130867503-Biochemistry-Board-Review-1.pdf

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The Behemoth Biochem Board ReviewPart IMay 16, 2000The Incomparable Roger LaneContract/ gunners nonymous

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 tissuesthat are anaerobic, but this is the one we will focus on. Certain muscle cells (whitemuscle cells), skin cells, WBCs, as well as the adrenal and renal medullas, are allanaerobic tissues. These tissues have to make due with a single metabolic pathwayat all times, anaerobic glycolysis (glucose pyruvate lactate). The ATP is

produced by substrate level phosphorylation . Bear in mind that this pathway doesnot require mitochondria or oxygen. This pathway would be activated during anischemic event (MI or stroke); the tissue would eventually die, but it would first tryto make energy via this pathway. The problem is that lactic acid will build up, andthis is toxic to tissues that are deprived of oxygen or a blood supply. So if you seethe term anaerobic ischemia , this is the pathway that theyre talking about.

In all of the other tissues, glucose pyruvate acetyl CoA CO 2. Glucose isused by all tissues in this state, but it is absolutely critical in the brain , and 3

pathways convert glucose totally to CO 2, which is exhaled.

1. (Aerobic) Glycolysis : glucose pyruvate 2. Pyruvate dehydrogenase complex: pyruvate acetyl CoA 3. Krebs Cycle : Acetyl CoA CO 2

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 , whilethe other 2 pathways occur within the mitochondria .

One of the major things that might be tested, without getting into any details yet, isthat thiamine is required in the PDH complex and also for Krebs cycle activity . Sothis is a high yield question, and thiamine deficiency is seen very often in thiscountry associated with chronic alcoholism, and the patient suffers from Wernicke-Korsakoffss syndrome . If the patient presents, and you put him on a 5% dextroseIV drip, you must also give him so thiamine along with that. The reason is thatnerve cells cannot use glucose for energy without thiamine, and that would be key ifthe patient is comatose and you put in a glucose line. Some of the symptoms of the

Wernickes component include a peripheral neuropathy, nystagmus, and

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 aremaking stuff glycogen, triglyceride and protein synthesis. The key hormone hereis insulin.

Glycogenesis is glycogen synthesis. It occurs in ALL tissues, but quantitativelymost important are liver and muscle .

Protein synthesis (translation ) occurs in ALL tissues. Amino acids are used tomake proteins. Most of the protein is contained in muscle. In the bottom right handcorner, 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 basicamino acids to make protein intracellularly. Only 10 of these are classified asdietarily essential for children and infants (PVT TIM HALL) and only 8 of these areessential for healthy adults. HA! histidine and arginine -- are required in adultsonly during times of growth (recovery from injury, etc). Recall that we get tyrosinefrom 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 malnutritiondevelops, and this is called Kwashiorkor . This leads to a negative nitrogenbalance . In normal, healthy people, we are in a normal nitrogen balance (no netgain or loss of body protein). Anything that causes protein to be broken down morethan 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. Adeficiency of the amino acid tryptophan can lead to a pellegra-like situation as well.

Fat Synthesis

What we are trying to do here is either degrade dietary fat and store it or to convertexcess dietary carbohydrate to fat and store it. There are 2 types of lipoproteins thatare elevated in the blood postprandially, VLDLs and chylomicrons . Thechylomicrons come from the intestine , and they are carrying dietary triglycerides(fat). VLDLs come from the liver and ca rry triglycerides made there. So bear inmind that if a patient has elevated blood TGs, it will be one or both of these (VLDLor 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 easytest 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 highchylomicron 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 pathwaysinvolved and the synthesis of fat is activated by insulin :

1. Glycolysis2. PDH complex3. Fatty Acid synthesis (cytosolic and this is also stimulated by the

presence of citrate, because this tells your body that levels of energyare high and therefore can be stored.)

What we are doing is breaking glucose down to acetyl CoA using glycolysis andPDH, and then taking the acetyl CoA and making fatty acids. The overall process isanabolic glucose is being made to make fats. Fatty acids and glycerol phosphate are used to make triglycerides. The triglyceride is exported into the blood inVLDLs. So something simple: if the patient is on a high fat, low carb diet, thenchylomicrons will be elevated in the blood. If the patient is on a low fat, high carbdiet, 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 thatdiabetics have increased lipids in their blood. . This degrades the triglycerides fromthe interiors of these particles, releasing fatty acids and glycerol. These thing shrinkand become chylomicron and VLDL remnants , which will be taken up by the liverand disposed of.

The fatty acids are taken up by the adipose tissue and stored as fat. This requiresglycerol phosphate, which the adipocytes CANNOT make. This can only be done inthe liver by an enzyme called glycerol kinase . So the glycerol released from theVLDL/chylomicron cannot be used for fat synthesis in the adipocyte because it

doesnt have the enzyme to convert this to glycerol phosphate. Adipocytes get theglycerol phosphate for this from glycolysis . So glucose is needed for TG synthesisin adipocytes.

