SUBJECT: BIOCHEMISTRY TOPIC: LIPID METABOLISM 3 (Cholesterol Sources & Biosynthesis & Degradation) LECTURER: Dr.

Laygo DATE: November 30, 2010

DIET – (Cholesterol) is found in animal fat BIOSYNTHESIS Primarily synthesized by the LIVER(hepatocytes) from Acetyl CoA (formed from oxidative decarboxylation of glucose) o Synthesis in: Cytoplasm (cytosol)  Membrane of ER  Inhibited by LDL uptake by the LIVER *LDL is formed from the lipoprotein VLDL, carries triacylglycerol(TAG), enters the circulation, TAG is acted by an enzyme stimulated by Apo CII(in VLDL) - activates lipoprotein lipase, release free fatty acid which is deposited in adipose tissue, VLDL becomes IDL further degradation LDL(contains cholesteryl ester) reuptake by LIVER receptor mediated endocytosis by Apo B100(ligand) (In absence of receptors or apo B-100, LDL remains in circulation = hypercholesterolemia) — lipid metab II DEGRADATION   Occurs in the LIVER Cholesterol  Not utilized by the cell  Precursor for steroid hormones synthesis (Anabolic) o e.g. glucocorticoids, meneralocorticoids, sex hormones (androgen/estrogen), Vit. D (Calcitriol or 1,25-dihydroxycholecalciferol)  Converted  Bile Acids (Catabolic) 
2 Acetyl‐CoA  
forms  Acetoacetyl‐CoA  catalyzed by  Thiolase 

  Found in the 

Cytoplasm  (Cytosol)  Addition of  another mole  of Acetyl‐CoA 

Important  intermediate, as  a precursor of the  synthesis of  different  intermediates 
     ↓Decrease  Synthesis of     Cholesterol 

  Embedded in  

ER membrane  (Rate Limiting  Step)

STEP 2 – Formation of Isoprenoid units
(See Figure 26-2, Harper’s Illustrated Biochemistry, p226)

BIOSYNTHESIS of CHOLESTEROL (All Carbon atoms from Acetyl CoA) STEP 1 – Biosynthesis of Mevalonate (at next column)
(See Figure 26, Harper’s Illustrated Biochemistry, p225)  

 Isoprene unit contains (5 Carbons)   Terpene  Polymerization of Isoprene units     
Monoterpene  Sesquiterpene  Triterpene  1 isoprene unit**  3 isoprene units  6 isoprene units  Isopentenyl  pyrophosphate  Farnesyl  pyrophosphate  Squalene 

3 phosphorylation reduction  
steps by utilizing (3)ATPs 

Removal of 1 CO2 (decarboxylation)  6‐carbon to 5‐carbon 

**Note: Many references state that Monoterpene is formed  with 2 isoprene units NOT 1 isoprene unit (e.i. Geranyl  pyrophosphate) contradicting what was indicated in the PPT:   A Monoterpene  Isopentenyl pyrophosphate 

Isoprene unit   (5 Carbon atoms) 

Isopentenyl pyrophosphate  Monoterpene** (terpenes = contain isoprene units)

Pyrophosphate  or Diphosphate 

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STEP 3 – Six Isoprenoid Units Form Squalene
(See Figure 26-2: Harper’s Illustrated Biochemistry, p226)

In the process, an epoxide intermediate (Squalene epoxide) is formed – catalyzed by Squalene monooxygenase/ Squalene epoxidase

3, 3 – dimethylallyl diphosphate an Isomer joins Isopentenyl pyrophosphate in a head to tail manner/head to tail condensation.

