CHOLESTEROL Amy M. Sharma PHM 142 REM University of Toronto Sept. 27 2012.

CHOLESTEROLAmy M. SharmaPHM 142 REMUniversity of TorontoSept. 27 2012

HDL• Stage 4 GOOD HDL : Reverse cholesterol transport and function

• 1. HDL is synthesized and secreted from the liver and the intestine. HDL contains 65% protein + free fatty acids, cholesterol, triacylglyceride and phospholipids.

• • 2 Function: • HDL picks up cholesterol released into the plasma from dying cells• and from membranes undergoing turnover and returns it to the liver

• 3. HDL contains cholesterol, cholesterol ester, phospholipid and Lecithin:Cholesterol Acyl Transferase (LCAT) - synthesised in the liver that catalyses :

• LECITHIN + CHOLESTEROL • LYSOLECITHIN + CHOLESTEROL ESTER

• LCAT is activated by apo-A1 and deficiency in LCAT means that HDL can’t take up cholesterol from tissue, therefore cholesterol and lecithin in tissue

Increasing HDL to decrease tissue Clt• Anacetrapib (Merck drug) NEW• Niacin best• Fibrate drugs• bile acid binding resins• Exercise , -3 fatty acids,red wine,orange juice,beans, soy,oat

bran• tr.fatty acids, high carbohydrate. decr. HDL

Cholesterol Synthesis • Essential molecule in many animals including humans• However not required from diet – all cells can synthesize it• Majority produced in the liver • Synthesis occurs in 4 stages:

• 1. Condensation of 3 acetate units to form 6-carbon intd, Mevalonate• 2. Mevalonate to activated isoprene units • 3. Polymerization of six 5-carbon isoprene units to form Squalene• 4. Squalene cyclization to form four rings of steroid nucleus followed by

more changes to form final cholesterol

Acetoacetyl CoA + Acetyl CoA + H2O

3-Hydroxy-3-methylglutaryl CoA (HMG CoA)

Mevalonate

synthase

NADPH

NADP+

rate-limitingstep

CHOLESTEROL or STATIN: feedbackinhibition bycholesterol

thiolase

Acetyl CoA+

*Acetoacetate

*-OH butyrate

NADH

*acetone (breath)

HMG CoA reductase

CH2 OH

CH2

C CH3OH

CH2

COO

Lyase

NAD+

COO

CH2

C CH3OH

CH2

C S CoA

O

Cholesterol Synthesis:

Step 1:Mitochondria

Committed and rate-limiting step:Reduction of HMG-CoA to Mevalonate

Cholesterol Synthesis:

Step 2:

Synthesis of isopentenyl pyrophosphate frommevalonate occurs in the PEROXISOMES

COO-

CH2

CHO CH3

CH2

CH2OH

COO-

CH2

CHO CH3

CH2

CH2O P O-

O-

O

COO-

CH2

CHO CH3

CH2

CH2O P O

O-

O

P

O-

O-

O

CH2

C CH3

CH2

CH2O P O

O-

O

P

O-

O-

O

ATP ADP

ATP

ADP + Pi + CO2

Mevalonate 5-phosphomevalonate

5-pyrophosphomevalonate

Isopentenylpyrophosphate

ATP

ADP

phosphotransferase

kinase

(pyrophosphate)

decarboxylase

Cholesterol Synthesis:

Step 3:

Synthesis of squalene occurs in the peroxisomesthen the endoplasmic reticulum

peroxisome

Squalene synthase DIMERIZATIONe.r.

peroxisome

H3C C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-

CH2

C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-CH2

CH

C

CH3

H3C

H3C C CH2

CH2

CH2

O P

O-

O

O

P

O

O-

O-

PPi

H3C C CH2

CH2

CH2

O P

O-

O

O

P

O

O-

O-

PPi

CH2

C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-CH2

CH

C

CH3

CH2

CH2

CH

C

CH3

H3C

Dimethylallyl pyrophosphate

Geranyl pyrophosphate

Farnesyl pyrophosphate

H3C C CH

CH3

CH2

H2C C

CH3

CH

CH2 CH2C

CH3

CH

H2C CH2

CH

C

CH3

CH3

2 2

Squalene

Farnesyl pyrophosphate + NADPH

NADP+ + 2PPi + H+

isopentenylpyrophosphate

isopentenylpyrophosphate

isomerase

prenyltransferase (head-to-tail)

prenyltransferase

CoQ, heme ADolichol-PPPrenylated proteins

Cholesterol Synthesis:

