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The Behemoth Biochem Board Review Part I May 16, 2000 The Incomparable Roger Lane Contract/gunners anonymous Metabolism Major metabolic Pathways in the Postprandial State (page 1) The major tissues we will talk about are RBCs, adipose tissue, muscle, liver and brain. The red cell is an anaerobic tissue; bear in mind that there are other tissues that are anaerobic, but this is the one we will focus on. Certain muscle cells (white muscle cells), skin cells, WBCs, as well as the adrenal and renal medullas, are all anaerobic tissues. These tissues have to make due with a single metabolic pathway at all times, anaerobic glycolysis (glucose pyruvate lactate). The ATP is produced by substrate level phosphorylation. Bear in mind that this pathway does not require mitochondria or oxygen. This pathway would be activated during an ischemic event (MI or stroke); the tissue would eventually die, but it would first try to make energy via this pathway. The problem is that lactic acid will build up, and this is toxic to tissues that are deprived of oxygen or a blood supply. So if you see the term anaerobic ischemia, this is the pathway that theyre talking about. In all of the other tissues, glucose pyruvate acetyl CoA CO2. Glucose is used by all tissues in this state, but it is absolutely critical in the brain, and 3 pathways convert glucose totally to CO2, which is exhaled. 1. 2. 3. (Aerobic) Glycolysis: glucose pyruvate Pyruvate dehydrogenase complex: pyruvate acetyl CoA Krebs Cycle: Acetyl CoA CO2

ophthalmoplegia. The Korsakoffs, component, which is a psychosis, would include symptoms such as amnesia and confabulation. The other thing that you should be aware of in this metabolic state is that you are making stuff glycogen, triglyceride and protein synthesis. The key hormone here is insulin. Glycogenesis is glycogen synthesis. It occurs in ALL tissues, but quantitatively most important are liver and muscle. Protein synthesis (translation) occurs in ALL tissues. Amino acids are used to make proteins. Most of the protein is contained in muscle. In the bottom right hand corner, you see the mnemonic of the essential amino acids: PVT TIM HALL. These should be remembered, and the main concept here is that you need 20 basic amino acids to make protein intracellularly. Only 10 of these are classified as dietarily essential for children and infants (PVT TIM HALL) and only 8 of these are essential for healthy adults. HA! histidine and arginine -- are required in adults only during times of growth (recovery from injury, etc). Recall that we get tyrosine from phenylalanine, and if we can make an amino acid in the body, it is classified as nonessential. If any of the amino acids are deficient (not absent) in the diet, protein malnutrition develops, and this is called Kwashiorkor. This leads to a negative nitrogen balance. In normal, healthy people, we are in a normal nitrogen balance (no net gain or loss of body protein). Anything that causes protein to be broken down more than it is synthesized is negative nitrogen balance. Kwashiorkor differs from Marasmus in that it is basically just a protein deficiency. Marasmus is a deficiency of calories AND protein. A deficiency of niacin is a common vitamin deficiency, which leads to the disease pellegra. This is characterized by the 3 Ds: dermatitis, dementia, and diarrhea. A deficiency of the amino acid tryptophan can lead to a pellegra-like situation as well. Fat Synthesis

In the case of aerobic tissue, much more ATP can be generated via oxidative phosphorylation (ox-phos). Recall that glycolysis takes place in the cytosol, while the other 2 pathways occur within the mitochondria. One of the major things that might be tested, without getting into any details yet, is that thiamine is required in the PDH complex and also for Krebs cycle activity. So this is a high yield question, and thiamine deficiency is seen very often in this country associated with chronic alcoholism, and the patient suffers from WernickeKorsakoffss syndrome. If the patient presents, and you put him on a 5% dextrose IV drip, you must also give him so thiamine along with that. The reason is that nerve cells cannot use glucose for energy without thiamine, and that would be key if the patient is comatose and you put in a glucose line. Some of the symptoms of the Wernickes component include a peripheral neuropathy, nystagmus, and

What we are trying to do here is either degrade dietary fat and store it or to convert excess dietary carbohydrate to fat and store it. There are 2 types of lipoproteins that are elevated in the blood postprandially, VLDLs and chylomicrons. The chylomicrons come from the intestine, and they are carrying dietary triglycerides (fat). VLDLs come from the liver and carry triglycerides made there. So bear in mind that if a patient has elevated blood TGs, it will be one or both of these (VLDL or chylomicrons) that are elevated. An increased carbohydrate diet will increase the VLDLs, while a diet high in fat will lead to an increase in chylomicrons. An easy test for increased chlyomicrons in the blood is to let the blood sit in the refrigerator

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overnight and see if a creamy layer precipitates out. This will be the high chylomicron fraction in the blood. You will not see this if the VLDLs are elevated. Be aware of how glucose is converted to fat in the liver. There are 3 pathways involved and the synthesis of fat is activated by insulin: 1. 2. 3. Glycolysis PDH complex Fatty Acid synthesis (cytosolic and this is also stimulated by the presence of citrate, because this tells your body that levels of energy are high and therefore can be stored.)

As far as you are concerned for the boards, amino acids are metabolized exclusively in the liver. They are degraded and their amino groups are removed and converted to urea via the urea cycle this is a liver-specific pathway. When the urea appears in the blood, it is referred to as the blood urea nitrogen (BUN). So if the BUN is lower than normal, one should suspect either generalized liver disease or a defect in the urea cycle itself. A higher than normal BUN is a classic clinical test for renal insufficiency. The other thing that may accumulate in kidney disease is creatinine (not creatine). Cholesterol synthesis and degradation Cholesterol synthesis (acetyl CoA cholesterol) occurs in most cells, but the primary site is again the liver. The acetyl CoA comes from carbohydrates. There are 27 carbons in cholesterol and they ALL come from acetyl CoA. The key enzyme here is HMG CoA reductase. In the liver, and only the liver, the cholesterol is converted to bile salts, which are then sent into the bile. This conversion of cholesterol to bile salts, though it is not totally degraded, should be thought of as degradation. This is a very common question regarding the treatment of hypercholesterolemia This very often due to a defect in the LDL receptor (takes up LDLs from the blood). LDL is the major way that cholesterol is transported in the blood. Some drugs work to upregulate LDL receptors in plasma membranes of liver cells (and therefore reduce the LDL concentration in the blood). In short, if I can lower the cholesterol level in the liver, I will lower the cholesterol level in the blood. As I lower the cholesterol in the liver, the number of LDL receptors will increase and lower the cholesterol level in the blood. Simply put, looking at the diagram of cholesterol synthesis in the liver, you want to stop its synthesis and increase its degradation. You stop synthesis with a statin (lovastatin, pravastatin, any statin). The key enzyme is HMG CoA reductase. To increase degradation, get the bile salts and prevent them from coming back in. thats done with a bile acid binding resin. The ones to remember are cholestyramine and cholestipol. These increase the conversion of cholesterol to bile salts because I am preventing bile salts from coming into the liver. A combination of both (decreasing synthesis and increasing degradation) is a good treatment for people who have a genetic defect in the LDL receptor (heterozygous for that protein) familial hypercholesterolemia. Insulin The key things to know here are how insulin gets glucose out of the blood to prevent hyperglycemia and how insulin gets fats out of the blood to prevent hyperlipoproteinemia (hypertriglyceridemia). The key enzyme getting fats out of the blood is Lpl, and that is stimulated by insulin. In an uncontrolled insulin-dependent

What we are doing is breaking glucose down to acetyl CoA using glycolysis and PDH, and then taking the acetyl CoA and making fatty acids. The overall process is anabolic glucose is being made to make fats. Fatty acids and glycerol phosphate are used to make triglycerides. The triglyceride is exported into the blood in VLDLs. So something simple: if the patient is on a high fat, low carb diet, then chylomicrons will be elevated in the blood. If the patient is on a low fat, high carb diet, VLDL levels will be elevated. Both of these particles are attacked in the capillaries by lipoprotein lipase (Lpl), which is stimulated by apoprotein CII as well as insulin. This is one reason that diabetics have increased lipids in their blood.. This degrades the triglycerides from the interiors of these particles, releasing fatty acids and glycerol. These thing shrink and become chylomicron and VLDL remnants, which will be taken up by the liver and disposed of. The fatty acids are taken up by the adipose tissue and stored as fat. This requires glycerol phosphate, which the adipocytes CANNOT make. This can only be done in the liver by an enzyme called glycerol kinase. So the glycerol released from the VLDL/chylomicron cannot be used for fat synthesis in the adipocyte because it doesnt have the enzyme to convert this to glycerol phosphate. Adipocytes get the glycerol phosphate for this from glycolysis. So glucose is needed for TG synthesis in adipocytes. This is a type of question that can be asked. Maybe not specifically about this enzyme, but in general, and that is that all diploid cells in the body have the full complement of 46 chromosomes. So even if a particular enzyme is not present in a specific tissue, the gene is of course there. So the problem is that the gene in not being expressed; its not being transcribed to produce mRNA and the mRNA is not being translated to produce the protein. The tests for DNA, RNA and protein are, in order, Southern Blot, Northern Blot, and Western Blot. Protein Metabolism