Hypothetical reactions (on formation of Geranyl pyrophosphate) 1. Ionization  Condensation 2. Ionization  Condensation  Ionization  Both molecules (with 5 carbon atoms) form a Geranyl pyrophosphate (a 10-carbon moiety)
Isopentenyl  pyrophosphate 

Lanosterol ‐ 1st intermediate in cholesterol  biosynthesis which contain sterol ring.  4 rings:  (3) 6‐membered &   (1) 5‐membered 

*sterol ring = cyclopentanoperhydrophenanthrene ring /sU-klb-pen-t^-nb-per-hUcdrb-fenc^-thrTn/ Antifungal – utilized in inhibition of the formation of the epoxide. STEP 5 – Formation of Cholesterol (19 different steps) Important characteristics:    Reduction reaction (utilize NADPH+H+ or NADH+H+) Molecular oxygen is utilized Cholesterol (27 Carbons) o 3 Carbons need to be removed from (30-C Moiety)    Formate (HCOOH) – step 4 Carbon dioxide (CO2) – step 9 Carboxylic group – step 14

Another mole of Isopentenyl pyrophosphate in head to tail manner will condense with Geranyl pyrophosphate forming Farnesyl diphosphate with (15 Carbons atoms) 3 isoprene units = Sesquiterpene.

Important Features of Cholesterol Carbon 3 Between 5&6 17 Feature Hydroxyl groups Double bond Aliphatic side chain Methyl groups

 2 moles of Farnesyl pyrophosphate (15-Carbon) will condense in a head to head manner forming Squalene (30-Carbon moiety ) 6 isoprene units =Triterpene) STEP 4 – Formation of Lanosterol
(See Figure 26-3: Harper’s Illustrated Biochemistry, p227)

18 and 19

Delocalization of the electrons of Squalene to assume a shape where electrons can easily move with the use of the enzyme 2,3- oxidosqualene-lanosterol cyclase.
6 isoprene units 

2,3‐ oxidosqualene‐ lanosterol cyclase 

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Hydroxyl group at C3 – Esterification of Fatty Acid Palmitic acid (most common fatty acid synthesized by eukaryotes) with the use of enzyme ACAT.

Utility of NADPH + H+ as source of electrons for reduction reaction at:  Steps: 7, 8, 9

BILE ACIDS FROM CHOLESTEROL  formed from cholesterol in the liver  stored in the gall bladder in bile as bile salts (sodium and potassium) o o Precursor molecules for synthesis of steroid hormones Geranyl pyrophosphate, Farnesyl pyrophosphate utilized in synthesis of:    Heme A Dolichophosphate Ubiquinone – (ETC) Coenzyme Q (channel electrons between complex I and III and between complex II and III) G protein – connected to inner leaflet of plasma membrane; connection to isoprene units making up prenylated protein (serves as anchor).

ACAT inhibitors act  here. Inhibit  formation of  Cholesterol/  Cholesteryl esters 
Demethylation reaction – liberating the carbon moiety (Carboxylic group & CO2); can be affected by the anti-fungal.

utilized during digestion of fats and other lipid substances (act as detergents) o Emulsification: Lipid droplets  Micelle

↓ (See Figure 26-3: Harper’s Illustrated Biochemistry, p227)

Rate limiting step for bile acid synthesis: Cholesterol 7-α hydroxycholesterol 7‐α‐hydroxylase  Attachment of hydroxyl group at Carbon number 7

Lanosterol 19 steps Cholesterol Release of carbon atoms via:    Step 4: Formate (HCOOH) Step 9: Carbon dioxide (CO2) (at reaction 9 to 10) Step 14: Carboxylic group (–COOH)

Further reduction using NADPH+H+ and CoA‐SH  forms Primary bile acids   (Cholyl‐CoA & Chenodeoxycholyl‐CoA)  * These 2 are the ONLY Primary bile acids. 

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↓(See FIGURE 26-7: Harper’s Illustrated Biochemistry, p.231)

  •

Taurodeoxycholate –8% Various lithocholate –4%

Fat digestion products are absorbed in the first 100 cm of small intestine o enzymes = hydrophilic o dietary lipase = hydrophobic o Emulsification: droplet formation = ↑ Surface Area o Bile salts and acids = ↓ Lipid Aqueous interface

The primary and secondary bile acids are reabsorbed almost exclusively in the ileum returning to the liver by way of the portal circulation (98 to 99%) o this is known as the enterohepatic circulation Less than 500 mg a day escapes reabsorption and is excreted in the feces.

