biochem: vitamins

the information in our vitamin lecture...

biochem: mark's medical biochem chapter 41- purines and pyrimidines

note: i smashed my laptop last week so some of these entries won't have the question/answer section.

purines and pyrimidines are two categories of bases from which nucleosides (base plus ribose sugar) and thus nucleotides (base plus ribose plus phosphate) are made. they are not obtained easily from the diet and as such need to be synthesized de novo. purine synthesis begins with ribosyl-5 phosphate and ATP:

1. ribosyl 5-phosphate and ATP combine to form 5-phosphoribosyl pyrophosphate (PRPP) via PRPP synthetase.
2. the pyrophosphate in PRPP is exchanged for an amine group from glutamine via amido phosphoribosyl transferase, forming 5-phospho ribosylamine.
3. a glycine molecule is added, forming glycinamide ribosyl 5-phosphate.
4. a series of C and N donations: N10 formyl FH4 donates carbon 8.
5. glutamine donates nitrogen 3.
6. N10 formyl FH4 donates carbon 2.
7. aspartate donates carbon 1.
8. CO2 donates carbon 6, forming the purine nucleotide inosine monophosphate (IMP)

IMP can be converted to the nucleotides GMP or AMP as well. the conversion to AMP involves the addition of an aspartate molecule to form adenylosuccinate, using one GTP (in a reaction similar to adding aspartate to citrulline in the urea cycle). adenylosuccinate then loses a fumarate in the subsequent reaction to form AMP. to form GMP, IMP is oxidized by IMP dehydrogenase to xanothine, which is then decarboxylated to GMP with the help of ATP.

regulation of purine synthesis occurs at four places: PRPP synthetase (step 1) is inhibited by ADP and GDP, while amidophosphoribosyl transferase (step 2) is inhibited by AMP and GMP. IMP dehydrogenase and adenylosuccinate synthetase are inhibited by the products they eventually form, GMP and AMP, respectively.

the purine nucleotide cycle occurs in the brain: IMP is converted to AMP by the process described above, and AMP is converted back to IMP by a deaminase reaction, the net result being the conversion of aspartate to fumarate and the production of ammonia (recall from the urea cycle chapter).

the purine salvage cycle (see diagram) is a system of enzymes that are designed to rebuild the purine nucleotides in the peripheral tissues from the free bases or nucleoside components that are delivered to them. except for adenosine, the general strategy is to convert nucleosides to free bases, and then to nucleotides. there are several types of enzymes involved which interconvert nucleosides, nucleotides, and free bases. 5'nucleotidases convert nucleotides to nucleosides by dephosphorylating the ribose. purine nucleotide phosphorylases cleave the nucleoside into a free base and a phosphorylated ribose. hypoxanthine guanine phosphoribosyl transferase enzymes add an phosphoribosyl unit from PRPP to the base, creating a nucleotide. deaminases can convert AMP to IMP. finally, adenosine kinase can directly phosphorylate adenosine into the nucleotide AMP (it is the only nucleoside that can be converted to a nucleotide directly via salvage enzymes)

purines can be degraded by salvage enzymes as well: AMP is deaminated to form IMP. the nucleotides IMP and GMP are then converted into nucleosides inosine and guanosine, which are then converted to the bases adenine and guanine. adenine and guanine are converted to xanthosine, which is converted to uric acid and excreted in the urine.


pyrimidine synthesis begins by building the ring structure and adding the phosphoribose later (as opposed to purines, which start with the phosphoribose):

1. glutamine, CO2, and 2ATP combine via carbamoyl phosphate synthetase II to form carbamoyl phosphate.
2. an aspartate is added, forming carbamoyl aspartate, via aspartate transcarbamoylase.
3. dihydroorotase adds an OH, forming dihydroorotate.
4. dihydroorotate is oxidized by dihydroorotate dehydrogenase into orotate, forming the ring structure.
5. a phosphoribosyl unit is added to orotate by orotate phosphoribosyl transferase, forming the nucleotide orotate monophosphate.
6. orotate monophosphate is decarboxylated by OMP decarboxylase to form the nucleotide UMP.
7. UMP can be phosphorylated to UTP.
8. UTP can be converted to the nucleotide CTP.

synthesis of pyrimidines is mainly regulated at the CPS II enzyme level (recall that CPS I is involved in the first step of the urea cycle, in the mitochondria as opposed to the cytosol in pyrimidine synthesis). this enzyme is activated by ATP and inhibited by UTP. pyrimidines are degraded similarly to purines; nucleotides converted to nucleosides converted to free bases cytosine, thiamine, and uracil. cytosine is deaminated to uracil, which is degraded further into CO2, ammonium, and beta-alanine, while thiamine is degraded into CO2, ammonium, and beta-aminoisobutyrate.

purines and pyrimidines can be converted to the deoxygenated form for use in DNA synthesis. the enzyme that catalyzes this reaction is ribonucleotide reductase, and the electron donor is thioredoxin (which is regenerated by the enzyme thioredoxin reductase and NADPH from the pentose phosphate pathway). ribonucleotide reductase regulation is complex in that it can be activated and inhibited towards specific molecules depending on what binds to it (see diagram).

biochem: mark's medical biochem chapter 40- FH4, vitamin B12, SAM

this chapter talked about folate, B12, and S-adenosyl methionine: three compounds that can either donate or accept single carbon groups and participate in a wide variety of essential reactions in the body. folate has three structural components: a bicyclic pteridine ring, para-amino benzoic acid (PABA), and a polyglutamate tail. folate can be obtained from the diet from animal products, leafy greens, legumes, fruits. after ingestion, folate is cleaved by brush border proteases to the monoglutamate form, reduced in intestinal epithelial cells and transported to the liver, where it is reconjugated into the polyglutamate form and stored.

folate can be reduced to dihydrofolate, and then to tetrahydrofolate, the form that is able to accept carbon groups. it can then accept carbons can attach to the N5 or N10 (on the pteridine ring and PABA, respectively), to form N10-formyl-FH4. this compound is converted to the bridge form N5,N10 methenyl FH4, which can be reduced to N5,N10 methylene FH4 and further reduced to folate's most stable form, N5 methyl FH4. this is the predominant form that folate is found in blood, and once formed, can not be re-oxidized to other forms of folate.

folate can accept carbons from a variety of different sources, including serine, formate, glycine, formaldehyde, histidine, choline, methionine. serine is the major carbon donor and forms glycine (recall from chapter 39 that this is the major pathway of glycine formation) and N5,N10-methylene FH4. the opposite reaction, donation of one carbon groups, is also possible: the formation of the nucleotide dTMP, which is required for DNA synthesis, involves transferring a carbon from N5,N10 methylene FH4 to dUMP to form dTMP and FH2, which is reduced back to FH4 by dihydrofolate reductase.

vitamin B12 is the second one carbon transfer compound: it is composed of a large corrin ring with a cobalt ion in the middle, which can form complexes with carbon molecules. vitamin B12 is synthesized by bacteria and as such needs to be ingested in the form of animal products. digestion depends on the form it is obtained in; it can come in a free form, or bound to dietary proteins. if ingested in the free form, it will be bound to haptocorrins in the mouth or stomach, then cleaved and bound to intrinsic factor in the duodenum. if bound to dietary proteins, folate will be cleaved in the stomach or intestine and also be bound to intrinsic factor. at this point it is absorbed and bound to transcobalamin II and transported to the liver.

vitamin B12 is involved in two important reactions: (see diagram) the conversion of homocysteine to methionine, and the conversion of methylmalonyl CoA to succinyl CoA (aiding in the conversion of propionyl CoA to succinyl CoA in fatty acid oxidation and amino acid synthesis). if vitamin B12 is deficient, then one of the two pathways for homocysteine conversion is blocked (see diagram), and homocysteine will begin to accumulate- a condition called hyperhomocysteinemia. this condition can also occur when the cystathione synthase enzyme is blocked, or if the enzyme that converts N5N10 methylene FH4 to N5 methyl FH4 is blocked.

one final carbon donor: SAM is s-adenosyl methionine, formed by methionine and ATP (see diagram). it can donate carbon groups to O or N, as opposed to folate, which donates to S or C. after donation, it forms s-adenosyl homocysteine, which can be converted to homocysteine.

questions
folate structure, sources, and digestion...
1. what are the structural components to folate?
2. what is the function of folate in chemical reactions?
3. where do the carbon groups attach to folate?
4. what does the reduction of folate yield?
5. most of the folate in the body is in the form of...
6. what are some sources of folate in the diet?
7. what is the difference between the folate in supplements vs. dietary sources?
8. what happens to dietary folate in the intestine?
9. what happens to folate in the liver?

one carbon pool...
10. what is the one carbon pool?
11. describe the reduction of the formyl group on FH4.
12. what is the major donor of the carbon group to folate in humans?
13. describe the interconversions of the one carbon units of FH4.
14. what does the reaction of serine and FH4 produce?
15. what is dTMP and how is it related to the one carbon pool?
16. what is DHFR?

vitamin B12...
17. what are the key structural characteristics of vitamin B12?
18. what are the two key reactions that B12 aids in?
19. how is vitamin B12 produced and what are some dietary sources?
20. what are the two forms that vitamin B12 will be present in dietary sources?
21. describe the digestion and absorption of vitamin B12.
22. what happens to vitamin B12 after it is absorbed into the ileum?

s-adenosylmethionine (SAM)...
23. what does SAM do?
24. how is SAM synthesized?
25. what does SAM form after it donates its methyl group?

methyl trap hypothesis...
26. what is the most stable form of folate in the folate cycle?
27. what is the only reaction that can extract the methyl from N5 methyl FH4?
28. what is the methyl trap hypothesis?
29. how is homocysteine synthesized?
30. how can a vitamin B12 deficiency lead to an hyperhomocysteinemia?
31. what are two other ways in which homocysteine can accumulate?
32. what is the connection between neural tube defects and folate?

answers
1. bicyclic pteridine ring, para-amino benzoic acid (PABA), and a polyglutamate tail.
2. it accepts a one carbon group.
3. N5 or N10.
4. dihydrofolate and tetrahydrofolate.
5. FH4.
6. green leafy vegetables, fruits, legumes. also synthesized by bacteria.
7. dietary sources have the reduced form whereas supplements have the oxidized form.
8. brush border conjugases convert it into the monoglutamate form, which is absorbed into the intestine, where it is converted into tetrahydrofolate. FH4 is then transported to the liver.
9. it is reconjugated and mostly secreted in the bile to be reabsorbed.

