Cholesterol for classs

cholesterol introduction , synthesis , degradation and functions.
different intermediate products , biochemical importance, fate of cholesterol: synthesis of bile acids (primary and secondary ) , synthesis of vitamin D and different steroid hormones
clinical significance of cholesterol: Hypercholesterolemia ANd hypocholesterolemia normal ranges and so on

1. Cholesterol metabolism Hari Sharan Makaju M.Sc. Clinical . Biochemistry 1 st year

2. Contents Introduction Cholesterol Biosynthesis Regulation of Cholesterol synthesis Degradation of Cholesterol Clinical Significance Summary References

3. Introduction The word cholesterol is derived from Greek words, chole = bile; steros = solid; ol = alcohol. Exclusively found in animal & most abundant animal sterol . Widely distributed in all cells & major component of membranes and Lipoproteins Cholesterol comes from two sources I. Body (specifically liver) Makes all the cholesterol we need (about 700 mg of cholesterol per day). II. Rest we get from foods from animal origin such as cheese, egg yolks, beef, pork, poultry, fish (about 300 mg of cholesterol per day).

4. Structure STRUCTURE 27 carbon compound Double bond at C5-C6 OH group at C3 8 carbon side chain at C17 Total 5 methyl group molecular formula of C 27 H 45 OH .

5. Properties Yellowish crystalline solid Isolated cholesterol is a white, flaky solid that is insoluble in aqueous environments [ Strongly hydrophobic ], Soluble in organic solvent like chloroform, benzene, ether etc

6. Overview of Cholesterol Metabolism

7. Absorption and transport of Cholesterol Cholesterol ester present in the diet is hydrolyzed by cholesterol-esterase. The free cholesterol is incorporated into bile salt micelle and absorbed into the mucosal cell , absorption needs micellar formation. There is a specific protein which facilitates the transport of cholesterol into the mucosal cell from the micelle. Inside the mucosal cell, cholesterol is re-esterified and incorporated into chylomicrons. The chylomicrons reach the blood stream through lymphatics (lacteals). This dietary cholesterol reaches the liver through chylomicron remnants.

8. Functions Structural component of cell membrane (providing greater stability and modulating its fluidity) Precursor for synthesis of other steroid- steroid hormone, Vit D and bile acids Essential ingredient in structure of Lipoprotein Transportation of fatty acid Insulating cover for transmission of electric impulse in nervous tissue

9. CHOLESTEROL BIOSYNTHESIS Site of Synthesis: Cholesterol synthesis can occur in all nucleated cells , including arterial walls. Major sites : Liver(50% of total), adrenal cortex, testes, ovaries and intestine. Minor sites : Adipose tissue, muscle, aorta & neural tissues Brain of newborn can synthesize cholesterol while the adult brain cannot synthesize. Site : Cytosol and microsomes , Enzymes are partly located in the endoplasmic reticulum and partly in the cytosol

10. Biosynthesis Complex biosynthetic pathway Principal precursor :- Acetyl-CoA Reducing equivalents are supplied by NADPH while ATP provides energy.

12. STEPS OF BIOSYNTHESIS Condensation of three acetate units to form a six-carbon intermediate, mevalonate Conversion of mevalonate to activated isoprene units Polymerization of six 5-carbon isoprene units to form the 30-carbon linear squalene Cyclization of squalene to form the four rings of the steroid nucleus. Ref: Lehninger Principle of biochemistry 4 th edition,

13. 1. Synthesis of Mevalonate from Acetyl CoA Irreversible & rate limiting enzyme

14. MITOCHONDRION Fatty acids (2) Acetyl CoA  -oxidation Acetoacetyl CoA HMG CoA HMG CoA synthase Thiolase Acetoacetate  -Hydroxybutyrate Ketone bodies (only synthesized in liver) HMG CoA lyase oxaloacetate Citrate Citrate Mevalonate CHOLESTEROL smooth endoplasmic reticulum HMG CoA reductase Acetoacetyl CoA HMG CoA cytoplasm HMG-CoA synthase Thiolase Synthesis of HMG-CoA in mitochondria (ketone bodies) and cytoplasm (cholesterol) Lyase (requires ATP) (2) Acetyl CoA

15. STEP 2: CONVERSION OF MEVALONATE TO TWO ACTIVATED ISOPRENES

16. Ref: Lehninger Principle of biochemistry 4 th edition,

17. Step 3: Condensation of 6 activated isoprene units to form squalene Isopentenyl pyrophosphate(IPP) and dimethylallyl pyrophosphate(DMPP) undergoes a head-to-tail condensation, in which one pyrophosphate group is displaced and a 10-carbon chain, geranyl pyrophosphate, is formed Geranyl pyrophosphate undergoes another head-to-tail condensation with isopentenyl pyrophosphate,yielding the 15-carbon intermediate farnesyl pyrophosphate. Finally, two molecules of farnesyl pyro -phosphate join head to head, with the elimination of both pyrophosphate groups, to form squalene .

