Lipoprotein metabolism 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)

Composition and properties of human lipoproteins Lipoprotein class Density (g/mL) Diameter (nm) Protein % of dry wt Phospholipid % 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 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 b-2-glycoprotein 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, apoC-II/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, apoA-II, 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-B-100/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-a-hydroxylase

Conversion of cholyl-CoA to glycocholic acid

Conversion of cholyl CoA to taurocholic acid

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

Conversion of glycocholic acid to deoxycholic acid 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

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: incr. chylomicrons, reduced HDL, absence of lipoprotein lipase; deficiency of apo CII (hyperchylomironemia) Type II-A: raised LDL; decreased catabolism of LDL (receptor deficiency or polygenic) Type II-B: raised VLDL + LDL; often reduced HDL; increased production of VLDL + impaired LDL catabolism Type 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: raised VLDL; often reduced HDL; impaired VLDL catabolism; dietary indiscretion ( formerly known as hyperprebetalipoproteinemia) Type 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

Bile sequestering resins

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 1-bromodecane 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

More plant sterols

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

Synthetic statins

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

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: ancillary: 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 precautions half life: 1.1 hours stimulates lipoprotein lipase interact with PPARa (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) 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) 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 (Schering- Plough 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

ACAT Inhibitors

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

The End