This is a type of question that can be asked. Maybe not specifically about thisenzyme, but in general, and that is that all diploid cells in the body have the fullcomplement of 46 chromosomes. So even if a particular enzyme is not present in aspecific 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, inorder, Southern Blot , Northern Blot , and Western Blot .

Protein Metabolism

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 convertedto urea via the urea cycle this is a liver-specific pathway. When the urea appearsin the blood, it is referred to as the blood urea nitrogen (BUN ). So if the BUN islower than normal, one should suspect either generalized liver disease or a defect inthe urea cycle itself. A higher than normal BUN is a classic clinical test for renalinsufficiency. 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 are27 carbons in cholesterol and they ALL come from acetyl CoA. The key enzymehere is HMG CoA reductase .

In the liver, and only the liver, the cholesterol is converted to bile salts , which arethen sent into the bile. This conversion of cholesterol to bile salts, though it is nottotally degraded, should be thought of as degradation. This is a very common

question regarding the treatment of hypercholesterolemia This very often due to adefect in the LDL r eceptor (takes up LDLs from the blood). LDL is the major waythat cholesterol is transported in the blood. Some drugs work to upregulate LDLreceptors in plasma membranes of liver cells (and therefore reduce the LDLconcentration 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 tostop 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. thatsdone with a bile acid binding resin . The ones to remember are cholestyramine andcholestipol . These increase the conversion of cholesterol to bile salts because I am

preventing bile salts from coming into the liver. A combination of both (decreasingsynthesis and increasing degradation) is a good treatment for people who have agenetic 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


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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, orRBCs (they lack the GLUT 4 transporter).

Insulin does, however, stimulate many processes in the liver. It stimulatesglycogenesis 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 de tails follow

Carbohydrate Pathways Postprandially (page 2)

There are3 you should concentrate on: glycolysis , HMP pathway , andglycogenesis . 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 isinvolved. To get to glycogen, we must first go through glucose-1-phosphate . Thekey enzyme that controls synthesis is glycogen synthase (activated by insulin) . Theopposing enzyme (for degradation) is glycogen phosphorylase (or just

phosphorylase; this is inhibited by insulin). They both work with an enzyme thateither 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 ondown. All the intermediate and enzymes are NOT important. What you should comeaway with is the degradation of glucose-6-phosphate through fructose derivatives totwo triose phosphates (glyceraldehyde-3-phosphate and DHAP). Those areinterconvertable, and it is then the G-3-P that continues through the remaining stepsto 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 thisenzyme 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 inglycolysis is phosphofructokinase-1 (PFK-1 ). This is the regulatory enzyme in

glycolysis. The last enzyme to remember is at the bottom: pyruvate kinase (PEPpyruvate ). 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 orwith an anaerobic condition (remember the ischemic condition we talked aboutearlier).

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 (AMPis high), the pathway is turned on by turning on PFK-1. Remember that everythingon the left of the sheet (down to pyruvate/lactate) occurs in the cytosol. So youranswer 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 thehemapoietic 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 cantmake enough ATP. If I dont have ATP, the Na +/K + ATPase stops working, there isan ionic imbalance, and the cell swells and bursts. How do we know the problem isin glycolysis? 1,3-BPG in the RBC is used to make 2,3-BPG, which is thendegraded back to 3-PG. So this is a RBC situation.

The major genetic deficiency in glycolysis is pyruvate kinase deficiency . Theintermediates in the pathway will back up, and this means that 2,3-BPG levels willincrease. So if the 2,3-BPG levels in the RBC are abnormal, then the hemolyticanemia 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 inCO 2 , 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 theleft , 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 theconversion of 2,3-BPG to 3-PG is done by a phosphatase . If I have an overactivemutase, 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 ac idsynthesis in the liver. The key thing here is fructose 2,6-bisphosphate , made fromfructose-6-phosphate by PFK-2 . PFK-2 is activated by insulin. So when insulinlevels 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 ac tivates PFK-1, increasing glycolysis. Again, this is all in the liver. The insulin effect overridesthe 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 alsoinsures 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 K m (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 clinicalsituation. 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 organsystems will be affected. Galactose is converted to galactose-1-phosphate and thenenters 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 alsoaccumulate, but it cant get out of the cells because its phosphorylated, so itaccumulates in the liver and brain. The galactose in the blood gets into the eye, andyou get the formation of cataracts . This will develop within the first couple ofweeks of the infant being put on a milk diet. This early appearance should clue in tothis 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 (thisis 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 isthe most common and most severe (and the most asked about). Galactose-1-

phosphate 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 1- phosphate). 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 becataracts, 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 fruitsor table sugar are introduced in the diet. Fructose 1-phosphate is thought to be thetoxic 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 asmuch as possible.