18. 10C 15C 30C Ref: Lehninger Principle of biochemistry 4 th edition ,

19. STEP 4: CONVERSION OF SQUALENE TO THE FOUR-RING STEROID NUCLEUS The action of squalene monooxygenase adds one oxygen atom from O2 to the end of the squalene chain, forming an epoxide. NADPH reduces the other oxygen atom of O2 to H2O. In animal cells, cyclization of epoxide results in the formation of lanosterol , which contains the four rings characteristic of the steroid nucleus Lanosterol is finally converted to cholesterol in a series of about 20 reactions that include the migration of some methyl groups and the removal of others.

20. Add one mol. Of O2 to end of squalene chain (hydroxylation) Series of 20 reaction in ER via intermediate- 14-desmethyl lanosterol , Zymosterol , cholestadienol, and desmosterol Ref: Lehninger Principle of biochemistry 4 th edition

21. The 3 additional methyl groups on carbon atoms 4 and 14 are removed to produce zymosterol . Then the double bond migrates from 8-9 position to 5-6 position, when desmosterol is formed. Desmosterol is present in fetal brain . It is absent in adult brain and re-appears in gliomas (brain tumor). Finally, the double bond in the side chain (between carbon 24- 25) is reduced by NADPH when cholesterol is formed.

22. ENERGETIC COST OF THE SYNTHESIS OF CHOLESTEROL Production of 1 mole of cholesterol requires 18 moles of acetyl-CoA, 36 moles of ATP 16 moles of NADPH

23. Mevalonate Active Isoprenoids (C 5 ) Squalene (C 30 ) 3ATP CO 2 Several Condensation Steps 3ADP NADPH NADP + Stage 2 Squalene (C 30 ) Cyclization Squalene epoxidase/ cyclase Lanosterol (C 30 ) (4-ring structure) O 2 NADPH NADP + Stage 3 Stage 4 Lanosterol (C 30 ) (20 steps) O 2 NADPH NADP + 3 CH 3 Cholesterol (C 27 ) Acetyl CoA (C 2 ) HMG-CoA HMG-CoA Reductase Mevalonate (C 6 ) NADPH NADP + Stage 1 Overview of cholesterol biosynthesis rate-determining step cholesterol activates proteolytic degradation hormonally controlled via phosphorylation

24. Smith- Lemli - Opitz syndrome (SLOS) Autosomal-recessive disorder of cholesterol biosynthesis Prevalence  1 in 20,000-60,000 births  (USA) Caused by a deficiency in 7dehydrocholesterol reductase , Enzyme that reduces the double bond in 7-dehydrocholesterol (7-DHC), thereby converting it to cholesterol. Characterized by: Slow growth before and after birth, small head (microcephaly), Mild to moderate mental retardation and multiple birth defects including particular facial features, cleft palate, heart defects, fused second and third toes, extra fingers SLOS is one of several multisystem, embryonic malformation syndromes associated with impaired cholesterol synthesis

25. Regulation of cholesterol synthesis Cholesterol production is regulated by different mechanisms: Sterol-dependent regulation of gene expression Sterol-independent phosphorylation/ dephosphorylation Hormonal Regulation Inhibition by drugs

26. STEROL-DEPENDENT REGULATION OF GENE EXPRESSION Regulation of HMG-CoA reductase synthesis by cholesterol is mediated by an elegant system of transcriptional regulation of the HMG-CoA gene This gene, along with more than 20 other genes encoding enzymes that mediate the uptake and synthesis of cholesterol and unsaturated fatty acids, is controlled by a small family of proteins called sterol regulatory element-binding proteins (SREBPs). SREBP binds DNA at the cis-acting sterol regulatory element (SRE ) of the reductase gene.