Step 4:

Synthesis of cholesterol occurs in the ER

CH3

CH3H3C

CH3

CH3

CH3

CH3

C

CH3

CH3H3C

CH3

CH3

CH3

OCH3

C

CH3

CH3H3C

CH3

CH3

H3C CH3

HO

CH3

Dehydrocholesterol

Squalene

er P450, O2, NADPH

cyclase

Squalene epoxide

Lanosterol

CHOLESTEROL

CH3

CH3H3C

HO

CH3

CH3

NADPHP450 reductase

sunlight

diet

Unsat. FA acyl CoAOC

O

R

Cholesterol ester

H2C

HO

CH3

H3C

H

CH3

H3C

Vitamin D3

+cholesterol acyltransferase(ACAT)

CH3

CH3H3C

HO

CH3

CH3

Drug therapy to decrease plasma CltStatins (HMG-CoA reductase inhibitors)• Inhibit cholesterol biosynthesis in liver to decrease plasma LDL

cholesterol and cut the risk of heart attacks and strokes by at least 33%

• HMG CoA reductase inhibitors can induce rhabdomyolysis (test for muscle/kidney damage)

• Lipitor, Zocor, Crestor lower risk of heart attack, death and stroke

OO-

OH

O

SCoA

OO-

OH

OH

O

O

Mevinolin (Fungal), a competitive inhibitor of HMG CoA reductase, resembles 3-hydroxy-3-methyl-glutaryl CoA, the substrate.

Mevinolin3-hydroxy-3-methyl-glutaryl CoA

cholesterol LDL receptors (induced) LDL uptake LDL risk of atherosclerosis

Endogenous synthesisof ubiquinone and cholesterol. Formationof mevalonate is therate limiting step insynthesis.

Isopentenyl-PP

Dimethylallyl-PP

Geranyl-PP

Farnesyl-PP

Squalene synthase

Squalene

Cholesterol

Decaprenyl-PP

TYROSINE

4-OH-benzoate

Decaprenyl-4-OHbenzoatetransf.

Decaprenyl-4-OH-benzoate

COENZYME Q

trans-prenyltransf.

But Statins may also decrease plasma ubiquinone antioxidant

Free Rad. Biol. Med. 29, 285-94 (2000)Lancet 356, 391-5 (2000)

HMG-CoA

Polyprenyl-PP

Dolichol N-glycosylates secretory

proteins = Export glycoproteins

ER, GolgiPe

roxis

om

e

Drug therapy to decrease plasma CltResins (hydrocarbon secretion)Bind bile acids to lower cholesterol• Cholesterol is reabsorbed from intestine by forming complexes with bile acids. Liver then replaces

bile acids by oxidizing cholesterol (catalysed by CYP7A).

Prescription therapeutic resins bind bile acids and prevent cholesterol reabsorption: problem of constipation, ↓absorption of fat sol. vitamin A,D,E,K• e.g. colestipol • cholestyramine• colesevelam

Nonprescription bulk forming laxatives (soluble fibres)• Psyllium husks (metamucil)• Ispaghula husks• Oat bran (-glucan binds bile acids )

• Action of bile acid binders• cholesterol excretion• hepatic cholesterol 7a hydroxylase (CYP7A) activity which oxidizes cholesterol to bile acids.

(feedback inhibitor is normally bile acids)

Drug therapy to decrease plasma CltHypolipidemic ie antihyperlipidemic fibrate drugs

• CLOFIBRATE: 2g/day• (also: Gemfibrozil)

Cl O C

CH3

CH3

C

O

O C2H5

1. ↑ lipoprotein lipase activity2. ↑ fatty acid oxidation by inducing PEROXISOMES serum triglycerides serum triglyceride-rich lipoprotein3.Antioxidant action prevent LDL oxidation

Perioxisomes• Peroxisomes (microbodies) • found in virtually all eukaryotic cells.• Involved in the catabolism of very long chain fatty acids,

branched chain fatty acids, D-amino acids, polyamines, and biosynthesis of plasmalogens, i.e. ether phospholipids critical for the normal function of mammalian brains and lungs.

