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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 Density Diameter Protein % Phospholi Triacylglycerol
class (g/mL) (nm) of dry wt pid % % of dry wt
HDL 1.063-1.21 5 – 15 33 29 8

LDL 1.019 – 18 – 28 25 21 4
1.063

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-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-α-
hydroxylase
NADPH + H+ NADP

HO 7a-hydroxylase HO OH

cholesterol 7a-hydroxycholesterol
12a-hydroxylase
O2; NADPH + H+ O2
2 CoA-SH NADPH + H+
2 CoA-SH
OH

C S CoA

C S CoA

O
HO OH
H

cholyl-CoA
HO OH
H
chenodeoxycholyl- CoA
Conversion of cholyl-CoA to
glycocholic acid

ΟΗ

C S CoA

O
glycine ΟΗ
CoA-SH
C N CH2 COOH

HO O
ΟΗ
H
cholyl-CoA

HO ΟΗ
H

glycocholic acid
ΟΗ
Conversion of
C S CoA
cholyl CoA to
O taurocholic acid

HO ΟΗ
H
cholyl-CoA
taurine

CoA-SH

ΟΗ H

C Ν CH2 CH2 SO3H

HO ΟΗ
H

taurocholic acid (primary bile acid)


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

COO COO
H3N C H + O2 + O2 - CO2
H3N C H H3N CH2
CH2 CH2 CH2
SH SO2 SO3
cysteine cysteine sulfinate taurine
C S CoA

tauro- and glyco-chenodeoxycholic acids


(primary bile acids)

HO OH
H deconjugation +
chenodeoxycholyl- CoA 7a-dehydrxylation
(catalyzed by microbial
enzymes)

COOH

HO
H

lithocholic acid (secondary bile acid)


ΟΗ

C N CH2 COOH

HO ΟΗ
H
glycocholic acid (primary bile acid)

deconjugation +
7α -dehydroxylation
Conversion of glycocholic
( catalyzed by microbial
acid to deoxycholic acid enzymes)

ΟΗ

COOH

HO Deoxycholic acid (secondary bile acid


H
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
CH3
HO
CH3
CH3
COOH
COOH
12 CH3 H
CH3 H

H H
H H
3 7
HO OH
H
H
CHOLIC ACID
CHOLANIC ACID

CH3
CH3 CH3
COOH
CH3
COOH CH3 H
CH3 H
H H
H H HO OH
H
HO OH
H
CHENODEOXYCHOLIC ACID (CHENODIOL) URSODEOXYCHOLIC ACID (URSODIOL)
(ACTIGALL)
(CHENIX)
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 HN
HN . n HCl . n HCl . n HCl
H H2 H H2N
C C C
. n HCl (CH2)6N(CH3)3 (CH2)9-CH3

OH
H2
C CH

H2C N(CH3)3

(CH2)6N(CH3)3 (CH2)9CH3
n . n HCl
H2N . n HCl . n HCl
HN . n HCl
HN
HN
CHOLESTYRAMINE

COLESEVELAM
Bile sequestering resins

HN CH2 CH2 N CH2 CH2 N CH2 CH2 N CH2 CH2 NH

CH2 CH2 CH2 CH2 CH2

H COH H COH H COH H COH COH

CH2 CH2 CH2 CH2 CH2

-CH2CH2 N CH2 CH2 N H N CH2 CH2 N H N CH2-CH2-

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 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 • methyldopa
level) • nicotinic acid
• aspirin
• penicillin G
• clindamycin
• propranolol
• clofibrate
• • tetracycline
furosemide
• glipzide • thiazide diuretics
• tolbutamide • digoxin
• phenytoin • hydrocortisone
• imipramine • phosphate supplements
PLANT STEROLS
CH3
CH3
H 3C
H 3C
CH3 CH3
CH3
CH3
CH2CH3 CH2CH3

CH3 CH3

HO HO

STIGMASTANOL BETA SITOSTEROL


CH3 CH3
H 3C H 3C
CH3 CH3
CH3 CH3
CH2CH3 CH3
CH3 CH3

HO HO
STIGMASTEROL CAMPESTROL

CH3

H3C
CH3
CH3

CH3
CH3

HO
More plant sterols
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

O - CoASH OH

H H

SCoA

NADPH NADPH

CH3 CH3
OH
COOH OH
COOH

OH O

H
SCoA H
O HO O

O O
O CH3
H

H3C H H
CH3
H3C
CH3

H
H3C
Lovastatin
CH3
-10
Ki = 10

HO O

HO
COOH
O

O
H
P
O-

F
HO Cl

N
Et
Cl

Ph
Ki = 10-10 Ki = 10 -11
HO O
HO O

O
O O
O

O
H3C H H O
CH3 H3C H H
CH3

H3C
MEVASTATIN
LOVASTATIN (MEVACOR)

HO O
HO
COOH
O
OH
O
O

O
O
H3C CH3 H
H3C H H
CH3
CH3

HO
SIMVASTATIN (ZOCOR)
PRAVASTATIN (PRAVACHOL)
Synthetic statins
HO
CO2Na

F
OH

OH OH
F
CO2Na

CH(CH3)2
N
H3CO

(H3C)2HC N CH(CH3)2

CERIVASTATIN
FLUVASTATIN
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 CH3
O (CH2)3 C COOH Cl O C COOEt

CH3 CH3
H3C
GEMFIBROSIL (LOPID) CLOFIBRATE (ATROMID-S)

Cl

CH3

iPrO2C C O

CH3 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
A water soluble vitamin of the B family;
nicotinamide is not active
N
Once converted to the amide, it is
NICOTINIC ACID (NIACIN)
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

F
O S 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

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 (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
CF3
OH

O O CF3
S
N

CF3

CO2H
T0901317 -- LXR agonist

LG268 -- RXR agonist


ACAT Inhibitors
H3C CH3 H3C CH3
CH H CH
O H2
O N C
S C
O
CH3 O CH3
H3C
CH CH CH

CH3 CH3 CH3

AVASEMIBE (CI-1011)
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