Cholesterol. Lipid – Soluble in organic solvents and nearly insoluble in water – Yield fatty...

Cholesterol

• Lipid – Soluble in organic solvents and nearly insoluble in water– Yield fatty acids on hydrolysis– Complex alcohols that combine with fatty acids to form

esters

• Lipoprotein – An association of a core lipids with coat phospholipid and

protein– For solubility

• Apolipoproteins – Protein components of lipoprotein.

Classification of Clinically Important lipids

Structure of cholesterol

Cholesterol biosynthesis

Esterification of cholesterol mediated by ACAT & LCA

Structure of a typical lipoprotein particle

• apo B-100—containing lipoproteins – VLDL, IDL, Lp(a), LDL

Contributors to total cholesterol in normal people

• LDL – Two thirds

• HDL – One third

• IDL and Lp(a)– 2 to 3 mg/dL (each)

• Total chol = VLDL chol + LDL chol + HDL chol

Classification of LDL.Total. andHDL Cholesterol (mg/dL)

• Reference methods• To establish the accuracy of lipid and

lipoprotein measurements• Using the same basis (protocol ) for accuracy

that had been used in developing the relationships between lipid and lipoprotein concentration and CHD.

• From studies, cut points for the risk characterization in patients were derived.

• The accuracy of existing or newly developed methods could be assessed.

Methods and procedures commonly used

• Reference Method (total cholesterol)– Chemical method• Hydrolyze the cholesteryl esters. alcoholic KOH• extracted from the mixture with hexane• dried in vacuo• acetic acid, acetic anhydride, and sulfuric acid• Color development, 620 nm• pure cholesterol as the calibrator.• Expressed as mg/dL = mmoI/L x 38.7

HDL cholesterol

V-LDL & LDL cholesterol• VLDL chol = [Total chol] – [d > 1.006g/mL chol]

• LDL cholesterol – Cholesterol infranate – cholesterol supernate

• (IDL, LDL, Lp(a), HDL— HDL fraction)

• The Friedewald Equation– [LDL choI] = [Total chol] - [HDL chol]-[Triglyceride]/5

• Unacceptable at TG concentrations > 400 mgldL

Methods

• Reference methods – are complex, time consuming, at least partially

manual, and require a high level of expertise for reliable operation

• Routine Methods– Enzymatic methods

Enzymatic method

• Interference – Competition with the oxidation reaction• bilirubin, ascorbic acid, and hemoglobin.

– adding substances such as bilirubin oxidase and dual wavelength readings to minimize the effects of hemolysis

• β-hydroxy sterols and plant sterols (e.g., β-sitosterol) can also react.– are generally at very low concentrations ( so,not significant)

Sources of Variation in Lipid and Lipoprotein Measurements

• Analytical Variation

• Physiological Variation– Contribute about 70 to 98% of the overall

variance– Variation for triglyceride is considerably higher

+, minimal to moderate increase

++, moderate to high increase

-, minimal to moderate decrease

-, moderate to high decrease

NC, essentially no change or trend.

Recommendations

• Cholesterol – Two serial samples obtained at least 1 wk apart be

used– Fasting or non-fasting

• Triglyceride, HDL and LDL cholesterol– Two to three serial specimens are recommended– 12-h fast, or 9-h

• Specimens – Serum or plasma (EDTA plasma)

• Sampling – in the same position on each occasion

• Specimen storage– Serum or plasma should be removed from cells

within 3 h– Specimens can be stored for up to 3 d at 4 °C– Up to several wk at —20 °C– at —70 °C or lower for longer periods