ulcerative colitis, Crohn's disease and coeliac disease,  f phosphates and phytates in diet or vitamin C deficiency  | requirement: periods of growth and pregnancy   • in children: rickets; present with soft, easily deformed  bones, short stature and failure to thrive   • secondary to vitamin D deficiency: vitamin D is  necessary for intestinal absorption of calcium and  phosphate   goitre can lead to symptoms of hypothyroidism: tiredness,  weight gain, anorexia, cold intolerance, constipation   • Describe the symptoms of the major metabolic and nutritional disorders   • Work out a differential diagnosis for the major metabolic and nutritional disorders   • Summarize the main points of history taking and examination with an emphasis on eliciting signs and  symptoms of metabolic disorders   • Understand the indications for and basic principles behind the routine investigations   • Understand how to interpret the results of routine investigations in order to make a thorough  assessment of the patient's nutritional status    This section deals with some examples of common pre-  senting complaints, and symptoms of metabolic disor-  ders. For each complaint, the major metabolic causes  are considered.   Remember, for each symptom there are also lots of  non-metabolic causes, which might be more common  but are beyond the scope of this book. For further  discussions of these, refer to a general medicine  textbook.   Fatigue is one of the most common general presenting  complaints and patients may describe: 'tiredness', 'lack  of energy' (one of the most common general symp-  toms), 'weakness' or 'exhaustion'.   It is important to obtain a clear history (when it  started, its progression, precipitating and relieving fac-  tors, and any associated symptoms) to help detect  the cause. Always clarify whether tiredness occurs on ef-  fort or rest. A good follow-up question to differentiate  psychological from physical causes is 'What slows you  down when you attempt doing anything strenuous?'.  The main metabolic causes of fatigue are described in  Fig. 12.1.   Weight loss is associated with many different condi-  tions. Only the main metabolic causes are considered  here.   A loss of 5% or more of the usual body weight over a  period of 6 months.   It is essential to take a clear and well-documented  history from the patient and to attempt to quantify true  weight loss over a specific time period and to establish  changes in diet and exercise. The difficulty of doing this  accurately is well recognized, unless the patient is con-  tinually weighed and monitored over a period of time.  Indicators of weight loss include:   Involuntary weight loss is often a clue that the  patient has a serious underlying disease such as  cancer. Figure 12.2 lists the common metabolic causes  of weight loss.   Anaemia occurs when the blood haemoglobin level is  below the normal range for the patient's age and sex.  The normal haemoglobin range in males is 13.5-  18.0 g/dL and in females is 1 1.5-16.0 g/dL.   The symptoms depend on the severity of the anae-  mia; a small reduction in haemoglobin is usually   • haemolytic anaemia, e.g. G6PDH or pyruvate kinase deficiency (see Fig. 12.3 for  other examples)   This may be due to an iodine deficiency or an auto-immune disease, Flashimoto's  thyroiditis   Protein-energy malnutrition (PEM): marasmus or kwashiorkor (see Chapter 9)  Tiredness, glucose intolerance, risk of hypertension    asymptomatic. Most symptoms are non-specific and re-  sult from a decreased oxygen supply to the tissues:   • Importantly, anaemia could unearth coronary heart  disease by precipitating angina (chest pain, brought  on by exercise, relieved by rest)   • Similarly, peripheral vascular disease may become  symptomatic (intermittent claudication: leg pain  precipitated by walking and quickly relieved by rest)   As these symptoms are relatively non-specific, to find  out the cause of the anaemia, a number of laboratory  tests may be performed, including:   • Full blood count, which includes the measurements of  haemoglobin concentration, the red cell count, and  several calculated indices, such as the mean cell vol-  ume and the mean cell haemoglobin (see Fig. 12.20)   • Malnutrition: common in de-  veloping countries, and in the  UK may be seen in the elderly   For iron deficiency anaemia, there are unique signs and  symptoms which help in clinical diagnosis. They are:  painless glossitis (smooth tongue), angular stomatitis  (sores at corner of the mouth), koilonychia (spoon-  shaped nails) and unusual dietary cravings such as pica  (soil-eating).   Mean cell volume (MCV) is a useful indicator of the  type of anemia and possible causes (see Fig. 12.3).    • Blood film, which provides information on the mor-  phology of cells   These tests and their results are discussed fully in  Chapter 1 1 .   There are many causes of anaemia, ranging from acute  blood loss to hereditary haemolytic anaemias such as  sickle cell anaemia. The main metabolic causes of anae-  mia are listed in Fig. 12.3.   Deficiency of erythrocyte enzymes such  as pyruvate kinase or G6PDH (see  Chapter 4)   Auto-immune condition in which  antibodies against intrinsic factors prevent  B 12 absorption in the terminal ileum,  leadingto B 12 deficiency (see Chapter 10)   Folic acid and vitamin B 12 required for  DNA synthesis; results in megaloblastic  anaemia (see Chapter 10)   This is commonly a problem in elderly patients and  assessment of volume status, detailed clinical skills and  investigation of prior events are required. Establishing  the duration and form of fluid loss from diuresis, diarrhoea  and vomiting, or bleeding can give an estimate of  the amount and type of fluid that needs replacement. If  accurate quantification of loss is needed; monitor inputs  and outputs (from bowel, bladder, or surgical drains).  Symptoms of volume depletion are usually secondary to  decreased tissue perfusion.   Usually non-specific. Lassitude, muscle cramps and diz-  ziness. Signs are more important and include low skin  turgor/ tension, dry mucous membranes, acute weight  loss, jugular venous pressure (JVP), sunken eyes, pos-  tural hypotension, increased heart rate and reduced  urine output.   Dehydrated patients tend to have an increased  plasma sodium concentration and elevated urea and  creatinine concentration. In the so-called pre-renal  uraemia (when a decrease of the glomerular filtration  rate is due to volume depletion rather than to nephron  destruction), plasma urea increase exceeds that of  creatinine.   This is an important state to recognize, especially in pa-  tients with heart failure or renal failure. It can be  detected from symptoms such as shortness of breath,  paroxysmal nocturnal dyspnoea (waking at night/when  lying flat due to shortness of breath) and signs includ-  ing: peripheral or sacral oedema (if sedentary; pitting),  crepitations on auscultation of the chest, raised JVP and  weight gain.   Fluid status is an important aspect of the examination  especially for cardiovascular, renal and surgical  patients.   All amino acid disorders are rare. Most present in in-  fancy as developmental delay, vomiting, failure to  thrive, mental retardation and seizures. The symptoms  are all non-specific, making the differential diagnosis  complex. All neonates are now screened for phenylke-  tonuria at a few days of age using the Guthrie test.  The other amino acid disorders must be considered  and eliminated when infants present with these symp-  toms without other adequate explanation; for example,  in the absence of infection (Fig. 12.4). Their diagnosis  depends on the measurement of metabolites in the  blood and urine.    'The history is usually the most important part of  the consultation.'   • Always introduce yourself, clarify the patient's  identity and cross-check patient data with case  notes   • Look around the bedside for clues such as monitors,  treatments, supportive equipment (glasses, walking  sticks and frames)   • Observe the patient initially from the end of the bed  and afterwards assess in more detail   • Look at their overall state (physical and psychiatric):  are they agitated/distressed/in pain or uncomfort-  able? This should take only a few seconds, and  should precede your introductory history questions  to ensure you address issues of pain/discomfort, and  avoid asking obvious or unsuitable questions before  proceeding with the history taking.   This is a basic plan designed for you to copy and take  with you when you first start clerking patients, which  should be memorized and become automatic after sev-  eral uses.   This should be a short statement of the symptoms the  patient is complaining of, in his or her own words.  For example, pain.   Try to get the patient to tell the story in his or her own  words from when he or she thought it began. For most  symptoms you will need to know:   • What is the time course/duration? When did the prob-  lem start or when did the patient first feel unwell?   • What is the nature of the complaint? And has it  changed? If it is pain: what is its site, radiation and    so on; if it is vomiting: how often does it happen  and how much is there? What's the colour? Is there  any visible blood?   • Does the symptom show a pattern? Is it continuous,  intermittent or continuous with acute exacerba-  tions?   • Are there any precipitating or relieving factors? For  example, is it related to meals or the type of food  eaten, or to stress? Is it helped by painkillers or  any other medication?   Exacerbation and relieving factors  Severity (e.g. on a scale of 1 to 10)    • Are there any other relevant or associated symp-  toms? For example, for chest pain, ask about palpi-  tations, sweating and nausea   • Has it happened before? Ask about any previous  treatment or investigations for the complaint.   Use simple terms as much as possible and avoid medical  jargon.   Some of these symptoms may have already been cov-  ered in the history of the presenting complaint. You  need to use your discretion about the extent of this en-  quiry. Good general systemic enquiry questions which  can help detect thyroid/metabolic disorders, infections,  malignancies, even lymphoma, include:   • Everything else - ask the patient if there is anyth-  ing else they would like to add that they have  noticed?    • Changes in exercise tolerance (quantify by stairs  climbed or distance walked before onset of  breathlessness)   • Remember: Dyspnoea is shortness of breath;  orthopnoea is breathlessness when lying down  flat (quantify in terms of number of pillows the  patient must sleep on to prevent dyspnoea); parox-  ysmal nocturnal dyspnoea is waking up at night  breathless   • Claudication (calf pain on walking) . Ask how far can  the patient walk without discomfort. Importantly,  does it happen when walking on the flat or on in-  clines and stairs   • Change in nature of stools: consistency, colour, mu-  cus, melaena (black tarry stools due to digested  blood), and do they float in the toilet pan  (steathorrhoea).   • Remember that the skin is frequently affected by  substances encountered at work and at home.   • Location: generalized aches and pains or discomfort  affecting a specific muscle?   • Diurnal variations in symptoms (i.e. with time of  the day)   • Pins and needles, paraesthesiae; especially ask about  sensation in the peripheries/glove and stocking  distribution   • Motor function of the limbs; power, tone, coordina-  tion, tremors/uncontrolled movements.   This should only be taken when relevant. For example,  'last menstrual period', should be checked when the  presenting complaint is abdominal pain to consider  ectopic pregnancy as a differential diagnosis.   Ask the patient about previous illnesses, hospital admis-  sions, operations and investigations, with dates.    You should always ask specifically about these condi-  tions, as well as anaemia and jaundice.   Ask about the patient's nutritional history. A lot of  metabolic and nutritional disorders present in infancy.  When dealing with children, ask the parents specifically  about problems during the pregnancy or birth, or when  their child was a neonate; for example, problems with  feeding, bowels or failure to thrive. Enquire about develop-  mental milestones: smiling, sitting, walking and talking.   Is the patient currently on any medication (either over-  the-counter or prescription). Ask about the dosage. If  relevant, ask about alternative therapies, herbal medi-  cine and recreational drugs. Also remember to ask fe-  male patients if they are on the oral contraceptive pill,  as many do not regard it as a drug.   Is the patient allergic to any medicines or foods that they  know of? Ask specifically about penicillin. If they say  they are allergic, ask about what happened when they  took the medicine or food.   How many per day and how long ago did the patient  start? If they say they have given up, you must also  ask when - it might be just yesterday! A useful way of  quantifying smoking is in pack years: 20 cigarettes  smoked per day for 1 year equals 1 pack year.   The CAGE questionnaire is a useful screening test for  alcoholism, with two or more positive answers  suggesting an alcohol problem. Have you ever:   How many drinks or units each week? An alcohol unit =  10 ml/8 g pure alcohol = 25 ml single measure of  whisky or a third of a pint of beer or half a standard  (175 ml) glass of red wine. The limits are 21 units for  males and 14 units for females per week.   Ask about any known illnesses in first-degree relatives,  in particular, diabetes and heart disease. Remember to  ask specifically about premature heart disease ('Has  there been any talk in the family about a lot of people  having heart disease at a young age?' The accepted def-  inition of premature disease is onset below age 55 in  men and 65 in women). It may help to make a quick  sketch of the family tree.   A number of inborn errors of metabolism are inher-  ited as autosomal recessive disorders and have a high    incidence amongst populations where marriages be-  tween first cousins are quite common, for example, Ash-  kenazi Jews. Be tactful when asking about a family  history of malignancy.   Ask about marital status, number of children, and the  type of accommodation. Ask about the patient's occupa-  tional history: current and previous jobs, exposure to  chemicals or asbestos and any time off work due to ill-  ness. Ask about financial and personal worries and any  risk-related behaviour, for example, taking illegal drugs  or any risky homosexual or heterosexual contacts.   This should be a brief recall of the main points. For ex-  ample: Jonathan Brown, a 4-year-old boy referred by  his general practitioner, presenting with a 6-week  history of increasing thirst, polyuria and weight loss.  His mother has insulin-dependent diabetes mellitus.  On examination . . .    Medical schools now place great emphasis on commu-  nication skills and their importance in the practice of  medicine. Examinations now feature some form of  structured clinical examination, e.g. OSCEs.   Communication skills contribute heavily to the  global score in clinical exams, so it is important to de-  velop a rapport with the patient or actor, especially as  in addition to giving you a good global score it will en-  courage them to divulge more information/clues. So  make sure you practice communicating with your  friends in clinical scenarios.   How you communicate is absolutely essential. Good  communication makes patients more comfortable and  greatly facilitates the correct diagnosis. On the other  hand, poor communication is the most common cause  of patients' complaints.   There are many factors that can make it difficult to talk  with patients and colleagues. It is important to be aware  of these and address the ones you can do something  about, while making allowances for those you can't.  For example:   • Noisy environment and lack of privacy - try to find a  quiet room or cubicle in which to see your patient, if  possible    • Pain - does the patient need analgesia now, rather  than after the history?   • Other medical factors - breathlessness, hearing im-  pairment and confusion (acute or chronic) can all  make communication difficult. Patience and persis-  tence are required in these situations   • Language and cultural barriers - if you encounter  problems, try to take the history with a member of  the family who can interpret. If this is not possible,  it may be possible to obtain the help of an interpreter.  In an acute situation, you may have to make do with  smiles, drawings and gestures to establish the impor-  tant points, such as the presence and site of pain   • Hostility - some people may feel (rightly or  wrongly) aggrieved by some aspect of the treatment  they had already received, ft is vital that you  do not take this personally or be drawn into a con-  frontation. Try to remain calm and civil, empathise  with the patient and apologise if appropriate. If all  else fails, politely explain that you don't feel any-  thing is being achieved and come back later.   It is important to put the patient at ease during a consul-  tation, although this is often easier said than done, as   people are often understandably concerned in the clin-  ical situation. The following are important skills:   • ALWAYS begin by checking very carefully the pa-  tient's identity, explaining who you are, and gaining  their consent to take a history   • Empathise with the patient: this means trying to un-  derstand their point of view, and is not the same as  sympathy. It is perfectly good practice to use such  phrases as 'I understand' or 'That must have been  very frightening', etc. when a patient is relating the  details of their history   • Use open questions to start, such as: "What made you  come to see a doctor today?' or 'ITave you any other  problems that have been worrying you?' Always ask  the old doctor's question: 'How do you feel gener-  ally?'; you will be surprised how many things you  will learn. It is a good idea to let the patient talk  freely for the first minute or so, before you focus  the history with closed questions such as: 'Does  the pain catch you when you breathe in?'   • Avoid using 'leading' questions, which direct the pa-  tient as to what to say, e.g. compare 'Does the pain  go anywhere?' an open-ended question with: 'Does  the pain shoot down your left arm?' a leading question   • Use interjectors ('I see') to maintain the flow of  conversation   • Check that you have understood what the patient  has told you by repeating a summary back to him  or her   • Reflect the terminology your patient uses for symp-  toms and diagnoses as appropriate.   A large proportion of our communication 'bandwidth'  is non-verbal. This includes body posture, facial expres-  sion and gestures; we are conscious of some of these  things, but most are subconscious. Non-verbal cues  are very important in a clinical setting, both in achieving  a rapport with patients and in gaining insight into their  condition. The following points may be helpful during a  consultation:   • Sit with the patient so that your eyes are on roughly  the same level, preferably without a desk as a barrier  between you. Maintain a comfortable distance, and  try to face them while you are talking. It is also useful  to make sure you have a comfortable position to  write when you are taking a history; kneeling by  the bedside is sometimes the best option   • Maintain good eye contact, even if the patient  doesn't. However, try not to stare   • Use non-verbal cues to show you are listening, and  encourage the patient: nodding, smiling and even  appropriate laughter can help to put the patient at  ease. Smiling is particularly important   • It is worth having practice sessions with friends be-  fore you go into an exam, as they can point out  any nervous habits that you might be unaware of.   I lave a mental checklist of objectives when you go into a  consultation - especially when this is part of an exam.  Once again, the key to this is practice, preferably on  the wards, but you can also run through mock scenarios  in a study group if you are short of time. You may also  need to produce this kind of list in a short-answer exam  paper or viva. For example:   • Find out why the patient has presented to you and  the impact of the condition.   • Find out what the patient understands about the  problem, and if they have their own theory as to  what has caused it; ensure you explore their main  concerns   • What are the patient's expectations of this consulta-  tion - what do they want from meeting with you  today?   • Explore the problem with history and examination,  and formulate a plan for further management, i.e.  investigations and treatment    • Explain your findings and plan to the patient as  clearly as possible   CALF (Check-Ask-Literature-Follow-up) is a good way  to use the last few minutes of an OSCE station  to your advantage to cover the last four points can be  remembered with the mnemonic 'CALF'.   • Ask if there is anything else the patient is concerned  about   • Follow-up - make sure the patient knows what the  next point of contact will be: e.g. an outpatient  appointment.   When interviewing the patient, remember to ask about  the patient's perspective; this can gain you extra points  in exams. Determine, acknowledge and appropriately  explore the patient's ICE; Ideas, Concerns and  Expectations. Also ask how their problems are affecting  their life, and encourage the patient to express their  feelings.    