↓(See FIGURE 26-6: Harper’s Illustrated Biochemistry, p.230)

Secondary bile acids  Glycocholic acid 1. condensation reaction from Cholyl CoA 2. Uses: glycine  Taurocholic acid 1. Taurine is formed by the decarboxylation of  cysteic acid, which in turn is made by oxidation of cysteine


decarboxylation Bile salts   detergent character of bile salts is due to the hydrophobic-hydrophilic nature of the molecules the presence of hydroxyl (or sulfate) and the terminal carboxyl group on the tail give the molecule its hydrophilic face the steroid ring with its puckered plane provides the hydrophobic face

Lithocholic acid o Formed from deconjugation + 7α-dehydroxylation of  Taurocholilc acid,  Glycocholic acid,and  Chenodeoxycholic acid. Deoxycholic acid o deconjugation + 7α-dehydroxylation from glycocholic acid

* deconjugation+7α-dehydroxylation(Catalyzed by microbial enzymes)

Function of bile salts •emulsification of fats due to detergent activity •aid in the absorption of fat-soluble vitamins (especially vitamin K)   Vitamin K – synthesized by normal bacterial flora (Lipid-soluble Vitamins (A, D, E & K)

Bile acids •cholic acid is the bile acid found in the largest amount in bile •bile acids are converted to either glycine or taurine conjugates (in humans the ratio of glycine to taurine conjugates is 3:1) APPROXIMATE COMPOSITION OF BILE SALTS      Glycocholate –24% Glycochenodeoxycholate –24% Taurocholate –12% Taurochenodeoxycholate –12% Glycodeoxycholate-16%

•accelerate the action of pancreatic lipase •have choleretic action –stimulate the liver to secrete bile •stimulate intestinal motility •keep cholesterol in solution (as micelles)

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↑ LDL containing cholesterol ester , coalesce with lysosome  contents LDL in form of endosome will be hydrolyzed cholesterol released in the cell = down regulate synthesis of protein receptors

Type II-B: ↑ VLDL + LDL; often ↓ HDL; ↑ production of VLDL + impaired LDL catabolism (from VLDL) Allows enzyme to attach and begin digestion. Interior TAG with bile salts around (facing aqueous medium) allowing the enzymes to attach & start digestion process. GALLSTONE THERAPEUTIC AGENTS •chenodeoxycholic acid (chenodiol; Chenix) •ursodeoxycholic acid (ursodiol; Actigall) •MOA (Mechanism of Action): –↓ hepatic secretion of cholesterol into bile –inhibition of HMG-CoA reductase (most important enzyme in cholesterol biosynthesis) = inhibit cholesterol biosynthesis –↑ cholesterol solubility Chenodiol and Ursodiol • both are effective in dissolving cholesterol stones in some patients • ursodiol is the 7-beta epimer of chenodiol • most effective in dissolving small (<5 mm) floating stones in a functioning gallbladder • cannot dissolve stones that are more than 4% calcium by weight Atherosclerosis • hardening of the arteries due to the deposition of atheromas o Atheroma: lipid deposits in the intima of arteries • heart disease is the leading cause of death • caused by the deposition of cholesteryl esters on the walls of arteries • atherosclerosis is correlated with ↑ LDL (bad cholesterol*) and ↓ HDL (good cholesterol)
* oxidized form of LDL= harmful

Type III: ↑ IDL (dysbetalipoproteinemia); abnormal apolipoprotein E; impaired catabolism of IDL; ↑ cholesterol and triglycerides (formerly known as broad beta disease) Type IV: ↑ VLDL; often ↓ HDL; impaired VLDL catabolism; dietary indiscretion ( formerly known as hyperprebetalipoproteinemia) Type V: ↑chylomicrons + VLDL; ↓HDL; ↓lipoprotein lipase + VLDL hypersecretion (formerly known as mixed lipemia) Factors promoting elevated blood lipids •age –men >45 years of age; women > 55 years of age •family history of CAD (Coronary Artery Disease) •smoking (α1-antitrypsin inhibits elastase  elastin) Oxidizes α1-antitrypsin specifically its methionin residue  methionin sulfoxide = α1-antitrypsin release from normal inhibition of elastase •hypertension >140/90 mm Hg •low HDL cholesterol •obesity > 30% overweight •diabetes mellitus •inactivity/ lack of exercise Mechanisms of action of drugs •bind to bile acids/cholesterol –inhibit absorption/reabsorption •increase peroxisomal FA oxidation •stimulate lipoprotein lipase •inhibit triglyceride lipase •inhibit HMG-CoA reductase •stimulates microsomal 7-α hydroxylase Drug Classification •systemic/non-sytemic •cholesterol lowering agents –bile acid sequestrants –sitosterols* –probucol* –d-thyroxin* –HMG-CoA reductase inhibitors •mixed activity (nicotinic acid) •triglyceride lowering agent –clofibrate (Atromid-S) –gemfibrosil (Lopid) –fenofibrate (Tricor) * No longer available commercially in the U.S