HMP Pathway (Pentose Phosphate pathway) (bottom right)

This pathway is also cytosolic, so its in all ce lls. It degrades glucose 6 -phosphate topentoses . 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 synthesizethings. The key enzyme to remember is the one that generates NADPH glucose 6-phosphate dehydrogenase (G6PD).

Without NADPH, the cells that are affected a re 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 becomeoxidatively 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, bloodin 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 cellgene, so you would expect it in North Africa, the Mediterranean, and Asia. Thehematocrit will be lowered. How do I differentiate this from something like a

pyruvate kinase deficiency? The 2,3- BPG levels wont be affected in G6PDdeficiency, and this is not chronic hemolytic anemia. Most importantly, a peripheral

blood smear in a G6PD deficient patient will show Heinz bodies . These aredenatured 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-linkedrecessive.

In the bottom part of the pathway is an enzyme called transketolase , and thiamineis required for its activity. This is the enzyme that is assayed in RBCs to determinethe thiamine status of a patient. So in a Wernicke- Korsakoffs patient, you will seelower than normal transketolase activity in red cells, which should increase with theaddition 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 becausethey cant think of anything that i s clinically relevant. Most people with a deficiencyin Krebs cycle, unless its very minor, will be DEAD.

So in Krebs cycle, acetyl CoA is being converted to CO 2. Acetyl CoA is the onlysubstrate for the cycle. The CoA group is recycled, and the acetyl group comes outas CO 2. Acetate combines with oxaloacetate to form citrate; thats how it gets intothe cycle. Then I go through a series of things to generate energy. The energy isgenerated in the form of NADH primarily. We get 3 molecules of NADH for everyacetyl CoA.

At the same time, some FADH 2 is produced. When it is produced, unlike NADH,this remains bound to the enzyme that produces it. We also get a GTP made fromsubstrate level phosphorylation. So for one acetyl CoA, 3 NADH, 1 FADH 2, and 1GTP 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 adiagram. You should try and remember the enzymes that generate NADH andFADH 2. Any enzyme that generates one of these is called a dehydrogenase . Thereare four of these: in order, isocitrate dehydrogenase (NADH), -ketoglutaratedehydrogenase (NADH), succinate dehydrogenase (FADH 2), and malatedehydrogenase (NADH).

They may ask you the order of the generation of NADH, FADH 2, and GTP. In thiscase, you may just want to learn the order of all the intermediates. The mnemonic isthis: C itrate Is K rebs C ycle Substrate F or M aking O xaloacetate. The stepscatalyzed by isocitrate dehydrogenase and -ketoglutarate dehydrogenase are calledoxidative decarboxylations , because NADH and CO 2 are both being produced

here. The substrate level phosphorylation is catalyzed by succinate thiokinase andsuccinate dehydrogenase, which makes FADH 2, is the only enzyme in Krebs cyclethat is membrane bound (inner mitochondrial membrane; its invaginated on an EMif 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 isinhibited 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 outof control in an effort to produce NADH. This leads to the liberation of a large

amount of CO 2 , leading to a metabolic acidosis in the patient.

-ketoglutarate dehydrogenase is notable because it is exactly like PDH. The mainthing to remember is that this enzyme also requires thiamine, which accounts forthe thiamine requirement for Krebs cycle.

Finally, a couple of connections. Remember the malate shuttle. This connectsKrebs cycle with gluconeogenesis. You should associate ci trate with fatty acidsynthesis.

The Electron Transport Chain (lower right)

NADH is produced in Krebs cycle in the mitochondrial matrix, and it is used tomake ATP. FADH 2 is also produced, again it is protein bound, but it can also beused 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 . FADH 2 goes also to the inner mitochondrialmembrane (its already bound there) and it goes looking for coenzyme Q (ubiquinone ). This latter is asked all the time: where does FADH 2 send itselectrons? Ubiquinone! Doesnt matter what my name is, if I have FADH 2, theelectrons go to ubiquinone.

Three complexes pump protons across the inner mitochondrial membrane from thematrix out to the intermembrane space: I, III, and IV . This sets up a protongradient. How does this happen?

NADH carries high energy electrons, which represents the energy in our food, tocomplex I, where it gives up its electrons and is converted to NAD +. The electronsare then transferred from I III IV . As the electrons are transferred, they loseenergy, which is used to pump the protons across the membrane. The electrons go tooxygen , which interacts with complex IV and is converted to H 2O.