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patient, Lpl will be underactive, so VLDL and chylomicrons will be elevated in the blood (high TGs). Insulin also stimulates glucose transport into 2 of these tissues, adipose and muscle, via the GLUT 4 transporter. It does not stimulate transport in the liver, brain, or RBCs (they lack the GLUT 4 transporter). Insulin does, however, stimulate many processes in the liver. It stimulates glycogenesis and fat synthesis. Thats what you want to do after a meal. So all 3 pathways in fat synthesis (glycolysis PDH fatty acid synthesis) are stimulated by insulin in the liver after a meal. This is all sort of an overview; the ugly details follow Carbohydrate Pathways Postprandially (page 2) There are3 you should concentrate on: glycolysis, HMP pathway, and glycogenesis. Also keep in mind that all the key enzymes active during the postprandial state are dephosphorylated to make them active. Glycogenesis (upper left) Note that glucose-6-phosphate is produced regardless of which pathway is involved. To get to glycogen, we must first go through glucose-1-phosphate. The key enzyme that controls synthesis is glycogen synthase (activated by insulin). The opposing enzyme (for degradation) is glycogen phosphorylase (or just phosphorylase; this is inhibited by insulin). They both work with an enzyme that either puts on branches (synthase) or takes off branches (phosphorylase). The branches are 1,6 branches. During storage (synthesis), UDP-glucose is used. This is an activated sugar. Glycolysis Glycolysis proceeds from glucos-6-phosphate to fructose-6-phosphate, and then on down. All the intermediate and enzymes are NOT important. What you should come away with is the degradation of glucose-6-phosphate through fructose derivatives to two triose phosphates (glyceraldehyde-3-phosphate and DHAP). Those are interconvertable, and it is then the G-3-P that continues through the remaining steps to generate ATP. Two high energy compounds (1,3 bisphosphoglycerate and PEP) are produced and used to generate ATP via substrate level phosphorylation. The enzymes you should remember are hexokinase (glc glc-6-P). [Note that this enzyme is not unique to glycolysis; it makes Glc-6-P, which can then proceed to glycolysis, as well as to the HMP pathway or glycogenesis.] The key enzyme in glycolysis is phosphofructokinase-1 (PFK-1). This is the regulatory enzyme in

glycolysis. The last enzyme to remember is at the bottom: pyruvate kinase (PEP pyruvate). These 3 enzymes catalyze the irreversible steps in glycolysis. Screw the other enzymes in the pathway! Questions on the board may have to due with a genetic deficiency of an enzyme or with an anaerobic condition (remember the ischemic condition we talked about earlier). Regulation of Glycolysis Glycolysis is regulated by energy. If energy (ATP) is abundant, PFK-1 is inhibited. Citrate also inhibits PFK-1 if I am in an aerobic cell. When energy is needed (AMP is high), the pathway is turned on by turning on PFK-1. Remember that everything on the left of the sheet (down to pyruvate/lactate) occurs in the cytosol. So your answer involving an ischemic tissue would be rising AMP levels to activate PFK-1 (as well as the other 2 important enzymes) and an increase in anaerobic glycolysis. Enzyme deficiencies of glycolysis Though glycolysis occurs in all cells, when there is an enzyme deficiency, it is the hemapoietic system that suffers. The major symptom will be chronic hemolytic anemia. There are lots of ways to get this, but one way is that the RBCs cant make enough ATP. If I dont have ATP, the Na+/K+ ATPase stops working, there is an ionic imbalance, and the cell swells and bursts. How do we know the problem is in glycolysis? 1,3-BPG in the RBC is used to make 2,3-BPG, which is then degraded back to 3-PG. So this is a RBC situation. The major genetic deficiency in glycolysis is pyruvate kinase deficiency. The intermediates in the pathway will back up, and this means that 2,3-BPG levels will increase. So if the 2,3-BPG levels in the RBC are abnormal, then the hemolytic anemia is due to a problem in glycolysis. 2,3-BPG lowers the affinity of hemoglobin for oxygen (it aids in the dumping of oxygen to the tissues). The binding of hemoglobin to oxygen shows a sigmoidal curve and 2,3-BPG serves to shift this curve to the right. The curve is also shifted to the right with an increase in CO2, temperature, or a decrease in the pH of the tissues. If the defect is in hexokinase, the intermediates, as well as 2,3-BPG concentrations, should drop. The oxygen dissociation curve for hemoglobin would then shift to the left, telling you the problem is high in the pathway. The conversion of 1,3-BPG to 2,3-BPG is catalyzed, by a mutase, and the conversion of 2,3-BPG to 3-PG is done by a phosphatase. If I have an overactive mutase, then 2,3-BPG levels will increase, and it would mimic a pyruvate kinase deficiency.

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In the liver, the regulation is a little different. Pyruvate is converted to acetyl CoA, which is then used for fatty acid synthesis. So glycolysis is essential for fatty acid synthesis in the liver. The key thing here is fructose 2,6-bisphosphate, made from fructose-6-phosphate by PFK-2. PFK-2 is activated by insulin. So when insulin levels are high in the blood, F 2,6-BP levels are high because PFK-2 is activated. This is how insulin acutely activates glycolysis in the liver: F 2,6 BP activates PFK1, increasing glycolysis. Again, this is all in the liver. The insulin effect overrides the high energy inhibition in the liver so that fatty acid synthesis can take place. Finally, in the liver, there is an isozyme of hexokinase called glucokinase. It also insures that the liver is taking up glucose ONLY after a meal (when insulin is high). When glucose falls, the liver puts glucose into the blood. Glucokinase has a high Km (low affinity) for glucose. So it works only when the substrate concentration is high. Glucokinase is activated by insulin (induction). This is only in the liver! Mono/Disaccharide Metabolism (still on page 2, on the right) The best chance for a question here involves genetic disease or some kind of clinical situation. Galactose is coming from milk sugar lactose. Galactose is metabolized primarily in the liver, but also in the brain. So if there is a defect, these 2 organ systems will be affected. Galactose is converted to galactose-1-phosphate and then enters glycolysis as glucose-6-phosphate. A defect in uridyl transferase (gal-1-P Glc-1-P) causes classic galactosemia. This will cause a backup and an accumulation of galactose in the blood (galactosemia) and in the urine (galactosuria). The galactose-1-phosphate will also accumulate, but it cant get out of the cells because its phosphorylated, so it accumulates in the liver and brain. The galactose in the blood gets into the eye, and you get the formation of cataracts. This will develop within the first couple of weeks of the infant being put on a milk diet. This early appearance should clue in to this genetic defect. Cataracts in the elderly can be due to diabetes. Galactose is converted to a dead-end product, galactitol, which builds up and forms cataracts. If cataracts are the only defect, then the defect in is the enzyme galactokinase (this is not classic galactosemia). However, if the cataracts are associated with liver and brain damage, then the problem is the uridyl transferase. Remember this one. It is the most common and most severe (and the most asked about). Galactose-1phosphate accumulation causes the liver cells to die, so there will be hepatomegaly, jaundice, and hypoglycemia. The nervous system is also affected, so there will be psychomotor problems and mental retardation. Fructose intolerance is a defect in the enzyme aldolase B (this cleaves fructose 1phosphate). This is a liver-specific pathway, so the problem will be ingestion of sucrose (table sugar) causing clinical problems affecting the liver. There will not be cataracts, it will not happen 1-2 weeks after birth, and there will be no neurological