10. a term which refers to the single carbons that are attached to N5 or N10 of folate.
11. the formyl group is formed by the addition of the carbon to N10; this carbon can form a ring structure with a double bond which connects to N5. the double bond can be reduced, and the ring can also be reduced, moving the carbon group to N5.
12. serine.
13. N10-formyl-FH4 forms N5,N10 methenyl FH4, which is reduced to N5,N10 methylene FH4, which is reduced to N5 methyl FH4.
14. serine donates its hydroxymethyl group to FH4, forming glycine and N5,N10 methylene FH4.
15. dTMP is a nucleotide required for the synthesis of DNA, that is formed from addition of dUMP and a carbon from N5,N10 methylene FH4. this forms dTMP as well as FH2.
16. dihydrofolate reductase, the enzyme that aids in the reduction of FH2 back to FH4, after reactions such as the dTMP reaction, which forms FH2.

17. the overall structure is a "corrin" ring with 4 pyrrole rings, out of which 2 are joined directly and 2 are joined by a methylene bridge. in the center of the ring is cobalt, which can bind to carbon.
18. donation of a carbon group from N5 methyl FH4 to homocysteine to form methionine. also, rearrangement of methylmalonyl CoA to form succinyl CoA.
19. it is only synthesized by bacteria and is consumed in the diet in the form of meat, eggs, diary, fish, poultry, seafood.
20. either bound to dietary proteins or in the free form.
21. the free form is bound to the haptocorrin protein, which is secreted in the mouth and stomach. the bound form is cleaved from its proteins by proteases in the stomach and small intestine, and then bound to haptocorrin. in the intestine, both forms have their haptocorrins removed by intestinal proteases, and then are bound to intrinsic factor, which facilitates their absorption into ileum enterocytes.
22. in the enterocyte cells, B12 complexes with transcobalamin II, and then is transported to the liver and other tissues (about 50% to the liver).

23. it is involved in the donation of a methyl group to oxygen or nitrogen- it is involved in at least 35 important reactions.
24. methionine and ATP.
25. homocysteine and adenosine.

26. N5 methyl FH4.
27. the methionine synthase reaction with the aid of vitamin B12.
28. a lack of vitamin B12 causes an accumulation of the N5 methyl F4 form of folate, causing a folate deficiency.
29. from s-adenosyl homocysteine, which is formed when s-adenosyl methionine donates a carbon group.
30. vitamin B12 is required for the conversion of homocysteine back to methionine. if this pathway is blocked, homocysteine will be used for the synthesis of cysteine. when cysteine accumulates, this pathway will also be blocked, leading to an accumulation of homocysteine.
31. malfunctioning of the enzyme that converts N5N10 methylene FH4 into N5 methyl FH4 can prevent homocysteine conversion to methionine and cause homocysteine accumulation. also, a defect in the cystathionine synthase enzyme will prevent homocysteine from being converted into cystathione and will cause it to accumulate.
32. folate deficiency during pregnancy has been shown to lead to neural tube defects in the developing fetus.

biochem: mark's medical biochem chapter 39- synthesis and degradation of amino acids

this chapter looked at the synthesis and degradation of different amino acids (put in bold). first it introduced the cofactors involved in these reactions: transamination reactions (such as the reaction involving glutamate introduced in chapter 38) require pyridoxal phosphate (PLP), which bonds its aldehyde carbon to the amino nitrogen and allows for different reactions to occur. FH4 is another cofactor that is involved in one carbon exchanges. BH4 is a cofactor that is involved in ring hydroxylations.

there are several amino acids that can be derived from the glycolytic intermediate, 3-phosphoglycerate. these include serine, cysteine, and glycine. serine is formed by oxidation, transamination to phosphoserine, then removal of the phosphate to form serine. it degrades in a separate pathway to form pyruvate. serine can then be converted to glycine in a reaction that involves both PLP and FH4. cysteine is also derived from serine- serine combines with homocysteine to form cystathione, which forms cysteine (and also succinyl CoA by way of alpha-keto butyrate- to be explained later).

amino acids can also be synthesized to TCA cycle intermediates. in particular, oxaloacetate and alpha-ketoglutarate. as we saw in the urea cycle, oxaloacetate can be transaminated to aspartate, which can be further converted into asparagate. alpha-ketoglutarate can be converted to glutamate in the glutamate dehydrogenase reaction. glutamate can then be converted to glutamine, or it can be converted to glutamate 5-semialdehyde. this compound can be converted to proline, or ornithine. ornithine can then be used to fuel the urea cycle, which produces arginine.

there are also several amino acids that can supply TCA cycle intermediates (an anaplerotic reaction); in particular, the intermediate succinyl CoA. the first was mentioned above- the homocysteine that reacts with serine to form cysteine actually is derived from methionine. in the last step of the cysteine synthesis pathway, the intermediate cystathione cleaves into cysteine and alpha-ketobutyrate, which is converted into propionyl CoA, and ultimately succinyl CoA.

valine and isoleucine are two branched chain amino acids that can replenish succinyl CoA. they are both transaminated into keto acids, then oxidatively decarboxylated into acyl CoA. these are oxidized in a way similar to fatty acid beta oxidation, ultimately producing FADH2, NADH, and propionyl CoA, which is converted to succinyl CoA.

finally, there are amino acids which are "ketogenic"- they produce ketone bodies, either acetyl CoA or acetoacetate, in their degradation. this includes leucine, isoleucine, tyrosine, phenylalanine, threonine, and tryptophan. isoleucine, as mentioned above, produces both succinyl CoA and acetyl CoA in its degradation, whereas leucine only produces acetyl CoA. phenylalanine is converted to tyrosine, which then produces acetoacetate and fumarate in its degradation. finally, tryptophan is an amino acid that produces formate, acetyl CoA, and alanine in its degradation.


questions
cofactors...
1. what are the three cofactors involved in amino acid metabolism?
2. how does pyridoxal phosphate aid in amino acid reactions?
3. how is FH4 involved in amino acid reactions? what is it derived from?
4. how is BH4 involved in amino acid reactions?

amino acids derived from glycolysis...
5. what are the amino acids derived from intermediates of glycolysis?
6. describe the synthesis of serine from glycolytic intermediates.
7. describe the degradation of serine.
8. how is serine synthesis regulated?
9. how is glycine synthesized?
10. how does glycine relate to kidney stones?

cysteine...
11. describe the synthesis of cysteine.
12. describe the regulation of the synthesis of cysteine.
13. how is methionine involved in cysteine synthesis?
14. describe the degradation of cysteine.
15. how is alanine synthesized?

amino acids related to TCA cycle intermediates...
16. which TCA cycle intermediates can be used to synthesize amino acids?
17. which TCA intermediates can be replenished by anaplerotic reactions via amino acids?
18. explain the statement "glutamate can be both derived from glucose and converted to glucose".
19. what are the three enzymes in the body that can "fix" free ammonia?
20. why is glutaminase important in the kidney?

alpha keto glutarate derived amino acids...
21. describe the synthesis of proline.
22. how is arginine synthesized?
23. what is the enzyme that transaminates glutamate 5-semialdehyde into ornithine?
24. describe the synthesis of aspartate and asparagine.

amino acids that supply succinyl CoA...
25. which amino acids degrade to form succinyl CoA?
26. describe the degradation of methionine to succinyl CoA.
27. describe the degradation of threonine to succinyl CoA.
28. where does most branched chain amino acid oxidation occur?
29. how are the degradations of valine and isoleucine both anaplerotic and energy producing?
30. what does degradation of leucine form?

ketogenic amino acids...
31. what are the main ketogenic amino acids?
32. describe how phenylalanine can produce ketone bodies.
33. what does tryptophan degradation produce?
34. what does degradation of lysine form?

answers
1. pyridoxal phosphate (PLP) (see chapter 38), FH4, and BH4.
2. the N on the amino acids bind to the aldehyde carbon of the PLP and pulls electrons away from the alpha carbon on the amino acid, allowing for different reactions to occur.
3. FH4 is required to donate or accept one carbon groups. it is derived from the vitamin folate.
4. BH4 is important for ring hydroxylation reactions.

5. serine, glycine, cysteine, and alanine.
6. 3-phosphoglycerate is oxidized to 3-phosphohydroxypyruvate, by 3-pg dehydrogenase. 3-phosphohydroxypyruvate is then transaminated to phosphoserine. the phosphate from phosphoserine is then removed to form serine.
7. serine is transaminated to hydroxypyruvate. hydroxypyruvate is reduced and phosphorylated to form 2-phosphoglycerate, which then forms PEP and pyruvate.
8. when serine levels fall, higher levels of 3-phosphoglycerate dehydrogenase are induced, and inhibition of phosphoserine phosphatase by serine is relaxed.
9. the major pathway of glycine synthesis is a conversion of serine, involving FH4 and PLP. the minor pathway is through the degradation of threonine in an aldolase-like reaction.

10. glycine can be converted to glyoxalate, which can be oxidized to oxalate- the accumulation of which can cause kidney stones.
11. homocysteine combines with serine to form cystathione. cystathione is cleaved to form propionyl CoA (which is converted to succinyl CoA) and cysteine.
12. cysteine inhibits the cystathione synthase; thereby inhibiting its own production.
13. methionine, an essential amino acid, provides the sulfur for cysteine synthesis. if methionine is in short supply, cysteine can not be synthesized de novo and becomes an essential amino acid.
14. degradation of cysteine produces pyruvate, NH4, and sulfate.
15. alanine is synthesized from the transamination of pyruvate via alanine aminotransferase (ALT).

16. oxaloacetate, alpha-keto glutarate.
17. oxaloacetate, alpha-keto glutarate, succinyl CoA, fumarate.
18. glutamate is derived from alpha-ketoglutarate, which is derived from glucose via the TCA cycle. in the liver, it can be degraded back into alpha-ketoglutarate, which leads to the formation of malate, which produces glucose via gluconeogenesis.
19. carbamoyl phosphate synthetase I (first reaction from the urea cycle), glutamate dehydrogenase, and glutamine synthetase.
20. glutaminase catalyzes the release of NH3 from glutamine, which then is secreted into the renal tubules and is the basis of the ammonia buffer system which aids in the excretion of H+.

21. glutamate is reduced to glutamate 5-semialdehyde, which then spontaneously forms a cyclical structure. this is then reduced to proline.
22. glutamate 5-semialdehyde can be converted to ornithine via a transamination reaction. ornithine can then be used to fuel the urea cycle, which produces arginine.
23. ornithine aminotransferase.
24. aspartate is transaminated from oxaloacetate (see chapter 38 notes). aspartate can be converted to asparagine by aspargine synthetase.