This section deals with the main signs on examination  that are caused by an underlying metabolic disorder,  which may be observed on examination. This is not a  comprehensive guide to clinical assessment, so you  should refer to a detailed clinical examination textbook  for this. Some of the signs mentioned are relatively non-  specific and may be related to other diseases, which may  indeed be a lot more common than the metabolic cause.  Others may be specific to, and in fact be diagnostic of, a  metabolic disorder. Just remember, a lot of metabolic  disorders are very rare and you may go through all your  working life without seeing them.   This section considers the main signs that are indicative  of an underlying metabolic cause. These signs are often  non-specific and, therefore, there may be other possible  non-metabolic causes that must be considered and  eliminated in the differential diagnosis. In any clinical  examination it is important that you are seen to gener-  ally inspect the patient. Stand at the foot of the bed and  observe the patient taking a breath in and out.   The main signs you need to look out for are: wasting,  cachexia and obesity (Fig. 12.5), pallor, jaundice (see  Fig. 12.10), respiratory distress (see Fig. 12.12) and  tremors (Fig. 12.6).   • When severe, patient has a thin  emaciated appearance, almost  skeletal, and it is referred to as  cachexia   • Cachexia implies serious underly-  ing cause, such as cancer or AIDS   • In developing countries wasting  probably due to malnutrition due to  insufficient intake causing by  marasmus   • Measure weight and height and cal-  culate body mass index (BMI)   • Compare with tables of ideal weight  for height (mid-arm circumference  and skin-fold thickness are rarely  useful in practice)   • BMI > 30 kg/m 2 is regarded as  obese (see Chapter 9)   Usually energy input is greater than  energy output  Obesity is also seen in:    Fig. 12.6 Assessment of tremors, and their significance in metabolic disorder. Four common tremors are covered below.   Patient holds arms outstretched  in front of them with hands flat  Place a piece of paper on them   Also seen in: hypoglycaemia,  alcoholism, hyperthyroid (thyrotoxic)  patients, and Wilson's disease   Flapping tremor; C0 2 retention  caused by hyperventilation may be  seen in people with diabetes or can be  seen with hepatic encephalopathy in  chronic liver disease    2. Position and expose the patient (at 45 degrees for  cardiovascular and respiratory examinations with  the chest exposed, and at 1 80 degrees with the abdo-  men exposed (xiphisternum to pelvis) for abdomi-  nal examinations)   3. Ask if they are in any pain and ensure they are  comfortable   4. General observations; stand back and observe  (appearance, level of consciousness, colour and  distress) at rest   5. Examine starting at the hands, assessing the limbs,  head and neck in turn   6. Move onto detailed examination (inspection, palpa-  tion, percussion, auscultation) of the relevant sys-  tem; respiratory, cardiovascular, abdominal,  neurological   Examination of the limbs for underlying metabolic dis-  order can be conveniently divided into assessment of  skin and joint problems associated with metabolic  disorders (Fig. 12.7) and vascular supply (Fig. 12.8).  Remember to check peripheral pulses.   Diabetic patients: Look specifically for ischaemic and  neuropathic damage leading to ulceration and defor-  mity of limbs (see Chapter 8).    Patients with peripheral vascular disease: Look for ar-  terial ulceration and, in extreme disease, gangrene.   There are a number of signs on the hands and nails in-  dicative of underlying metabolic disorder. They are of-  ten subtle (Fig. 12.9). Clubbing, which is a common  exam question, may be indicative of liver cirrhosis  due to a number of causes, some of which are listed  in Fig. 12.9. However, it is most commonly caused by  suppurative lung disease or infective endocarditis and  therefore these must always be highest on your list of  differential diagnoses. It may also be congenital.   • Assessment of vascular supply. The quickest way to  do this is to feel the pulses (Fig. 12.8). You should also  observe colour, assess capillary filling time, feel for  temperature and look for ankle oedema   • Neurological assessment. Both motor and sensory  systems are particularly important in patients with  diabetes.   Tendon xanthomata, which are observed usually on  the Achilles tendon or extensor tendons on the back of  the hand, are often diagnostic of dyslipidaemias.   Also note there are cardiovascular and  other causes including thyroid disease,  and even congenital causes.   Coeliac disease, Crohn's disease, and  Cirrhosis of the liver caused by:   This can affect any joint in the body. In an acute at-  tack, look for a red, inflamed, painful joint. In  chronic gout, look for gouty tophi: deposits of urate  crystals around joints, tendons and the cartilage of  ear lobes, causing yellow discolouration of the over-  lying skin.   A number of metabolic and nutritional disorders  result in clinical signs evident on the face. For ease,  the signs observed are divided into those affecting ei-  ther the eyes (Fig. 12.10) or the lips and mouth  (Fig. 12.11).   With the exception of iodine deficiency and thyroid dis-  ease, there are very few metabolic or nutritional disor-  ders that manifest as signs in the neck.   book at the patient's neck and ask the patient to swallow.  You will often observe a prominent goitre (a diffuse en-  largement of the thyroid gland). Goitre, can be due to io-  dine deficiency and can also be seen in thyroid diseases  such as Graves' disease and Hashimoto's thyroiditis. All  thyroid lumps ascend on swallowing because they are  attached to the trachea.   Structure to examine a lump (to help differentiate  nature of the lump; benign or malignant).   Inspection: The 5 S’s: Site, Size, Shape, Surface,  Surroundings (state of adjacent tissues and  overlying skin).   Extras: Colour, edge, composition, reducibility,  pulsatility, and any findings on percussion and  auscultation.   Always mention the need to assess neurovascular  status in more detail.    Fig. 12.8 Peripheral pulses provide a quick assessment of vascular supply in the limbs.   J. or absent peripheral pulses (may also hear bruit  over the femoral artery, indicating turbulent  blood flow caused by stenosis of arteries)   May occur secondary to endocrine or renal  disease or to obesity in 95% of cases, cause  of high BP is unknown; 'essential'  hypertension   The signs associated with thorax can be divided  into those related to respiratory and cardiovascular  systems.   Few metabolic disorders result in obvious respiratory  signs. Therefore, only a brief discussion is included here  on acid base disorders.    Checklist for examination of the respiratory  system (Figs 12.12 and 12.13)   Use the generic formula for examinations outlined at  the start of this examination section. Specifically for a  respiratory examination:   • Observe any respiratory distress, the level of con-  sciousness, chest expansion (is it equal on both  sides?), tachypnoea, and so on.   Remember for examination of any system follow  the sequence: inspection, palpation, percussion and  auscultation.   As with the respiratory system, few metabolic disorders  present with cardiovascular signs. However, anaemia of  any cause can precipitate angina and eventually cause  heart failure and, in extreme cases, shock.   Metabolic signs that can be observed during a cardiovas-  cular examination are listed in Fig. 12.14.   Anaemia of any cause can lead to an innocent ejection  systolic murmur. For heart failure, you may hear a third  heart sound. Checklist for auscultation:   • Feel the pulse (carotid on one side) simultaneously  to listening to identify systolic and diastolic sounds   • Are there two heart sounds present? The first heart  sound is due to the closure of the mitral and tricus-  pid valves. The second heart sound is due to the  closure of the aortic and pulmonary valves. Listen  over all four areas (mitral, triscupid, aortic and  pulmonary)   on examination of the limbs: skin and joint problems associated with   • Achilles tendon and finger extensors on back of  hand leading to thickening   • Fat deposits in palmar creases of hand called  palmar xanthomata   • Deposits of urate crystals around joints, tendons  and cartilage of ear lobes    Listen for extra third and fourth heart sounds  Listen for murmurs. Murmurs are caused by turbu-  lent blood flow. They are classified into systolic, di-  astolic or continuous, depending on their timing  with the cardiac cycle   Listen over the carotid, renal and femoral arteries for  bruits. These indicate turbulent blood flow caused  by stenosis of arteries; they are heard in patients with  disseminated atherosclerosis.   Identify murmurs with their timing (systolic or  diastolic?), location - area where loudest, radiation,  character, pitch, changes with respiration or position/  movement or other accentuating manoeuvres.   Most metabolic disorders that produce abdominal signs  do so as a result of excessive deposition of a metabolite  or nutrient in organs such as the liver, or in arteries or  the skin. This interferes with the correct functioning  of the organ. For example:   • In glycogen storage diseases, the deposition of glyco-  gen in the liver causes hepatomegaly   • Hepatomegaly may be caused by fat deposition  (steatosis) in patient with dyslipidaemia   • In atherosclerotic disease, the deposition of fat in the  walls of arteries leads to atherosclerotic plaque for-  mation, resulting in turbulent blood flow which  may be heard as bruits over carotid or renal arteries.  Aortic aneurysm may cause a palpable pulsation in  the abdomen (be very careful when examining    Fig. 12.10 Observation of the eyes for signs associated with metabolic disorder.   Yellow discoloration of sclerae is  a more sensitive indicator of  jaundice than skin colour in  mild-moderate disease (sclerae  turn yellow first). With severe  jaundice, skin is yellow-green   • haemolytic anaemia (e.g. due to G6PDH or  pyruate kinase deficiency)   Anaemia:  observe colour of  conjunctiva (pull down the  lower eyelid) or buccal  mucosa (most sensitive  indicator: mucous  membranes)   Pale/pink colour  N.B. normal skin colour varies  according to skin thickness,  circulation and pigmentation  So paleness may be normal for  patient or indicative of anaemia  N.B. it is a poor indicator of  anaemia   Non-tender yellow fatty lumps  in the skin particularly around  the eyelids   • They may or may not indicate dyslipidaemia (see  Chapter 5)   • Significant in patients < 35 years old, as it may in-  dicate hyperlipidaemia, such as FH or familial  combined hyperlipidaemia (see Chapter 5)   Kayser-Fleischer rings:  examine corneal-sclera  junction for rings; N.B. they  can only been seen with a  slit lamp    Fig. 12.11 Observation of the mouth and tongue for signs associated with metabolic disorder.   