involvement. There will be liver disease, and it will occur when fruit juices or fruits or table sugar are introduced in the diet. Fructose 1-phosphate is thought to be the toxic agent, and it causes hepatomegaly, jaundice, and hypoglycemia. The treatment in both of these cases is to get the offending sugar out of the diet as much as possible. HMP Pathway (Pentose Phosphate pathway) (bottom right) This pathway is also cytosolic, so its in all cells. It degrades glucose 6 -phosphate to pentoses. The main thing to note is that this is a pathway for the generation of NADPH. This is not used for ATP production; the products are used to synthesize things. The key enzyme to remember is the one that generates NADPH glucose 6phosphate dehydrogenase (G6PD). Without NADPH, the cells that are affected are RBCs (and maybe WBCs). NADPH is needed in red cells to protect them against oxidative damage. Without NADPH, I cant have reduced glutathione, and, without this, red cells will become oxidatively damaged. In this case, there will be hemolytic anemia. In most instances, it wont be chronic. It will be induced by oxidative stress. Most cases will be patients suffering from an acute episode of hemolytic jaundice, back pain, blood in the urine, etc. The oxidative stresses are 3-fold: infection, sulfa drugs (anti-malarial drugs), favism (ingestion of fava beans). The gene for G6PD travels with the sickle cell gene, so you would expect it in North Africa, the Mediterranean, and Asia. The hematocrit will be lowered. How do I differentiate this from something like a pyruvate kinase deficiency? The 2,3-BPG levels wont be affected in G6PD deficiency, and this is not chronic hemolytic anemia. Most importantly, a peripheral blood smear in a G6PD deficient patient will show Heinz bodies. These are denatured clumps of hemoglobin that show up as dark spots. Heinz bodies are a pretty much dead giveaway for a G6PD deficiency. G6PD deficiency is X-linked recessive. In the bottom part of the pathway is an enzyme called transketolase, and thiamine is required for its activity. This is the enzyme that is assayed in RBCs to determine the thiamine status of a patient. So in a Wernicke-Korsakoffs patient, you will see lower than normal transketolase activity in red cells, which should increase with the addition of thiamine to the test mix. Dont worry about what transketolase does. Ribose 5-phosphate can be used for nucleotide biosynthesis. The pentose phosphate pathway provides this. Krebs Cycle and Oxidative Phosphorylation (page 3)

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Krebs Cycle Most questions involving Krebs cycle and ox-phos will be recall questions because they cant think of anything that is clinically relevant. Most people with a deficiency in Krebs cycle, unless its very minor, will be DEAD. So in Krebs cycle, acetyl CoA is being converted to CO2. Acetyl CoA is the only substrate for the cycle. The CoA group is recycled, and the acetyl group comes out as CO2. Acetate combines with oxaloacetate to form citrate; thats how it gets into the cycle. Then I go through a series of things to generate energy. The energy is generated in the form of NADH primarily. We get 3 molecules of NADH for every acetyl CoA. At the same time, some FADH2 is produced. When it is produced, unlike NADH, this remains bound to the enzyme that produces it. We also get a GTP made from substrate level phosphorylation. So for one acetyl CoA, 3 NADH, 1 FADH2, and 1 GTP are produced. Remember the PDH complex (pyruvate acetyl CoA) and its thiamine requirement. In terms of the other enzymes to remember here, they may show you a diagram. You should try and remember the enzymes that generate NADH and FADH2. Any enzyme that generates one of these is called a dehydrogenase. There are four of these: in order, isocitrate dehydrogenase (NADH), -ketoglutarate dehydrogenase (NADH), succinate dehydrogenase (FADH 2), and malate dehydrogenase (NADH). They may ask you the order of the generation of NADH, FADH2, and GTP. In this case, you may just want to learn the order of all the intermediates. The mnemonic is this: Citrate Is Krebs Cycle Substrate For Making Oxaloacetate. The steps catalyzed by isocitrate dehydrogenase and -ketoglutarate dehydrogenase are called oxidative decarboxylations, because NADH and CO2 are both being produced here. The substrate level phosphorylation is catalyzed by succinate thiokinase and succinate dehydrogenase, which makes FADH2, is the only enzyme in Krebs cycle that is membrane bound (inner mitochondrial membrane; its invaginated on an EM if they give you that). All other enzymes are found in the mitochondrial matrix. Succinate dehydrogenase is also known as Complex II of the respiratory chain. Regulation of Krebs Cycle Isocitrate dehydrogenase is the key regulatory enzyme in the cycle. The cycle is inhibited by high energy (ATP, NADH) and stimulated by a need for energy (ADP). Bear in mind that very low levels of NADH can cause the cycle to spin out of control in an effort to produce NADH. This leads to the liberation of a large amount of CO2, leading to a metabolic acidosis in the patient.

-ketoglutarate dehydrogenase is notable because it is exactly like PDH. The main thing to remember is that this enzyme also requires thiamine, which accounts for the thiamine requirement for Krebs cycle. Finally, a couple of connections. Remember the malate shuttle. This connects Krebs cycle with gluconeogenesis. You should associate citrate with fatty acid synthesis. The Electron Transport Chain (lower right) NADH is produced in Krebs cycle in the mitochondrial matrix, and it is used to make ATP. FADH2 is also produced, again it is protein bound, but it can also be used to make ATP. NADH is the major substrate for ox-phos. So the NADH that is produced in the mitochondrial matrix goes to the inner mitochondrial membrane, where it looks for Complex I. FADH2 goes also to the inner mitochondrial membrane (its already bound there) and it goes looking for coenzyme Q (ubiquinone). This latter is asked all the time: where does FADH2 send its electrons? Ubiquinone! Doesnt matter what my name is, if I have FADH 2, the electrons go to ubiquinone. Three complexes pump protons across the inner mitochondrial membrane from the matrix out to the intermembrane space: I, III, and IV. This sets up a proton gradient. How does this happen? NADH carries high energy electrons, which represents the energy in our food, to complex I, where it gives up its electrons and is converted to NAD+. The electrons are then transferred from I III IV. As the electrons are transferred, they lose energy, which is used to pump the protons across the membrane. The electrons go to oxygen, which interacts with complex IV and is converted to H2O. What you need to know here are the connectors. Something greasy connects I and III and also connects II and III, and that is coenzyme Q. So thats how electrons get from I to III or II to III (recall that complex II is succinate dehydrogenase). Cytochrome c connects III and IV. You should be aware of something called Mitchells Chemiosmotic hypothesis. It says that as electrons flow through I, III, and IV, energy is released, the energy is used to drive protons from the mitochondrial matrix out into the intermembrane space, and this sets up a proton gradient that collapses and produces ATP. The collapse is shown at the top left. The protons flow back through the complex, from outside to in, that makes ATP. This is complex IV, also known as ATP synthase. So the energy that was in my foodstuffs was transferred to NADH in the form of electrons and the energy was then put in the form of a proton gradient which drives the formation of ATP which can then be used for energy in cellular processes and reactions.