25. methionine, valine, isoleucine, threonine.
26. see question 13. methionine is converted to S-adenhosylhomocysteine, which is converted to homocysteine, which combines with serine to form cystathione. cystathione cleaves to produce cysteine and alpha-keto butyrate, which can be converted to propionyl CoA and ultimately succinyl CoA.
27. threonine is converted to alpha-keto butyrate by a hydratase, using PLP as a cofactor. alpha-keto butyrate is converted to succinyl CoA in the same path as for methionine degradation to succinyl CoA.
28. in muscle.
29. in both degradation pathways, they are transaminated to the alpha keto acids, then oxidatively decarboxylated to acyl CoA's. at this point they are oxidized just like fatty acyl CoA's using beta oxidation, producing NADH, FADH2, and propionyl CoA, which can be converted to succinyl CoA. thus the NADH and FADH2 provides energy while the succinyl CoA replenishes the TCA cycle.
30. leucine, the third branched chain amino acid, does not form succinyl CoA- instead it just produces acetoacetate and acetyl CoA (and thus is "ketogenic")

31. phenylalanine, tyrosine, isoleucine, threonine, tryptophan.
32. phenylalanine is converted to tyrosine, which is ultimately converted into acetoacetate (a ketone body) and fumarate.
33. alanine, formate, and acetyl CoA.
34. acetyl CoA, and NADH, FADH2.

biochem: mark's medical biochem chapter 38- urea cycle

this chapter looked at two aspects of nitrogen metabolism; the urea cycle in the liver, and supply of urea / nitrogen from different sources in the body.

the metabolism of amino acids releases carbon which can be used for ATP generation, as well as nitrogen in the form of NH4+/NH3. NH4+ is ammonium ion, which dissociates into NH3 and H+. at physiologic pH, NH4 predominates (it remains undissociated). NH4+/NH3 is toxic to the body and needs to be converted to urea in the liver; this chapter explains how the body transports nitrogen from amino acids to the liver for conversion in the urea cycle.

the most common reactions that frees nitrogen from amino acids are transamination reactions with glutamate. in this reaction, the amino group from the amino acid is transferred to an alpha keto-glutarate, forming glutamate and an alpha keto acid. glutamate can then receive another nitrogen and form glutamine, which is then released into the bloodstream. nitrogen in glutamine is one of the main transport mechanisms for nitrogen in the blood; the other is alanine, which is formed by transamination of pyruvate into alanine by glutamate.

glutamate is a crucial molecule which serves as an intermediate in several different pathways in nitrogen metabolism. in the glutamate dehydrogenase reaction, glutamate is oxidized to alpha-keto glutarate via the enzyme glutamate dehydrogenase, releasing NH4+ (basically the opposite of the transamination reaction without the transfer to an amino acid). the glutaminase reaction was mentioned above- the enzyme glutaminase cleaves glutamine into glutamate and NH4+. the opposite reaction, glutamine synthetase, creates glutamine from glutamate. thus glutamate can serve as both the nitrogen acceptor and the nitrogen donor, aiding in amino acid degradation as well as synthesis.

when the nitrogen in the form of glutamine or alanine reaches the liver, it is cleaved into carbon, which can be used for metabolism or gluconeogenesis, and nitrogen in the form of ammonia. in the mitochondria, the urea cycle begins:

1. ammonium combines with bicarbonate, forming carbamoyl phosphate via carbamoyl phosphate synthetase I
2. carbamoyl phosphate combines with mitochondrial ornithine, forming citrulline via ornithine transcarbamoylase.
3. citrulline is transported out of the mitochondria into the cytoplasm, where it combines with aspartate to form argininosuccinate via argininosuccinate synthetase.
4. argininosuccinate is cleaved by argininosuccinate lyase into arginine and fumarate.
5. arginine is then cleaved into urea and ornithine via arginase.
6. ornithine is transported back into the mitochondria in exchange for citrulline formed in step 2.

in step 4, the fumarate that is created can be acted upon by the same enzymes from the TCA cycle- fumarase catalyzes the hydration reaction to malate, which is then oxidized by malate dehydrogenase into oxaloacetate. oxaloacetate can then be transaminated into aspartate. thus the net result is the recycling of fumarate back into aspartate, where it can be reused in the urea cycle in step 3.

the urea cycle is regulated by a "feed forward" positive feedback mechanism in which the relative activity of the urea cycle is dependent of substrate availability. thus in conditions of high amino acid availability such as fasting or a high protein diet, more NH4 will be transported to the liver, and the urea cycle will be stimulated. another regulatory mechanism is the allosteric activation of CPSI, the enzyme in the first step of the urea cycle, by a protein NAG. NAG is synthesized in response to high arginine levels- when arginine levels are high, this indicates the need for the urea cycle to be activated further.

questions
1. what is the main way by which nitrogen is removed from amino acids in the body?
2. describe the formation of an alpha keto acid from an amino acid.
3. which amino acids do not undergo transamination reactions?
4. what is the cofactor in transamination reactions?
5. what is another way that nitrogen is removed from amino acids?
6. what is the relative concentration of ammonia to ammonium in the body and why?
7. which can cross cell membranes, ammonia or ammonium?

8. which reaction does glutamate dehydrogenase catalyze?
9. how is histidine deaminated?
10. how are serine and threonine deaminated?
11. how are glutamine and asparagine deaminated?
12. why is the deamination of glutamine important in the kidney?
13. what occurs in the brain and muscle that allows NH4+ to be released from amino acids?

14. how does glutamate aid in amino acid synthesis?
15. how does glutamate supply nitrogen to the urea cycle?
16. what are the two transporters of nitrogen in the blood?
17. how is alanine formed in the muscles?
18. what happens to alanine in the liver?
18b. what is the glucose alanine cycle and when is it used?
19. what does the enzyme glutamine synthetase do?
20. describe the production of glutamine in the tissues.
21. what is glutamine used for in the liver, intestines, and kidney?

urea cycle...
22. describe the first step of the urea cycle. where does it occur?
23. describe the formation of citrulline.
24. describe the transport of citrulline.
25. describe the formation of argininosuccinate.
26. describe the formation of fumarate and arginine.
27. how is fumarate recycled?
28. describe the formation of urea from arginine.

29. how is the urea cycle regulated?
30. describe the regulation of CPSI.
31. describe the regulation of the urea cycle via induction of urea cycle enzymes.
32. why is urinary excretion of urea high during fasting?
33. why does urea excretion decrease during prolonged fasting?

answers
1. through transamination reactions.
2. via a transamination reaction where the amino group from the amino acid is transferred to alpha keto-glutarate, which forms an alpha keto acid from the amino acid, and glutamate from the alpha keto glutarate.
3. lysine and threonine.
4. pyridoxal phosphate.
5. through the release of ammonia and ammonium.
6. about one hundred times more ammonium, because the pKa of the dissociation reaction (NH4+ -> NH3 + H+) is 9.3. this means that at pH 9.3, 50% of the NH4+ is dissociated into ammonia, but at the lower body pH of 7.4, most of the NH4+ is undissociated.
7. ammonia (recall the ammonia buffer system from the kidney)

8. the oxidative deamination of glutamate, producing ammonium and alpha keto glutarate.
9. directly deaminated to form NH4 and urocanate.
10. dehydration reactions that require pyridoxal phosphate as a cofactor and are catalyzed by serine dehydratase, releasing NH4+ in both cases. serine forms pyruvate, threonine forms alpha-ketobutyrate.
11. they both contain R group amides that may be released as NH4+ by deamidation.
12. because it produces ammonium, which is secreted directly into the renal tubules, which forms salts with metabolic acids and aids in their excretion.
13. the purine nucleotide cycle.

14. glutamate supplies nitrogen for amino acid synthesis; either from the glutamate dehydrogenase reaction or the transamination reaction. it then donates the amino group to an alpha keto acid to create an amino acid.
15. it supply nitrogen through NH4 produced in the glutamate dehydrogenase reaction, or it can transaminate oxaloacetate to aspartate, which then enters the urea cycle.
16. alanine and glutamine.
17. the metabolism of glucose in the muscles produces pyruvate, which can be transaminated by glutamate (see question 2) into alanine.
18. alanine is transaminated back to pyruvate, and the nitrogen will be used for urea synthesis. pyruvate can be used for gluconeogenesis and the glucose can be transported back to the muscles.
18b. the glucose/alanine cycle is the production of alanine via transamination of pyruvate in the muscles, and the breakdown of the alanine in the liver to ammonium and glucose. the cycle can take place in exercise, when the muscles "use blood borne glucose".
19. it is a cytoplasmic enzyme present in all cells that catalyzes the conversion of glutamate to glutamine.
20. in conditions of rapid amino acid degradation, glutamine is formed from glutamate via glutamine synthetase.
21. in the liver, glutamine is used in the urea cycle. in the intestines glutamine is used as fuel. in the kidney the NH4 released from glutamine degradations aids in excretion of metabolic salts.

22. in the mitochondria of liver and kidney cells: ammonium combines with bicarbonate and forms carbamoyl phosphate via CPSI and 2 ATP.
23. the carbomoyl phosphate formed in step one combines with ornithine to form citrulline, via ornithine transcarbamoylase.
24. citrulline is transported out of the mitochondria in exchange for cytoplasmic ornithine. =
25. citrulline in the cytoplasm combines with arginine (see question 15) to form argininosuccinate via argininosuccinate synthetase. this reactions costs 1 ATP
26. argininosuccinate is cleaved by argininosuccinate lyase to form fumarate and arginine.
27. fumarate is converted to malate via fumarase, which can be converted to oxaloacetate, which can then be transaminated to reform aspartate.
28. arginine is cleaved by arginine lyase into urea

29. by availability of substrates, regulation of CPSI, regulation of urea cycle enzymes.
30. high arginine levels stimulates NAG production from acetyl CoA, which activates CPSI.
31. during conditions of high protein metabolism (ie. high protein diet or fasting/starvation) causes urea cycle enzymes to be inducted.
32. because during fasting amino acids are used to supply substrates for gluconeogenesis; when an amino acid is converted to pyruvate, nitrogen is released and therefore excreted in the urine as urea.
33. during prolonged fasting ketone bodies are used for metabolism, sparing the need for amino acids as gluconeogenic precursors, which leads to less urea excretion.

biochem: mark's medical biochem chapter 37- protein digestion

this chapter looked at the digestion, absorption, and transport of proteins. digestion of proteins begins in the stomach, when the zymogen pepsinogen (recall that this is secreted by the parietal cells in the gastric pits) is autocatalytically cleaved to pepsin. pepsin works as an endopeptidase, randomly cleaving peptide bonds within the protein.