Central cyanosis: purple-blue colour because  of excess methaemoglobin in the tissues   • Since methaemoglobin cannot carry  oxygen, this leads to poor perfusion of  tissues and cyanosis   Angular stomatitis: inflamed, cracked corners  of mouth; cracks may become infected with  Candida albicans   Common in elderly due to iron  deficiency or deficiency of B-group  vitamins   Signs of respiratory distress  (rate, depth and rhythm of  breathing are observed)   E.g. tachypnoea, use of accessory  muscles of respiration, nasal flare and  sternal recession   In starvation, severe muscle wasting  can eventually cause wasting of the  diaphragm, leading to respiratory  distress and death   • Pigeon chest, pectus carinatum:  prominent sternum often accompanied  by indrawing of softened ribs along  attachment of diaphragm, Harrison's  sulcus   • Rickety rosary: expansion or swelling of  ribs at costochondral junctions   • Peripheral cyanosis: observe  purple-blue colour of extremities  (fingers and toes) caused by increased  level of deoxygenated blood   Inadequate perfusion of tissues caused  by peripheral vascular disease may be  seen in diabetics patients   Respiratory rate: count for a  minute; is it fast or laboured?  normal is 15-20/min (for an  adult)   • Deep Kussmaul respiration; deep,  sighing breathing with rapid respiratory  rate   Metabolic acidosis is a state that arises  with a diabetic ketoacidosis or uraemia,  and affects the respiratory system by  causing a respiratory compensation - to  correct the pH and acidosis — > this is  through increased respiratory rate and  Kussmaul breathing   • Anaemia from iron/folate/B 12 /vitamin C deficiency wet beri-  beri - B1 deficiency    Fig. 12.14 Clinical signs that may be observed during cardiovascular examination.    Fig. 12.15 Clinical signs that can be observed during an abdominal examination, and their underlying metabolic causes.   Abdominal distension: note  shape, symmetry, size of any  bulge or mass   Single, central arteriole feeding a  number of small branches in a radial  manner, with blanching (turning  white) on pressure    this!). Think of an abdominal aortic aneurysm in  elderlym male patients presenting with an abdom-  inal or back pain. This is a medical emergency.   • The patient should be lying as flat as possible, with  arms by his or her sides   • The patient should be exposed from the nipples to  the knees; however, in the interest of privacy, it is  best to expose in stages, beginning with xiphister-  num to pubis   • Kneel beside the bed so that you are at the same level  as the patient   • As with any system of the body go through the se-  quence of inspection, palpation, percussion and  auscultation.   Observe the general symmetry and shape of the  abdomen. The clinical signs that can be observed  during an abdominal examination, and their underly-  ing metabolic causes, are listed in Fig. 12.15. It is partic-  ularly relevant to comment on observing nutritional  and hydration status in an abdominal examination.   • Before you start, ask the patient 'I lave you any pain  anywhere in your abdomen?'. If the answer is 'yes',  begin your palpation furthest from the pain   • The abdomen is divided either into nine areas  (Fig. 12.16)    • Palpate all regions of the abdomen lightly then with  greater pressure (deep palpation), then palpate the  liver, spleen, while the patient takes deep breathes  to feel the organs move with respiration. Ask the pa-  tient to breath normally again as you palpate in the  midline for the abdominal aorta (to identify a path-  ological expansive mass indicating an aneursym;  NB: a pulsative aorta can be normal in slim pa-  tients). Finally, ballot the kidneys   • The clinical signs with their underlying metabolic  causes that can be detected on palpation of the abdo-  men are listed in Fig. 12.17.   • To outline the liver size. The liver is 'dull' to percus-  sion and therefore, it is useful in determining the de-  gree of hepatomegaly   • In the presence of abdominal distension, to deter-  mine whether it is due to solid, gas or free fluid (as-  cites) in the abdomen. The technique 'shifting  dullness' can be used to distinguish the presence  of ascites from solid or gas. Ascites is seen in conges-  tive heart failure, liver cirrhosis and secondary to wet  beriberi and kwashiorkor.    Also use percussion to demarcate the size of the spleen  and bladder.   The clinical signs that can be detected on auscultation of  the abdomen, with their underlying metabolic causes,  are listed in Fig. 12.18.   Fig. 12.16 Examination of the abdomen. A  schematic representation of the abdomen showing  nine areas    Fig. 12.17 Clinical signs that can be detected on palpation of the abdomen, with their underlying metabolic causes.   site (for clarity ask the patient to point  to where the pain is)   Acute, severe upper abdominal  pain ± guarding, rigidity and  rebound tenderness (= cardinal  signs of peritonism)   • Acute pancreatitis most commonly  due to gallstones and alcohol, but also  seen in type 1 familial lipoprotein lipase  deficiency orapoC-ll hyperlipidaemia  (see Chapter 5)   Spleen supposedly has a palpable  notch on its medial side but it is  very difficult to feel   • Lower pole of right kidney can be felt  in very thin or wasted people   Listen for bruits in the aorta (midline, above umbilicus),  and for renal and femoral bruits   Fig. 12.18 Clinical signs that can be detected on auscultation of the abdomen, and their underlying metabolic causes. (Figure  taken from http://faculty.washington.edu/alexbert/MEDEX/Fall/ABDAscultation.jpg)   Clinical signs can be a combination of any of the following:   • Pallor; which indicates anaemia, possibly caused by an iron, vitamin B 12 or folate deficiency   • Specific effects of vitamin deficiency; for example, deficiency of vitamin A causes Bitot's spots on the eyes,  or deficiency of vitamin D and calcium causes rickets in children and osteomalacia in adults   The clinical signs of nutritional disorders are summa-  rized in Fig. 12.19.    This section describes selected aspects of clinical inves-  tigation relevant to metabolic disorder. It is not a com-  prehensive description of laboratory tests used in  clinical practice. The main tests used every day to assess  metabolic function can be divided into:    • 'First-line' tests, that is, the tests most frequently  requested and often important for planning initial  management   With all routine investigations, the results should not be  interpreted separately; for example, in the diagnosis of  anaemia, the haematology and clinical biochemistry  tests are all part of the comprehensive patient assessment.   The simplest, first-line haematology test is the full blood  count (FBC). This measures red cell count and indices,  total and differential white cell count and platelets. The  'second-line' tests include clotting studies and assess-  ment of serum iron status and bone marrow iron stores  (Fig. 12.20).   First-line tests include urea and electrolytes (U&E), blood  glucose, liver function tests (EFTs) and troponin (used in  the diagnosis of myocardial infarction) (Fig. 12.21). Sec-  ond-line tests include thyroid function tests, glycated  haemoglobin, serum magnesium, ferritin, folate, lipid  profile and C-reactive protein (important in the diagno-  sis and monitoring of infection). Other specialized tests  include the measurements of vitamin and trace element  concentrations performed in patients who receive total  parenteral nutrition, and the specific diagnosis of genetic  metabolic defects in paediatrics. The measurement of  hormone levels in blood and tumour markers is a sub-  stantial part of specialized biochemistry testing.   You do not need to learn normal range values for  routine investigations as they will be given in exams.  However, a familiarity with the common tests is helpful  in appreciating the degree of abnormality when a result  is abnormally high or low.   Urine is commonly tested for glucose, protein and ke-  tones (dipstick tests). Although not as sensitive as blood  tests, urinalysis provides a quick and easy method of in-  vestigation (Fig. 12.22). Note that urinary ketones are  important in the diagnosis of diabetic ketoacidosis.   These tests are usually performed to confirm a diagno-  sis, usually after simpler biochemical tests have been  done (Fig. 12.23), and are often performed together  with medical imaging tests.   An example of an immunopathological investigations is  listed in Fig. 12.24.   Medical imaging has an increasingly important role in  medical investigations and diagnosis, with increased  use of MRI, due to the advantageous safety profile.    Fig. 12.21 First- and second-line biochemical investigations on blood or serum.    carcinoma of head of the pancreas (fairly non-  specific test of liver function)   potassium-sparing diuretics  Low: renal or intestinal loss surgical drainage of the  bowel, vomiting insulin treatment of diabetic  ketoacidosis hyperaldosteronism, diuretics   FHigh: renal damage  Urinary tract infections  (leucocytes and nitrites  may also be present)   Wilson's disease: increased copper  deposition leading to liver cirrhosis  Flaemochromatosis: iron deposition  may cause cirrhosis which may  progress to hepatocellular  carcinoma   Gout: the presence of yellow,  needle-shaped negatively  birefringent monosodium urate  crystals    There are many medical imaging tests used, from  simple radiographs and ultrasound to magnetic reso-  nance imaging and positron emission tomography  scanning. Figure 12.25 illustrates some examples.   Any patient presenting to casualty will require a  combination of first-line tests. Including:   • Blood glucose. Bedside blood glucose can be life  saving if a patient comes in unconscious or confused:  he or she may be drunk or severely hypoglycaemic   • If there are signs of infection, blood, urine, sputum,  wound swabs and CSF (if appropriate) samples  must be taken for culture, and the measurement of  C-reactive protein is useful   • Electrocardiogram and chest radiograph if necessary  •Troponin measurement if myocardial infarction is   Diagnosis of heart failure: anaemia of any cause, ischaemic heart disease, hypercalcaemia, or iron  overload, can all result in heart failure; on a Chest X-ray this can show up as: (reference: oxford  handbook of clinical medicine; 7th edition, permissions granted)   Defective bone mineralization: in rickets and osteomalacia: defective mineralization seen in  pelvis, long bones and ribs in the early stages   Used to identify abnormalities and to exclude focal lesions due to tumours or infection; essential  tool in the diagnosis of space-occupying lesions   The measurement of blood glucose is used to confirm or  reject a diagnosis of diabetes mellitus or impaired glu-  cose tolerance and to monitor the control of blood glu-  cose in diabetic patients.   