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Fatty Acid Synthesis (page 4) Structurally, ATP synthase has two parts: F0 and F1. F1 is the part that makes the ATP the active site. The ATP can not be made unless protons flow through the membrane, that is unless respiration is occurring. The protons flow through F0; it is the proton channel. F0 is the only way protons can get back into the matrix; the rest of the membrane is impermeable to protons. How might they test some of this stuff? They might be nasty enough to give you a diagram of the ETC and you might have to identify some parts of it. Know that cytochromes contain heme groups with iron. Know that cytochromes c1 and b are in complex III and cytochromes a and a3 are in complex IV. They will also ask you about inhibitors and uncouplers. Uncouplers are anything that allow protons to flow from outside to inside quickly, except that theyre not going through the ATP synthase (they increase the permeability of the membrane to protons). So in this case, ATP is not being made, but the energy is still being liberated, only now in the form of heat. So uncouplers make you warm. Also note that if I inhibit respiration (electron flow), I automatically inhibit ATP synthesis. By the same token, if I stop ATP synthesis first, then respiration stops. They are coupled, except when you use uncouplers. ATP synthesis will stop, but respiration will increase. This will be shown as an increase in oxygen consumption, as well as NADH consumption. 2,4-dinitrophenol (DNP) is a classic uncoupling agent, and an endogenous uncoupler is thermogenin. Thermogenin is found in brown adipose tissue, which is designed to keep newborns warm. Thermogenin allows the brown adipose tissue to produce heat. Other uncouplers include high levels of aspirin, as well as succinyl choline malignant hyperthermia. A metabolic acidosis also occurs here as well. As far as inhibitors are concerned, they might want you to know what inhibitors act at which point in the ETC. The best chances for inhibiting ATP synthase are going to be at complexes I and IV because I is the target for barbiturates (i.e. amytal/amylbarbitol), which slow respiration by directly inhibiting the respiratory center as well as inhibiting the mitochondrial flow of electrons. Complex IV (cytochrome oxidase) can be inhibited by CO and cyanide. CO might be seen in some sort of space heater scenario or someone in a closed garage, and the antidote for this is an oxygen mask oxygen at high concentrations. Cyanide might be a factor in things like house fires (upholstery and things like that generate a lot of CN ) and you may remember that an antidote for cardiac failure or hypertensive crises is infusion of nitroprusside. An excess of nitroprusside will generate cyanide. The treatment for cyanide poisoning is initially amylnitrite followed by thiosulfate. F0 is so-called because oligomycin blocks the chain here at F0. No protons, ATP synthesis dies, respiration dies. In humans, fatty acid synthesis occurs in the liver. It is the 3 rd of three pathways to go from glucose to fatty acids. Fatty acid synthesis occurs in the cytoplasm (along with glycolysis), but the PDH complex is located in the mitochondrial matrix. When pyruvate is made, it just goes right into the matrix where it is converted to acetyl CoA. But acetyl CoA cannot exit the mitochondrion, so it must be converted to citrate first (remember the citrate shuttle that links Krebs cycle with fatty acid synthesis). The citrate is then converted back to acetyl CoA in the cytosol. Note that this is occurring in the postprandial state, when citrate levels are high in the liver cytosol. The pathway itself starts with acetyl CoA and proceeds through a key intermediate, the only one you might be tested on, malonyl CoA, and of course ends with a fatty acid. The key enzyme is the first one, acetyl CoA carboxylase (acetyl CoA malonyl CoA. The carboxylase uses CO2, consumes ATP, and the cofactor for ALL carboxylases in intermediary metabolism is biotin. This is the key regulatory enzyme and since this is happening in the postprandial period, insulin is high and activates this enzyme. Citrate levels are also high, which also serves to activate the enzyme. Biotin is a B vitamin, but there is no known major problem with a biotin deficiency. It is made by intestinal flora, so even if one is deficient in biotin in the diet, it is impossible to get a biotin deficiency normally. There is, however, a protein in egg white called avidin, which avidly binds biotin in the gut and screws up its absorption. You must eat these raw, and you must eat at least 12 a day for this to have any effect. This is improbable, but has been known to happen, and when it does, you get a scaly dermatitis and you lose your hair (alopecia). Acetyl CoA carboxylase makes malonyl CoA which feeds into a huge multienzyme complex. You dont have to remember any of these enzymes, just know that it is called fatty acid synthase. A key point is that NADPH is consumed here. NADPH is a major cofactor in fatty acid synthesis. Enzymes that use NADPH are invariably called reductases. The NADPH here is produced by the HMP shunt. At the end of all this, a fatty acid is produced and in humans, that fatty acid is palmitate or palmitic acid. It has 16 carbons and no double bonds (16:0). It is released from the fatty acid synthase and then all other fatty acids are made from it. In fatty acid synthase, the fatty acid is built up on a protein that is part of this huge complex. This is called acyl carrier protein. When fatty acids are attached to something, they are called fatty acyl groups. There is nothing genetic here, nothing really clinically relevant. Remember the insulin/citrate stories.

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At the bottom of this page is cholesterol synthesis. There are a lot of intermediates, but you dont want to remember anything except HMG CoA and the key enzyme, HMG CoA reductase. What do reductases use? NADPH! Keep in mind that statins inhibit here. Mevalonic acid is produced, and if you see some of these crazy names, they are just things in cholesterol biosynthesis. Dont worry much about any of this except the HMG CoA stuff. At the bottom, dont forget that bile salts are made in the liver from cholest erol (which is really the only degradative pathway for cholesterol), and steroid hormones are made in the endocrine glands. Vitamin D3 is made in the skin from cholesterol as well, in the presence of sunlight. Vitamin D3 is eventually converted to calcetriol (the active form) also known as 1, 25-dihyroxycholecalciferol. The two organs, in sequence, for this conversion are the liver and the kidney. Dont forget about vitamin D deficiency. Vitamin D is needed to maintain calcium levels. It helps parathyroid hormone by stimulating calcium reabsorption from bone as well as stimulating the reabsorption of calcium in the kidney tubules. It does something that PTH cannot do, however. It stimulates intestinal transport of calcium. A vitamin D deficiency in children is called rickets and in adults it is called osteomalacea, which differs from osteoporosis in that both the matrix and calcium are lost in osteoporosis, but only calcium is lost in osteomalacea. Lipoprotein metabolism (page 5) The VLDL exits the liver carrying triglyceride made from dietary glucose. Chylomicrons are also being presented carrying dietary triglyceride, and Lpl gets rid of them. Page 5 is a good summary of lipoprotein metabolism. We know that our two carriers of TGs are chylomicrons (from the intestine) and VLDL (from the liver). What theyre going to ask you is what are apoproteins good for and also about a couple of the hyperlipoproteinemias. So first of all, chylomicrons come out with ApoB-48 and VLDL come out with ApoB-100. The major job of both of these is to get these out of the gut and liver, respectively. So if I have a deficiency of B-48, I cant get chylomicrons out of the gut, so triglycerides will stay in the gut and the problem will be fat malabsorption. If there is a problem with B-100, the fats will be trapped in the liver and I will have a fatty liver. Both of these lipoproteins have to pick up 2 more apoproteins: CII and E. They get these from HDL. HDL is the apoprotein shuffler. CII activates Lpl, which is found in the capillaries of adipose tissue. The hormone that activates it is insulin. So in diabetes (type I), there is no insulin, and therefore the Lpl cannot be activated and chylomicrons, VLDL and TGs accumulate in the plasma. When they are

metabolized by Lpl, they become remnants. VLDL remnants are also called IDL (intermediate density lipoprotein). E is good for getting remnants out of the circulation, both chylomicron remnants and VLDL remnants, and into the liver via an ApoE receptor. Where do LDL and HDL come into this? The sequence in the blood is VLDL IDL LDL. In order to get IDL to LDL, the main thing to note is that there are cholesterol esters being transported from HDL particles to IDL particles to convert them to LDLs. This accounts for the fact that most of the cholesterol is carried by LDLs as cholesterol esters. Where does the HDL get this stuff from? HDL is good cholesterol because it is taking cholesterol from the extrahepatic tissues (i.e. arteries) and disposing of it eventually in the liver. This is reverse cholesterol transport. The LDL has two choices: it can go to the liver, where it is relatively harmless or it can go to the extrahepatic tissues. This is good because all cells need cholesterol, but its bad because if you put too much in your arteries you get atherosclerosis. So you would ideally like to have low LDL levels and high HDL levels. The last thing to note is the conversion of cholesterol to cholesterol esters on HDL particles by a plasma membrane enzyme called LCAT (lecithin cholesterol acyl transferase). This is activated by AI, which is also associated with HDL. Note also that the only apoprotein associated with the LDL particle is B-100. That must mean that B-100 interacts with the LDL receptor (the B-100 receptor). 2 things to be aware of here: first of all is a common genetic disease called familial hypercholesterolemia. This is a high yield USMLE question; it is asked a lot! The defect is in the LDL receptor (though a defect in B-100 would cause the same symptoms). If I dont have receptors, LDL will accumulate in the blood high cholesterol. It is autosomal dominant, which means that the inheritance of one bad allele is enough to cause clinical symptoms. So theres no such thing as a car rier of this. So what you most likely will see is a heterozygote, someone in middle age suffering from signs of heart disease. The frequency is about 1/500 and the gene is going to be coming down one side of the family, and you will see that at least one parent of the patient will have this as well. A homozygote for this disease will be DEAD by in childhood. A heterozygote will have a cholesterol around 400 (about double the normal limit) and a homozygote (very rare) will have a cholesterol over 1000 (mg/dL). What are the symptoms? Pain in the leg after exercise or angina, but the main things to note are xanthomas (mainly in the Achilles tendon) and also deposition of cholesterol in the retina (bleached retina) and signs of artery disease. Remember: ApoAI activates LCAT

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ApoB-48 gets chylomicrons out of the gut ApoB-100 gets VLDL out of liver and LDL into tissues ApoCII activates Lpl ApoE gets remnants out of the blood Also remember that if there is a Lpl deficiency, that is a selective TG accumulation problem. That does not cause coronary artery disease! Accumulation of TGs only in the blood cause pancreatitis. Major Metabolic Pathways in the Postabsorptive State (page 6) In this case the glucose level is low and what were trying to do is maintain blood glucose. A normal fasting blood glucose is about 70 110 mg/dL. If I am in the 160 range, think diabetes. If I am in the 40-45 range, I have a hypoglycemia problem, and they should test you on both. In terms of what is happening here, the two pathways to remember are gluconeogenesis and glycogenolysis, both occurring in the liver. These are the pathways to put glucose into the blood. Glycogen breakdown in the liver does this, glycogen breakdown in the muscle DOES NOT. Glycogen breakdown in the muscle is good for exercise. Gluconeogenesis requires substrates, amino acids from muscle protein breakdown. The key amino acid is alanine. The urea cycle is working here to get rid of alanine to get rid of nitrogen groups. Lactate from anaerobic glycolysis is another major substrate, as is glycerol from the breakdown of fat. The idea is to keep the brain happy and supplied with glucose. Fat breakdown is occurring in the adipocytes. The key enzyme here is hormone sensitive lipase. This is controlled by insulin, but it is the opposite of Lpl. Hormone sensitive lipase is activated by a drop in insulin levels and inhibited by high insulin levels (this is why fatty acids and ketone bodies are NOT found in the blood postprandially). Fatty acids are transported as fatty acid albumins and in order to provide energy for these other aerobic tissues, fatty acids are degraded in a pathway called -oxidation (fatty acids acetyl CoA). This occurs in all tissues except anaerobic tissues and the brain (because of the blood-brain barrier). Fatty acids are never used as fuels in the brain. The acetyl CoA from this degradation can then be fed into Krebs cycle to provide lots of energy. Note that in the liver, the fatty acids do not get converted into glucose. Also note that acetyl CoA can be converted to ketone bodies in the liver via ketogenesis. The greater the rate of fat breakdown in adipose tissue, there greater the rate of -oxidation, and also the greater the rate of ketogenesis and their output into the blood. The liver cannot use ketone bodies for energy, so it exports them into the blood for use by muscle and the kidney via ketogenolysis.