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 getfrom 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 calledMitchells Chemiosmotic hypothesis. It says that as electrons flow through I, III,and IV, energy is released, the energy is used to drive protons from themitochondrial matrix out into the intermembrane space, and this sets up a protongradient 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 theform 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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Structurally, ATP synthase has two parts: F 0 and F 1. F1 is the part that makes theATP the active site. The ATP can not be made unless protons flow through themembrane, that is unless respiration is occurring. The protons flow through F 0; it isthe proton channel. F 0 is the only way protons can get back into the matrix; the restof the membrane is impermeable to protons.

How might they test some of this stuff? They might be nasty enough to give you adiagram of the ETC and you might have to identify some parts of it. Know thatcytochromes contain heme groups with iron. Know that cytochromes c1 and b are incomplex III and cytochromes a and a 3 are in complex IV.

They will also ask you about inhibitors and uncouplers. Uncouplers are anythingthat allow protons to flow from outside to inside quickly, except that theyre notgoing 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 beingliberated, 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, butrespiration will increase. This will be shown as an increase in oxygen consumption,as well as NADH consumption. 2,4-dinitrophenol (DNP ) is a classic uncouplingagent, and an endogenous uncoupler is thermogenin . Thermogenin is found in

brown adipose tissue, which is designed to keep newborns warm. Thermogeninallows the brown adipose tissue to produce heat. Other uncouplers include highlevels of aspirin, as well as succinyl choline malignant hyperthermia. Ametabolic acidosis also occurs here as well.

As far as inhibitors are concerned, they might want you to know what inhibitors actat 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 respiratorycenter as well as inhibiting the mitochondrial flow of electrons. Complex IV(cytochrome oxidase) can be inhibited by CO and cyanide . CO might be seen insome sort of space heater scenario or someone in a closed garage, and the antidotefor this is an oxygen mask oxygen at high concentrations. Cyanide might be afactor 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 isinfusion of nitroprusside. An excess of nitroprusside will generate cyanide. Thetreatment for cyanide poisoning is initially amylnitrite followed by thiosulfate .

F0 is so-called because oligomycin blocks the chain here at F 0. No protons, ATP

synthesis dies, respiration dies.

Fatty Acid Synthesis (page 4)

In humans, fatty acid synthesis occurs in the liver. It is the 3 rd of three pathways togo from glucose to fatty acids. Fatty acid synthesis occurs in the cytoplasm (alongwith 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 acetylCoA. But acetyl CoA cannot exit the mitochondrion, so it must be converted tocitrate first (remember the citrate shuttle that links Krebs cycle with fatty acidsynthesis). The citrate is then converted back to acetyl CoA in the cytosol. Note thatthis is occurring in the postprandial state, when citrate levels are high in the livercytosol.

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 fattyacid. The key enzyme is the first one, acetyl CoA carboxylase (acetyl CoA malonyl CoA. The carboxylase uses CO 2, consumes ATP, and the cofactor for ALLcarboxylases in intermediary metabolism is biotin . This is the key regulatoryenzyme and since this is happening in the postprandial period, insulin is high andactivates 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 isimpossible to get a biotin deficiency normally. There is, however, a pro tein in eggwhite called avidin , which avidly binds biotin in the gut and screws up itsabsorption. You must eat these raw, and you must eat at least 12 a day for this tohave any effect. This is improbable, but has been known to happen, and when itdoes, you get a scaly dermatitis and you lose your hair (alopecia).

Acetyl CoA carboxylase makes malonyl CoA which feeds into a huge multi-enzyme 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 areinvariably 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 ispalmitate or palmitic acid . It has 16 carbons and no double bonds (16:0). It isreleased 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 hugecomplex. This is called acyl carrier protein . When fatty acids are attached tosomething, they are called fatty acyl groups.

There is nothing genetic here, nothing really clinically relevant. Remember theinsulin/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 statinsinhibit 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 o f thisexcept 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 steroidhormones are made in the endocrine glands. Vitamin D 3 is made in the skin fromcholesterol as well, in the presence of sunlight. Vitamin D 3 is eventually convertedto calcetriol (the active form) also known as 1, 25-dihyroxycholecalciferol . Thetwo organs, in sequence, for this conversion are the liver and the kidney.

Dont forget about vitamin D deficiency. Vitamin D is needed to maintain calciumlevels. It helps parathyroid hormone by stimulating calcium reabsorption from

bone as well as stimulating the reabsorption of calcium in the kidney tubules. Itdoes 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 iscalled osteomalacea, which differs from osteoporosis in that both the matrix andcalcium 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 ridof them. Page 5 is a good summary of lipoprotein metabolism. We know that ourtwo carriers of TG s are chylomicrons (from the intestine) and VLDL (from theliver).

