Lipoproteins

PSC 3110 Fall 2004

Lipoprotein metabolism By Henry Wormser, Ph.D. Professor of Medicinal Chemistry

Lipoproteins • particles found in plasma that transport lipids including cholesterol • lipoprotein classes • chylomicrons: take lipids from small intestine through lymph cells • very low density lipoproteins (VLDL) • intermediate density lipoproteins (IDL) • low density lipoproteins (LDL) • high density lipoproteins (HDL)

Lipoprotein class

Density (g/mL)

Diameter (nm)

Protein % of dry wt

Phospholi pid %

Triacylglycerol % of dry wt

HDL

1.063-1.21

5 – 15

33

29

8

LDL

1.019 – 1.063

18 – 28

25

21

4

IDL

1.006-1.019

25 - 50

18

22

31

VLDL

0.95 – 1.006

30 - 80

10

18

50

chylomicrons

< 0.95

100 - 500

1-2

7

84

Composition and properties of human lipoproteins most proteins have densities of about 1.3 – 1.4 g/mL and lipid aggregates usually have densities of about 0.8 g/mL

Lipoprotein structure

LDL molecule

The apolipoproteins • major components of lipoproteins • often referred to as aproteins • classified by alphabetical designation (A thru E) • the use of roman numeral suffix describes the order in which the apolipoprotein emerge from a chromatographic column • responsible for recognition of particle by receptors

HELICAL WHEEL PROJECTION OF A PORTION OF APOLIPOPROTEIN A-I

LIPOPROTEINS • spherical particles with a hydrophobic core (TG and esterified cholesterol) • apolipoproteins on the surface • large: apoB (b-48 and B-100) atherogenic • smaller: apoA-I, apoC-II, apoE

• classified on the basis of density and electrophoretic mobility (VLDL; LDL; IDL;HDL; Lp(a)

Apoproteins of human lipoproteins • A-I (28,300)- principal protein in HDL • 90 –120 mg% in plasma; activates LCAT

• A-II (8,700) – occurs as dimer mainly in HDL • 30 – 50 mg %; enhances hepatic lipase activity

• B-48 (240,000) – found only in chylomicron – <5 mg %; derived from apo-B-100 gene by RNA editing; lacks the LDL receptor-binding domain of apo-B-100

• B-100 (500,000) – principal protein in LDL • 80 –100 mg %; binds to LDL receptor

Apoproteins of human lipoproteins • C-I (7,000) – found in chylomicron, VLDL, HDL • 4 – 7 mg %; may also activate LCAT

• C-II (8,800) - found in chylomicron, VLDL, HDL • 3 – 8 mg %; activates lipoprotein lipase

• C-III (8,800) - found in chylomicron, VLDL, IDL, HDL • 8 15 mg %; inhibits lipoprotein lipase

• D (32,500) - found in HDL • 8 – 10 mg %; also called cholesterol ester transfer protein (CETP)

• E (34,100) - found in chylomicron, VLDL, IDL HDL • 3 – 6 mg %; binds to LDL receptor

• H (50,000) – found in chylomicron; also known as β-2glycoprotein I (involved in TG metabolism)

Major lipoprotein classes • Chylomicrons (derived from diet) – – – –

density <<1.006 diameter 80 - 500 nm dietary triglycerides apoB-48, apoA-I, apoA-II, apoA-IV, apoCII/C-III, apoE – remains at origin in electrophoretic field

Chylomicron • formed through extrusion of resynthesized triglycerides from the mucosal cells into the intestinal lacteals • flow through the thoracic ducts into the suclavian veins • degraded to remnants by the action of lipoprotein lipase (LpL) which is located on capillary endothelial cell surface • remnants are taken up by liver parenchymal cells due to apoE-III and apoE-IV isoform recognition sites

Chylomicron metabolism

Major lipoprotein classes • VLDL – – – – – –

density >1.006 diameter 30 - 80nm endogenous triglycerides apoB-100, apoE, apoC-II/C-III prebeta in electrophoresis formed in the liver as nascent VLDL (contains only triglycerides, apoE and apoB)

