LIPID PROFILE IN HEALTH AND DISEASE

Guide: Dr. M.M. Khandekar

HEADINGS

Overview Lipoproteins, apolipoproteins and related proteins Classification Lipid transport and Lipoprotein Metabolism Lipid and lipoprotein measurement NCEP guidelines Lipids, lipoproteins and disease Recent issues Management

OVERVIEW
Lipids are ubiquitous in body tissue and have important role in virtually all aspects of life :Hormones or hormone precursors Aiding in digestion Providing energy storage and metabolic fuels Functional &structural component of membranes Insulation

Lipids
Triacylglycerols (TAG) Phospholipids (PL) Free cholesterol (CH) and cholesterol esters (CHE) Free fatty acids (FFA) Cholesterol and triglycerides are the most important lipids in study and management of coronary heart disease risk

lipids
Soluble in nonpolar organic solvents but relatively insoluble in polar solvents such as water Cholesterol and triglycerides travel in plasma not as free-floating molecules ,but as part of water soluble macromolecules called lipoproteins

LIPOPROTEIN STRUCTURE

CLASSIFICATION
Based on Density Gofman et al in 1954 Electrophoretic mobility Frederickson et al in 1967 Apolipoproteins Alaupovic et al in 1972

Classification of lipoproteins

PLASMA LIPOPROTEINS

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

CHYLOMICRONS
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

ISD

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 lecithincholesterol acyl transferase (LCAT) to form HDL3

 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

APOLIPOPROTEINS
Protein component of Lp complex Apo-A to H & (a) various types in various lipoproteins control their metabolic fate functions: Activate enzymes in Lp metabolic pathway Facilitates Lp uptake through specific cell surface receptors Participate in the exchange of lipids between particles Maintain structural integrity of Lp complex

APOLIPOPROTEINS
Chilomicrons: apoB-48, A, C, E VLDL: apoB-100, C, E LDL: apoB-100 HDL: apoA, C, D, E Lp(a) :apo(a), apoB-100

All particles containing apoB (apoB-100 or apoB-48) are atherogenic

Apo B-100
-Single translation product -4536 a.a -Synth. in liver -Part of VLDL,LDL

Apo B-48
-Post transcriptional modification of apoB100 -2512 a.a -Synth. in intestine -Part of chylomicrons

LIPOPROTEIN METABOLISM
Pathways are complex and divided into
Exogenous pathway Endogenous pathway

LDL-receptor pathway
HDL-reverse cholesterol transfer pathway

Lipids are insoluble (does not dissolve) in water but are soluble (dissolves) in alcohol and other solvents. When dietary fats are digested and absorbed into the small intestine, they eventually re-form into triglycerides, which are then packaged into lipoproteins. Dietary fats, including cholesterol, are absorbed from the small intestines and transported into the liver by lipoproteins called chylomicrons. Chylomicrons are large droplets of lipids with a thin shell of phospholipids, cholesterol, and protein. Once chylomicrons enter the bloodstream, an enzyme called lipoprotein lipase breaks down the triglycerides into fatty acid and glycerol. After a 12- to 14-hour fast, chylomicrons are absent from the bloodstream. Thus, individuals who are having a lipid profile done should fast overnight to ensure that chylomicrons have been cleared. The liver removes the chylomicron fragments, and the cholesterol is repackaged for transport in the blood in very low-density lipoproteins (VLDLs), which eventually turn into low-density lipoproteins (LDL). LDL cholesterol (LDL-C)the "bad cholesterol" consists mainly of cholesterol. Most LDL particles are absorbed from the bloodstream by receptor cells in the liver. Cholesterol is then transported throughout the cells. Diets high in saturated fats and cholesterol decrease the uptake of LDL particles by the liver. LDL particles are also removed from the bloodstream by scavenger cells, or macrophages, which are white blood cells that bury themselves in blood vessels such as arteries. Scavenger cells prevent cholesterol from reentering the bloodstream, but they deposit the cholesterol in the inner walls of blood vessels, eventually leading to the development of plaque. High-density lipoproteins (HDLs) are a separate group of lipoproteins that contain more protein and less cholesterol than LDL. HDL cholesterol (HDL-C) is also called "good cholesterol." HDL is produced primarily in the liver and intestine, and it travels in the bloodstream, picks up cholesterol, and gives the cholesterol to other lipoproteins for transport back to the liver.