What theyre going to ask you is what are apoproteins good for and also about acouple of the hyperlipoproteinemias. So first of all, chylomicrons come out withApoB-48 and VLDL come out with ApoB-100 . The major job of both of these is toget these out of the gut and liver, respectively. So if I have a deficiency of B-48, Icant 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 betrapped 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 getthese from HDL . HDL is the apoprotein shuffler. CII ac tivates Lpl, which is foundin the capillaries of adipose tissue. The hormone that activates it is insulin. So indiabetes (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 remnantsand VLDL remnants, and into the liver via an ApoE receptor.

Where do LDL and HDL come into this? The sequence in the blood is VLDLIDL LDL . In order to get IDL to LDL, the main thing to note is that there arecholesterol esters being transported from HDL particles to IDL particles to convertthem to LDLs. This accounts for the fact that most of the cholesterol is carried byLDLs as cholesterol esters . Where does the HDL get this stuff from? HDL is goodcholesterol 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 itcan 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. Soyou 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 acyltransferase). 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. Thatmust mean that B-100 interacts with the LDL receptor (the B-100 receptor). 2things 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 a sked a lot! Thedefect is in the LDL receptor (though a defect in B -100 would cause the samesymptoms). If I dont have receptors, LDL will accumulate in the blood highcholesterol. 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 ofthis. So what you most likely will see is a heterozygote, someone in middle agesuffering from signs of heart disease. The frequency is about 1/500 and the gene isgoing to be coming down one side of the family, and you will see that a t least one

parent of the patient will have this as well. A homozygote for this disease will beDEAD by in childhood. A heterozygote will have a cholesterol around 400 (aboutdouble the normal limit) and a homozygote (very rare) will have a cholesterol over1000 (mg/dL).

What are the symptoms? Pain in the leg a fter exercise or angina, but the main thingsto note are xanthomas (mainly in the Achilles tendon) and also deposition ofcholesterol 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 gutApoB-100 gets VLDL out of liver and LDL into tissuesApoCII activates LplApoE 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 bloodglucose. A normal fasting blood glucose is about 70 110 mg/dL . If I am in the160 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 aregluconeogenesis 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 muscleis 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 alanineto get rid of nitrogen groups. Lactate from anaerobic glycolysis is another majorsubstrate, as is glycerol from the breakdown of fat. The idea is to keep the brainhappy 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. Hormonesensitive 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 pathwaycalled -oxidation (fatty acids acetyl CoA). This occurs in a ll tissues exceptanaerobic tissues and the brain (because of the blood-brain barrier). Fatty acids arenever used as fuels in the brain.

The acetyl CoA from this degradation can then be fed into Krebs cycle to providelots of energy. Note that in the liver, the fatty ac ids do not get converted intoglucose. Also note that acetyl CoA can be converted to ketone bodies in the livervia ketogenesis . The greater the rate of fat breakdown in adipose tissue, theregreater 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 exportsthem 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 uncontrolleddiabetic (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), a nd ketone bodies will be coming out of theliver at a high rate, resulting in diabetic ketoacidosis . To correct this, you givethem 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 diabeticsituation, not only can you not get glucose out of the blood, glucose is being put intothe blood due to overactivation of glycogenolysis and gluconeogenesis. Insulininhibits 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. Fastinghypoglycemia means one of 3 things: a problem in glycogenolysis, a problem ingluconeogenesis (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 thereforehypoglycemia. 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 aglucagon challenge . If this raises the blood glucose, that says that glycogendegradation is alright. If it does not raise blood glucose, glycogenolysis is notalright, 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 themajor way that glucose gets into the blood in the postprandial state. How do I knowif the problem is in gluconeogenesis? I look at the lactic ac id levels. If thehypoglycemia 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 candifferentiate 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. Bothof these pathways put glucose out into the blood, and in order to do that, they need aspecific 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 inthe liver, because i ts 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 theliver, hepatomegaly, hypoglycemia, and lactic acidosis.

Glycogenolysis

Under these conditions (fasting), the key enzyme being activated here isphosphorylase (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 muscleglycogenolysis.

Gluconeogenesis

Alanine, lactate, and glycerol are your major substrates. I am converting them toglucose. You need to know that alanine and lactate enter a t the very end of the

pathway. Pyruvate is converted to PEP, to the triose phosphates, and then back toglucose. There are 3 enzymes you need to remember, plus a shuttle. The 3 enzymesare (in addition to glucose 6 phosphatase):

1. Pyruvate Carboxylase : pyruvate oxaloacetate (requires biotin )2. PEP carboxykinase : oxaloacetate PEP3. Fructose 1,6-bisphosphatase : F 1,6-P F 6-P

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, itwill not raise blood glucose levels if there is a problem in gluconeogenesis. Itdoesnt matter where the defect is.

Another thing to remember is fructose. Although it is not a major substrate, fructosecan 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 inthe patient, then you know the defect has to be above that point in the pathway, so itwill 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 byhigh levels of fructose 2,6-bisphosphate. That is compliments of insulin. Thereforeit will be activated when F 2,6-BP levels fall, thank you glucagon. This explainswhy insulin acutely turns off gluconeogenesis and glucagon acutely turns offhepatic gluconeogenesis.