You should think of this metabolic state as what is happening in an uncontrolled diabetic (type I). Insulin levels are low, so hormone sensitive lipase is overactive, fatty acids will accumulate in the blood (hyperlipidemia), Lpl will be underactive (TGs will accumulate in the blood), and ketone bodies will be coming out of the liver at a high rate, resulting in diabetic ketoacidosis. To correct this, you give them some insulin, which will inhibit hormone sensitive lipase and eventually fix the ketoacidosis. The other thing to note is glucose in the blood hyperglycemia. In a diabetic situation, not only can you not get glucose out of the blood, glucose is being put into the blood due to overactivation of glycogenolysis and gluconeogenesis. Insulin inhibits glycogenolysis and gluconeogenesis. These are stimulated by glucagon. The liver is really the only tissue in humans that is sensitive to glucagon. Epinephrine has the same effect as glucagon. The may test you with something like a fasting hypoglycemia question. Fasting hypoglycemia means one of 3 things: a problem in glycogenolysis, a problem in gluconeogenesis (or both), or a problem in -oxidation. If there is a problem in oxidation, the liver will not have the energy to drive gluconeogenesis, and therefore hypoglycemia. You need ATP for gluconeogenesis, it comes from fatty acids. How do I find out where the problem is in someone who is fasting? I give a glucagon challenge. If this raises the blood glucose, that says that glycogen degradation is alright. If it does not raise blood glucose, glycogenolysis is not alright, and glycogen will accumulate in the liver and I will get hepatomegaly. A problem with glycogenolysis will present much earlier in the fast because this is the major way that glucose gets into the blood in the postprandial state. How do I know if the problem is in gluconeogenesis? I look at the lactic acid levels. If the hypoglycemia is accompanied by lactic acidosis, the problem is in gluconeogenesis. Additionally, this will occur later in the fast (10-12 hours) and ketone bodies will be normal or increased. However, if there is a problem in -oxidation, this will also produce hypoglycemia with lactic acidosis later in the fasting period. I can differentiate these by looking at the ketone body levels. If the defect is in oxidation, ketone levels will be abnormally low. This stuff is asked quite often. Carbohydrate Pathways of the Postabsorptive State (page 7) On the left, at the top, is glycogenolysis, and in the middle is gluconeogenesis. Both of these pathways put glucose out into the blood, and in order to do that, they need a specific enzyme. The enzyme is that same in both pathways, and its glucose 6 phosphatase. You must have this to get glucose out in to the blood! This is found in the liver, because its the only tissue sending glucose out into the blood. If you have a defect in this enzyme, there will be a problem in BOTH of these pathways. That is

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classic Von Gierckes disease. There will be an accumulation of glycogen in the liver, hepatomegaly, hypoglycemia, and lactic acidosis. Glycogenolysis

Lastly, glucocorticoids (cortisol) will stimulate gluconeogenesis. These will chronically stimulate gluconeogenesis by stimulating the synthesis of PEP carboxykinase. Muscle Glycogenolysis

Under these conditions (fasting), the key enzyme being activated here is phosphorylase (dont forget that it works with a debranching enzyme). This is activated by glucagon, which also inhibits the synthase enzyme for glycogenesis. Remember that this is only in the liver. Glucagon has no effect on muscle glycogenolysis. Gluconeogenesis Alanine, lactate, and glycerol are your major substrates. I am converting them to glucose. You need to know that alanine and lactate enter at the very end of the pathway. Pyruvate is converted to PEP, to the triose phosphates, and then back to glucose. There are 3 enzymes you need to remember, plus a shuttle. The 3 enzymes are (in addition to glucose 6 phosphatase): 1. 2. 3. Pyruvate Carboxylase: pyruvate oxaloacetate (requires biotin) PEP carboxykinase: oxaloacetate PEP Fructose 1,6-bisphosphatase: F 1,6-P F 6-P

In muscle, glycogen breakdown is not stimulated by glucagon, but by epinephrine and Ca2+. These activate the phosphorylase and the debrancher. Muscle glycogen breakdown does not result in glucose in the circulation because there is no glucose 6-phosphatase in muscle cells. Glucose enters glycolysis and is degraded anaerobically to lactate to produce ATP via substrate level phosphorylation. A deficiency in phosphorylase produces McCardles disease, which is an exercise intolerance disease. This is due to a lack of energy for exercise. Glycogen will accumulate in the muscle, the lactate levels will not rise during exercise. You will also see an increase in blood creatine kinase levels as well as myoglobin levels, as certain isoforms of these are found in skeletal muscle. Know these glycogen storage disease (von Gierckes and McCardles). Fats (page 8) Fatty Acid -oxidation This is on the left. Fatty acids are oxidized in the post absorptive period to provide energy for the extrahepatic tissues to spare glucose for the brain. Fatty acids enter from the blood (carried as albumins) and enter the cytosol where they are attached to CoA (derived from the B vitamin pantothenic acid) to make a fatty acyl CoA. This is fatty acid activation. -oxidation occurs in the mitochondrial matrix, so the fatty acyl must be taken in attached to something called carnitine. This is very high yield! Identify carnitine and the carnitine shuttle with -oxidation. Without carnitine, no -oxidation hypoglycemia and low ketone body levels. The one enzyme you need to identify is at the beginning: acyl CoA dehydrogenase. It generates FADH2, which remains bound to the enzyme and goes looking for coenzyme Q in the ETC. The most common enzyme defect in -oxidation is a defect in this enzyme, specifically a medium chain acyl dehydrogenase (MCAD). Again, the symptoms are fasting hypoglycemia, lactic acidosis, and very low ketone body levels. Dont worry about the other enzymes in the pathway. Ketone Body synthesis and degradation (on the right) My ketone bodies are acetoacetate, acetone (at pathological levels),and hydroxybutyrate. The liver makes acetoacetate and -hydroxybutyrate and sends them out into the blood. Acetone is volatile and appears on the breath, and the only

Remember the malate shuttle. This connects Krebs cycle and gluconeogenesis. A simple test for a problem in gluconeogenesis: If I infuse alanine into a patient, it will not raise blood glucose levels if there is a problem in gluconeogenesis. It doesnt matter where the defect is. Another thing to remember is fructose. Although it is not a major substrate, fructose can enter gluconeogenesis, and it does so at PEP (about the middle). You can do a fructose challenge test. If you infuse fructose and it does not raise blood glucose in the patient, then you know the defect has to be above that point in the pathway, so it will be either fructose 1,6-bisphosphatase or glucose 6 phosphatase. If it does raise blood glucose, the defect will be below that point. Regulation The main thing to remember is fructose 1,6-bisphosphatase. This is inhibited by high levels of fructose 2,6-bisphosphate. That is compliments of insulin. Therefore it will be activated when F 2,6-BP levels fall, thank you glucagon. This explains why insulin acutely turns off gluconeogenesis and glucagon acutely turns off hepatic gluconeogenesis.