VLDL • nascent VLDLs then interact with HDL to generate mature VLDLs (with added cholesterol, apoC-II and apoC-III) • mature VLDLs are acted upon by LpL to generate VLDL remnants (IDL) • IDL are further degraded by hepatic triglyceride lipase (HTGL) to generate LDLs

VLDL metabolism

Major lipoprotein classes • IDL (intermediate density lipoproteins) – – – – –

density: 1.006 - 1.019 diameter: 25 - 35nm cholesteryl esters and triglycerides apoB-100, apoE, apoC-II/C-III slow pre-beta

Major lipoprotein classes • LDL (low density lipoproteins) – – – – – –

density: 1.019 - 1.063 diameter: 18-25nm cholesteryl esters apoB-100 beta (electrophoresis) < 130 LDL cholesterol is desirable, 130-159 is borderline high and >160 is high

Major lipoprotein classes • HDL (high density lipoproteins) – – – – –

density: 1.063-1.210 diameter: 5-12nm cholesteryl esters and phospholipids apoA-I, apoA-II, apoC-II/C-III and apoE alpha (electrophoresis)

HDLs • Several subfamilies exist – Discoidal HDL : • contains cholesterol, phospholipid, apoA-I, apoAII, apoE and is disc shaped; • it is formed in liver and intestine • It interacts with chylomicra remnants and lecithin-cholesterol acyl transferase (LCAT) to form HDL3

HDLs – HDL3 • composed of cholesterol, cholesterol ester, phospholipid and apoA and apoE • interacts with the cell plasma membranes to remove free cholesterol • reaction with LCAT converts HDL3 to HDL2a (an HDL with a high apoE and cholesterol ester content) • cholesterol ester-rich HDL2a is then converted to triglyceride-rich HDL2b by concomitant transfer of HDL cholesterol esters to VLDL and VLDL triglycerides to HDL

HDL metabolism

Functions of HDL • • • •

transfers proteins to other lipoproteins picks up lipids from other lipoproteins picks up cholesterol from cell membranes converts cholesterol to cholesterol esters via the LCAT reaction • transfers cholesterol esters to other lipoproteins, which transport them to the liver (referred to as “reverse cholesterol transport)

Lipoproteins (a)- Lp(a) • another atherogenic family of lipoproteins(at least 19 different alleles) • they consist of LDL and a protein designated as (a) • the apoA is covalently linked to apoB-100 by a disulfide linkage • unusual in that it contains a kringle protein motif/domain (tri-looped structure with 3 intramolecular disulfide bonds – resembling a Danish pretzel) • high risk association with premature coronary artery disease and stroke

Cholesterol and lipid transport by lipoproteins

Cholesterol and lipid transport by lipoproteins

The LDL receptor • characterized by Michael Brown and Joseph Goldstein (Nobel prize winners in 1985) • based on work on familial hypercholesterolemia • receptor also called B/E receptor because of its ability to recognize particles containing both apos B and E • activity occurs mainly in the liver • receptor recognizes apo E more readily than apo B-100

Representation of the LDL receptor (839 aa) extracellular domain is responsible for apo-B100/apo-E binding intracellular domain is responsible for clustering of LDL receptors into coated pit region of plasma membrane

Cholesterol sources, biosynthesis and degradation • diet – only found in animal fat

• biosynthesis – primarily synthesized in the liver from acetyl CoA – biosynthesis is inhibited by LDL uptake by the liver

• degradation – only occurs in the liver – cholesterol is converted to bile acids

Biosynthesis of cholesterol - synthesis of acetoacetyl CoA

Biosynthesis of cholesterol - synthesis of mevalonate

rate-limiting step and step subject to inhibition by statins

Biosynthesis of cholesterol -synthesis of isopentenyl -pyrophosphate

A monoterpene

Synthesis of farnesyl pyroposphate

Biosynthesis of cholesterol - synthesis of squalene

a sesquiterpene

a triterpene

Synthesis of squalene

Biosynthesis of cholesterol - synthesis of lanosterol

the allylamine antifungals interfere with the epoxidation step (naftidine, terfinabine)