Lastly, glucocorticoids (cortisol) will stimulate gluconeogenesis. These willchronically stimulate gluconeogenesis by stimulating the synthesis of PEPcarboxykinase .

Muscle Glycogenolysis

In muscle, glycogen breakdown is not stimulated by glucagon, but by epinephrine

and Ca 2+. These activate the phosphorylase and the debrancher. Muscle glycogen breakdown does not result in glucose in the circulation because there is no glucose6-phosphatase in muscle cells. Glucose enters glycolysis and is degradedanaerobically to lactate to produce ATP via substrate level phosphorylation.

A deficiency in phosphorylase produces McCardles disease , which is an exerciseintolerance disease. This is due to a lack of energy for exercise. Glycogen willaccumulate in the muscle, the lac tate levels will not rise during exercise. You willalso see an increase in blood creatine kinase levels as well as myoglobin levels, ascertain isoforms of these are found in skeletal muscle. Know these glycogen storagedisease (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 provideenergy for the extrahepatic tissues to spare glucose for the brain. Fatty ac ids enterfrom the blood (carried as albumins) and enter the cytosol where they are attachedto 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 thefatty acyl must be taken in attached to something called carnitine . This is very highyield! Identify carnitine and the ca rnitine shuttle with -oxidation. Withoutcarnitine, no -oxidation hypoglycemia and low ketone body levels.

The one enzyme you need to identify is at the beginning: acyl CoA dehydrogenase .It generates FADH 2, which remains bound to the enzyme and goes looking forcoenzyme Q in the ETC. The most common enzyme defect in -oxidation is adefect 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 sendsthem out into the blood. Acetone is volatile and appears on the breath, and the only


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time thats significant is in diabetic ketoacidosis . The patient will have a metabolicacidosis with a wide anion gap.

In terms of synthesis, remember that it is in the liver. In terms of degradation, thetissues you should think of are the muscle, kidney, and eventually the brain, butonly during prolonged starvation. All of this occurs in the mitochondrial matrix. Theone 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 mitochondrialmatrix.

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 fattyacids 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 thatfor 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 carryout these reactions ( transaminations ) are called transaminases . In the postprandial

period, amino acids are degraded for energy or converted to fat, so you can get fateating too much protein. In the postabsorptive state in the liver, the -keto acids areconverted to glucose by gluconeogenesis. Only 2 amino acids CANNOT beconverted 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 sameoption 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 ofglutamate. Be aware of this transamination, and the fact that all amino groups arefunneled into glutamate. Also be aware of the fact that transaminases, as well asalmost 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 generatingammonia in the liver and half of them generating aspartate. The enzyme thatgenerates ammonia in the liver from Glu is glutamate dehydrogenase ; it uses NADand 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 convertedto 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 Krebscycle.

Ammonia is also generated in the extrahepatic tissues (lower left). Extrahepatictissues do not have the urea cycle (its liver specifi c), so they detoxify ammonia bythe enzyme glutamine synthetase . This generates glutamine from glutamate by

putting the ammonia on the Glu side chain. If a patient has elevated ammonia levelsin 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 theintestine and the kidney. In normal people, the intestine is the principle site, butthats 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 thenervous system.

In the kidney, this enzyme is most important for ac id-base balance. The release ofammonia 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 highestconcentration of nitrogen containing compounds (in the urine) is urea itself (85% ormore). Ammonia itself is present in small amounts (


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The key regulatory enzyme is CPS-I. It is activated by N-acetyl glutamate . Sowhen amino acids are being degraded, Glu rises, N -acetyl glutamate rises and tellsthe urea cycle to go faster.

The rest of the story is the diseases. The most common diseases they will ask are adeficiency 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 below. Ammonia will be high, as will glutamine. The c linical problem is due to thehyperammonemia, which is a nervous system toxin and will also causehyperventilation, leading to a respiratory alkalosis. What you will see is that withinabout a day or two of delivery, the kid becomes lethargic, irritable, vomits, becomeshypothermic 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 inany of these other enzymes later in the pathway, the citrulline levels will bedecreased.

How do you differentiate between the two? Orotic acid ! If the orotic acid level ishigh in the blood and urine (orotic aciduria), the defect is in ornithinetranscarbamoylase. If it is normal, CPS-I. The most common defect in real lifeinvolves ornithine transcarbamoylase (its X-linked recessive males only!).

Know the sequence of intermediates so you can tell what will be high if a particularlevels will be high. You treat problems in the urea cycle by limiting protein in thediet put them on a high carb diet. This will stimulate insulin release, which willinhibit protein degradation, another source of nitrogens. In most cases, except withan arginase deficiency, supplement with arginine. Two drugs used to relieve thehyperammonemia are sodium benzoate and sodium phenylacetate .