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

NCEP Classification (adults) mg/dl

Category
Desired N/A desir Borderline

LDL
<100 100-129 130-159

Total chol.
<200

HDL
<40

TG
<150

200-239

150-199

High
V.High

160-189
190

240

60

200-499
500

Pathway of Exogenous Cholesterol Metabolism

INTESTINAL LUMEN BILIARY TRACT Bile acids Free cholesterol
Synthesis CE

Plaque formation

Remnants

DIET
Cholesterol

Unstirred water layer Brush Border

BLOOD
ENTEROCYTE
FC biosynthesis

Micelles

FC

ACAT

Cholesteryl Ester (CE)

CE

Chylomicrons

LYMPH

DISORDERS OF LIPOPROTEIN METABOLISM

PRIMARY
Isolated hypercholesterolemia Familial hypercholesterolemia Familial defective apo-B100 Polygenic hypercholesterolemia Isolated hypertriglyceridemia Familial hypertriglyceridemia Deficient LPL activity Deficient apo CII Hypercholesterolemia & hypertriglyceridemia Combined hyperlipidemia Dysbetalipoproteinemia

SECONDARY CAUSES

DIABETES MELLITUS
Type 1 diabetes mellitus (DM) is associated with overproduction of VLDL and LPL deficiency secondary to insulinopenia. Improves with diet control of the diabetes In type 2 DM,insulin resistance & obesity combine to cause mild to moderate hypertriglyceridemia and low HDL cholesterol levels and elevated small, dense LDL Therapy should not be delayed in patients with type 2 DM, as they are at increased risk for CHD Treatment goal is to reduce their LDL to <2.6 mmol/L (<100 mg/dL)

HYPOTHYROIDISM
Second to DM as a cause of secondary hyperlipidemia Levels of LDL cholesterol is elevated even in subclinical disease where TSH levels are elevated but other thyroid function tests are normal Hypertriglyceridemia can occur if obesity is present Also associated with increased levels of HDL cholesterol due to reduced HTGL activity

FREDRICKSON CLASSIFICATION
Type I- Extremely elevated TG Type IIa- Elevated LDL Type IIb- Elevated LDL & VLDL Type III- Elevated Choles.TG;presence of -VLDL Type IV- Elevated VLDL Type V- Elevated VLDL & presence of CM

Contd.

FH
Bad prognosis if:
Decreased HDL or Increased TG or Increased Lp(a) or Co inheritance of apo E 4 genotype

Familial defective apo B100:

Substitution of glutamine for arginine at 3500 a.a residue of apo B100

FAMILIAL COMBINED HYPERLIPIDEMIA

1 in 50 Mutation in promoters of LPL gene or its activator & inhibitor, apo-CII, or CETP gene or insulin resistance may be co-inherited High hepatic VLDL secretion,so is LDL Increased flux of CE from HDL to TG rich Lp High risk for atherosclerosis T/t Fibrates/omega-3FA + Statins

DYSBETALIPOPROTEINEMIA
Broad disease/floating disease/ remnant removal disease Raised CM remnant & IDL Striate palmar & tuberoeruptive xanthomata DNA test apo-E 2 homozygosity 90% Weight reduction + fibrates

LIPID CONCENTRATIONS AND CHD Total cholesterol (TC) is a sum of HDL cholesterol, LDL cholesterol and 20% of the triglyceride value. TC level is an excellent predictor of CHD. Since atheroselerosis begins early in life cholesterol levels in young adults predict CHD risk 30 to 40 years later. Cholesterol measurement will thus reduce the long-term risk for CHD. Although the role of high triglycerides as an independent factor in the development of CHD remains controversial, data from several prospective studies suggest that triglycerides are probably an important risk factor. Hypertriglyceridaemia is often associated with increased plasminogen activator inhibitor levels and impaired fibrinolysis. This is especially of importance in the Indian context since triglyceride levels are considered to be significantly elevated in Indians. However, recent data show that cholesterol levels are also significantly elevated in the Indian population. The relation between VLDL and CHD is unclear at present. However, elevated VLDL levels occur quite commonly in persons afflicted by premature CHD. Moreover, VLDL gives rise to LDL, which has been undoubtedly proven to be atherogenic. LDL cholesterol is highly atherogenic, hence high levels of LDL increase the risk of CHD. LDL = TC - (HDL + triglyceride/5) HDL cholesterol has been found to be inversely related to subsequent development of CHD, i.e. as HDL cholesterol increases, CHD risk decreases. Cardiac risk ratio i.e. Total cholesterol/ HDL, is an extremely potent predictor of CHD Lp (a), an LDL particle to which a large plasminogen-like protein, termed apo(a) has been linked via a disulfide bond is an atherogenic lipoprotein. There has been speculation that apo(a) could competitively inhibit the binding of plasminogen to its reeeptor and thus decrease plasmin formation and thrombolysis

Effective control of the blood lipid levels reduced cardiovascular morbidity and mortality both in patients with established CHD and in those at risk of developing CHD. Hence knowledge of the various aspects of the lipid profile and the significance of each of the parameters is vital and is essential part of management of CHD and people at risk of CHD. Drugs that reduce blood cholesterol levels also cut heart patients' long-term risk of dying. Researchers now report that aggressive treatment to reduce blood fats (lipids) in patients with chest pain or those who have just had a heart attack can reduce their risk of dying by as much as 60%.