• Major function: breakdown of very long chain fatty acids through beta-oxidation.

• converted to medium chain fatty acids to mitochondriacarbon dioxide and water.

PEROXISOME (numerous genetic diseases)Peroxisomal fatty acid b-oxidation forms H2O2 which is removed by catalase located in the peroxisomes. Medium-chain fatty acids (C8-18) prefer mitochondrial b-oxidation that doesn’t form H2O2.

Acylcarnitine

Fatty acyl CoA

SynthaseAcetyl CoA

O2 H2O2

oxidase*

heat

Shorter-chain fatty acid

MITOCHONDRIAb-oxidation

NADH+

Acetyl CoA

CholesterolCoQ10*

Bile acidsH2O + O2

catalase

H2O2 also formed by peroxisomal glycolate/glyoxylate oxidases,xanthine oxidase, uricase

* Peroxisomes induced by peroxisome proliferators via a cytosolic receptor (PPAR)e.g., hypolipidemic drugs, e.g., clofibrate; plasticizers, e.g., phthalate (DEHP); endogenous steroids formed by the adrenal glands e.g., dehydroepiandrosterone.

Ann Rev Biochem. 61, 157-97 (1992)Ann Rev Nutr. 14, 343-70 (1994)

Long chain or 3 or branched fatty acids

Drug therapy to decrease plasma CltNiacin (Vitamin B3) • Deficiency causes pellagra (rough photo-sensitive skin, dementia, etc) Flour now fortified with niacin; B 1 thiamine; B2 riboflavin

tryptophan niacin nicotinamide NAD NADP NADPHderivatives NAD, NADH, NAD+, and NADP play essential roles in energy

metabolism and DNA repair.

• Niacin 1.5-3g/day ↓ plasma LDL cholesterol & triglycerides; best for ↑ HDL But early hot flashes so use slow delivery formula

Rare hepatotoxicity or hyperglycemia

Drug therapy to decrease plasma CltBlocking intestinal cholesterol permease

Ezetimibe - drug that blocks cholesterol uptake by inhibiting intestinal sterol permease (packaged with a statin).

Natural therapy to decrease plasma CltPlant sterols/stanols

Ezetimibe - drug that blocks cholesterol uptake by inhibiting intestinal sterol permease (packaged with a statin).

• Sitosterol , Sitostanol• Clt lowering action of plant sterols on the diet

Plant sterol not absorbed by gut (2g/day) so inhibits gut absorption of cholesterol from diet. “functional margarine”)e.g. Becel pro-activ.in Loblaws

Natural therapy to decrease plasma Clt• Chitosan (shellfish exoskeleton) (LIBRACOL is polychitosamine:

amine groups bind cholesterol)• Policosanol (sugar cane wax or rice wax alcohol ie.

Octacosanol) CH3(CH2)26CH2OH)

DIETARY WAYS OF DECREASING THE ATHEROSCLEROSIS RISK:

1. cholesterol and saturated fatty acids plant stanols (2g/margarine day)

2. polyunsaturated fatty acids which cholesterol oxidation to bile acids

LDL catabolism cholesterol excretion into intestine

3. smoking, obesity, lack of exercise, low Ca2+

4. high HDL in premenopausal women protects but not after menopause.

Summary:

Summary:

Dietary mechanisms to decrease cholesterol are additive (e.g., use in patients resistant or intolerant to statins).

1. Decrease intestinal bile acids by binding them to viscous fibres, e.g., oats (b-glucan), barley, psyllium (metamucil), egg plant,ochra.

Glucan is also a soluble fibre & an antioxidant which prev. oxidn of PUFA & cholesterol. Amer.J.Clin.Nutrition 75(2002)834-9.