Frederickson – WHO classification Type I: ↑ chylomicrons, ↓ HDL, absence of lipoprotein lipase; deficiency of apo CII activates lipoprotein lipase. A deficiency of Apo CII will result in accumulation of chylomicrons and triacylglycerols (Hyperchylomicronemia) Type II-A: ↑ LDL; ↓catabolism of LDL (Receptor deficiency or polygenic)  Receptors synthesized thru Transcription & Translation

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BILE SEQUESTERING RESINS   Cholestyramine Colesevelam (WelChol) o polyalkylamine hydrochloride) cross linked with  epichlorohydrin and alkylated with 1‐bromodecane  and (6‐bromohexyl) trimethylammonium bromide  available as a 625 mg tablet  same mechanism of action as colestipol and  cholestyramine 

3. Gene expression: cholesterol levels control the amount of mRNA  Down regulation process of transcription and translation (↓synthesis of receptors and enzyme molecules)

o o

Colestipol (Colestid)

They try to bind bile acids to intestine preventing absorption into enterohepatic circulation  more cholesterol transform to bile acids.      Po (per orem/per os - via the mouth; orally), safest, non systemic bind to bile acids and inhibit reabsorption ↑ 7-α hydroxylase activity leading to cholesterol degradation ↓plasma LDL problems: – abdominal discomfort, bloating, constipation – decreases drug absorption; wait 4 hrs after administration of BAS to give drugs drug interactions (decreased serum level) aspirin clindamycin clofibrate furosemide glipzide tolbutamide phenytoin imipramine methyldopa nicotinic acid penicillin G propranolol tetracycline thiazide diuretics digoxin hydrocortisone phosphate supplements Left side reactions: Glucagon & Epinephrine

Right side reactions: Insulin

                 

Left side reactions     During starvation (low energy level) Predominance of Glucagon and Epinephrine Inhibits Cholesterol biosynthesis HMG-CoA reductase inactivates when it is phosphorylated with the use of an ATP and a kinase HMG-CoA reductase kinase which also requires an activation by coenzyme HMG-CoA reductase kinase kinase and phosphate from ATP. HMG-CoA reductase kinase kinase attaches a phosphate from ATP to  HMG-CoA reductase kinase (becomes activated) attaches another phosphate from ATP to  HMG-CoA reductase (becomes inactive) = Inhibition of cholesterol biosynthesis Insulin predominance Allows cholesterol biosynthesis
(right top of the figure) HMG-CoA reductase kinase phosphatase by dephosphorylation inactivates HMG-CoA reductase kinase by removing the phosphate with the use of H2O (at the bottom of the figure) HMG-CoA reductase phosphatase by dephosphorylation activates HMGCoA reductase by removing also the phosphate with the use of H2O = allowing biosynthesis of cholesterol.

HMG CoA reductase  3 different regulatory mechanisms are involved: 1. Covalent modification: phosphorylation(attachment of phosphate, predominating glucagon and epinephrin) by cAMP-dependent protein kinases inactivate the reductase. This inactivation can be reversed by 2 specific phosphatases Lipid is catabolized and not synthesized during starvation/in need of energy. Predominance of Insulin: remove phosphate group  activity is stimulated  synthesis of cholesterol 2. Degradation of the enzyme–half-life of 3 hours and the half-life depends on cholesterol levels ↑ LDL endocytosed (receptor mediated endocytosis) coalesce with lysosome  degrade contents, release free cholesterol  triggers enzyme degradation Protein molecules degraded thru Ubiquitination/Ubiquitin cycle