Reference ranges for blood glucose levels are shown  in Fig. 12.26.   The estimation of blood glucose uses the glucose oxi-  dase and peroxidase reaction.   The test is based on the reaction catalysed by the en-  zymes glucose oxidase and peroxidase, and a peroxidase  substrate (a dye). Glucose oxidase oxidizes glucose pre-  sent in a deproteinized blood sample to gluconolactone  and hydrogen peroxide. The hydrogen peroxide reacts  with a dye to form a coloured complex, absorbance of  which is read in a spectrophotometer. Under standard  conditions, the amount of glucose in the unknown  blood sample is equal to the amount of coloured prod-  uct formed. Standardized solutions of glucose are pro-  cessed at the same time in order to construct a  calibration curve. Therefore, the amount of glucose in  the unknown blood sample can be read off from the  curve.   The test is specific for glucose. A similar enzyme reaction  is found in commercially available self-monitoring re-  agent strips: these are commonly used at home by pa-  tients with type 1 diabetes.   The fasting blood glucose sample is usually used to  diagnose diabetes mellitus in an asymptomatic pa-  tient or if the random blood glucose results are  borderline.   The patient should be fasted overnight (at least  10 hours) and have their blood glucose tested the fol-  lowing morning. Interpretation of fasting glucose re-  sults is shown in Fig 12.26.   The oral glucose tolerance test (OGTT) is a reference  method to diagnose disturbances in glucose homeo-  stasis. However, fasting blood glucose provides very  similar information and should be used first.  The use of OGTT is restricted to the detection of bor-  derline cases.    Fig. 12.26 Diagnostic intervals of fasting glucose for diabetes and impaired fasting glucose (IFG) and following this, diagnostic  intervals for impaired glucose tolerance (IGT) from an oral glucose tolerance test (OGTT).   Fasting blood glucose > 7.0 mmol/L and/or 2 h value >11.1 mmol/L in an OGTT   Fasting plasma glucose < 7.0 mmol/L and 2 h value between 7.8 and 11.1 mmol/  L in an OGTT   Patients should make sure that they eat a normal diet,  containing adequate carbohydrate, for the preceding  3 days. This ensures that the enzymes involved in  glucose metabolism are present at normal levels. Af-  ter an overnight fast, an initial basal blood sample is  taken and the blood glucose concentration is deter-  mined; 75 g of glucose in 250-300 mL of water is  drunk and the blood glucose is measured every  30 min for the next 2 h. The blood glucose concen-  tration is determined by the glucose oxidase method.  Patients must sit comfortably during the test because  stress can lead to cortisol release, which antagonizes  the action of insulin, therefore, increasing blood glu-  cose concentration. Figs 12.26 and 12.27 illustrate  the results of an OGTT.   The concentration of glycated haemoglobin (HbA lc )  provides a measure of the average blood glucose con-  centration over the preceding 4-6 weeks, that is, the  half-life of erythrocytes. This is useful for diabetic  patients to show how well their blood glucose concen-  tration has been controlled over a period of time and  for the doctor to determine the true level of control - it  is a good check on the validity of glucose values en-  tered by patients in their glucose result log book.  The higher the value of HbA lc the less well-controlled  the diabetes.   Fig. 12.27 Oral glucose tolerance test. Please note that  persons with impaired glucose tolerance can have a normal  fasting glucose concentration   Glucose irreversibly attaches non-enzymatically to adult  haemoglobin (I lbA) over the lifetime of erythrocytes.  The extent to which this occurs is proportional to the  blood glucose concentration. The amount of glycated  haemoglobin in the blood can be measured by a number  of methods including high-pressure chromatography.    Fig. 12.28 Reference values for HbA 1c expressed  as a percentage of total haemoglobin.   < 6.5% (< 7.5% in patients at risk  of severe hypoglycaemia)    electrophoresis and immunoassay. It is usually expressed  as a percentage of total haemoglobin (Fig. 12.28); report-  ing in mmol/mol units is now being introduced.   Coronary heart disease is a major cause of death in the  UK. Plasma cholesterol levels are monitored routinely  in 'at-risk' groups and when necessary in the rest of  the population. 'At-risk' groups include:   • Patients with coronary heart disease (angina, post-  myocardial infarction, post-angioplasty or coronary  artery bypass graft), those with peripheral or cerebro-  vascular disease, and patients with diabetes mellitus   • Patients with dyslipidaemias and their families, and  patients with a family history of premature cardio-  vascular disease   • Patients with multiple cardiovascular risk factors; for  example, patients with high blood pressure, or high  cholesterol (cardiovascular risk assessment charts  can be found in the British National Formulary (BNF).   Screening for cholesterol levels can be done on a non-  fasting sample. If a raised cholesterol is found, a full  lipid profile is performed, which measures the total cho-  lesterol, high-density lipoprotein (FIDL) cholesterol,  and triacylglycerols (triglycerides). Blood taken for lipid  studies is obtained after an overnight fast. Reference  values for fasting plasma lipid concentrations are shown  in Fig. 12.29.    Fig. 12.29 Desirable values for fasting plasma lipid  concentrations in males cf and females?.    Low-density lipoprotein (LDL) cholesterol levels can  also be obtained by calculation using the Friedewald  equation. This is only valid if triacylglycerol levels are  less than 4.5 mmol/L.   Triacylglycerol levels of greater than 10 mmol/L are  associated with an increased risk of pancreatitis.   An enzyme assay is used for the diagnosis of glucose-  6-phosphate dehydrogenase deficiency, the most  common erythrocyte enzyme defect (see Chapter 4).  It allows patients with glucose-6-phosphate dehydro-  genase deficiency to be detected in between haemolytic  attacks, such that diagnosis is not just dependent on  the blood picture during an attack. An enzyme activity  of <2% of the normal level may be seen in very  severe cases.   Pyruvate kinase assay is used for the diagnosis of eryth-  rocyte pyruvate kinase deficiency (see Chapter 4).   The production of pyruvate is coupled to the reduction of  a dye, with the colour change monitored spectrophoto-  metrically. A decreased production of ATP can also be  measured when radioactively labelled 32 P is added to  erythrocytes and its incorporation into ATP is monitored.   Patients typically need to have an enzyme level of  5-25% of the normal level to show clinical features.   Both galactose and fructose are reducing sugars and are  detected in the urine using alkaline copper (Cu 2+ ) re-  agents, such as Benedict's reagent.   Galactosaemia is caused by a deficiency of the enzyme  galactose- 1 -phosphate uridyl transferase.   • Test for galactosuria: Clinitest tablets or reagent  strips contain copper citrate, which is reduced by ga-  lactose. The colour change observed is clear blue to  green to brown to a brick red precipitate if the reduc-  tion is complete. The presence of galactose in the  urine along with positive symptoms should lead to  the withdrawal of galactose and lactose from the diet  until a diagnostic test can be performed   • Diagnostic test: assay erythrocytes for decreased  galactose- 1 -phosphate uridyl transferase activity.   The absence of fructokinase leads to a combination of a  high fructose concentration in the blood and fructose  accumulation in the urine. Both must be present to  form a diagnosis. Fructose, like galactose, is a reducing  sugar and its presence in urine can be detected with cer-  tain tablets. Bear in mind that this is a rare diagnosis,  virtually always seen in paediatric practice.   The measurement of thyroid -stimulating hormone (TSH)  levels is used as a screening test for suspected hyperthy-  roidism and hypothyroidism. Both hyperthyroidism  and hypothyroidism influence the basal metabolic rate  by over-stimulating or under-stimulating lipid and carbo-  hydrate metabolism respectively. Elevated TSH levels sig-  nify an inadequate thyroid hormone production, while  suppressed levels signify excessive unregulated produc-  tion of thyroid hormone. If TSH is abnormal, decreased  levels of thyroid hormones, T3 and T4, may be present;  these may be measured to confirm the diagnosis.   Other diagnostic endocrine tests you should be  aware of include the dexamethasone suppression test,  measuring ACTH in Cushing's syndrome (excess corti-  sol), and the Synacthen test in suspected Addison's dis-  ease (primary adrenal insufficiency), tests of levels of  TSH, LH, FSH, cortisol, and use of the insulin tolerance  test in the investigation of hypopituitarism.   Every neonate is now screened for phenylketonuria as  part of the neonatal screening (Guthrie test). Diagnosis  is based on a high concentration of phenylalanine in the  blood (see Chapter 6 for a full discussion).   The most severe and devastating form of  hypothyroidism is seen in young children with  congenital thyroid hormone deficiency. If the  condition is not corrected by supplemental therapy  through oral administration of synthetic thyroid  hormone soon after birth, the child will suffer from  cretinism, a form of irreversible growth and mental  retardation.   It is worth knowing the different criteria for the  diagnosis of diabetes and glucose intolerance as they  are frequently asked in exams.   A sample of capillary blood is taken from a heel-prick at  5-10 days after birth. The delay allows sufficient time  for feeding, and therefore, for protein intake to be estab-  lished and for the effect of the mother's metabolism to  subside. This test used to be based on a microbiological  technique, using a strain of Bacillus subtilis which only  grows if excess phenylalanine is present. However, it  is now based on chromatography. Increased plasma  phenylalanine levels are indicative of phenylketonuria.  The neonatal screening also includes the measurement  of thyroid stimulating hormone (TSH), to screen babies  for hypothyroidism.    For any individual, adequate nutrition is essential to  maintain growth and development and recovery from  illness. It is especially important in newborn babies, in-  fants and during pregnancy, when nutritional defi-  ciency can lead to wasting, severe mental retardation  and even death. Malnutrition must be recognized and  accurately assessed to enable decisions to be made  about treatment and re-feeding methods. Assessment  is divided into:   The main aspect of this is the dietary history, but often  weight loss and poor nutrition are related to medical,  psychological or financial factors (see Fig. 12.2).   • Symptoms of hyperthyroidism: weight loss, in-  creased appetite, irritability, heat intolerance   • Relevant past medical history, periods of weight loss  and gain in the past; use of laxatives   • Psychiatric history, especially if there is the possibil-  ity of depression or an eating disorder (e.g. anorexia  nervosa) .   • Malnutrition may be related to the poor socio-  economic status of a family   • Elderly people ('tea and biscuit brigade') living alone  who are unable to cook or shop   • Young pregnant mothers who live off a staple diet of  chips, pizzas and so on   In developing countries, nutritional deficiency may be  related to war, poor crops and the poor socio-economic  status of the entire country.   • Do you watch what you eat; are you on any particu-  lar diet?   Patients are often asked to keep a food diary. This is usu-  ally more accurate than simply questioning the patient,  although it relies on the patient's compliance to fill the  diary in, and also their willingness to provide accurate  information (patients with eating disorders are not usu-  ally willing to do so).   The cause of weight loss or malnutrition is often not as  simple as 'not eating enough'. You must eliminate  serious underlying illnesses (such as cancer) before you  consider psychiatric illnesses (depression or anorexia  nervosa) or social/economic causes.   • Calculation of the body mass index (BMI): weight  (kg)/height (m) 2   • Mid-arm circumference: a measure of skeletal mus-  cle mass (less frequently used)   • Skin-fold thickness. This helps to assess the amount  of subcutaneous fat stores (needs to be done in a  standardized way and is rarely used in routine  practice).   For infants, it is difficult to measure skin-fold thickness  accurately and it is therefore, of little value. The World  Health Organization recommends that nutritional sta-  tus is expressed as:   • % Weight/height: Measures wasting as an index of  acute malnutrition   • % Height/ age: Measures growth retardation/stunting  as an index of chronic malnutrition.   In infants, regular growth measurements are very valu-  able in assessing their nutritional status. Therefore, all  infants have their height and weight plotted on a growth  chart (Fig. 12.30), which allows a decrease in the rate of  growth to be easily recognized and monitored as an  early sign of malnutrition.   • Pallor, which indicates anaemia, possibly caused by  an iron, vitamin B 12 or folate deficiency   • Specific effects of vitamin deficiency; for example,  deficiency of vitamin A causes Bitot's spots on the  eyes, or deficiency of vitamin D and calcium causes  rickets in children and osteomalacia in adults    Fig. 12.30 Example of a growth chart often used to help in  assessment of nutritional status in children. The line plotted here  shows that the patient's height lies along the 50th centile, i.e.  average height.   The tests for individual nutrients, vitamins and minerals  are dealt with in Chapter 8. Here, the types of tests  which can be employed are considered.   These are the measurements of the concentration of a  nutrient or a metabolite in body fluid, usually in the se-  rum or urine.   Such measurements may exploit the activation of an  enzyme by a vitamin. For example, thiamine is a cofac-  tor for erythrocyte transketolase. In thiamine deficiency,  the erythrocyte enzyme activity can be measured before  and after the addition of thiamine pyrophosphate (the  active form of thiamine). Addition of thiamine pyro-  phosphate to erythrocytes should lead to an increase  in enzyme activity, proving thiamine deficiency.    Thiamine can also be measured directly using high-  pressure liquid chromatography (HPLC).   • A decrease in the dietary intake of a nutrient leads to  the mobilization of that nutrient from its stores to  maintain a normal plasma concentration   • Usually, only in severe deficiency does the plasma  concentration drop significantly   • In some cases, by measuring a decrease in the body  stores we can detect a deficiency earlier.   For example, the best way to assess iron deficiency is to  measure a decrease in bone marrow iron stores (serum  ferritin reflects iron stores and is low in iron deficiency),  and the best way to assess vitamin C deficiency is to  measure a decrease in white cell vitamin C content  (Fig. 12.31).   A plasma albumin of less than 30 g/L is often used  as an index of malnutrition. However, albumin level  is very much affected by fluid and electrolyte disor-  ders, and in these patients, albumin is not an accu-  rate index of nutritional status. The diagnosis of  severe malnutrition is usually made on the basis of  clinical assessment.    Fig. 12.31 Some biochemical tests for nutrients (FBC, full  blood count; MCV, mean cell volume).   FBC, ferritin, MCV etc., serum  transferrin Best estimate is a fall in  bone marrow iron stores   This is used in patients with high LDL cholesterol  thought to be at risk of familial hypercholesterolae-  mia, in which many patients have mutations in the  LDLR gene that encodes the LDL receptor protein. Ge-  netic testing is relevant for risk estimation and    management as patients who have one abnormal  copy (heterozygotes) of the LDLR gene carry a risk  of developing premature cardiovascular disease at  the age of 30 to 40, and having two abnormal copies  (homozygotes) may cause severe cardiovascular dis-  ease in childhood.   c. Modulation of enzyme function is a major cellular  regulation strategy.   c. Is exemplified by glucagon release following  elevation of blood glucose.   a. The Na + /K 1 ATPase imports 2 K + ions for every  3 Na + ions exported.   b. The Na + /K + ATPase exemplifies secondary active  transport.   e. Steroid hormones require specialised transport  mechanisms to cross cell membranes.   a. Reduction is associated with a molecule losing a  hydrogen atom.   d. Possesses a thiol group within the CoA component  of the molecule.   c. Receive electron pairs from complexes in the  electron transport chain.   e. As long as one is present in sufficient quantity, the  other is not necessary.   a. 12 ATP are generated (indirectly and directly) per  'turn' of the cycle.   a. Rate-limiting enzymes of the TCA cycle exhibit  reduced activity in the presence of Ca + 1 ions.   b. Rate-limiting enzymes of the TCA cycle are  allosterically inhibited by ATP.   d. An increased rate of respiration imposes a reduced  rate of TCA cycle activity.   13. Which of the following does not require a TCA cycle   c. Electrons enter the electron transport chain within  NADH + H+ or FADH 2 .   d. The term is a misnomer as it does not require  oxygen.   e. Is less productive in terms of ATP generation than  substrate-level phosphorylation.   b. Results in sequential reduction, then oxidation of  each complex of the ETC.   c. Terminates with transfer of electrons from oxygen  to complex IV.   b. Only complex I and II 'accept' electron pairs from  NADH + H+ and FADH 2 .   a. Is in the direction high^low from the cytoplasm to  the mitochondrial intermembrane space.   c. Is of no functional importance in terms of cell  metabolism.   e. Plays no role in sustaining a cytoplasmic pool of  NAD + .   a. Sucrose is a triose sugar consisting of lactose and  fructose.   a. Glucose directly diffuses across phospholipid  bilayers in the direction of the concentration  gradient.   b. When the concentration gradient is unfavourable  for glucose entry into cells, ATP hydrolysis directly  "powers" glucose import.   d. Facilitated diffusion transporters are the main  mechanism via which glucose enters  gastrointestinal cells.   d. Produces one pyruvate molecule for every glucose  molecule entering the pathway.   a. There are three rate-limiting enzymes, catalysing  reaction 1,3, and 9.   b. Phosphorylation is an important regulation  mechanism used to modulate activity of the rate-  limiting enzymes.   c. ATP allosterically activates PFK-1 , thus increasing  pathway rate when ATP is abundant.   e. PFK-2 is one of the rate-limiting enzymes of the  glycolysis pathway.   b. Catalyses the conversion of pyruvate to acetyl CoA  via a number of steps.   c. Requires CoA, riboflavin and lipoic acid as cofactors  to function.   d. Is allosterically activated by NADFI + H + and acetyl  CoA.   e. Is an unusual enzyme in that phosphorylation does  not affect activity.   a. Is the production of glucose from non-  carbohydrate molecules, and occurs in all cell  types.   b. Occurs in hepatocytes, partly in the cytoplasm and  partly in the mitochondria.   d. Is the first-line mechanism to activate to raise  plasma glucose in response to falling plasma  glucose.   gluconeogenesis is a two-step reaction, as  opposed to PEP — > pyruvate conversion in  glycolysis which is a single reaction.   d. Conditions in which the glycolysis pathway is highly  active also promote high activity in the  gluconeogenesis pathway.   a. Glucose generated by glycogenolysis in muscle or  hepatocytes is released into the bloodstream to  maintain plasma glucose levels.   b. The structure is composed exclusively of glucose  monomers linked by (1 —4) bonds.   c. Synthesis occurs in the mitochondria, and requires  UDP-glucose, ATP and a primer.   e. Glycogen phosphorylase, which mediates  glycogenolysis, is inactivated by phosphorylation by  PKA.   b. Consists of an initial, irreversible, oxidative phase  and a reversible non-oxidative phase.   c. The proportion of different intermediates generated  by the pathway is relatively insensitive to cellular  requirements.   d. The PPP generates NADP 1 , which is required for  regeneration and reactivation of glutathione.   a. Fructose is absorbed into enterocytes via the GLUT-  1 transporter, and then leaves via the same  transporter.   b. Fructokinase deficiency and fructose-1 -phosphate  aldolase deficiency are both X-linked genetic  afflictions.   c. Galactose enters enterocytes from the gut lumen via  the sodium/glucose symport.   d. Increased sorbitol production has no negative  consequences for a cell.   d. Exist primarily in the non-ionised form at  physiological pH, i.e. margaric acid rather than  margarate.   b. 16 carbons and three double bonds at C4, C5  and C14.   c. 15 carbons and double bonds at C4, C5 and C14.   d. 14 carbons and double bonds at C3, C4 and C5.   d. Consists of two completely disparate pathways, one  for saturated FA and one for unsaturated FA.   e. First requires acetyl CoA to export from  mitochondria into cytoplasm via the citrate shuttle.   