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time thats significant is in diabetic ketoacidosis. The patient will have a metabolic acidosis with a wide anion gap. In terms of synthesis, remember that it is in the liver. In terms of degradation, the tissues you should think of are the muscle, kidney, and eventually the brain, but only during prolonged starvation. All of this occurs in the mitochondrial matrix. The one intermediate to remember in the synthesis is HMG CoA. I make this with a synthase and degrade it with a lyase. Where have I seen HMG CoA before? Cholesterol synthesis, but cholesterol synthesis doesnt occur in the mitochondrial matrix. In order to degrade ketone bodies, namely acetoacetate, I have to attach CoA. Here, the CoA comes from succinyl CoA. So in order for fats to be degraded, either fatty acids or ketone bodies, they must be activated by attachment to CoA. No genetic defects here to worry about that Im aware of. Nitrogen Metabolism (page 9) In the top left is the flow of nitrogen atoms in the liver. You should be aware that for amino acids to be converted to their carbon skeletons, the amino groups have to be removed. Carbon skeletons are called -keto acids, and the enzymes that carry out these reactions (transaminations) are called transaminases. In the postprandial period, amino acids are degraded for energy or converted to fat, so you can get fat eating too much protein. In the postabsorptive state in the liver, the -keto acids are converted to glucose by gluconeogenesis. Only 2 amino acids CANNOT be converted to glucose in the liver, and those are the ones that start with L: leucine and lysine. These are strictly ketogenic. If these happen to appear in the same option list, leucine is your first choice. In transamination reactions, the amino acid donates its amino group to ketoglutarate (which is in Krebs cycle), the -keto acid is left behind and the ketoglutarate becomes glutamate. -ketoglutarate is the carbon skeleton of glutamate. Be aware of this transamination, and the fact that all amino groups are funneled into glutamate. Also be aware of the fact that transaminases, as well as almost all of enzymes dealing with amino acid metabolism, require PLP (pyridoxal phosphate) as a cofactor. It contains vitamin B6 (pyridoxine). The amino groups from Glu funnel in 2 directions, with half of them generating ammonia in the liver and half of them generating aspartate. The enzyme that generates ammonia in the liver from Glu is glutamate dehydrogenase; it uses NAD and generates some NADH. The other enzyme is a transamination by aspartate transaminase (AST), where Glu gives the amino group to oxaloacetate and is

recycled to -ketoglutarate. When oxaloacetate gets an amino group, it is converted to aspartate (oxaloacetate is the -keto acid of aspartate). It is the aspartate nitrogen and the ammonia nitrogen that enter the urea cycle. When the aspartate goes in, it generates fumarate, which should be considered a product of the urea cycle. This is also the link between the urea cycle and Krebs cycle. Ammonia is also generated in the extrahepatic tissues (lower left). Extrahepatic tissues do not have the urea cycle (its liver specific), so they detoxify ammonia by the enzyme glutamine synthetase. This generates glutamine from glutamate by putting the ammonia on the Glu side chain. If a patient has elevated ammonia levels in the blood (hyperammonemia), glutamine will be elevated in the blood as well. Glutaminase is the enzyme that hydrolytically releases the ammonia from the Gln. Although a small amount is in the liver, the 2 principle organs that do this are the intestine and the kidney. In normal people, the intestine is the principle site, but thats ok because the ammonia can get to the liver via the portal circulation. So the portal circulation will have a higher ammonia concentration than the systemic circulation, and that is good because high levels of ammonia are toxic to the nervous system. In the kidney, this enzyme is most important for acid-base balance. The release of ammonia will suck up H+ ions (protons) and is used to combat metabolic acidosis. The ammonia is then just put out in the urine. In a normal individual, the highest concentration of nitrogen containing compounds (in the urine) is urea itself (85% or more). Ammonia itself is present in small amounts (<3%). The ammonia will increase in an acidotic patient, thank you glutaminase. Urea Cycle This is shown on the right. They sometimes like to show pathways, and this usually means Krebs and the urea cycle. Ammonia enters the cycle as carbamoyl phosphate. Ammonia is combined with CO2 via carbamoyl phosphate synthetase I (CPS-I) to form carbamoyl phosphate. This then combines with ornithine (which is the carrier) to form citrulline. [remember that something that starts with O (oxaloacetate) is the carrier for acetyl CoA in Krebs cycle and they form Citrate. The carrier here also starts with O (ornithine) and they from citrulline (sounds like citrate)]. From then on its the making and breaking of argininosuccinate via a synthetase and a lyase. Then its the actual hydrolysis of arginine that produces urea. You might want to have a general idea of how the cycle works in case they show you a picture.

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The key regulatory enzyme is CPS-I. It is activated by N-acetyl glutamate. So when amino acids are being degraded, Glu rises, N-acetyl glutamate rises and tells the urea cycle to go faster. The rest of the story is the diseases. The most common diseases they will ask are a deficiency of CPS-I or ornithine transcarbamoylase. The top right tells you that both of these enzymes are in the mitochondrial matrix. The rest of the enzymes are outside in the cytoplasm. If there is a defect in either of these enzymes, urea will be low, so the BUN will be low. Ammonia will be high, as will glutamine. The clinical problem is due to the hyperammonemia, which is a nervous system toxin and will also cause hyperventilation, leading to a respiratory alkalosis. What you will see is that within about a day or two of delivery, the kid becomes lethargic, irritable, vomits, becomes hypothermic and goes into convulsions. If the defect is in one of these two enzymes (which is most probable), the citrulline level will be lower than normal. If it is in any of these other enzymes later in the pathway, the citrulline levels will be decreased. How do you differentiate between the two? Orotic acid! If the orotic acid level is high in the blood and urine (orotic aciduria), the defect is in ornithine transcarbamoylase. If it is normal, CPS-I. The most common defect in real life involves ornithine transcarbamoylase (its X-linked recessive males only!). Know the sequence of intermediates so you can tell what will be high if a particular levels will be high. You treat problems in the urea cycle by limiting protein in the diet put them on a high carb diet. This will stimulate insulin release, which will inhibit protein degradation, another source of nitrogens. In most cases, except with an arginase deficiency, supplement with arginine. Two drugs used to relieve the hyperammonemia are sodium benzoate and sodium phenylacetate.

deficiency of this can cause PKU as well (you would supplement this cofactor as well in this case). Tetrahydrobiopterin is required for the synthesis of catecholamine neurotransmitters, as well as serotonin, so its important to have this cofactor to prevent neural deficits. Two other things to remember: the kid will have a mousy or musty odor. These kids will also be pale, with blue eyes and light hair because this deficiency also interferes with the production of melanin. Avoid aspartame, an artificial sweetener, a dipeptide consisting of phenylalanine and aspartate. Lastly, maternal PKU. PKU is autosomal recessive, so anyone can get it. They are put on a Phe diet, which they hate, but they can get off of after 6 years of age because the nervous system is developed. Phe levels will go through the roof while off the diet, and if she gets pregnant, then the high Phe levels will cross the placenta and impair nervous system development in the fetus. So the mother has to go back on the diet. Tyrosine is converted to melanin, so albinism is a problem with inhibition of that pathway (or in PKU). Alcaptonuria is the accumulation of homogentisic acid due to a defect in an oxidase. This is one of the few, if not the only one, of the amino acidurias that DOES NOT cause neurological problems. This is black urine disease inky diapers. As the person gets older, there may be a b lack spot in the eye. You dont treat this. The problem is ochronotic arthritis the cartilage darkens (ochronosis). This predisposes the person to premature arthritis. On the left, we are talking about the branched chain amino acids ( isoleucine, valine, and leucine) and methionine. Know that there is a disease called MSUD (maple syrup urine disease). The urine smells good, and its a defect in the branched chain -keto acid dehydrogenase. The branched chain amino acids and their corresponding -keto acids are elevated in the blood and urine. This enzyme is like PDH and requires thiamine. Therapeutic does of oral thiamine may help some of these patients. Due to the accumulation of the -keto acids, these patients suffer from crises of -keto acidosis. This can lead to mental retardation, so I restrict the branched chain amino acids in the diet. Methionine is converted to succinyl CoA via a couple of intermediates called homocysteine and cystathionine. As its degraded it produces SAM, the methyl man. This is the major donor of biological methyl groups. Homocysteine is formed, and this can go either back to Met or continue on down the degradative pathway to cystathionine. The disease that is important here is called homocystinuria. Cystathionine synthase takes homocysteine and converts it to cystathionine. In the opposite direction, homocysteine methyl transferase converts homocysteine back to Met. A deficiency of EITHER of these enzymes cause homocystinuria. It can also be caused by a defect in any of 3 vitamins (all Bs): B12 (cobalamine), PLP,

Amino Acid Degradation (page 10) Here you just need to be aware of the genetic diseases associated with this. First of all, phenylketonuria. As a group, the amino acidurias affect the nervous system and cause psychomotor delay, hypotonia and eventually mental retardation. As a group, they are treatable by restricting the offending amino acids in the diet (not eliminating them, because they are essential). Classic PKU is the inability to degrade phenylalanine to tyrosine (a deficiency of phenylalanine hydroxylase). Under these conditions, phenylalanine is elevated in the blood and urine. This is the only way we can make tyrosine, so the treatment is restriction of Phe in the diet and supplementation with Tyr (because Tyr is now by definition essential). There is a cofactor here called tetrahydrobiopterin, so a