Right side reactions   

STATINS     Mevastatin Lovastatin (Mevacor) Simvastatin Pravastatin

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Synthetic Statins:   Fluvastatin Cerivastatin

o o

inhibits TG synthesis stimulates catabolism of VLDL

 Indicated primarily for hypertriglyceridemia  Same side effects and precaution as in other fibric acid compounds  Half-life: 20 hours  Dose: 67-201 mg/day with meals Nicotinic Acid (Niacin)      A water soluble vitamin of the B family; nicotinamide is not active (vitamin B3) Once converted to the amide, it is incorporated into NAD (NADH/NADPH) In order to be effective, it has to be dosed at the rate of 1.5 to 3.5 gm daily. A sustained release dosage form is available Adverse effects: o GI disturbances (erosion and ulceration) o red flush especially in the face and neck area o caused by vasodilation of capillaries MOA  dual plasma triglyceride and cholesterol lowering o decreases VLDL and LDL  decreases TG lipase in adipose tissue  increases lipoprotein lipase in adipose tissue Precaution  transient cutaneous flush  histamine release  potentiates BP effect of antihypertensives Rosuvastatin (Crestor) New statins: rosuvastatin (ZD4522)  Nicknamed” superstastin/ gorilla statin” because of its powerful effect on LDL cholesterol Ezetimibe (Zetia) This drug blocks the intestinal absorption of cholesterol. A dose of 10 mg qd leads to a 19% reduction of LDL; shows real promise in combo product with statins (ScheringPlough and Merck) INVESTIGATIONAL DRUGS Acyl-CoA Cholesterol Acyltransferase inhibitors (ACAT Inhibitors) –Orphan nuclear receptors: •LXR –“oxycholesterol receptor” ---enhanced cholesterol efflux •FXR –“bile acid receptor” ----decreased cholesterol conversion to bile salts •Avasemibe (CI-1011) *These drugs do not have a common name yet. Squalene synthase inhibitors     squalestin 1, a fermentation product derived from Phloma species (Coelomycetes) a potent inhibitor of squalene synthase produces a marked decrease in serum cholesterol and apoB levels may represent an alternative clinical therapy to hypercholesterolemia ║END OF TRANSCRIPTION.

Statin group of drugs – inhibitors for HMG-CoA reductase  These drugs are administered during mal-elevation of serum amino transferases: alanine aminotranferease (ALT) and aspartate aminotransferase (AST)
*AST also = SGOT (serum glutamic-oxaloacetic transaminase)  Normally found in liver, heart, muscle, kidney, and brain *ALT also = SGPT (serum glutamic-pyruvic transaminase)  Normally found largely in the liver Increased Serum amino transferases released in the blood are indicators of liver damage.

Precaution: •mild elevation of serum aminotransferase (should be measured at 2 to 4 month intervals) •minor increases in creatine kinase (myopathy, muscle pain and tenderness) •do not give during pregnancy

FIBRIC ACID DERIVATIVES Gemfibrosil (Lopid) MOA  stimulates lipoprotein lipase  interact with PPAR(peroxisome proliferator-activated receptors)  inhibits triglyceride lipolysis in adipose tissue  decreases FFA uptake by the liver  decreases hepatic VLDL/TG synthesis  slight cholesterol lowering effect Precautions  similar to clofibrate  myositis (voluntary muscle inflammation)  GI (indigestion, abdominal pain, diarrhea)  cholelithiasis (increased cholesterol biliary secretion) Half life: 1.1 hours Clofibrate (Atromid-S) Primary activity on triglycerides MOA:  ↑ lipoprotein lipase  ↓ VLDL  ↑ peroxisomal FFA oxidation  inhibits cholesterol biosynthesis  ↑ biliary secretion of cholesterol Ancillary (secondary effect):  decreases platelet adhesiveness/fibrinogenbad effect Precautions  enhances coumarin activity  renal/hepatic injury contraindication  pregnancy/nursing  cholelithiasis  most commonly reported ADR are GI related  liver malignancies (not very common; but has led to scant usage) Fenofibrate (Trecor)  A relatively new fibric acid derivative (micronized form of the drug)  Lowers plasma TG

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