in the intestine, protein digestion continues with enzymes secreted from the pancreas, in zymogenic (inactive) form: trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase. trypsinogen is cleaved first by enteropeptidase into trypsin. trypsin both works to digest proteins directly by cleaving peptide bonds adjacent to carboxyl groups contained by arginine or lysine, and more importantly, it activates the other digestive enzymes.

it converts chymotrypsinogen to chymotrypsin, which then cleaves peptide bonds adjacent to carboxyl groups from acidic or hydrophobic amino acids. it converts proelastase to elastase, which then cleaves peptide bonds in elastin as well as those next to carboxyl groups contained by small chain amino acids such as alanine, glycine, and serine. finally, it activates carboxypeptidase from procarboxypeptidase, which acts an exopeptidase; cleaving amino acids one at a time from the outside edges of the protein fragments created by digestion via the other pancreatic enzymes.

in addition to pancreatic enzymes, the intestinal epithelial cells also contain protein digesting enzymes; these include aminopeptidases, which also act as exopeptidases, and intracellular peptidases, which break down protein fragments that have been absorbed by the enterocytes.

the amino acids produced from degradation of proteins are transported into enterocytes via Na+ cotransporters, similar to the glucose / Na+ cotransporters seen in carbohydrate digestion. these transporters are powered by the low Na+ concentration in the cells which is manufactured by the Na+/K+ pumps. thus amino acids are absorbed via secondary active transport- and then they diffuse out of the serosal side via facilitated transport proteins.

the "intracellular amino acid pool" is a measure of how much amino acid there is at any given moment within a cell. this is a dynamic balance between proteins acquired from the diet vs. the degradation of proteins. the "half life" of a protein refers to the point at which 50% of the protein has been degraded; proteins in muscle cells, digestive enzymes, or hemoglobin, are all examples of proteins which have a short half life and therefore a high "turnover" rate.

a few notes about intracellular protein digestion: cells can digest proteins by the process of "autophagy", in which proteins in extracellular vesicles fuse with lysosomes, which contain proteases which degrade the proteins into amino acids, which then are absorbed into the cytoplasm. another method of protein digestion is through the ubiquitin / proteasome pathway, in which ubiquitin is attached to proteins, "tagging" them for digestion via proteasomes, which are large barrel shaped proteins with multiple internal proteolytic sites.

questions
1. what form are the protein digesting enzymes secreted in?
2. where are parietal and chief cells located and what do they secrete?
3. how is pepsinogen activated?
4. describe the action of pepsin on proteins.
5. what are the zymogens that are secreted by the pancreas into the intestine?
6. describe trypsinogen's role in protein digestion in the intestine.

7. how is trypsinogen activated?
8. how does trypsin directly digest proteins?
9. describe the digestive action of chymotrypsin on proteins.
10. describe the digestive action of elastase on proteins.
11. describe the action of carboxypeptidase on proteins.
12. what is the difference between carboxypeptidase A and B?
13. where are aminopeptidases located and what do they do?
14. what are intracellular peptidases?

15. describe the transport of amino acids from the intestinal lumen into enterocytes.
16. describe the transport of amino acids from the enterocyte to the portal vein.
17. describe the diversity of amino acid transport proteins on the apical side of enterocytes.
18. how do amino acids get transported into cells of peripheral tissues?
19. in what way does transport and absorption of amino acids into peripheral tissues differ from that of carbohydrates?

20. what is the half life of a protein?
21. what are some examples of proteins that undergo extensive synthesis and degradation in the body?
22. how much of the cells lining the intestinal wall are replaced each day?
23. what percentage of proteins that are absorbed from the intestines are excreted?

24. what is autophagy?
25. which enzymes in lysosomes aid in protein digestion?
26. what is the ubiquitin-proteasoms pathway?
27. how does ubiquitin "tag" proteins?
28. what is a proteasome?

answers
1. zymogenic: an inactive, larger form of the enzyme that is activated by proteolytic cleavage once in the digestive tract.
2. in the gastric pits / epithelium of the stomach. parietal cells secrete HCl and chief cells secrete pepsinogen.
3. the acidity in the stomach allows pepsinogen to be cleaved to its active form, pepsin.
4. pepsin acts as an endopeptidase, cleaving peptide bonds at random intervals within the denatured protein.
5. trypsinogen, pepsinogen, proelastase, procarboxypeptidase.
6. trypsin (the activated form of trypsinogen) catalyzes the activation of the other pancreatic enzymes to their active forms, as well as directly aiding in the digestion of proteins.

7. through enteropeptidase.
8. trypsin cleaves peptide bonds adjacent to carboxyl groups that are provided by lysine or arginine.
9. chymotrypsin cleaves peptide bonds next to residues that contain hydrophobic or acidic amino acids.
10. elastase cleaves peptide bonds within elastase as well as bonds next to residues with small side chains (alanine, glycine, serine)
11. carboxypeptidase acts an exopeptidase, removing amino acids one at a time from the carboxyl end, from the smaller peptides resulting from breakdown of the other pancreatic enzymes mentioned above.
12. A preferentially cleaves hydrophobic amino acids while B preferentially cleaves basic amino acids.
13. they are located on the epithelial wall of the intestine and act as exopeptidases, removing one amino acid at a time.
14. the enzymes within cells that break down peptides which are absorbed.

15. a Na+/K+ pump in the enterocyte creates a low concentration of Na+ in the enterocyte. The resulting influx of Na+ is coupled with amino acid transport, this is called secondary active transport.
16. the amino acids in the enterocytes are transported into the portal vein via "facilitated transporters"
17. there are at least 6 such transport proteins which have overlapping specificities for different types of amino acids.
18. mainly through Na+ cotransporters.
19. amino acids are transported into cells mainly by Na+ cotransporters wheras in peripheral tissues carbohydrates are transported by facilitated transporters (recall the GLUT transporters). in the intestine and renal cells the absorption of both amino acids and carbohydrates are Na+ coupled.

20. the point at which 50% of the protein in a cell has been degraded.
21. hemoglobin, muscle proteins, digestive enzymes.
22. roughly 1/4th.
23. roughly 6%.

24. the process by which cells digest proteins using lysosomal enzymes.
25. the cathepin family of proteases.
26. a method of intracellular protein digestion using ubiquitin tagging and degradation via proteosomes.
27. by covalently binding to the epsilon-amino group of lysine residues.
28. a cylindrical 20S protein complex with multiple internal proteolytic sites.

biochem: mark's medical biochem chapter 24- reactive oxygen species

this chapter looked at the production of different reactive oxygen species, the damage they produce in the body, and the different ways that the body deals with them.

the reactive oxygen species (ROS) produced in the body are dangerous because they have a single electron in the outer orbital of the oxygen atom, which gives them a high capacity for extracting electrons from other molecules in order to complete the orbital. the main ROS are superoxide anion, hydrogen peroxide, and hydroxyl radical. the most potent ROS is hydroxyl radical, which is formed by two different pathways: the haber weiss and the fenton reaction. in the haber weiss reaction, superoxide (O2-) combines with hydrogen peroxide to form O2, H2O, and OH•. in the fenton reaction, hydrogen peroxide is reduced via a transition metal catalyst to a hydroxide ion and hydroxide radical.

in the body, ROS's are particularly attracted to cellular membranes, where they end up oxidizing the double bond of polyunsaturated fatty acids in the membranes. this starts a free radical chain reaction, where one oxidized molecule oxidizes another, etc. etc. in addition, they can cause base pair substitutions in DNA or strand cleavage. finally, they can also cause proteins to either cleave or crosslink excessively.

nitric oxide can also produce reactive oxygen species. normally used in small amounts in neurons and for vasodilation, it can be synthesized in large quantities by cells of the immune system in order to fight pathogens. for example, a macrophage might assemble a nitric oxide synthase complex, which synthesizes nitric oxide, in its phagolysosome, where the pathogen is housed. the nitric oxide can then create reactive oxygen species, such as peroxynitrite or nitric oxide, both of which can be used to destroy microbes.

this synthesis of reactive oxygen species by phagocytic cells in the immune system is called the "respiratory burst", and includes other ROS's as well: superoxide anion is formed by NADPH oxidase, which donates an electron to O2. superoxide can be converted to hydrogen peroxide, which can be converted to the potent hydroxyl radical or hypochlorous acid, both highly damaging to microbes.

the body uses several strategies to deactivate reactive oxygen species. the first is through enzymes such as superoxide dismutase, catalase, and the enzymes that control glutathione activity. superoxide dismutase is one of the body's main defense against antioxidants, since superoxide anion is such a strong initiator of free radical chain reactions- it converts superoxide back to hydrogen peroxide and oxygen. catalase is present in peroxisomes, cellular vesicles that house certain reactions that produce hydrogen peroxide (such as very long chain fatty acid oxidation) and reduces hydrogen peroxide into water. finally, glutathione acts as an antioxidant by reducing peroxides. in this reaction 2 molecules of glutathione combine to form one glutathione disulfide, which can be reduced back into glutathione by glutatione reductase and NADPH (from the pentose phosphate pathway).

some other antioxidants that the body uses: vitamin E, vitamin C (which regenerates vitamin E's antioxidative capacity), carotenoids, and flavonoids.

questions
1. what is a free radical molecule and how does it cause damage?
2. what type of radical is an oxygen atom?
3. oxygen can accept a total of how many electrons? what is it converted to?
4. describe the reduction of oxygen to water.
5. which ROS is the most potent?
6. what is the haber-weiss reaction?
7. what is the fenton reaction?
8. what percentage of consumed oxygen turns into ROS?
9. describe the formation of superoxide from coenzyme Q.
10. how do drugs and alcohol potentially create more ROS?
11. how is fatty acid oxidation related to ROS production?
12. how is eicosanoid synthesis related to ROS production?
13. how does cosmic radiation produce ROS?
14. describe the chain reaction that occurs during lipid membrane damage via ROS.
15. describe the effect of reactive oxygen species on proteins and peptides.
16. describe the potential damage to DNA by ROS.

17. how is nitric oxide synthesized in the body?
18. what are the three isotypes of nitric oxide synthase?
19. how are the activities of the different NOS isotypes regulated?
20. describe how NO can be directly toxic to cells.
21. describe how NO can be indirectly toxic to cells.
22. what does NO form during inflammation?
23. compare the effects of ROS that contain nitrogen vs ROS that do not.

24. what is the respiratory burst?
25. describe the production of superoxide anion during the respiratory burst.
26. describe the production of hypochlorous acid from H2O2.