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and methyl FH4(tetrahydrofolate, from the vitamin folic acid). PLP is required for cystathionine synthase and the B12 and methyl FH4 are required for homocysteine methyl transferase. A characteristic feature of folate or B12 deficiency is megaloblastic anemia (a peripheral smear will show large RBCs and hypersegmented neutrophils). This is a quick way to determine where the problem is involving homocystinuria. If it is accompanied by megaloblastic anemia, then the problem is with the homocysteine methyl transferase. Without anemia, the problem is in the synthase. The synthase defect is classic homocystinuria. You might give these people B6 (PLP) to help them out. With homocystinuria, you will see vascular problems, strokes, deep vein thromboses, and dislocation of the lenses of the eyes. Elevated levels of homocysteine are a definite heart disease risk factor. Lastly, at the bottom, we go through proprionyl CoA through methyl malonyl CoA to succinyl CoA. Note that biotin and another B12 are required down here. Proprionyl CoA is converted to methyl malonyl CoA by a carboxylase (the biotin requirement) and then to succinyl CoA via a mutase (B12 requirement). The bottom line is a defect of either folate OR B12 cause megaloblastic anemia and homocystinuria, but ONLY a defect of B12 also causes methyl malonic aciduria. A defect of only the mutase will cause only methyl malonic aciduria. The accumulation of methyl malonic acid causes the neurological deficits seen in B12 deficiency (not seen in folate deficiency). Thats called pernicious anemia, caused by a defect in intrinsic factor, a transport protein for B12 made by parietal cells in the stomach. So bear in mind that stomach resections, ileal disease, peptic ulcers, thing like that, can cause pernicious anemia. Also note that you have enough B12 in your liver to last 5-7 years, so you wont get deficiency without an absorption problem. Dont forget the folate story in pregnant women. Folate deficiency has been associated with neural tube defects, so take folate as soon as you can. Heme Biosynthesis and Degradation (page 11) On the left is the pathway of heme biosynthesis. What do I need? I need protoporphyrin (a ring) and I will put iron into the ring. This gives me heme. At the beginning, I have to start with glycine and succinyl CoA, but the main thing to note is that the enzyme here is called ALA (aminolevulinic acid) synthase, and this requires PLP. With a defect in this pathway, you should think anemia. But it wont be megaloblastic, the cells here will be small and pale (hypochromic microcytic anemia). This can be due to an iron deficiency, or an inability to make the ring structure (B6 deficiency). This can be differentiated by the amount of iron. In the latter case, we have plenty of iron but no ring to put it in, so the anemia will also be

characterized as sideroblastic. This is due to accumulation of iron in the mitochondria of macrophages. This is also a great place for a lead poisoning scenario. Lead inhibits ALA dehydratase as well as ferrocheletase (the last step). So lead will also cause an anemia, but it will also cause a rise in the levels of -ALA and protoporphyrin. -ALA is a nervous system toxin; it is thought to cause the neurological and abdominal/peripheral problems with lead poisoning. If any of this gets through, there will also be accumulation of zinc protophorphyrin in red cells. Of these other diseases on here, I would be concerned with congenital erythropoietic porphyria. In this case, you would expect to see hypochromic microcytic anemia, uroporphyrinogen I will accumulate, and this will cause severe skin photosensitivity. The very first disease is called acute intermittent porphyria. This is a disease that does not affect the blood system or the skin; it causes abdominal problems and neuropsychological problems. So its unusual. These people present with nerve damage, psychotic behaviors, and pain in the abdomen. This is due to the accumulation of -ALA, but it can be differentiated from lead poisoning by a rise in PPG (phorphobilinogen) levels. You treat this with some derivative of heme hemitin or hemin. This knocks out ALA synthase, and reduces flux through the pathway. You do not give barbiturates! This stimulates this pathway and makes their symptoms worse. Oral contraceptives are contraindicated for the same reason. These both rev up the CYP450 system. Protoporphyria is down here at the bottom. It is essentially the same as congenital erythropoietic porphyria, except the symptoms will not be as severe. Heme Degradation (on the right) We degrade heme as we destroy millions of red cells everyday. The degradation of heme results in bilirubin formation. The destruction is basically hemolysis. The bilirubin is greasy and is carried in the blood as albumin. It is dropped off in the hepatocyte and converted to a conjugated form called diglucuronide. The enzyme is called a glucuronyl transferase. This converts the insoluble bilirubin to a soluble form. Clinically, the first stuff is called indirect; the stuff that has been conjugated is called direct. The major things here are the diseases. Prehepatic jaundice is going to be some situation where there is massive hemolysis (hemolytic jaundice). This is production of bilirubin faster than the hepatocyte can take it up and conjugate it. So the problem will be accumulation, preferentially, of the indirect bilirubin in the blood. This will not be seen in the urine; it is water insoluble.

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The next problem is a problem with the enzymes in the liver. Neonatal jaundice means that young kids do not have fully induced glucuronyl transferase activity, so they are put under fluorescent lights to destroy the indirect so that it can be eliminated as a more soluble form in the bile and urine. There are a couple of disease here. The one thats a real killer is Crigler-Najjar syndrome, which is a total absence of glucuronyl transferases. Again, the indirect bilirubin will be elevated in the blood, none in the urine, but the indirect bilirubin has a very high affinity for nerve cell membranes. This accumulates in the brain and is fatal. That is called kernicterus (the deposition of unconjugated bilirubin in the brain and nervous tissue). Gilberts disease is a partial deficiency of this enzyme, so the patients can survive. As a treatment, try to induce whats left. Thats done with phenobarbitol. Toxic (hepatic) jaundice means destruction of liver cells. The liver cannot take up bilirubin and as you lose liver cells, it cannot be conjugated. So there is an elevation of both indirect and direct, with the proportions changing depending on the nature of the liver disease. Bilirubin will be in the urine here. Posthepatic jaundice is a secretion problem. Dubin-Johnson syndrome is a problem in the genes encoding the proteins getting the conjugated from the liver into the bile. Posthepatic jaundice can also be caused by gall stones and pancreatic tumors. You should note that the water-soluble bilirubin will regurgitate through the hepatic veins rather than coming through the bile, and appear in the blood and urine. So preferential elevation of direct bilirubin in the blood and urine is indicative of posthepatic jaundice, some kind of obstructive jaundice. If there is a problem here, the stools will have a chalky, light colored appearance because Im not getting bile pigments into the stools. Purine and Pyrimidine Biosynthesis (page 12) Purines (adenine and guanine) The major thing to note here is that I need a sugar [ribose (R)] and a phosphate (P) and a base. That is a nucleotide. To get the P and the R, we start out with something called PRPP. We will replace the PP with a base to get the nucleotide. Purine rings are the big rings, and you dont need to remember where all the atoms are coming from. You need 2 amino acids to make both kinds of nucleotides. They give me nitrogens, and those are glutamine and aspartate. I make something called PR-NH2, I replace the PP with an amino group, and then I use 9 steps to make the purine ring. There is an amino acid used for purine synthesis that is not used for pyrimidine synthesis, and that is glycine. You need folate to make both purines but only 1 of the pyrimidines (thymine).