LAB EVALUATION
Blood sampling & storage Estimation of plasma lipids: Enzymatic methods
Estimation of plasma lipoproteins Estimation of apolipoproteins

BLOOD SAMPLING & STORAGE

Fasting (12hrs.) sample Collected in sitting posture Avoid prolonged tourniquet application Either plasma or serum can be used Plasma preferred if Lp estimation by ultracentri or electrophoresis EDTA preferred Storage of plasma/serum Long period- -70C or lower Short period- -20C

HDL-Cholesterol
Phosphotungstic acid + MgCl2 LDL, VLDL Chylomicron HDL Cholesterol Total Cholesterol

HDL-C = Cholesterol in filtrate x dilution factor


Serum 250 ml Precipitating reagent 25 ml

10
4,000 rpm 10

HDL-C


*
Blk Std

Total Cholesterol

HDL-C

Unk

Unk

Cholesterol reagent (ml) Standard (ml)

1.0 -

1.0 20

1.0 -

1.0 -

Sample (ml)
Time interval

1.0

20

20*

Incubate RT 20

DW (ml)

1.0

1.0

1.0

OD 500 nm Blank 0


Au Total Cholesterol (mg/dl) = X Cs As Au HDL-Cholesterol (mg/dl) = X Cs X 1.1 As Cs = 200 mg/dl

Triglyceride
Lipases

Triglycerides + 3H2O Glycerol + ATP Glycerol-3-P + O2 2 H2O2 + 4-AAP + DHBS

GK

Glycerol + Fatty acids Glycerol-3-P + ADP Dihydroxyacetone-P + H2O2 Quinonemime + HCl + 4H2O

GPO Peroxidase

Total Cholesterol
Cholesterol Esterase

Cholesterol Ester + H2 Cholesterol + O2

Peroxidase

Cholesterol + Fatty acid Cholesterol-3-One + H2O2 Quinonemime dye + 4H2O

Cholesterol Oxidase

2 H2O2 + PSA + 4-AAP

ESTIMATION OF LIPOPROTEINS
Ultracentrifugal method: Reference method Polyanion precipitation method +Enzymatic methods Adsorption Gel filtration Affinity chromatography Electrophoresis in different media Immunochemical procedures

APOLIPOPROTEINS ANALYSIS
RIA ELISA & Fluorescence immunoassay RID EIA Immunonephelometry

RECENT ADVANCES
Small dense LDL:
Cholesterol depleted LDL (LDL III) More susceptible to oxidation Associated with high TG & low HDL Results from variation in gene for apo-B Hyperapobetalipoproteinemia High risk for atherosclerosis & CHD Direct measurement not available currently

Contd. Lp(a):
Structurally related to LDL Unlike LDL contain apo-(a), a specific marker of Lp(a) Apo(a) exhibits size polymorphism & homology with plasminogen Smaller isoforms high risk for atherosclerosis & CHD

SITES OF ACTION OF ANTIHYPERLIPIDEMIC DRUGS

I. Stimulation of cholesterol excretion II. Stimulation of LPL activity III. Inhibition of VLDL production IV. Inhibition of cholesterol biosynthesis V. Stimulation of cholesterol secretion into bile fluid VI. Stimulation of cholesterol conversion to bile acids. VII. Increased plasma clearance of LDL

COMBINATION THERAPY
Bile acid-binding resins + reductase inhibitors effective for isolated elevations of LDL cholesterol Reductase inhibitors + niacin, or resins + niacin high LDL and low HDL cholesterol levels Reductase inhibitor + gemfibrozil very high LDL cholesterol with concomitant hypertriglyceridemia Reductase inhibitors with either niacin or gemfibrozil - best CHD and combined hyperlipidemia.

HEALTH BENEFITS OF OMEGA-3 FATS

Lowers TC,LDL-C,TG & increase HDL-C Lowers B.P Reduces clotting Increase immune response Decrease inflammation Sources: Blue fish,Salmon,Halibut etc.

REFERENCES
Burtis & Ashwood, Tietz Textbook of Clinical Chemistry 4th ed. Philadelphia: WB Saunders;2006 Henry ed. Clinical Diagnosis and Management by Laboratory Medicine 20th ed. Philadelphia: WB Saunders;2001 Durrington P. Dyslipidemia.The Lancet 2003: 717-731 Executive Summary of Third Report of NCEP. JAMA 2001: 2486-2499 Angelo M. Lp(a) Lipoprotein-Coping with Heterogeneity. NEJM 2003: 2089-2090 Todd Hurst R Increased Incidence of Coronary Atherosclerosis in Type 2 Diabetes Mellitus Ann Intern Med. 2003 824-834

Thank you