Formation of the sterol ring system

Biosynthesis of cholesterol The demethylation of lanosterol is also a useful step for drug design – i.e. azole antifungals

ACAT inhibitors act here

Biosynthesis summary

Bile acids from cholesterol • formed from cholesterol in the liver • stored in the gall bladder in bile as bile salts (sodium and potassium) • utilized during digestion of fats and other lipid substances (act as detergents) • rate limiting step is the conversion of cholesterol to 7-alpha cholesterol by 7-αhydroxylase

NADPH + H+ NADP HO

HO

7a-hydroxylase

OH 7a-hydroxycholesterol

cholesterol 12a-hydroxylase O2; NADPH + H+ 2 CoA-SH

O2 NADPH + H+ 2 CoA-SH

OH C

S

CoA

O C HO

O

OH H cholyl-CoA HO

OH H chenodeoxycholyl- CoA

S

CoA

Conversion of cholyl-CoA to glycocholic acid ΟΗ C O

HO H cholyl-CoA

S

CoA

glycine

ΟΗ CoA-SH

C O

ΟΗ

HO H

ΟΗ

glycocholic acid

N

CH2 COOH

ΟΗ C

S

CoA

O

HO H cholyl-CoA

Conversion of cholyl CoA to taurocholic acid

ΟΗ taurine

CoA-SH H

ΟΗ C O

HO H

ΟΗ

taurocholic acid (primary bile acid)

Ν

CH2 CH2 SO3H

Taurine • Taurine is formed by the decarboxylation of cysteic acid, which in turn is made by oxidation of cysteine COO H3N

C

H

COO + O2

H3N

C

H

+ O2

- CO2

H3N

CH2

CH2

CH2

CH2

SH

SO2

SO3

cysteine

cysteine sulfinate

taurine

C

S

CoA

O

tauro- and glyco-chenodeoxycholic acids (primary bile acids) HO

OH H

deconjugation + 7a-dehydrxylation (catalyzed by microbial enzymes)

chenodeoxycholyl- CoA

COOH

HO H lithocholic acid (secondary bile acid)

ΟΗ C

N

CH2 COOH

O

HO

ΟΗ

H

glycocholic acid (primary bile acid) deconjugation + 7α -dehydroxylation ( catalyzed by microbial enzymes)

Conversion of glycocholic acid to deoxycholic acid ΟΗ

COOH

HO H

Deoxycholic acid (secondary bile acid

Bile acids • cholic acid is the bile acid found in the largest amount in bile • cholic acid and chenodeoxycholic acid are referred to as primary bile acids • 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% Taurodeoxycholate – 8% Various lithocholate – 4%

Bile acids • fat digestion products are absorbed in the first 100 cm of small intestine • 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%) • this is known as the enterohepatic circulation • less than 500 mg a day escapes reabsorption and is excreted in the feces

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 gives the molecule its hydrophilic face • the steroid ring with its puckered plane provides the hydrophobic face

Function of bile salts • emulsification of fats due to detergent activity • aid in the absorption of fat-soluble vitamins (especially vitamin K) • accelerate the action of pancreatic lipase • have choleretic action –stimulate the liver to secrete bile • stimulate intestinal motility • keep cholesterol in solution (as micelles)

Mixed micelle formed by bile salts, triacylglycerols andf pancreatic lipase

BILE ACIDS CH3 HO CH3

CH3 CH3 12 H

CH3 H

3

COOH

COOH H

CH3

7

H

H

H HO

OH

H

H

CHOLIC ACID

CHOLANIC ACID

CH3 CH3

CH3 COOH

CH3 COOH

H

CH3

H

CH3

H H HO H

H OH

CHENODEOXYCHOLIC ACID (CHENODIOL) (CHENIX)

HO

H OH

H

URSODEOXYCHOLIC ACID (URSODIOL) (ACTIGALL)