You first make IMP (inosine monophosphate). Then from there you make G and A. Pyrimidines (cytosine, uracil, and thymine). Forget the numbering system, forget where things are coming from. Dont forget that you still need glutamine and aspartate. There are 2 key intermediates to remember: carbamoyl phosphate and orotate. Carbamoyl phosphate is also an intermediate in urea cycle as well. CUT are your pyrimidines. First I make the U and then I make the C and the T from the U. Folate is required to make the T only. Thats critical because T is found in DNA (not U) and if I have a folate deficiency, then I have trouble making DNA. This messes up cell division and you get megaloblastic anemia and hypersegmented neutrophils. The rest of this is not critical, but note that if I want to knock out ribonucleotide reductase, I use hydroxyurea, so I cant make deoxy stuff, and that prevents me from making DNA. That inhibits the cell cycle. Thymidylate synthase is inhibited by fluorouracil. That prevents the production of T, and again will inhibit DNA synthesis and the cell cycle. Dihydrofolate reductase is inhibited by methotrexate, trimethoprin, and pyrimethamine. In order these inhibit the eukaryotic enzyme, the prokaryotic enzyme, and the protozoal enzyme. Purine degradation (page 13) This also shows purine synthesis: PRPP IMP AMP or GMP. The key enzyme here is amidotransferase; thats the key enzyme in purine synthesis. This is inhibited by AMP and GMP and IMP. Now what do I do with the base/sugar/phosphate? I take it off! You take off the phosphate, leaving a nucleoside: adenosine or guanosine. Adenosine loses its identity and becomes inosine. The enzyme that does this is adenosine deaminase. This is deficient in SCID (servere combined immunodeficiency). It was also the first successful attempt at gene therapy in the early 90s. You get the accumulation of A stuff, and these are toxic to T and B cells. Now you have the base and sugar. You cut it apart; guanosine is still guanosine. It becomes guanine and the base in inosine is hypoxanthine. These are just bases. I can do 1 of 2 things with these bases. 90% of the time, I salvage them. I take the base an put it right back on PR, I release PP and get a nucleotide. The enzyme that does this is HGPRT. 10% of the time I oxidize hypoxanthine, convert guanine to xanthine, and then convert this to uric acid and send it out in the urine. The key enzyme here is xanthine oxidase. Associate this with gout. Gout is an overproduction of uric acid

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or too little excretion of uric acid. the key drug would be allopurinol, which is converted to oxypurinol (alloxanthine) by xanthine oxidase. The product, oxypurinol, inhibits xanthine oxidase, so its called a suicide inhibitor. This is done, not during an attacke of gout, but chronically to prevent gout. If you have a person with an attack of gout, you give them colchicine or indomethacin (preferred because its an NSAID). A deficiecny in HGPRT is called Lesch-Nyhan syndrome. Hypoxanthine and guanine will be poured into uric acid, so these perople have hyperuricemia. They dont have gout so much, but the uric acid gets deposited into the kidneys , and they get kidney failure, uric acid stones. The classic thing here is aggressive behavior, mental retardation, and self mutilation. This is X-linked recessive, so this affects mainly males.
We have covered all of the water soluble vitamins. We didnt touch on C really, but be aware of scurvy (vitamin C deficiency): loose teeth, bleeding gums, that sort of stuff. In terms of the fat-soluble vitamins, remember K for coagulation, A is for vision night blindness and dry skin and dry eyes (xerophthalmia). Vitamin E, one word, antioxidant. This prevents oxidation of membranes and other cellular components. It also prevents oxidation of LDL particles, which can lead to the formation of atherosclerotic plaques.

From here on is basically stuff that he didnt go over last year. This is the last 3 pages of the handout, and I will only include stuff that he adds to the handout, not stuff that is already on the handout or stuff that was previously covered in the review. Major Biochemical Diseases Lactose intolerance is very common. The deficiency is in the intestinal disaccharidase lactase. It causes abdominal distention, diarrhea, cramping, etc. after the ingestion of dairy and milk products. Get lactose out of the diet or add lactase, known as lactaid. The test for this is an H2 breath test because if you dont have lactase, the lactose will be digested by the flora in your intestine, some of which will produce hydrogen gas. Hartnup Disease is a problem with the aromatic or branched-chain amino acids. The thing to remember here is that one of the aromatics is trp, so this person can present with a pellegra-like syndrome, treat with niacinamide. Cystinuria is a problem with renal stones due to the accumulation of cysteine because of a problem with the transport system. The thing to note here is that the basic amino acid transport system is also knocked out. The mnemonic to remember here is COAL Cysteine, Ornithine, Arginine, Lysine. Pernicious Anemia causes a B12 deficiency due to a B12 transport problem. The problem is in a transport protein called intrinsic factor, secreted by the gastric mucosa. It binds the B12 from the diet and absorbs it from the distal ileum. So for problems with B12 deficiency and transport, think 1 st stomach,

then ileum (Crohns dz, Celiac dz, resection), then pancreas, which is needed to release the B12 from foods (think CF). All 3 can give you problems. Celiac disease is an autoimmune response to wheat gluten. Hemolytic anemia associated with a defect in glycolysis is mainly due to a deficiency in pyruvate kinase. The change is going to be in BPG. Fructose intolerance is an aldolase B deficiency. Galactosemia is a defect in uridyltransferase. Acute (induced) hemolytic anemia associated with the pentose phosphate pathway is due to a G6PD deficiency. It is induced by stress infection, sulfa drugs, antimalarials, etc. Chronic hemolytic anemia may also be seen with a G6PD deficiency and these people sometimes show symptoms of chronic granulomatous disease. Von Gierkes Disease is Type I glycogen storage disease and is caused by a deficiency in glucose 6-phosphatase. So glycogenolysis and gluconeogenesis are both affected. McCardles is a deficiency in muscle phosphorylase, causing exercise intolerance. Coris Disease is a debranching enzyme defect. Pompes Disease is a problem with a lysosomal glucosidase. It leads to a cardiomyopathy. Tay-Sachs disease is a lysosomal storage defect. The defective enzyme is hexosaminidase A, leading to an accumulation of ganglioside. This affects the nerve cells directly, so mental retardation, early blindness, and cherry red maculae. Gauchers disease is a defect in glucosyl cerebrosidase and glucocerebrosides accumulate. Symptoms include hepatomegaly and neurologic problems, as well as bone problems (pancytopenia and bone erosions). In Niemann-Pick disease, sphingomyelin causes the problems. Symptoms here include hepatomegaly and neurologic deficits. Hyperchylomicronemia does not cause CV problems. It causes abdominal pain after a fatty meal and pancreatitis. Forget about cystathionuria under letter M. Its not important. Primary gout means that I have a genetic defect. No one knows for sure, but maybe an overproduction of PRPP synthetase. Secondary Gout is when I cant get rid of excess uric acid, probably most important in the population. Orotic aciduria is to remind you that you can get megaloblastic anemia without a B12 or a folate deficiency. Ascorbic Acid is Vitamin C, involved in collagen biosynthesis. It is also an important antioxidant. A deficiency is scurvy, characterized by vascular lesions. So there is bleeding, not due to clotting problems, but due to fragile capillaries. Therefore, tests such as the PT would be normal. The key enzyme here that will be deficient is proline hydroxylase.

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Beriberi and Wernicke-Korsakoff are essentially the same, except that beriberi has a cardiopathic component. Riboflavin is the vitamin that is most light-sensitive. It is destroyed by light. What do you do with kids with neonatal jaundice? P ut them under lights. Thats to get rid of the bilirubin, so you might want to give some riboflavin to these kids undergoing this light treatment. Folate deficiency during pregnancy has been implicated in neural tubes defects such as spina bifida. Biotin means carboxylations. Pantothenate means coenzyme A. Vitamin A comes in a variety of forms: retinol, retinoic acid, Retin-A, retinal. I can also get it from plants by eating beta-carotene. If eaten too much, the skin will look orange. It is needed for vision. Night-blindness is an early sign of a deficiency. The other forms are needed for maintenance of healthy epithelial tissues. Xeropthalmia is important. There is infection and kids go blind. Also goose flesh (keratomalacea). Never give vitamin A to a pregnant woman! Calciferol is vitamin D and it is used to keep adequate calcium levels in the blood. If calcium levels are low, then bone mineralization will be defective. In kids, thats called rickets. These are little kids with bow legs and skeletal deformities. In adults, its osteomalacea, where there is demineralization of the bone, causing fractures. It is not osteoporosis! Tocopherol is E. It is an antioxidant that protects against membrane damage. One of the membranes it gets into are RBC membranes, to prevent hemolysis. It also gets into nerve cell membranes and protects from neurological damage. Also, in terms of vitamin E, think that this is the fat soluble vitamin that I can eat as much of as I want. It just wont hurt you. It is thought to protect a gainst heart disease by preventing the oxidation of LDL. Remember K for coagulation. Hemorrhage and bruising are the classic signs. Think newborns with sterile guts and antibiotics that wipe out the intestinal flora. If I have too much iron, that is hemochromatosis. Primarily seen in males, it affects multiple organs: the liver, heart, kidney. The treatment is phlebotomy, or if you dont want to do that, deferoxamine. A deficiency of copper is Menkes disease. This is a defect in a transport protein for copper. I cant get it absorbed. Free copper in the blood is low, ceruloplasmin is also low in the blood. There will be problems in collagen biosynthesis, osteoporosis, and bleeding. The also have neurologic problems and characteristic kinky, steely hair. Copper is required to make melanin. Treat with a copper histidine complex. Wilsons disease is copper toxicity, also due to a defect in a transport protein. The problem is in getting copper from the liver into the bile, so the 1 st organ affected will be the liver. There is liver necrosis and then it spills over and causes neurologic deficits. The treatment is penicillamine.