Lab. 8

The major lipids present in the plasma are: fatty acids, Triglycerides, cholesterol and phospholipids.

Other lipid-soluble substances, present in much smaller amounts (e.g. steroid hormones ).

Elevated plasma concentrations of lipids, particularly cholesterol, are related to the pathogenesis of atherosclerosis.

Lipids are carried in the bloodstream by complexes known as lipoproteins.

This is because these lipids are not soluble in the plasma water.

Thus they travel in micelle-like complexes composed of phospholipids, cholesterol and protein on the outside with cholesteryl esters, and triglycerides on the inside.

The four main types of lipoproteins are chylomicrons, VLDL, LDL, and HDL

Cholesterol and triglycerides, like many other essential components of the body, attract clinical attention when present in abnormal concentrations.

Increased or decreased levels usually occur because of abnormalities in the synthesis, degradation, and transport of their associated lipoprotein particles.

Increased or decreased plasma lipoproteins are named hyperlipoproteinemia & hypolipoproteinemia respectively.

Clinical chemistry laboratories offer many tests for lipid disorders.

One of the most common tests is the lipid profile. This panel of tests includes measurement of

triglycerides and cholesterol in the form of lipoprotein-cholesterol molecules,

low density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

The results of testing for these lipids provide measures of risk for coronary artery disease.

It is therefore clear that lipid measurements should be made in all patients known to have vascular disease, and in those at increased risk.

Thus plasma lipids should be measured in the individuals with the following: CHD (and cerebrovascular and peripheral

vascular disease) a family history of premature coronary disease

(occurring at age <60 years) other major risk factors for CHD (e.g. diabetes

mellitus, hypertension)patients with clinical features of

hyperlipidaemia patients whose plasma is seen to be lipaemic.

Positive Risk FactorsPositive Risk Factors

• Age: ≥45 years for men; ≥55 years or premature menopause for women

• Family history of premature CHD

• Current cigarette smoking

• Hypertension (blood pressure ≥140/90 mm Hg or taking antihypertensive medication)

• LDL cholesterol concentration ≥160 mg/dL , with ≤1 risk factor

• LDL cholesterol concentration ≥130 mg/dL , with ≥2 risk factors

• LDL cholesterol concentration ≥100 mg/dL , with CHD or risk equivalent

• HDL cholesterol concentration <40 mg/dL

• Diabetes mellitus = CHD risk equivalent

Negative Risk Factors

• HDL cholesterol concentration ≥60 mg/dL

• LDL cholesterol <100 mg/dL

8

Glycerol backbone with FA attached by ester bonds

Sources of Triglycerides:Exogenous source: DietaryEndogenous : Liver and tissue

storageH

CH OH

C

C

H OH

H

H OH

Glycerol

H

CH O

C

C

H O

H

H O

CO

CO

CO (CH2)n

(CH2)n

(CH2)n

CH3

CH3

CH3

Triglyceride

Serum triglycerides measurements are done for the following clinical reasons:• Hypertriglyceridemia increases the risk

for pancreatitis.• Hypertriglyceridemia is associated with

the following clinical findings: eruptive xanthoma, lipemia retinalis, hepatomegaly, splenomegaly, depressed HDL-cholesterol.

• in characterizing risk of CVDs• For the estimation of LDL-cholesterol,

using the Friedewald equation

Serum, Plasma (EDTA) Certain anticoagulants, such as fluoride, citrate, and

oxalate, cause large shifts of water from the red blood cells to the plasma, which result in the dilution of plasma components ( as much as 10%)

Fasting sample (from 12 to 16 h) is essential for triglyceride analysis

Samples drawn from nonfasting patients are not suitable for analysis, since elevated TG levels caused by normal absorption of food cannot be distinguished from elevated TG resulting from abnormal lipid metabolism or inborn errors of metabolism.

Storage and stability

Glycerol, released from triglycerides after hydrolysis with lipoprotein lipase,

Tranformed by glycerolkinase into glycerol-3-phosphate

which is oxidized by glycerolphosphate oxidase into dihydroxyacetone phosphate and hydrogen peroxide.

In the presence of peroxidase, the hydrogen peroxide oxidizes the chromogen 4-aminophenazone/ESPT to form purple quinoneimine whose intensity is proportional to the concentration of triglycerides in the sample.

Triglycerides Glycerol + 3 fatty acids

Glycerol + ATP Glycerol-3 phosphate + ADP

Glycerol-3 phosphate dihydroxyacetone + H2O2 phosphate

H2O2 + 4-aminophenazone/ESPT Quinoneimine

Lipoprotein lipase

glycerolkinase

glycerolphosphate oxidase

peroxidase

Cholesterol is a sterol compound that is found in all animal tissues

Serves many important physiological functions including: synthesis of bile acids, steroid hormones, and cell membranes.

Cholesterol also appears to be involved in atherosclerosis; thus cholesterol measurement is one of the most common laboratory tests used today.

Serum, Plasma (EDTA, Heparin) Certain anticoagulants, such as fluoride,

citrate, and oxalate, cause large shifts of water from the red blood cells to the plasma, which result in the dilution of plasma components.

Storage and Stability 7 days at 20 – 25 °C 7 days at 4 – 8 °C 3 months at -20 °C

Determination of cholesterol after enzymatic hydrolysis and oxidation.

The colorimetric indicator is quinoneimine which is generated from 4-aminoantipyrine and hydroxybenzoate by hydrogen peroxide under the catalytic action of peroxidase

Cholesterol Esterase

Cholesterol oxidase

Peroxidase

Cholesterol-3-one

HDL is a fraction of plasma lipoproteins It is composed of:

50% protein, 25% phospholipid, 20% cholesterol, and 5% triglycerides

Evidence suggests that high-density lipoprotein (HDL) cholesterol is a primary coronary heart disease (CHD) risk factor.

Ultracentrifugation Polyacrylamide Gel Electrophoresis Precipitation

based on the ability of various agents to precipitate selectively the major lipoprotein fractions, except HDL

Immunological Antibodies against human lipoproteins are

used to form antigen-antibody complexes with LDL, VLDL and chylomicrons in a way that only HDL-cholesterol is selectively determined by an enzymatic cholesterol measurement

In the plasma, cholesterol is transported by three lipoproteins: high density lipoprotein, low density lipoprotein, and very low density lipoprotein

HDL lipoproteins are assayed, after precipitation of LDL and VLDL lipoproteins with polyethylene glycol (PEG) 6000.

HDL is left in the supernatant solution for cholesterol quantitation.

[LDL-chol] = [Total chol] - [HDL-chol] - ([TG]/2.2)

Where all concentrations are given in mmol/L (note that if calculated using all concentrations

in mg/dL then the equation is: [LDL-chol] = [Total chol] - [HDL-chol] - ([TG]/5)

The quotient ([TG]/5) is used as an estimate of VLDL-cholesterol concentration.

It assumes, first, that virtually all of the plasma TG is carried on VLDL, and second, that the TG:cholesterol ratio of VLDL is constant at about 5:1

The Friedewald equation should not be used under the following circumstances: when chylomicrons are present when plasma triglyceride concentration

exceeds 400 mg/dL in patients with dysbetalipoproteinemia

(type III hyperlipoproteinemia