34 . The following is a correct description of part of the   a. Acetyl CoA transfer to cytoplasm, conversion to  malonyl CoA, assembly of malonyl and acetyl  groups on ACP, dehydration, condensation,  reduction, dehydration.   b. Acetyl CoA transfer to cytoplasm, conversion to  malonyl CoA, assembly of malonyl and acetyl  groups on ACP, condensation, dehydration,  reduction, dehydration.   c. Acetyl CoA transfer into mitochondria, conversion  to malonyl CoA, assembly of malonyl and acetyl  groups on ACP, condensation, reduction,  dehydration, reduction.   d. Acetyl CoA transfer to cytoplasm, conversion to  malonyl CoA, assembly of malonyl and acetyl  groups on ACP, condensation, reduction,  dehydration, reduction.   e. Acetyl CoA conversion to malonyl CoA, transfer of  malonyl CoA to the cytoplasm, assembly of malonyl  and acetyl groups on ACP, condensation, reduction,  dehydration, reduction.   b. Can be synthesised from glycerol and FA in a three-  stage process.   e. Catabolism (lipolysis) involves hydrolysis of the ester  bond at C2.   a. Requires activated FA to access the cytoplasm  from the mitochondrial matrix via the carnitine  shuttle.   c. Commences with fatty acyl CoA being reduced by  fatty acyl-CoA reductase.   d. Involves conversion of a single bond between the  Cl and C2 to a double bond.   e. Includes thiolytic cleavage (releasing an acetyl CoA)  prior to oxidation.   38 . The following do not play an important role in   d. Ingestion of cholesterol has a greater impact on  blood cholesterol than ingestion of saturated fat.   e. Cholesterol regulates its own synthesis via allosteric  activation of HMG-CoA reductase.   a. Lipoprotein outer shells are hydrophobic on  account of the phospholipid orientation.   c. Chylomicrons transport dietary lipids from  enterocytes to the liver via the hepatic portal vein.   a. Familial chylomicronaemia is a very common  condition resulting for absence/deficiency of LPL.   b. Acquired hypercholesterolaemia is very  uncommon and is not detrimental to health.   c. The mechanism by which statins lower blood  cholesterol is inhibition of HMG-CoA reductase.   d. High levels of plasma cholesterol are essential for  effective neurotransmission in waterfowl.   e. In placental mammals, atherosclerotic plaques are  of no medical significance.   a. Acetoacetate, acetone and 3-epigallobutyrate  are the three biologically significant ketones in  humans.   b. Hepatocytes, natural killer cells and enterocytes  are all capable of ketogenesis.   d. Reduced TCA cycle activity increases ketogenesis  by allowing more acetyl CoA to be diverted to  ketogenesis.    e. Predominant catabolism (rather than synthesis) of  lipids and high gluconeogenesis activity represses  ketone synthesis.   c. Hydrogen bond interactions operate between C  atoms of the C = 0 component of one amino acid  and the amino N atom of another amino acid.   d. Transamination and oxidative deamination both  perform the same function; removal of the carboxyl  group from an amino acid.   e. Oxidative deamination allows any amino acid to  convert to alanine, glutamate or aspartate.   a. Only amino acids that cannot be synthesis  endogenously are deemed essential.   e. Absence of phenylalanine hydroxylase renders  tyrosine an essential amino acid in individuals with  phenylketonuria.   a. Serotonin is synthesised from tryptophan by  hydroxylation followed by decarboxylation.   b. Adrenaline, noradrenaline and dopamine are all  synthesised from the amino acid methionine.   c. Tyrosine residues within thyroglobulin are  fluorinated, ultimately allowing formation of T3  and T4.   d. Thyroid hormone generation is mediated by the  enzyme thyroid transferase.   d. Arginine generated by transamination (during  transdeamination) enters the urea cycle.   a. The urea cycle processes nitrogen (from amino acid  catabolism and ammonia) for excretion as urea.    b. It occurs in cells of the renal cortex, across both the  cytoplasm and the mitochondrial matrix.   c. Malate generated by cleavage of arginosuccinate is  unable to participate in the TCA cycle.   d. Aspartate in the urea cycle must be generated in one  of two ways: either by transamination of  oxaloacetate or cleavage of arginosuccinate.   e. Urea can cause acidosis if the levels in the blood rise.   a. Is reduced from folate by the action of dihydrofolate  reductase.   d. The methionine salvage pathway is essential for  maintaining a continual supply of THF   e. Folate deficiency can occur secondary to vitamin B 12  deficiency.   a. Is caused by a deficiency of the salvage enzyme,  adenine phosphoribosyl transferase (APRT).   c. Causes patients to develop severe mental  retardation and to self-mutilate.   d. The salvage pathway for guanine and hypoxanthine  is virtually inactive.   e. Increased levels of guanine and hypoxanthine result  in hyperuricaemia and gout.   b. May be caused by low levels of hypoxanthine-  guanine-phosphoribosyl transferase (HCPRT).   c. Clinical features include chronic arthritis of the hips  and knees.   b. Initially, gout is characterized by swelling of a single  joint.   a. May be caused by increased excretion of uric acid by  the kidneys.   b. May result from a partial deficiency of  hypoxanthine-guanine phosphoribosyltransferase  (HGPRT-).   c. May result from overproduction of 5-  phosphoribosyl 1 -pyrophosphate (PRPP).   57 . The main metabolic changes found in diabetes mellitus  are listed below: select the one false statement:   a. HBA 1C is a good marker of average blood glucose  concentration.   b. The oral glucose tolerance test involves drinking  75 g of glucose in 250-300 mL of water.   c. 7.9 mmol/L is a normal fasting glucose level  according to the WHO criteria.   d. Serum fructosamine is a good marker of average  blood glucose concentration.    e. Impaired glucose tolerance is normally diagnosed by  an oral glucose tolerance test.   59 . Secondary diabetes can occur in all of the following   a. HbAlc provides a marker for the preceding  12 months of control.   b. Impaired fasting glucose is defined as fasting plasma  glucose 4-5 mmol/L.   c. Impaired glucose tolerance is defined as fasting  plasma glucose <7.0 mmol/L and 2 h value  between 7.8 and 11.1 mmol/L in an OGTT.   d. Diagnosis of diabetes always requires the patient to  be symptomatic.   e. To be diagnosed with diabetes you always need to  have both a fasting blood glucose >7.0 mmol/L  and 2 h value >11.1 mmol/L in an OGTT.   c. Eventually leads on to type 2 diabetes in 90%  women.   d. Is more likely to result in type 2 diabetes in patients  with a low body mass index.   b. Is always caused by a lack of protein and energy.   e. Usually occurs when a child is weaned from  breastfeeding because of the arrival of a second  child.   a. Iodine deficiency is now rare due to fortification of  bread.   b. Triiodothyronine production has a negative  feedback effect on the hypothalamus and anterior  pituitary.   d. The Guthrie test is only used on babies born to  hypothyroid mothers.   a. Can occur when energy intake is equal to energy  expenditure.   c. Is more common in low socio-economic class in  the West.   d. Often occurs in children at the age below  18 months.   b. Is indicated when a patient is eating less than 70%  of their recommended daily intake.   e. Is most often given through a central venous  catheter into the superior vena cava.   a. Is a cofactor for proline and lysine hydroxylases  involved in collagen synthesis.   d. Deficiency results in pellagra, characterized by  swollen, sore gums and poor wound healing.   c. The nutritional requirement for niacin is decreased  when the diet contains large amounts of protein.   d. The clinical features of niacin deficiency include  dermatitis, diarrhoea and dementia.   e. Nicotinic acid can be used in the treatment of  hyperlipidaemia.   c. Deficiency can be induced by eating lots of raw egg  whites.   b. Is the cofactor for methylmalonyl CoA mutase,  involved in the breakdown of odd-numbered fatty  acids.   c. Intrinsic factor released by gastric parietal cells is  required for its absorption.   b. Vitamin B 12 , C and E are all antioxidants.   c. Vitamin A, D, E and C are all fat-soluble  vitamins.    e. Both vitamin K and vitamin B 12 are synthesized by  intestinal bacteria.   a. Is the most common cause of vitamin B 12  deficiency.   a. Is a coenzyme for clotting factors III, V, IX and X.   b. Needs to be taken in the diet regularly to avoid  deficiency.   d. Deficiency will cause a decreased activated partial  thromboplastin time (APTT).   c. Results in defective mineralization of the long bones  visible on X-ray.   a. Its absorption is favoured in the ferrous state  (Fe 2+ ).   d. Is active in its ionized form, Ca 2 + .   e. In overload, it is deposited in the eyes producing  Kayser-Fleischer rings on the cornea.   b. A 2-month course of chelation therapy normally  resolves the problem.   b. Iron is absorbed in the ferrous (Fe 2+ ) form.   88. Common symptoms of anaemia include all of the  following except:   d. Of the flapping type is observed with outstretched  arms and hands flat.   b. Is defined as increased angle between the nail and  nail bed.   e. Is commonly seen as a result of glycogen storage  disorder.   a. Iron deficiency will cause a decrease in haem and  erythrocyte production.   b. Macrocytic anaemia may be caused by a deficiency  of erythrocyte enzymes.   c. Pernicious anaemia is an auto-immune condition  causing deficiency of B 12 and folate.   d. Lead poisoning inhibits three enzymes of haem  synthesis, leading to anaemia.   94. Classic signs and symptoms of peritonism in an  abdomen examination include all of the following  except:   b. A red cell count of 8 x 10 12 /L is consistent with  polycythaemia.   For each of the following, select the SINGLE most appropri-  ate response from the above list.   3. This enzyme has another isoform (glucokinase) in  pancreatic islet cells and hepatocytes.   4. This enzyme catalyses a rate-limiting reaction which  includes a phosphorylation, using ATP as the phosphoryl  donor.    For each of the following, select the SINGLE most appropri-  ate response from the above list.   2. This enzyme catalyses the reaction which reduces FAD  to FADH 2 .   3. A rate-limiting enzyme that mediates a reaction that  does not generate carbon dioxide as a product.   4. This enzyme catalyses the decarboxylation reaction that  produces acetyl CoA from pyruvate.   For each of the following, select the SINGLE most appropri-  ate response from the above list.   2. Branched chain amino acid that is both ketogenic and  glucogenic.   For each scenario described below, choose the single most  likely option from the list of options.   Each option may be used once, more than once or not  at all.   B. Autoimmune disease with peak age of onset at  15-20 years.   F. Disease with typical age of onset from 60 years  onwards.   