GALLSTONE THERAPEUTIC AGENTS • chenodeoxycholic acid (chenodiol; Chenix) • ursodeoxycholic acid (ursodiol; Actigall) • MAO: – reduce hepatic secretion of cholesterol into bile – inhibition of HMGCoA reductase: inhibit cholesterol biosynthesis – increase 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 • heart disease is the leading cause of death • caused by the deposition of cholesteryl esters on the walls of arteries • atherosclerosis is correlated with high LDL and low HDL

Photograph of an arterial plaque

Frederickson -WHO classification Type I: I incr. chylomicrons, reduced HDL, absence of lipoprotein lipase; deficiency of apo CII (hyperchylomironemia) Type II-A: II-A raised LDL; decreased catabolism of LDL (receptor deficiency or polygenic) Type II-B: II-B raised VLDL + LDL; often reduced HDL; increased production of VLDL + impaired LDL catabolism Type III: III raised IDL (dysbetalipoproteinemia); abnormal apolipoprotein E; impaired catabolism of IDL; elevated cholesterol and triglycerides (formerly known as broad beta disease)

Frederickson -WHO classification Type IV: IV raised VLDL; often reduced HDL; impaired VLDL catabolism; dietary indiscretion ( formerly known as hyperprebetalipoproteinemia) Type V: V raised chylomicrons + VLDL; reduced HDL; reduced 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 • smoking • 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-alpha hydroxylase

Drug Classification • systemic/non-sytemic • cholesterol lowering agents – – – – –

bile acid sequestrants sitosterols* probucol* d-thyroxin* HMG Co-A reductase inhibitors

* No longer available commercially in the U.S

Drug Classification • mixed activity (nicotinic acid) • triglyceride lowering – clofibrate (Atromid-S) – gemfibrosil (Lopid) – fenofibrate (Tricor)

Bile sequestering resins HN H C

H2 C

H2 C

H2N

H C

HN

. n HCl

. n HCl

. n HCl (CH2)6N(CH3)3

HN

. n HCl

(CH2)9-CH3

OH CH H2C

N(CH3)3

n H2N

CHOLESTYRAMINE

(CH2)6N(CH3)3

. n HCl HN

. n HCl HN

COLESEVELAM

. n HCl

(CH2)9CH3

HN

. n HCl

Bile sequestering resins HN

CH2 CH2 N

CH2 H

-CH2CH2

CH2

COH CH2 N

CH2 CH2 N

H

COH

CH2 H

CH2 CH2 CH2 N

CH2 CH2 N

COH CH2

H

N

H

CH2 CH2 NH

CH2

CH2

COH

COH

CH2

CH2

CH2 CH2 N

H

N

CH2-CH2n

COLESTIPOL (COLESTID)

Bile acid sequestrants • po, safest, non systemic • bind to bile acids and inhibit reabsorption • increase 7-alpha hydroxylase activity leading to cholesterol degradation • decreases plasma LDL • problems: – abdominal discomfort, bloating, constipation – decreases drug absorption; wait 4 hrs after administration of BAS to give drugs

Colesevelam (WelChol) • polyalkylamine hydrochloride) cross linked with epichlorohydrin and alkylated with 1bromodecane and (6-bromohexyl) trimethylammonium bromide • available as a 625 mg tablet • same mechanism of action as colestipol and cholestyramine

Bile sequestering resins • 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

PLANT STEROLS CH3

CH3 H 3C

H 3C CH3

CH3

CH3

CH2CH3 CH3

CH3

HO

HO STIGMASTANOL

BETA SITOSTEROL

CH3 CH2CH3

CH3 H 3C

CH3 H 3C

CH3

CH3

CH3

CH2CH3

CH3

CH3 CH3

CH3

HO

HO CAMPESTROL

STIGMASTEROL CH3 H3C

CH3

CH3 CH3

CH3

More plant sterols

HO CH3 ALPHA1-SITOSTEROL

HMG CoA reductase • 3 different regulatory mechanisms are involved: • covalent modification: phosphorylation by cAMP-dependent protein kinases inactivate the reductase. This inactivation can be reversed by 2 specific phosphatases • degradation of the enzyme – half life of 3 hours and the half-life depends on cholesterol levels • gene expression: cholesterol levels control the amount of mRNA