27. describe how superoxide anion is neutralized and what products are formed.
28. how is hydrogen peroxide neutralized?
29. which cells of the body have the highest peroxisomal content?
30. how does glutathione act as an antioxidant?
31. what role does selenium play in antioxidant activity?
32. what role does NADPH play in antioxidant activity?
33. what is the common structural characteristic of non enzymatic free radical scavengers?
34. how does vitamin E act as an antioxidant?
35. how does vitamin C help in antioxidation?
36. what are the antioxidants contained in red wine, tea, and chocolate?
37. what is the major antioxidant present in extracellular fluid, plasma, mucus membranes, and one of the only antioxidants in the upper airways?
38. what is an antioxidant that is primarily a hormone?

answers
1. a molecule with a single unpaired electron which can extract an electron from another molecule to complete its orbital ring.
2. biradical
3. four electrons, which reduces it to water.
4. oxygen accepts one electron and forms superoxide anion (a thermodynamically unfavorable reaction). superoxide accepts one electron and forms hydrogen peroxide. hydrogen peroxide accepts one electron and forms water and the hydroxyl radical. hydroxyl radial accepts one electron and becomes water.
5. the hydroxyl radical.
6. superoxide and hydrogen peroxide form oxygen, water, and the hydroxyl radical
7. hydrogen peroxide reduced into hydroxyl radical and hydroxyl ion via a transition metal catalyst
8. 3-5%.
9. coenzyme Q in the mitochondrial membrane can accidentally transfer an electron to O2, forming superoxide anion.
10. they induce cytochrome 450 enzymes which are involved in transferring single electrons to O2 and organic substrates, from which accidental leakage of free radical intermediates might occur.
11. oxidation of very long chain fatty acids occurs in the peroxisomes, where the first step oxidation involves formation of H2O2 via oxidase enzymes.
12. some eicosanoid synthesis pathways involve lipid peroxide intermediates, including leukotriene and prostaglandins.
13. it is powerful enough to split H2O into the hydroxide and hydrogen radicals, as well as organic radicals.
14. an initiator such as hydroxyl radical extracts a single electron from the double bond of a polyunsaturated fatty acid, creating a chain reaction that creates lipid peroxides and causing membrane damage.
15. ROS can cause different effects on proteins or peptides; they can cause some proteins to degrade proteolytically and others to undergo more crosslinking and thus be more difficult to degrade.
16. the Fe2+ bound on DNA can catalyze the formation of hydroxyl radical (see question 7) and other reactive oxygen species. these molecules can either cause base substitutions or even strand cleavage.

17. arginine is converted to NO via nitric oxide synthase, via NADPH reduction.
18. neuronal (nNOS), inducible (iNOS), and endothelial (eNOS)
19. neuronal and endothelial and regulated by Ca2+ levels whereas inducible is regulated by gene transcription.
20. they can bind to iron containing compounds such as the complexes in the electron transport chain.
21. NO can be indirectly toxic by catalyzing the formation of other compounds such as peroxynitrite.
22. NO combines with superoxide to form peroxynitrite (ONOO-) or with O2 to form N2O3.
23. N containing ROS can be just as, if not more, damaging to cells, by causing the same sort of oxidative damage to membranes and proteins, and also nitrating and nitrosylating compounds.

24. the production of ROS during inflammation and infection to destroy pathogens.
25. NADPH oxidase is assembled and transported to the phagolysosomes, where it produces superoxide anion, which can be further converted into other ROS to combat the endocytosed pathogens.
26. neutrophils contain myeloperoxidase, which converts H2O2 into hypochlorous acid, which is highly antimicrobial.

27. superoxide anion is converted to hydrogen peroxide and oxygen via superoxide dismutase.
28. hydrogen peroxide is reduced to water by catalase, mainly in the peroxisomes.
29. kidney and liver.
30. two molecules of glutathione reduce one molecle of hydrogen peroxide via glutathione peroxidase, in the process forming glutathione disulfide.
31. selenium is required for the glutathione peroxidases, which are the main enzymes that catalyze antioxidant activity in the mitochondria and cytosol of cells.
32. NADPH is required to reduce glutathione disulfide back into glutathione via glutathione reductase, so that it can perform antioxidative activity again.
33. they a conjugated double bond system that might be in the form of an aromatic ring.
34. it can be oxidized by free radicals, because the high energy intermediates are stabilized by the resonance of the double bonds. it can be oxidized twice and acts as a terminator for the free radical chain reactions.
35. by reducing vitamin E back to its original form.
36. flavonoids.
37. uric acid.
38. melatonin.

biochem: mark's medical biochem chapter 35- eicosanoids

this chapter was a brief introduction to the synthesis and metabolism of eicosanoids. eicosanoids are 20 carbon long polyunsaturated fatty acids derived from membrane phospholipids, mainly arachadonic acid. arachadonic acid can not be synthesized de novo, and is mainly converted from linoleate from the diet. in general, eicosanoids are short lived molecules that affect virtually all tissues and have a multifaceted regulatory role in diverse processes that occur in the body (and by this i mean i have no idea what they do yet)

conversion of arachadonic acid in the phospholipid membrane into the eicosanoids can take several pathways. one is the production of prostaglandins and thromboxanes, which have important, opposing roles in mediating inflammation. prostaglandins are produced on vascular endothelium, inhibit platelet aggregration and stimulate vasodilation. thromboxanes are produced by enzymes on platelets, stimulate platelet formation as well as vasoconstriction.

the synthesis of prostaglandins and thromboxanes occurs when the cyclooxygenase enzyme converts arachadonic acid in the membrane into an unstable endoperoxide, PGG2. PGG2 is then reduced to PGH2, which can be converted by different enzyme isoforms to prostaglandins or thromboxanes. antiinflammatory drugs often target the first enzyme in the synthesis of these inflammation mediating enzymes, cyclooxygenases. these drugs are called COX-2 inhibitors, referring to the two types of cyclooxygenases present in the body. COX-1 is involved in normal physiologic function, whereas COX-2 is used in inflammation.

some other pathways of eicosanoid synthesis: leukotrienes and lipoxins are formed by adding hydroperoxides to the double bonded carbons (which shifts the double bonds over and changes their configuration), followed by conversion to leukotrienes and lipoxins by other enzymes. leukotrienes and lipoxins are involved in increasing vascular permeability, bronchoconstriction, and cytokine production.

isoprostanes are created by free radical modification of arachadonic acid in the phospholipid membrane, which is then cleaved by phosphlipase A2. they are used to assess the level of oxidative damage a person is exposed to. finally, endocannabinoids are synthesized when arachadonic acid is transferred to the amino group on ethanolamine, which is then cleaved from the cell membrane by phospholipase D. endocannabinoids can act as analgesics, by acting retroactively on the presynaptic membranes from which they are released, stimulating continual neurotransmitter release.


questions
1. eicosanoids are synthesized from...
2. what is the major precursor for eicosanoids and where does it come from?
3. describe how arachidonic acid gets released from membrane phospholipids.
4. what are the three major pathways for eicosanoid synthesis?

5. what are the structural characteristics of prostaglandins?
6. what is the difference between the PGE and PGF family of prostaglandins?
7. what are the structural characteristics of thromboxanes?
8. what is the "2 series" of of prostaglandins and thromboxanes?
9. describe the synthesis of the 2 series of prostaglandins from arachadonic acid.
10. how are thromboxanes synthesized?
11. what is PGH2 converted to in the vascular endothelium vs. the platelets?
12. what are the two types of cyclooxidase enzymes and when/where are they present/active?
13. how is aspirin related to prostagladin production?
14. describe the inactivation of prostaglandins.
15. describe the inactivation of thromboxanes.

16. describe the formation of leukotrienes and lipoxins from arachadonic acid.
17. what do lipoxins do?
18. what do leukotrienes do?
19. describe the synthesis of isoprostanes.
20. describe the synthesis of endocannabinoids.
21. what are isoprostanes used as a measure of?
22. what are endocannabinoids used for and why?
23. what does the cytochrome450 pathway produce and what role do the molecules play in the body?

answers
1. 20 carbon long polyunsaturated fatty acids derived from membrane phospholipids.
2. arachidonic acid, which is esterified to membrane phospholipids. it can not be synthesized de novo, so it must be converted from dietary sources, mainly linoleate.
3. arachadonic acid gets released by the activation of phospholipase A2 or C via agonists such as histamine or cytokines.
4. cyclooxygenase, lipoxygenase, cytochrome P450.

5. 20 carbon atoms long, an internal 5 carbon ring, hydroxyl group at C15, double bond at C13.
6. the substituents on the internal ring; PGE has a carboxyl group on carbon 9 and a hydroxyl on carbon 11, whereas PGF has alpha-hydroxyl groups on both carbons 9 and 11.
7. similar to prostaglandins, but the internal ring is 6 membered, with 5 carbons and 1 oxygen. the most common thromboxane, TXA2, also contains an oxygen atom in the middle of the ring.
8. molecules with double bonds between carbons 13 and 14, and 5 and 6.
9. arachadonic acid is converted to an unstable endoperoxide, PGG2 by cyclooxygenase. PGG2 is reduced to PGH2. PGH2 can then be acted on by different isomerases which synthesize different series 2 prostaglandins (PGD synthase creates PGD2, PGE synthase creates PGE2, etc).
10. PGH2 can be converted to thromboxane, TXA2, by TXA synthase.
11. in platelets it is converted to thromboxane, which causes vasoconstriction and platelet aggregation. in vascular endothelium it is converted to PGI2, which inhibits platelet aggregation and causes vasodilation.
12. COX-1 and COX-2. COX-1 is the main enzyme in healthy tissues and COX-2 is present in inflamed tissue.
13. cyclooxidase enzymes are blocked by antiinflammatory drugs such as aspirin.
14. prostaglandins are inactivated within seconds or minutes by oxidation of the 15-OH to a ketone and reduction of the double bond at C13. this creates a dicarboxylic acid that is excreted in the urine.
15. TXA2 is metabolized to the inactive TXB2 by cleavage of the oxygen bridge in the internal ring into two hydroxyl groups.

16. lipoxygenases add a hydroperoxide to one of the double bonded carbons in arachadonic acid, forming 5, 12, or 15-HPETE. this shifts the double bond one carbon further away and changes the configuration from cis to trans. the unstable hydroperoxide group can then be metabolized to form leukotrienes or lipoxins.
17. induce chemotaxis and stimulate superoxide anion production in leukocytes.
18. increase vascular permeability, T-cell proliferation, leukocyte aggregation, cytokine production.

