Monday, January 26, 2009

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.

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