2 NADPH

2 NADP

CH3

CH3

OH

OH

COOH

COOH

- CoASH

O

OH H

H

SCoA

NADPH

NADPH

CH3 OH

CH3 COOH

OH

COOH

OH

O

H SCoA

H

HO

O

O

O

O CH3

O

H

H3C

H

H

CH3

CH3

H3C

H H3C

Lovastatin CH3

Ki = 10 HO

-10

O HO COOH

O

O

H

P O-

F HO

Cl

N Cl

Ki = 10-10

Et Ph

Ki = 10

-11

HO

O HO

O

O O

O O O

H

H3C

H

O

CH3

H

H3C

H CH3

H3C

MEVASTATIN

LOVASTATIN (MEVACOR)

HO

O

HO COOH

O

OH

O

O O

H3C

CH3

O

H CH3

SIMVASTATIN (ZOCOR)

H3C

H

H CH3

HO PRAVASTATIN (PRAVACHOL)

Synthetic statins HO CO2Na F

OH

OH

OH

F CO2Na

N

CH(CH3)2 H3CO

(H3C)2HC

FLUVASTATIN

N

CERIVASTATIN

CH(CH3)2

HMG CoA reductase inhibitors • 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

Selected hypolipidemic products

FIBRIC ACID DERIVATIVES CH3 CH3 O

(CH2)3 C

CH3 COOH

Cl

O

CH3 H3C

CLOFIBRATE (ATROMID-S)

Cl

CH3 C

O

CH3

COOEt

CH3

GEMFIBROSIL (LOPID)

iPrO2C

C

O

FENOFIBRATE (TRICOR)

Clofibrate (Atromid-S) • 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)

CLOFIBRATE • Primary activity on triglycerides • MOA: • • • • •

increases lipoprotein lipase lowers VLDL increases peroxisomal FFA oxidation inhibits cholesterol biosynthesis increases biliary secretion of cholesterol

• ancillary: • decreases platelet adhesiveness/fibrinogen

Gemfibrosil (Lopid) • MOA • stimulates lipoprotein lipase • interact with PPARα (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

Fenofibrate (Tricor) • a relatively new fibric acid derivative (micronized form of the drug) • lowers plasma TG – 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

Now also available as a 200 mg tablet

NICOTINIC ACID (Niacin) COOH

N NICOTINIC ACID (NIACIN)

A water soluble vitamin of the B family; nicotinamide is not active Once converted to the amide, it is incorporated into NAD

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: GI disturbances (erosion and ulceration) red flush especially in the face and neck area caused by vasodilation of capillaries

Nicotinic acid (Niacin) • MOA • dual plasma triglyceride and cholesterol lowering – 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

Advicor® • niacin-extended-release and lovastatin tablets • reduces LDL-C, TC, TG and increases HDL-C • available as 500/20, 750/20 and 100/20 mg tablets

Rosuvastatin (Crestor) • New statins: rosuvastatin (ZD4522) CH3 O

S

F

O

N H

COOH iPr

OH

OH

nicknamed” superstastin/ gorilla statin” because of its powerful effect on LDL cholesterol

Ezetimibe (Zetia) OH OH

N F

O

F

EZETIMIBE

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 • acylCoA: cholesterol acyltransferase inhibitors – Orphan nuclear receptors: • LXR – “oxycholesterol receptor” --- enhanced cholesterol efflux • FXR – “bile acid receptor” ---- decreased cholesterol conversion to bile salts

ACAT Inhibitors CF3 OH

O

O

CF3

S N

CF3 CO2H

T0901317 -- LXR agonist LG268 -- RXR agonist

ACAT Inhibitors H3C

CH3

H3C

CH

CH

H O O

H2 C

N S

CH3

C

O CH3 CH CH3

O H3C

CH3 CH CH3

AVASEMIBE (CI-1011)

CH CH3

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