27. STEROL-DEPENDENT REGULATION OF GENE EXPRESSION When cholesterol levels in the cell are low, the SREBP-SCAP complex is sent out of the ER to the Golgi complex. In the Golgi, SREBP is sequentially acted upon by two proteases , which generate a soluble fragment that enters the nucleus, binds the SRE, Functions as a transcription factor activates transcription of its target genes, including HMG-CoA reductase, the LDL receptor protein, and a number of other proteins needed for lipid synthesis This results in increased synthesis of HMG CoA reductase and, therefore, cholesterol synthesis is increased

28. SREBP activation .

29. Sterol-accelerated enzyme degradation If sterols are abundant , however, they bind SCAP at its sterol-sensing domain and induce the binding of SCAP to yet other ER membrane proteins (insulin induced gene ( Insig )). This results in the retention of the SCAP-SREBP complex in the ER, thus preventing the activation of SREBP, and leading to down-regulation of cholesterol synthesis. SCAP and Insig act as sterol sensors. Intracellular levels of cholesterol, when elevated, inhibit both the activity as well as the synthesis of HMG CoA reductase.

30. STEROL-INDEPENDENT PHOSPHORYLATION/ DEPHOSPHORYLATION Short-term regulation of HMG CoA reductase activity is controlled covalently through the actions of adenosine monophosphate (AMP)–activated protein kinase (AMPK ) and a phosphoprotein phosphatase The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active.

31. Hormonal regulation Insulin and Thyroxine : An increase in insulin and thyroxine favors up-regulation of the expression of the gene for HMG CoA reductase . increase the activity of HMG CoA reductase favoring cholesterol synthesis. Glucagon and the glucocorticoids : Decrease the activity of HMG CoA reductase favoring decreased cholesterol synthesis .

32. Inhibition of Drugs Competitively inhibitor of HMG CoA reductase Control Hypercholesterolemia Statin drugs (atorvastatin, fluvastatin , lovastatin, pravastatin, rosuvastatin , and simvastatin)

33. circadian rhythm of cholesterol biosynthesis Hepatic synthesis of cholesterol peak at about 6 hours after dark and at a minimum some 6 hours after exposure to light. This rhythm is the result of corresponding changes in HMG-CoA reductase activity. Exploiting this, the drugs inhibiting cholesterol synthesis ( statins ) are usually taken at night to ensure maximal effect. The mechanism of control of HMG-CoA reductase in these circumstances is poorly understood, although dietary pattern plays a part.

34. Synthesis of cholesterol esters Cholesterol ester is more hydrophobic The ester is transported in lipoprotein to other tissue for storage or use Intracellular Intravascular

35. Cholestrol Ester Enter Cells by (elucidate by Michael brown & J. Goldstein) Receptor- Mediated Endocytosis ApoB 100 of LDL is recognized by specific receptor ‘ LDL receptors ’ Endocytosis endosome Fuse with lysosome, enzyme hydrolyze the cholesterol esters, releasing cholesterol and fatty acid into cytoplasm. ApoB 100 is degraded to amino acid while LDL receptor escape the degradation and return to cytoplasm which can again function in LDL uptake Receptor mediated endocytosis Cholesterol released may incorporate into membrane or reesterified by ACAT for storage Absent of the LDL receptor leads hypercholesterolemia

36. Familial Hypercholesterolemia Genetic disease that arises from any one of many mutant alleles for the LDL receptor gene. LDL receptor gene is located on the short arm of chromosome 19 Receptor mediated uptake of cholesterol by LDL into cells is defective, which results in cholesterol accumulation in the blood. Homozygotes: 680 mg/ dL (atherosclerosis in childhood) Heterozygotes: 300 mg/ dL ( atheroclerosis in middle age) Cholesterol deposited in various tissue due to high LDL-C in plasma Treated with inhibitors of de novo cholesterol biosynthesis and Cholestyramine. Ref: Lubert Stryer , Biochemistry, 4 th edition

37. 5 major classes of FH due to LDLR mutations: Class I : LDLR is not synthesized at all. Class II: LDLR is not properly transported from the endoplasmic reticulum to the Golgi apparatus for expression on the cell surface. Class III : LDLR does not properly bind LDL on the cell surface because of a defect in either apolipoprotein B100 (R3500Q) or in LDL-R. Class IV: LDLR bound to LDL does not properly cluster in clathrin -coated pits for receptor-mediated endocytosis (pathway step 2). Class V : LDLR is not recycled back to the cell surface (pathway step 5).

38. This finding might add biochemical evidences of atherogenicity of these lipoproteins. Measuring CEOOH level in these lipoproteins need to be investigated for the risk assessment of the cardiovascular disease

39. Cholesteryl ester storage disease (CESD) Rare an autosomal recessive genetic disorder Due to deficiency of the lysosomal acid lipase characterized by: Alterations of blood lipoprotein profile; Patients present hypercholesterolemia, hypertriglyceridemia, HDL deficiency with abnormal lipid deposition in many organs.