19. isoprostanes are created by free radical damage to arachadonic residues in phospholipid membranes, which are then cleaved and released by phospholipase A2.
20. arachidonic acid group is transferred from the 2-position to the free amino group on ethanolamine, and the modified ethanolamine is then cleaved by phospholipase D.
21. the amount of oxidative stress the person is exposed to.
22. analgesics, because they are released from neurons and work retroactively on the presynaptic membrane to increase neurotransmitter release.
23. epoxides, certain HETE's, which have actions in ocular, vascular, endocrine, and renal systems, the exact role of which is unknown.

biochem: mark's medical biochem chapter 34- cholesterol

this chapter looked at the synthesis, processing, and usage of cholesterol in the body.

cholesterol is an alicyclic compound made of 4 carbon rings totaling 27 carbons, with an eight carbon tail, 2 methyl groups, and has a hydroxyl group on carbon 3 that can be esterified with a fatty acid. the esterified form is the predominant form that appears in the body (2/3), and is more hydrophobic than the free form. cholesterol synthesis can be broken down into four steps:

1. synthesis of mavelonate from acetyl CoA [see answer to question 10 for greater detail]
2. conversion of mavelonate to activated isoprenes [question 16]
3. combining activated isoprenes to form squalene [question 18]
4. converting the squalene chain into the cholesterol ring structure. [question 20]

the first step contains the rate limiting reaction of cholesterol synthesis, which is the reduction of HMG-CoA to mavelonate via HMG CoA reductase. this enzyme is regulated by at least three different mechanisms. first is called transcriptional control: high cholesterol levels inhibit the SREBP (sterol regulatory element binding protein) transcription factors that facilitate the transcription and therefore synthesis of cholesterol. second is proteolytic degradation of HMG-CoA reductase, which can occur when high sterol levels alter the HMG-CoA reductase's ability to sense and reduce HMG-CoA (thus inhibiting sterol synthesis). third is by covalent modification: high glucagon or AMP levels activate a protein kinase that phosphorylates HMG-CoA reductase into its inactive form, and high insulin levels activate a phosphatase that dephosphorylates the reductase into its active form-- the implication here being that the fasting or energy poor state inhibits cholesterol synthesis and the fed state induces cholesterol synthesis.

once cholesterol is synthesized (mainly in liver cells), they are either packaged into lipoprotein particles or converted into bile salts to aid in digestion (see chapter 32 notes). bile salts are created by modification of cholesterol:

1. addition of hydroxyl group to carbon 7 via 7-hydroxylase
2. reduction of the double bond on carbon ring 3
3. addition of several more hydroxyl groups to the sterol ring
4. cleavage of the cholesterol carbon chain to a 5 carbon chain ending in a carboxyl group.

the compounds that are created fall into two families: cholic acids have carboxyl groups on carbons 3,7, and 12, while chenocholic acids have carboxyl groups on carbons 3 and 7 only. these bile salts have a pKa of ~6, similar to that of the intestinal lumen, meaning that in the intestines they 50% in the dissociated form. the bile salts can also be "conjugated", which is the addition of an amine group onto the carboxyl on the end of the carbon chain. conjugating the bile salts decreases the pKa's significantly such that the salt will appear in the dissociated form in a greater proportion in the intenstinal lumen. once in the intestinal lumen, these bile salts act as a detergent, surrounding hydrophobic lipid droplets and forming "micelles" (see chapter 32). after lipids are absorbed into enterocytes, the bile salts are mostly recycled back into the liver (95%) to be stored back in the gall bladder, although they can also be de-conjugated and de-hydroxylated by intestinal bacteria and excreted.

cholesterol that is synthesized in the liver is also packaged in lipoprotein particles, enter into circulation, and used by peripheral tissues for cellular membrane synthesis, vitamin D biosynthesis, and sterol molecule synthesis. lipoproteins are, in general, hydrophilic phospholipid spheres that contain hydrophobic lipids and cholesterols, allowing these compounds to be transported through the blood. one class of lipoprotein, the chylomicron (discussed in chapter 32), is used to package and circulate the TG's derived from the diet (exogenous lipoprotein pathway). the VLDL is a similar vehicle, packaging mostly triacylglycerides and other lipids that are produced endogenously by the liver (and adipose tissue during fasting). both contain the apoproteins CII and E, which allow them to bind to lipoprotein lipase (LPL) on the endothelium, which cleaves their contents and allows the lipids to be metabolized by tissues. once VLDL's deliver their contents to tissues, they become IDL's (intermediate density lipoproteins), which can also be converted to LDL's by removal of triglycerides. LDL's, which are mostly cholesterol, are either returned back to the liver and endocytosed (60%), or transported to tissues for use in sterol synthesis.

HDL's are high density lipoproteins and are functionally distinct from the other lipoproteins. they are created in nascent form either in the liver or grown from apoproteins in circulation, and continue to mature as they gather phospholipids and cholesterol esters from endothelial cells. HDL's engage in "reverse cholesterol transport", which is the uptake of cholesterol from cholesterol laden cells (such as endothelium) into the lipoprotein, where it travels back to the liver to be degraded. HDL accomplishes this by collecting the free cholesterol molecules into the lipoprotein and then converting it to the esterified form, which is essentially trapped in the center of the lipoprotein particle. the HDL then travels to the liver, where it binds to a "scavenger receptor" that induces its transport into hepatocytes.

HDL's also interact with other lipoproteins in the bloodstream. HDL's transfer apoproteins CII and E to both chylomicrons and VLDL's, allowing them to be recognized and cleaved by LPL on endothelium (thus converting them to the "mature" form). they also transfer cholesterol esters to VLDL's in exchange for triacylglycerides, when the concentration of high density lipoproteins is high. this occurs via transferring the lipid contents on a protein intermediate called "cholesterol ester transfer protein".

a few notes about the development of atherosclerosis and how cholesterol is involved: certain risk factors such as smoking or high blood pressure lead to the increased concentration of modified/oxidized high density lipoproteins. macrophages have scavenger receptors that bind to these HDL's, inducing phagocytosis. when a collection of macrophages that are engorged with lipid accumulates, a "fatty streak" develops within the sub-endothelial space of the artery. this eventually bulges out into the lumen of the artery and is susceptible to fissure, which can cause acute thrombitis and occlusion of the artery.

questions
introducing cholesterol...
1. how much cholesterol is produced by the liver and how much is ingested from dietary sources?
2. what percentage of cholesterol in the lumen of the gut is actually absorbed?
3. what regulates cholesterol absorption in the gut besides diffusion processes?
4. what is phytosterolemia?
5. describe the major physical characteristics of cholesterol.
6. what are the free and esterified forms of cholesterol and what is the relative proportion that cholesterol appears in naturally in the body?
7. what is the major building block for cholesterol and where does it come from?

synthesis...
8. where does cholesterol synthesis occur?
9. what is the rate limiting step of cholesterol synthesis?
10. describe the formation of mevalonate from acetyl CoA in the cytosol.
11. what is the enzyme that facilitates the reduction of HMG-CoA and where is it located?
12. what are the three methods of HMG-CoA reductase regulation?
13. describe transcriptional control of HMG-CoA reductase.
14. describe proteolytic degradation control of HMG-CoA reductase.
15. describe covalent modification control of HMG-CoA reductase.

16. how are the activated isoprenes formed from mevalonate?
17. what are the intermediates in the formation of squalene from activated isoprenes?
18. describe the formation of squalene from activated isoprenes.
19. how many carbons are in squalene?
20. what are the major steps in the formation of cholesterol from squalene?

cholesterol fate...
21. where does most cholesterol synthesis take place?
22. what are the three major forms of cholesterol produced by the liver?
23. what is the ACAT enzyme and what does it do?
24. in which form is cholesterol more hydrophobic, free or esterified?
25. what are three uses for cholesterol in the peripheral tissues?

bile...
26. what are the two types of bile salts?
27. describe the production of bile salts from cholesterol.
28. what is the enzyme that catalyzes the addition of alpha-hydroxy to cholesterol and what is it regulated by?
29. what is the difference between the two classes of bile salts?
30. what is the pKa of bile and how it relevant?
31. what is conguation of bile salts and what effect does it have?
32. what effect can intestinal bacteria have on bile salts?
33. how much bile is recycled by the body? how is it recycled?

lipoproteins...
34. why are lipoproteins hydrophilic?
35. what are some defining characteristics of chylomicrons?
36. what are the apoproteins on chylomicrons?
37. what are the fates for the TG's in chylomicrons?
38. what are chylomicron remnants and what happens to them?

39. describe VLDL production, composition, fate.
40. how are IDL's and LDL's formed? what are their compositions?
41. what is the fate of LDL's?
42. what are the three ways in which nascent HDL can be synthesized?
43. describe the maturation of nascent HDL's.

44. what is reverse cholesterol transport?
45. describe the uptake of cholesterol from the cells to the HDL.
46. why are HDL's considered to be "vasculoprotective"?
47. how are HDL's taken up by the liver cells?
48. describe the interaction of HDL with VLDL's/chylomicrons in the circulation in reference to apoprotein
exchange.
49. what happens after the apoproteins are used by VLDL and chylomicrons?
50. what is the CETP? what does it do?
51. what is an HDL2 vs. an HDL3?

52. describe the endocytosis of an LDL by liver cells.
53. how is the regulation of synthesis and activity of LDL receptors and HMG-CoA synthesis similar?
54. describe the structure of an LDL receptor.
55. decrease in the levels of LDL receptors can lead to...
56. what is hypercholesteremia and what is it caused by?
57. what is the LDL receptor-related protein?

58. where are scavenger receptors found and what do they do?
59. what is a fatty streak?
60. what are some vascular risk factors for atherosclerosis?
61. describe what happens when a fatty streak becomes acute thrombitis.

62. what are the five classes of steroid hormones synthesized from cholesterol?
63. how are steroids transported in the blood?
64. where does the cholesterol for steroid synthesis come from?
65. where is aldosterone produced? describe the synthesis pathway and what it is initiated by.
66. describe the conversion of cholesterol to progesterone.
67. where does cortisol synthesis take place?

answers
1. 1g synthesized by the liver and 300mg ingested per day.
2. 55%
3. the ABC protein family (ABC 1,5,8), which uses the energy from ATP hydrolysis to remove cholesterol from the enterocyte cell back into the lumen, where they are excreted.
4. a rare genetic disease that leads to the lack of ABC 5,8, which leads to excessive serum cholesterol levels.
5. an alicyclic compound with 4 rings, 27 carbons, a hydroxyl group on carbon 3, two methyl groups, an eight carbon chain off of carbon 17.
6. free cholesterol is the structure just described, whereas esterified cholesterol has a fatty acid esterified to the hydroxyl group on carbon 3. 2/3 of cholesterol is esterified and 1/3 is free.
7. acetyl CoA, which comes from beta oxidation of fatty acids, or oxidation of pyruvate via pyruvate dehydrogenase, or oxidation of ketogenic amino acids.