2. Competitive inhibition of cholesterol absorption from the gut, e.g., plant sterols margarine, almonds, flaxseed.

3. Increase LDL receptor-mediated LDL cholesterol uptake and degradation, e.g., soy proteins, soy milk.

4. Decrease oxidized LDL using antioxidants, e.g., almonds (Vit E), soy proteins (isoflavones).

Bile: function & importance Bile (gall) - fluid produced by the liver of most vertebrates • Composed of water, bile salts, mucus and pigments, fats,

inorganic salts and cholesterol• Main functions:

• Essential in the process of digestion of lipids from the small intestine • For protection of small intestines from oxidative damage• For excretion of endogenous xenobiotic compounds

Also:• Aids in absorption of fat-soluble vitamins A, D, E, K• Bile salts are also bactericidal (protect from microbial products

in foodstuff)

Bile: dysfunction & implications • Drugs that slow/block bile flow from the liver to the gallbladder

& gut may cause liver failure. e.g. chlorpromazine,prochlorperazine, penicillin

ampicillin,estradiol, nitrofurantoin, sulindac.

• Symptoms include dark urine, pale stool, jaundice, fever/rash persistent itching.

MUST discontinue the drug.

Bile: potential for toxicity • Bile acids are cytotoxic to hepatocytes (can cause liver failure

via free radical formation).• Mechanism:• Reduces Fe3+ which reduces H2O2 to form hydroxyl radicals and

reactive oxygen species (ROS)• Radicals oxidize nucleic acids, proteins, and unsaturated lipids

to form other radicals.• BUT…• Antioxidants & blockers of mitochondrial permeability

transition prevent apoptosis

Cholestasis defined

Cholestasis - any condition in which the flow of bile from the liver is blocked.

i.e. where bile cannot flow from the liver to duodenum

• Obstructive cholestasis - mechanical blockage in the duct system such i.e. as a results of gallstone or malignancy

• Metabolic cholestasis - disturbance in bile formation due to genetic defects or as an adverse effect of a drug

Extra-hepatic cholestasisCholestasis occurring outside the liver

• Caused by blockage of bile duct or ducts• Possible causes:- bile duct tumors - Pancreatic tumor or pseudocyst- Cysts- Narrowing of the bile duct- Stones- Pancreatitis- Pressure on an organ due to tumor or nearby mass

Intra-hepatic cholestasisCholestasis occurring within the liver

• Caused by significant blockage of small ducts or by disorders, such as hepatitis, that impair the body's ability to eliminate bile

• Possible causes:- Alcoholic liver disease- Amyloidosis- Bacterial abscess- Lymphoma

• Drugs- Primary biliary cirrhosis- Tuberculosis- Sepsis,- Viral hepatitis

Biochemistry. 31, 4737-49, (1992)

G) BILE ACID SYNTHESIS BY LIVEREndoplasmic reticulum (except CYP27)

COO-OH

HOHOH

COO-OH

HOHOH

Cholesterol

cholesterol 7 hydroxylaseCYP7A

er, CYP12

erside chainoxidation

erthioesterase (ligase)acetylCoA

CHOLIC ACID(Bile acid, "detergent")

LIVER CIRRHOSIS AND DEATH

CYP27mitoch.

CYP7Ber

side chain oxidation

CHENODEOXYCHOLIC ACID

er, NADPHinhibited by bile acids or fasting; induced by cholesterol or thyroxine

er

Cholyl CoA

3

er, UGT

UDPGA

OGluc3

7

CHOLESTASISTOXICITY

CYTOSOL

blocked bile duct

Hepatocyte death

28

Then efflux into bile and stored in gall bladder.Then released by bile duct into upper-small intestine (ileum).Then metabolised (deconjugation (CO2), dehydroxylation) by anaerobic bacteria of colon to deoxycholate, lithocholate, urodeoxycholate.Then actively reabsorbed and recirculates via liver 8 times / day.

Ga) BILE ACID SYNTHESIS BY LIVER (cont)

OHH

OH

OH NH

O

taurine

Cholyl CoA

N-acetyltransferase+ taurine

CYTOSOL

Glycocholate

SULT2A1

O3SO

sulfotransferase(SULT2A1)+ PAPS

3 TAUROCHOLATE

N-acetyltransferase+ glycine

sulfate

Cholic Acid

NHglycine

Endoplasmic reticulum