40. Degradation of Cholesterol Conversion to bile acid Secretion of cholesterol to bile Precursor for synthesis of steroid hormones, vitamin D Modification by intestinal bacteria to form coprostano l and cholestanol

41. Synthesis of bile Acids

42. Synthesis of bile acid Bile acids are Amphipathic in nature Serves as emulsifying agents in intestine Synthesis occurs in liver by series of reactions ~ 500 mg/day of cholesterol get converted into bile In the bile the conjugated bile acid exist as Na and K salts, which are known as Bile salts Approximate composition of bile salts Glycocholate – 24% Glycochenodeoxycholate – 24% Taurocholate – 12% Taurochenodeoxycholate – 12% Glycodeoxycholate - 16% Taurodeoxycholate – 8% Various lithocholate – 4%

43. Enterohepatic Circulation Bile salts are secreted in intestine through bile duct, which help in digestion and absorption of fats. In gut the glycine and taurine residues are removed. About 90%, except lithocholic acid are reabsorbed in ileum by active transport Albumin c arries the bile to liver through portal blood where they are effectively removed by liver parenchymal cells In liver bile acid are conjugated with glycine & taurine and again secreated in bile. •This process of secretion from the liver to the gallbladder, to the intestines and finally re- absorbtion is termed the enterohepatic circulation. 15- 30 gm of bile salts are secreted from liver into duodenum each day, around 0.5 gm is lost in feces. Appox 0.5 gm is synthesised per day to replace the lose.

44. Clinical Significance of Bile Acid Synthesis Bile acids perform following physiologically significant functions: Their synthesis and subsequent excretion in the feces represent the only significant mechanism for the elimination of excess cholesterol. Bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the precipitation of cholesterol in the gallbladder. They facilitate the digestion of dietary triacylglycerols by acting as emulsifying agents that render fats accessible to pancreatic lipases. They facilitate the intestinal absorption of fat-soluble vitamins. Stimulate intestinal motility

45. Cholelithiasis Bile salt and phospholipids are responsible to keep cholesterol in bile in soluble state Due to their deficiency (particularly Bile salt), cholesterol get precipitated in gall bladder or duct resulting cholelithiasis “ Cholesterol Gall Stone Disease ” Causes: Malabsorption of bile acid from the intestine, as seen in patient with ileal disease Obstruction of the Biliary tract, interrupting the enterohepatic circulation Severe hepatic dysfunction leading to decrease synthesis of bile etc Treatment Surgical removal of gall bladder Some patient may respond to administration of chenodeoxycholic acid, commonly called chenodiol , for gradual (month to year) dissolution of gall stones

46. Synthesis of Steroid Hormone Cholesterol is precursor for 5 classes of steroid hormone Glucocorticoids (e.g. Cortisol) Affects protein and carbohydrate metabolism; suppresses immune response , inflammation, and allergic responses Mineralocorticoids (e.g. Aldosterone) Regulate reabsorption of Na + , Cl - , HCO - in the kidney. Progestins (e.g. Progesterone) prepares the lining of the uterus for the implantation of an ovum and maintains the pregnancy Androgens (e.g. Testosterone) Responsible for the development of male secondary sex characteristics Estrogens ( e.g. Estradiol ) Responsible for the development of female secondary sex characteristics

47. Synthesis of Steroid hormones C 27 C 21

48. Vitamin D synthesis 7-Dehydrocholesterol, an intermediate in the synthesis of cholesterol, is converted to cholecalciferol (vitamin D3) by ultraviolet rays in the skin. Plays essential role in calcium and phosphorous metabolism

49. Fate of cholesterol

50. Intermediates in Cholesterol Biosynthesis Isopentenyl pyrophosphate ,an intermediate in cholesterol biosynthesis is the activated precursor of a huge array of biomolecules with diverse biological role. E.g. vitamins A, E, and K; plant pigments such as caroten ; dolichols , ubiquinone and plastoquinone etc. Protein prenylation is another important role for the isoprene derivatives of the pathway to cholesterol

51. Plasma Cholesterol Clinical Importance In healthy individual total plasma cholesterol is in the range of 150-250 mg/dl In newborn it is less than 100 mg/dl & rises to about 150 mg/dl with in an year Women have relatively lower cholesterol level Cholesterol level increases with age ( in women particularly after menopause) and also in pregnancy

52. Estimation of Serum cholesterol Reference method CDC reference method (gold standard), Abell et al 1.Hydrolysis of cholesterol esters - 0.5 ml serum is treated with 5 ml of alc. KOH 2. Extraction of total cholesterol with 10 ml of hexane for 15 min 3.The extract is dried in Vacuo 4. The dry residue is estimated by mixture of acetic acid, acetic anhydride and sulfuric acid ( Libermann - Burchard reagent) at 620 nm.