For each of the following, select the SINGLE most appropri-  ate option from the above list.   3. The initial treatment of most patients with Type 2  diabetes.   5. Method for measuring blood glucose control in the past  month.   For each of the following, select the SINGLE most appropri-  ate vitamin or mineral from the above list.   For each of the following, select the SINGLE most appropri-  ate response from the above list.   2. Wernicke-Korsakoff syndrome can arise in alcoholics as  a result of deficiency in this.   3. Increased risk of bruising and bleeding in alcoholics is  due to deficiency in this.   4. Oedema can arise from severely depleted circulating  levels of this.   5. This compound has been shown to have a beneficial  effect on heart attacks and strokes when ingested in  moderate amounts.    For each of the following, choose ONE option from the list   1. A 10-year-old boy presents with painful joints in the  hands and feet, jaundice and anaemia. Fie also has  splenomegaly and his blood film shows target cells.   2. A 50-year-old man presents with macrocytic anaemia.  Fie drinks alcohol regularly.   B. A 25-year-old Creek lady presents with anaemia and  jaundice following anti-malarial treatment. She is noted  to have Fleinz bodies.   4. A 10-month-old baby presents with anaemia and failure  to thrive. FHis blood film shows target cells, hypochromic  and microcytic cells. HbF is still present.   5. A 40-year-old woman presents with lethargy and a sore  red tongue. Her blood film shows hypersegmented  polymorphs, an MCV >110 fL and a low haemoglobin.   I. A 15-year-old girl complains of not being able to see  very well at night.   2. A 45-year-old man with poor diet complains that his  gums are bleeding easily.   opthalmoplegia and ataxia. Fie is known to drink at least  40 units of alcohol a week.   4. A 20-year-old woman with chronic diarrhoea complains  of prolonged bleeding after a small cut to her index  finger. The prothrombin time is increased.   5. A 25-year-old man was recently on anti-TB treatment and  now presents with dermatitis, diarrhoea and depression.   I. The doctor suspects that the patient has been  misleading him regarding the control of her blood  glucose levels.   2. The patient, a teetotal, has macrocytic anaemia, despite  eating a healthy and sensible diet.   3. The newborn baby had a blood sample taken from a  heel-prick around 7 days after birth.   4. The patient is known to have a family history of  coronary heart disease.   5. The patient complains of a painful and swollen big toe,  following a heavy protein meal.   I. A 6-month-old baby presents with seizures and failure  to thrive. His parents are advised to follow a food  restriction diet.   2. The patient presents with painful joints and complains  that his urine turns black on standing.   3. The patient presents with whitish hair, grey-blue eyes  and pale skin. Fie is advised to use high sun protection  when going out.   4. A 1 -month-old baby presents with metabolic acidosis,  hypoglycaemia and seizures. His parents complain that  his urine smells sweet.   5. A 4-month-old baby presents with self-mutilation,  spasticity and kidney stones. His parents complain of an  'orange' nappy.   4. a. The Na + /K + ATPase imports 2 K + ions for every 3   11. b. 1 FADH 2 and 3 NADH + H + are generated per   gluconeogenesis is a two-step reaction, as  opposed to PEP — > pyruvate conversion in  glycolysis which is a single reaction.   32. b. 16 carbons and three double bonds at C4, C5   malonyl CoA, assembly of malonyl and acetyl  groups on ACP, condensation, reduction,  dehydration, reduction.   49. a. Is caused by a deficiency of the salvage enzyme,   54. a. May be caused by increased excretion of uric acid   plasma glucose <7.0 mmol/L and 2 h value  between 7.8 and 11.1 mmol/L in an OGTT.   62. b. Is always caused by a lack of protein and energy.   71. e. Is safe to use in pregnancy, even in excess.   74. d. The stores of vitamin B 12 are very small.   76. d. A deficiency of vitamin B 12 results in anaemia.   78. a. Is the most common cause of vitamin B 12   glycolysis is reaction 10; the conversion of  phosphoenolpyruvate (PEP) to pyruvate.  Transfer of the phosphoryl group from PEP  to ADP results in pyruvate and ATP synthesis.  This is catalysed by pyruvate kinase. The AG 0 of  this reaction is -31.4 kJ/mol.   glycolysis; the conversion of glucose to glucose-  6-phosphate (Glc-6-P). The main functional  difference is that hexokinase is allosterically  inhibited by the reaction product Glc-6-P, whilst  glucokinase is in sensitive to such inhibition. The  isoforms also differ in affinity; low-affinity  glucokinase requires higher [glucose] to perform  equally as effectively as the high-affinity  hexokinase dose at lower [glucose],   these enzymes meet the question's criteria, i.e.  catalysing rate-limiting glycolytic reactions  where ATP is the phosphate donor in the  reaction. Hexokinase catalyses reaction 1 of  glycolysis, whilst phosphofructokinase-1  catalyses reaction 3 of glycolysis.   phosphorylation reactions in glycolysis. The first  is catalysed by phosphoglycerate kinase  (reaction 7) and the second is catalysed by  pyruvate kinase (reaction 10).   1 . E. Succinyl CoA synthetase. This enzyme catalyses  the conversion of succinyl CoA to succinate in a  substrate-level phosphorylation that directly  generates GTP.   the oxidation of succinate to fumarate. The  coupled redox reaction is the reduction of FAD to  FADH 2 .   of the TCA are reactions 1, 3 and 4; catalysed by  citrate synthase, isocitrate dehydrogenase and a-  ketoglutarate dehydrogenase respectively.  However reaction 1 is the only reaction of these  three that does not generate C0 2 , making citrate  synthase the answer.   Phosphoglucoisomerase. These two enzymes are  enzymes of glycolysis, therefore these two are the  only members of the list not associated with the  TCA cycle.   acid arginine is a reaction within the urea cycle;  arginosuccinate is cleaved by arginosuccinate lyase  to form arginine and fumarate.   discussed in this text) have branch-structure  R-groups. These are Leucine, Isoleucine and Valine.  However, of these three, only Isoleucine has a  structure allowing catabolism to generate  intermediates that can participate in both  ketogenesis and gluconeogenesis.   phenylalanine, resulting from deficiency or  phenylalanine hydroxylase, characterises the  autosomal recessive condition phenylketonuria.   4. H. Tyrosine. This amino acid is used as a substrate in   importance containing sulphur in their R-groups are  methionine and cysteine. However, methionine is  not present in the list.   1 . B. Autoimmune disease with peak age of onset at   15-20 years. Type 1 diabetes, also known as  insulin-dependent diabetes or juvenile-onset  diabetes, typically occurs in childhood or puberty.   there is a complete deficiency of insulin that can  only be corrected by life-long insulin treatment.   typically associated with obesity, and diet  management is often the only treatment necessary  when patients are first diagnosed. Weight loss can  prevent or delay the development of type 2  diabetes.   autonomic symptoms, such as sweating, nausea  and palpitations, and more severe neuroglycopenic  symptoms as a result of a decrease in glucose supply  to the brain: drowsiness, unsteadiness, confusion  and coma.   concentration of glycated haemoglobin provides a  measure of the average blood glucose  concentration over the preceding 4-6 weeks, that  is, the lifetime of an erythrocyte.   1. J. Vitamin D. The main role of vitamin D is in calcium   homeostasis, where a severe deficiency causes  rickets in children and osteomalacia in adults.   of beri-beri: wet beri-beri which results in oedema  and heart failure, and dry beri-beri which causes  muscle wasting and peripheral neuropathy.   3. H. Vitamin A. Deficiency of vitamin A results in an   4. C. Folic acid. A deficiency in folic acid results in   decreased synthesis of purines and pyrimidines,  leading to a decrease in nucleic acid synthesis and  cell division. This shows up mostly in cells that  rapidly divide such as red blood cells, presenting as  large, immature red blood cells.   bone mass, due to the effects of oestrogen  deficiency in post-menopausal women, and calcium  deficiency increases the risk of osteoporosis.   pigmentation of skin or sclerae of the eyes due to a  raised plasma bilirubin level.   3. L. Vitamin K. Vitamin K is a coenzyme required for   the y-carboxylation of clotting factors II, VII, IX  and X, activating them and thus, the clotting  cascade.   fluid may escape into tissues to cause oedema or  into body cavities to cause ascites or pleural  effusions.   amounts of alcohol, i.e. one glass of red wine a day  has been found to have a protective effect against  heart attacks and strokes.   occurs when the microcirculation is obstructed by  sickled erythrocytes, causing ischaemic injury to  joints, resulting in pain. The spleen can undergo  a sudden very painful enlargement due to pooling  of large numbers of sickled cells. This  phenomenon is known as splenic sequestration  crisis.   secondary folate deficiency, which leads to  decreased production of DNA and defective cell  division. As a result, the developing erythrocytes are  megaloblastic, where the nuclei mature more slowly  than the cytoplasm.   Anti-malarial treatment is a precipitating  factor here, resulting in oxidative stress and  haemolysis of erythrocytes. Damage to the  haemoglobin causes its oxidation to  methaemoglobin, and the globin chains are  precipitated as Heinz bodies.   resulting in a deficiency of (3-globin chains. As a  result, fetal haemoglobin (HbF) persists in the  circulation.   1. C. Vitamin A. Vitamin A is required for proper function   of 11 -cis retinal, which binds to opsin to form  rhodopsin, the visual pigment of rod cells in the  retina involved in night vision.   2. I. Vitamin C. A deficiency in vitamin C results in scurvy   which typically presents with swollen, sore, spongy  gums with bleeding and loose teeth.   4. K. Vitamin K. Vitamin K is a coenzyme required for the   y-carboxylation of clotting factors II, VII, IX and X,  activating them and thus, the clotting cascade.   inhibits nicotinic acid, which results in pellagra  which presents with dermatitis, diarrhoea and  depression.   glycated haemoglobin provides a measure of the  average blood glucose concentration over the  preceding 4-6 weeks, that is, the half-life of an  erythrocyte.   absorption of vitamin B 12 and is usually used to  diagnose pernicious anaemia.   3. F. Guthrie test. A sample of capillary blood is taken   from a heel-prick at 5-10 days after birth and  screened for phenylketonuria and hypothyroidism. 
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