8. cytosol
9. the synthesis of the intermediate mevalonate.
10. 2 acetyl coA's in the cytosol combine to form acetoacetyl CoA, to which is then added a third molecule of acetyl CoA, forming beta-hydroxy beta-methyl glutaryl CoA (HMG-CoA). HMG-CoA is then reduced to mevalonate via NADPH from the pentose phosphate pathway.
11. HMG-CoA reductase, embedded within the endoplasmic reticulum
12. transcriptional control, proteolysis, phosphorylation.
13. normally, SCAP proteins on the ER membrane release S2P proteins, which activate the SREBP (sterol regulatory element binding proteins) to increase the rate of transcription of the mRNA of cholesterols. high sterol levels in the blood inactivate the SCAP proteins, thus leading to a decrease in the rate of transcription of cholesterol mRNA.
14. proteolytic degradation occurs when rising cholesterol and bile salt levels in cells that synthesize them alter the "oligomerization state of the membrane domain" of HMG-CoA, thereby reducing its ability to sense HMG-CoA, inhibiting its reduction activity.
15. HMG-CoA reductase can be inactivated by phosphorylation and activated by dephosphorylation. when glucagon or AMP levels are high, an AMP dependent protein kinase is activated, which phosphorylates and deactivates HMG-CoA reductase. when insulin levels are high, HMG-CoA reductase is dephosphorylated and activated.

16. a pyrophosphate is added to mevalonate, forming 5-pyrophosphate mevalonate. another phosphate is added to the hydroxyl group on the third carbon, forming 3-phospho 5-pyrophosphomevalonate. the COO- on carbon 1 plus the phosphate on carbon 3 are then removed, forming the first activated isoprene: ∆3 isopentenyl pyrophosphate, which can be isomerized to the second activated isoprene, dimethylallyl pyrophosphate.
17. activated isoprenes, geranyl pyrophosphate, farnesyl pyrophosphate, squalene.
18. the activated isoprenes: ∆3 isopentenyl pyrophosphate and dimethylallyl pyrophosphate combine in a head to tail fashion, cleaving the pyrophosphate from the dimethylallyl pyrophosphate, forming the 10 carbon chain geranyl pyrophosphate. another ∆3 isopentenyl pyrophosphate is added to the geranyl pyrophosphate, resulting in the 15 carbon farnesyl pyrophosphate. farnesyl pyrophosphate then combines with another molecule of farnesyl pyrophosphate in a head to tail fashion, creating squalene.
19. 30
20. squalene is reduced to squalene 2,3 epoxide, which is converted to lanosterol (which contains the 4 ring structure) by a series of reactions, which is then converted to cholesterol through another series of reactions.
21. in the liver cells.
22. cholesterol esters, biliary cholesterol, or bile acids.
23. acyl-CoA-cholester acyl transferase, which transfers the fatty acid from CoA onto the hydroxyl group of a cholesterol, esterifying the cholesterol.
24. esterified
25. synthesis of cell membranes, formation of steroid hormones, synthesis of vitamin D.

26. cholic acid and chenocholic acid.
27. a hydroxyl group is added to carbon 7, the double bond in the B ring is reduced, more hydroxyl groups are added to the sterol ring and the side chain is cleaved into a 5 carbon fragment ending in a carboxyl group.
28. 7-alpha hydroxylase, inhibited by bile salts.
29. cholic acids have hydroxyl groups on carbons 3,7, and 12, whereas chenocholic acids have hydroxyl groups on 3 and 7 only.
30. the pKa is 6, which is the same as the intestinal lumen, which means that 50% of the bile salts are present in dissociated form, allowing it to aid in digestion.
31. conjugation is activation of the carboxyl group on the side chain of bile salts which form amides that have a much lower pKa, allowing more of the bile salts to be in dissociated form in the intestinal lumen.
32. they can de-conjugate them and de-hydroxylate them, which significantly decreases the solubility and therefore leads to excretion of the bile salts.
33. 95% of the bile salts is resorbed in the ileum, circulated back to the liver via enterohepatic circulation, and stored back in the gallbladder.

34. because of the interactions between the N on the phospholipid membrane of the lipoproteins, as well as some hydrophilic apoproteins.
35. the largest of the lipoproteins, least dense because of the high triacylglyceride content, synthesized within enterocytes, and enter the bloodstream via the lympathic system through the left subclavian vein.
36. apoB-48, CII, and E.
37. LPL on the endothelium of blood vessels in peripheral tissue cleaves the TG's in the chylomicrons and releases the fatty acids for oxidation in muscle, TG formation in adipose tissue, and milk formation in the lactating breast.
38. chylomicrons that have been depleted of their TG's, are reabsorbed in the liver.

39. VLDL's are the packaging of the lipids produced in the endogenous pathway (the TG's synthesized by the liver and adipose as opposed to dietary intake). they contain TG's, cholesterol, phospholipids, and the apoproteins apoB-100, CII, and E. they are released into the bloodstream from the liver during excess calorie consumption, or from the adipose during the fasting state. the contents are cleaved by LPL in endothelium of muscle or adipose, and the remnant is recycled in the liver.
40. IDL's are formed from VLDL remnants by removal of additional TG's. LDL's are formed by removal of TG's from IDL's in hepatocyte cells. LDL's are made up mainly of cholesterols.
41. 60% are returned to the liver and endocytosed in hepatocytes. 40% are circulated to peripheral tissues where the cholesterols are used for steroid synthesis, vitamin D synthesis, or cell membrane synthesis.
42. either by liver and intestinal cells, packaged containing cholesterol, phospholipids, and a very small amount of TG's, or from budding of apoproteins from VLDL or chylomicrons that have been cleaved by LPL, or from free floating apoprotein AI.
43. nascent HDL's absorb cholesterol esters and phospholipids from endothelium, forming a more globular shape and turning into mature HDL's.

44. the process in which HDL absorbs cholesterol from cells with an excess of cholesterol, and returns it to the liver.
45. cholesterol is moved to the outer plasma membrane of cells by the ABC protein (the action of ABC proteins in the intestinal lumen is mentioned in question 3), and is then picked up by the HDL and modified to a cholesterol ester by LCAT, trapping the cholesterol ester in the middle of HDL so it doesn't return to the cells.
46. because they transport excess serum cholesterol to the liver, preventing the formation of cholesterol induced plaque on the vessel walls.
47. they are bound to the scavenger receptors on liver cells, which cause them to be taken up into the liver cells.
48. HDL transfers the apoproteins CII and E to VLDL and chylomicrons, which turns them into mature lipoproteins and allows their contents to be cleaved by LPL for use in tissues.
49. they are returned back to HDL.
50. the cholesterol ester transfer protein exchanges the cholesterol in HDL for TG's in VLDL's or VLDL fragments when blood levels of VLDL's are high.
51. HDL3 is a mature high density lipoprotein, and HDL2 is an HDL3 that has exchanged cholesterol for TG's as described in the previous question.

52. liver cells have surface receptors for the apoproteins on LDL, contained on "coated pits", which then invaginate the LDL and form endocytic vescicles. these fuse with lysosomes and the contents of the lipoprotein are degraded to free cholesterols and fatty acids. the cholesterols are then re-esterified by the actions of ACAT as described in question 23.
53. see question 13; both are inhibited by sterols- high cholesterol levels in cells with LDL receptors interfere with the SREBP's that aid in the transcription and synthesis of LDL's. to sum up: high cholesterol levels in the cell leads to less uptake of cholesterol.
54. a membrane protein with 6 different domains.
55. increased serum levels of LDL and atherosclerosis.
56. a genetic abnormality from mutation of one allele that codes for the LDL receptor which alters its ability to bind LDL, inhibiting the autofeedback mechanism described in question 53 and therefore raising serum cholesterol levels.
57. a LDL like receptor that has broader specificity for ligands and is not regulated by intracellular levels of sterols.
58. they are found on liver cells and macrophages and bind to oxidatively modified (damaged) HDL's in addition to other lipoproteins (recall that they are used to transport HDL's into hepatocytes in question 45)
59. an accumulation of lipid-laden macrophages in the sub-endothelial space of blood vessels.
60. smoking, high serum lipoprotein levels, high arterial pressure, high angiotensin II levels.
61. macrophages that have ingested high lipid levels accumulate in the subintimal layer and begin to bulge out into the lumen of the blood vessel. the covering over this "plaque" can be degraded and lead to formation of a thrombus, which can completely occlude the artery.

62. glucocorticoids, mineralcorticoids, androgens, estrogens, progestins.
63. because they are hydrophobic they have to be transported in a serum protein such as albumin.
64. either from intracellular cholesterol esters, or from cholesterol containing lipoproteins, or via cholesterol synthesis from acetyl CoA.
65. angiotensin II stimulates the synthesis of aldosterone in the zona glomerulosa of the adrenal cortex. cholesterol is hydroxylated to DOC, which is then oxidized to aldosterone.
66. cholesterol has its side chain cleaved, forming pregnenolone, which is converted to
67. cortisol synthesis takes place in the zona fasciculata of the adrenal cortex.

biochem: mark's medical biochem chapter 33- fatty acid synthesis

this chapter looked at the synthesis, processing, and packaging of fatty acids in the liver and adipose tissue. the first section looked at where the precursors for fatty acid synthesis come from: acetyl CoA is required for fatty acid synthesis and is created in the mitochondria from pyruvate via pyruvate dehydrogenase, then combined with oxaloacetate to form citrate. citrate is shuttled out into the cytosol and cleaved back into acetyl CoA and oxaloacetate (which is then recycled back to pyruvate).

the synthesis of fatty acids begins with the conversion of acetyl CoA to malonyl CoA via the enzyme acetyl CoA carboxylase. (recall from chapter 23 that malonyl CoA is an inhibitor of beta oxidation of fatty acids). this is the rate limiting step of fatty acid synthesis and is regulated by a number of different factors. first, it is inhibited by the products of fatty acid synthesis, malonyl CoA, palmitate, and palmitoyl CoA, and also stimulated by a buildup of reactants- acetyl CoA, citrate, glucose. secondly, acetyl CoA carboxylase is inhibited by a high insulin level, which activates a phosphatase that inactivates the enzyme, while stimulated by high AMP levels (signaling the need for more energy), which activates a protein kinase that activates the enzyme.

the steps for the synthesis of fatty acids:

1. an acetyl moeity attaches to the sulfhydryl group (short arm) of the fatty acid synthase complex and then is transferred to the cysteine-sulfhydryl group (long arm).
2. a malonyl moeity attaches to the short arm and undergoes a condensation reaction with the acetyl CoA from step 1.
3. the keto-acyl that is formed in step two is reduced to an alcohol, forms a double bond via removal of water, and reduced again to remove the double bond. the net result of steps 2 and 3 is the addition of 2 carbons to the ∆-end of the fatty acid chain.
4. the keto-acyl chain is transferred back to the long arm, and another malonyl moeity is attached to the short arm.
5. the keto-acyl chain and malonyl combine via a condensation reaction and the elongation process continues.
6. when the fatty acyl chain reaches 16 carbons, hydrolysis occurs and palmitate is released.

palmitate can then be elongated and desaturated. elongation occurs in a process similar to fatty acid synthesis, except the fatty acyl chain attaches to coenzyme A rather than the ACP sulfhydryl group. a common elongation reaction is that of palmityl CoA to stearyl CoA (C18). desaturation occurs in the endoplasmic reticulum and requires molecular oxygen, NADH, and cytochrome b5. in humans, this process can only occur up to carbon ∆9; thus carbons ∆10 through ∆16 on the palmitate produced by fatty acid synthesis can not be de-saturated. omega 3 and 6 fatty acids fit into this category (3 and 6 carbons from the opposite end correspond to carbons ∆14 and ∆11) and thus must be obtained from plant oils and fish oils. plant oils contain linoleic acid and alpha-linolenic acid, both of which are used as precursors for eicosanoids synthesis.


the next section talks about what happens to fatty acids after they are synthesized in the liver. fatty acids are then combined into triacylglycerols- the glycerol backbone is created either through phosphorylation of glycerol via glycerol kinase, or through the glycolytic intermediate G3P. the process of forming a triacylglycerol is as follows: two fatty acyl CoA's are combined with a glycerol 3-phosphate molecule to form phosphatidic acid. phosphatidic acid is then dephosphorylated to form a diacylglycerol, to which another fatty acyl CoA is added, forming a triacylglycerol. the TG's are then packaged into very-low-density-lipoproteins (VLDL), which are similar to chylomicrons (recall from lipid digestion) in that they are hydrophilic droplets containing TG's, cholesterol, and apoproteins. The nascent VLDL is released into the bloodstream, where it matures upon receiving apoproteins CII and E from a HDL molecule.

the fate of VLDL's is discussed: in circulation, VLDL encounters the enzyme lipoprotein lipase (LPL), located on the endothelium basement membrane, which cleaves the TG's in the VLDL's into glycerol and fatty acids. the LPL in skeletal muscle has a high affinity for the TG's, allowing skeletal muscle to metabolize fatty acids even if the concentration is low. in contrast, the LPL in adipose tissue has a lower affinity for the TG's, and is consequently activated during the fed state when the concentration of VLDL's in circulation is high. once the TG's have been removed from the VLDL, it becomes an intermediate or low density lipoprotein. in adipose tissue, the glycerol backbone is released back into the bloodstream and back to the liver to be recycled, since adipose tissue does not have the glycerol kinase necessary to reuse glycerol for TG synthesis. during the fed state, the high insulin/glucagon ratio stimulates synthesis of LPL, ushering fatty acids into the adipose cells, where they are repackaged into TG's in a similar fashion to what was described above. in the fasting state, a glucagon sensitive lipase cleaves the stored TG's into fatty acids, which are then released into circulation, where they can be metabolized for energy (see chapter 23 notes).



questions
introduction...
1. when are fatty acids synthesized?
2. where does fatty acid synthesis take place?
3. where is the fatty acid synthase complex located?
4. what are the two possible fates for pyruvate in the mitochondria?
5. describe the production of acetyl coA for fatty acid synthesis.
6. why does acetyl CoA need to be produced in the mitochondria? why is it converted into citrate?
7. where does the NADPH required for fatty acid synthesis come from?
8. describe the recycling of oxaloacetate back to pyruvate in the cytosol.
9. acetyl coa synthesis in the mitochondria is stimulated by...
10. why doesn't citrate just get used in the TCA cycle after being synthesized in the mitochondria?

fatty acid synthesis...
11. describe the synthesis of malonyl CoA.
12. what is the rate limiting enzyme of fatty acid synthesis?
13. what is acetyl coA carboxylase regulated by?
14. describe the first steps of fatty acid synthesis on the fatty acid synthase enzyme.
15. describe how the fatty acid chain is elongated.
16. at what point is the fatty acid released from fatty acid synthase?
17. where does the elongation of palmitate occur?
18. what is the main difference between the processes of fatty acid synthesis and fatty acid elongation?
19. what is the main elongation reaction that occurs in the body?

desaturation...
20. what does desaturation of fatty acids require?
21. what are the most common desaturation reactions that take place in the body?
22. what is the limitation in the body's ability to unsaturate fatty acids?
23. where do we obtain w6 and w3 fatty acids?
24. what are linoleic and alpha-linolenic converted to in the body?
25. where do the w3 and w6 fatty acids in fish oil come from?

packaging...
26. where does G3P come from in the liver?
27. where does G3P come from in adipose tissue?
28. describe the synthesis of a triacylglycerol.
29. what is glyceroneogenesis? where and when does it occur? what is it regulated by?
30. what is a VLDL?
31. what is the major apoprotein in VLDL's?
32. how do VLDL's differ from chylomicrons?
33. how do VLDL's become mature?

fate...
34. what is the fate of VLDL's?
35. what is LPL activated by?
36. describe the difference in Km of LPL between muscle and adipose tissue.
37. what happens to the VLDL after the TG's have been removed?
38. describe what happens to fatty acids in adipose tissue in the fed state.
39. what happens to glycerol in the adipose tissue?
40. describe what happens to TG's in adipose tissue in the fasting state.





answers
1. whenever an excess of calories is consumed.
2. mostly in the liver, also in the adipose tissue.
3. in the cytosol
4. conversion to oxaloacetate via pyruvate carboxylase or acetyl coA via pyruvate dehydrogenase.
5. pyruvate in the mitochondria is converted to acetyl coA and combined with oxaloacetate to form citrate. citrate is shuttled out into the cytosol to and split back into oxaloacetate and acetyl coA via citrate lyase.
6. because pyruvate dehydrogenase is only found in the mitochondria, and acetyl coA can not directly cross the mitochondrial membrane.
7. from the pentose phosphate pathway and also from the recycling of oxaloacetate back to pyruvate in the cytosol.
8. oxaloacetate is reduced by malate dehydrogenase into malate. malate is oxidatively decarboxylated into pyruvate via malic enyzme. NADPH is formed in the second step.
9. a high insulin/glucagon ratio.
10. because of allosteric inhibition of isocitrate dehydrogenase.

11. acetyl CoA is oxidatively decarboxylated to malonyl CoA via acetyl CoA carboxylase.
12. acetyl CoA carboxylase.
13. acetyl CoA carboxylase is active in the dephosphorylated form; when insulin levels are high, it is dephosphorylated by a phosphatase. when energy supplies are low, an AMP-dependent protein kinase phosphorylates it back into the inactive form. also, malonyl CoA and palmitoyl CoA (an intermediate and a product of fatty acid synthesis) inhibit while acetyl CoA and citrate (reactants) stimulate the enzyme.
14. an acetyl CoA moiety is transferred to the cysteine-sulfhydryl group, then transferred to the sulfhydryl group. malonyl coA attaches to the sulfhydryl group and then undergoes a condensation reaction with the acetyl, forming a 4 carbon keto-acyl chain and releasing CO2.
15. the carboxyl at the omega end of the four carbon keto-acyl chain is then reduced to an alcohol, has water removed to form a double bond, and reduced again to form a single bond. the keto-acyl chain is then transferred to the cysteine-sulfhydryl group and malonyl is added to it in the same fashion as the first step.
16. when the fatty acid is 16 carbons long (palmitate), it is released via hydrolysis.
17. in the endoplasmic reticulum.
18. in fatty acid elongation, the fatty acyl attaches to a CoA on the fatty acid synthase complex, as opposed to a phosphopantetheinyl group.
19. elongation of palmitate (C16) to stearate (C18).

20. molecular oxygen, NADH, and cytochrome b5.
21. introduction of a double bond in position ∆9 as in the conversion of palmitic acid to palmitoleic acid and stearic acid to oleic acid.
22. the body can only introduce a double bond up to carbon ∆9. thus it can not produce omega-3 or 6 unsaturated fatty acids, which involve carbons beyond that point.
23. mainly from dietary plant oils: linoleic acid (18:2, ∆9,12), alpha-linolenic acid (18:3, ∆9,12,15)
24. arachidonic acid, eicosapentaenoic acid, precursors for eicosanoids.
25. the phytoplankton that they consume.

26. phosphorylation of glycerol via glycerol kinase, or from reduction of DHAP from glycolysis.
27. only from glucose via reduction of DHAP (there is no glycerol kinase enzyme in adipose)
28. two fatty acids combine with the glycerol 3-P to form phosphatidic acid, which is then phosphorylated to form diacylglycerol. a third fatty acid is added to diacylglycerol to form a triacylglycerol.
29. glyceroneogenesis is the formation of new glycerol molecules from gluconeogenic precursors such as alanine, aspartate, and malate. it occurs in the adipose tissue during the fasting state and is regulated by the presence of PEPCK enzyme.
30. a very low density lipoprotein particle which is packaged in the golgi apparatus, contains TG's, cholesterol, phospholipids, proteins, and released by the liver into circulation.
31. apoB-100, related to the apoB-48 in chylomicrons.
32. they are more dense because they contain a smaller proportion of TG's.
33. upon acquisition of apoproteins CII and E from HDL particles in circulation.

34. the TG's in the VLDL's get cleaved by lipoprotein lipase present in the endothelium basement membrane.
35. the C-II apoprotein.
36. muscle tissue LPL has a low Km, allowing muscle to use fatty acids even with concentration of VLDL's are low. adipose tissue LPL has a high Km and is most active in the fed state, when the concentration of circulating fatty acids is high.
37. they form intermediate-density lipoproteins or low density lipoproteins.
38. during the fed state, high insulin levels stimulate synthesis of LPL in adipose tissue capillaries, which releases fatty acids from VLDL's (and chylomicrons). fatty acids are activated and form triacylglycerols using the same pathway as in the liver.
39. since adipose tissue has no glycerol kinase, it can't use glycerol to produce more TG's. thus glycerol travels back into circulation to the liver.
40. glucagon activates a hormone sensitive lipase, which cleaves fatty acids off of TG's, which are released into the bloodstream along with glycerol.

real questions
where do fatty acid elongation and desaturation occur in the body?
why can't double bonds be produced beyond carbon 9 during unsaturation of fatty acids?