53. Estimation of Serum cholesterol Other methods: Libermann - Burchard Reaction Zak’s Ferric Chloride Method Enzymatic Method

54. Estimation of Serum cholesterol Enzymatic method Cholesterol ester + H 2 O Cholesteryl ester hydrolase Cholesterol + fatty acid Cholesterol + O 2 cholesterol oxidase Cholest-4-en-3-one + H 2 O 2 H 2 O 2 + phenol + 4-aminoantipyrine peroxidase quinoneimine dye + 2H 2 O Color complex is Measured at 500nm This method interfere from other colored compound or those that compete with oxidation reaction, such as bilirubin, ascorbic acid and Hb Assay is linear up to 600 to 700 mg/dl

55. Clinical Significance Hypercholesterolemia Plasma Cholesterol Diabetes mellitus Hypothyroidism Obstructive jaundice Nephrotic Syndrome

56. Clinical Significance Hypocholesterolemia Decrease in plasma cholesterol Less common Hyperthyroidism Pernicious anemia Malabsorption syndrome Hemolytic jaundice Massive parenchymatous liver damage Familial hypobetalipoproteinaemias Therapautic reduction during clofibrate , cholestyramina , nicotinic acid etc

57. Control of Hypercholesterolemia Dietary restriction egg, meat, diet rich in carbohydrate Dietary fiber Consumption of PUFA PUFA helps in transportation of Cholesterol By LCAT mechanism Cottonseed oil, Sunflower oil, corn oil, soya been oil and fish oil are good source of PUFA while ghee and coconut oil are bad source 4. Use of drugs Lavastatin :- inhibit the HMG CoA reductase Cholestyramine & cholestipol- binds with bile and decrease intestinal absorption Clofirate:- increase activity of lipoprotein lipase and reduce plasma cholesterol and TG

58. Hypercholesterolemia is associated with atherosclerosis & Coronary Heart Disease Coronary Heart Disease

59. Atherosclerosis & Coronary Heart Disease When the sum of cholesterol synthesis and obtain from diet exceed the rate of degradation Accumulation of cholesterol in blood vessels resulting obstruction( atherosclerosis) Hardening of arteries due to accumulation of lipids particularly cholesterol, collagen, fibrous tissue, proteoglycans, ca++ deposits etc Coronary artery are most commonly affected leading Coronary heart disease Leading cause of death in developed country

60. Atherosclerotic Plaque Atherosclerotic Plaque Lubert Stryer , Biochemistry, 4 th edition

61. Atherosclerosis & Coronary Heart Disease Associated with diabetes mellitus, hyperlipoproteinemias, nephrotic syndrome, hypothyroidism etc Other risk factors are Obesity High consumption of saturated fat Excessive smoking Lack of physical exercise Hypertension Stress etc

62. Young adult exposures to elevated systolic BP, diastolic BP and LDL were associated with increased CVD risks in later life, independent of later adult exposures

63. Atherosclerosis & Coronary Heart Disease Classification Total cholesterol (mg/dl) LDL-Cholesterol (mg/dl) Desirable < 200 <130 Boderline to high risk 200 – 239 130 - 159 High risk > 240 > 160 Coronary heart disease risk classification based on total cholesterol and LDL cholesterol

64. Summary Cholesterol is both our friend and foe It is synthesized in our body; non essential lipid It is a precursor for many compounds e.g. bile acids and salts, vitamin D, steroid hormones etc Increased level of cholesterol is very dangerous and cause atherosclerosis , myocardial infarction etc.

65. References Lehninger , Principle of biochemistry, 4th edition, Martin A. Crook, Clinical chemistry and Metabolic Medicine, 7th edition Lippincott’s Illustrated Reviews: Biochemistry, 2nd Edition Tietz , Textbook of Clinical Chemistry and Molecular Diagnostics, 4th edition Tietz , Fundamentals of Clinical Chemistry, 2nd edition Kaplan, Method in clinical chemistry Lubert Stryer , Biochemistry, 4th edition Ranjna Chawla, Practical clinical Biochemistry, methods & Interpretation, 3rd edition Web.indstate.edu/ thcme / mwking /home.html www.gwu.edu www.med.unibs.it https://ghr.nlm.nih.gov/gene/LDLR#location

66. Thank you