Amino Acid Degradation (page 10)

Here you just need to be aware of the genetic diseases associated with this. First ofall, phenylketonuria . As a group, the amino acidurias a ffect the nervous systemand cause psychomotor delay, hypotonia and eventually mental retardation. As agroup, they are treatable by restricting the offending amino acids in the diet (noteliminating them, because they are essential).

Classic PKU is the inability to degrade phenylalanine to tyrosine (a deficiency ofphenylalanine hydroxylase ). Under these conditions, phenylalanine is elevated inthe 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 bydefinition essential). There is a cofactor here ca lled tetrahydrobiopterin , so a

deficiency of this can cause PKU as well (you would supplement this cofactor aswell in this case). Tetrahydrobiopterin is required for the synthesis of catecholamineneurotransmitters, 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 mousyor musty odor. These kids will also be pale, with blue eyes and light hair becausethis deficiency also interferes with the production of melanin. Avoid aspartame , anartificial 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 whileoff the diet, and if she gets pregnant, then the high Phe le vels will cross the placentaand impair nervous system development in the fetus. So the mother has to go backon 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 thatDOES NOT cause neurological problems. This is black urine disease inkydiapers. As the person gets older, there may be a b lack spot in the eye. You donttreat 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 branchedchain -keto acid dehydrogenase . The branched chain amino acids and theircorresponding -keto acids are elevated in the blood and urine. This enzyme is likePDH and requires thiamine. Therapeutic does of oral thiamine may help some of

these patients. Due to the accumulation of the -keto acids, these patients sufferfrom 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 calledhomocysteine and cystathionine . As its degraded it produces SAM , the methylman. 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 tocystathionine. The disease that is important here is called homocystinuria .

Cystathionine synthase takes homocysteine and converts it to cystathionine. In theopposite direction, homocysteine methyl transferase converts homocysteine back

to Met. A deficiency of EITHER of these enzymes cause homocystinuria. It canalso be caused by a defect in any of 3 v itamins (all Bs): B12 (cobalamine ), PLP ,


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and methyl FH 4(tetrahydrofolate , from the vitamin folic acid). PLP is required forcystathionine synthase and the B12 and methyl FH 4 are required for homocysteinemethyl transferase.

A characteristic feature of folate or B12 deficiency is megaloblastic anemia (a peripheral smear will show large RBCs and hypersegmented neutrophils). This is aquick way to determine where the problem is involving homocystinuria. If it is

accompanied by megaloblastic anemia, then the problem is with the homocysteinemethyl transferase. Without anemia, the problem is in the synthase. The synthasedefect is classic homocystinuria. You might give these people B6 (PLP) to helpthem out.

With homocystinuria, you will see vascular problems, strokes, deep veinthromboses, and dislocation of the lenses of the eyes. Elevated levels ofhomocysteine are a definite heart disease risk factor.

Lastly, at the bottom, we go through proprionyl CoA through methyl malonyl CoAto 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 bottomline is a defect of either folate OR B12 cause megaloblastic anemia andhomocystinuria, but ONLY a defect of B12 also causes methyl malonic aciduria .A defect of only the mutase will cause only methyl malonic aciduria. Theaccumulation of methyl malonic acid causes the neurological deficits seen in B12deficiency (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 inthe 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 tolast 5- 7 years, so you wont get deficiency without an absorption problem. Dont

forget the folate story in pregnant women. Folate deficiency has been associatedwith 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 needprotoporphyrin (a ring) and I will put iron into the ring. This gives me heme. Atthe beginning, I have to start with glycine and succinyl CoA, but the main thing tonote is that the enzyme here is called ALA (aminolevulinic acid) synthase, and thisrequires 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 microcyticanemia ). 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 thelatter 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 themitochondria 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 ananemia, 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 andabdominal/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 hypochromicmicrocytic anemia, uroporphyrinogen I will accumulate, and this will cause severeskin photosensitivity.

The very first disease is called acute intermittent porphyria . This is a disease thatdoes not affect the blood system or the skin; it causes abdominal problems andneuropsychological problems. So its unusual. These people present with nervedamage, 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 inPPG (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 makestheir symptoms worse. Oral contraceptives are contraindicated for the samereason. These both rev up the CYP450 system.

Protoporphyria is down here at the bottom. It is essentially the same as congenitalerythropoietic 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 ofheme 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 thehepatocyte and converted to a conjugated form called diglucuronide . The enzymeis called a glucuronyl transferase . This converts the insoluble bilirubin to a solubleform. Clinically, the first stuff is called indirect; the stuff that has been conjugated iscalled direct.

The major things here are the diseases. Prehepatic jaundice is going to be somesituation where there is massive hemolysis (hemolytic jaundice). This is productionof 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, sothey are put under fluorescent lights to destroy the indirect so that it can beeliminated as a more soluble form in the bile and urine. There are a couple ofdisease here. The one thats a real killer i s Crigler-Najjar syndrome , which is atotal absence of glucuronyl transferases. Again, the indirect bilirubin will beelevated 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 iscalled kernicterus (the deposition of unconjugated bilirubin in the brain andnervous tissue).

Gilberts disease is a partial deficiency of this enzyme, so the patients can survive.As a treatment, try to induce whats l eft. 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 elevationof both indirect and direct, with the proportions changing depending on the natureof 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 liverinto the bile. Posthepatic jaundice can also be caused by gall stones and pancreatictumors. You should note that the water-soluble bilirubin will regurgitate through thehepatic 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 withsomething 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 atomsare 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-NH 2, I replacethe PP with an amino group, and then I use 9 steps to make the purine ring. There isan 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 andA.

Pyrimidines (cytosine, uracil, and thymine).

Forget the numberin g system, forget where things are coming from. Dont forget

that you still need glutamine and aspartate. There are 2 key intermediates toremember: carbamoyl phosphate and orotate . Carbamoyl phosphate is also anintermediate in urea cycle as well. CUT are your pyrimidines. First I make the Uand 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 getmegaloblastic 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 preven ts mefrom 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 enzymehere is amidotransferase; thats the key enzyme in purine synthesis. This isinhibited 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 thefirst successful attempt at gene therapy in the ea rly 90s. You get the accumulationof 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. Ican 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 thatdoes 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 isxanthine 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 isconverted 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 personwith 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 andguanine will be poured into uric acid, so these perople have hyperuricemia . Theydont have gout so much, but the uric acid gets deposited into the kidneys , and theyget kidney failure, uric acid stones. The classic thing here is aggressive behavior,mental retardation, and self mutilation. This is X-linked recessive, so this affectsmainly 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 othercellular components. It also prevents oxidation of LDL particles, which can lead to the formation ofatherosclerotic plaques.

From here on is basically stuff that he didn t go over last year. This is the last 3pages 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 inthe review.

Major Biochemical Diseases

Lactose intolerance is very common. The deficiency is in the intestinaldisaccharidase lactase . It causes abdominal distention, diarrhea, cramping, etc.after the ingestion of dairy and milk products. Get lactose out of the diet or addlactase, known as lactaid. The t est for this is an H 2 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 thatthe basic amino acid transport system is also knocked out. The mnemonic toremember here is COAL C ysteine, O rnithine, Arginine, L ysine.

Pernicious Anemia causes a B12 deficiency due to a B12 transport problem.The problem is in a transport protein called intrinsic factor , secreted by thegastric mucosa. It binds the B12 from the diet and absorbs it from the distal

ileum. So for problems with B12 deficiency and transport, think 1st

stomach,

then ileum (Crohns dz, Celiac dz, resection), then pancreas, which is needed torelease 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, sulfadrugs, antimalarials, etc.

Chronic hemolytic anemia may also be seen with a G6PD deficiency andthese people sometimes show symptoms of chronic granulomatous disease .

Von Gierkes Disease is Type I glycogen storage disease and is caused by adeficiency in glucose 6-phosphatase . So glycogenolysis and gluconeogenesisare both affected.

McCardles is a deficiency in muscle phosphorylase, causing exerciseintolerance.

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 thenerve cells directly, so mental retardation, early blindness, and cherry redmaculae .

Gauchers disease is a defect in glucosyl cerebrosidase and glucocerebrosidesaccumulate. Symptoms include hepatomegaly and neurologic problems, as wellas bone problems (pancytopenia and bone erosions).

In Niemann-Pick disease , sphingomyelin causes the problems. Symptoms hereinclude 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 vascularlesions. So there is bleeding, not due to clotting problems, but due to fragilecapillaries. 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. Thatsto get rid of the bilirubin, so you might want to give some riboflavin to thesekids undergoing this light treatment.

Folate deficiency during pregnancy has been implicated in neural tubes defectssuch 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 skinwill look orange. It is needed for vision . Night-blindness is an early sign of adeficiency. The other forms are needed for maintenance of healthy epithelialtissues. Xeropthalmia is important. There is infection and kids go blind. Alsogoose 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. Inkids, thats called rickets . These are little kids with bow legs and skeletaldeformities. 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 caneat as much of as I want. It just wont hurt you. It is thought to protect a gainstheart 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 intestinalflora.

If I have too much iron, that is hemochromatosis. Primarily seen in males, itaffects 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 c opper. 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 problemsand 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 organaffected will be the liver. There is liver necrosis and then it spills over andcauses neurologic deficits. The treatment is penicillamine .

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