Preksha Meditation & Human Health: 5.3.2 Estimation of Cholesterol in Serum (Tarbutton, 1974)

(a) Principle

Cholesterol esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidative couples with 4-aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance maximum at 510 nm. The intensity of the red colour is proportional to the amount of total cholesterol in the specimen.

* Abbreviation: CHE = Cholesterol esterase, CHO = Cholesterol oxidase, POD = Peroxidase

(b) Specimen collection

Arterial blood of subject was collected by sterilized syringe, in a clean and dry glass container. Serum was separated from the cells with the help of centrifuge machine at the earliest possible (within 30 minutes).

(c) Procedure

Pipette into clean, dry tubes labeled Blank (B), Standard (s) and Test (T) and add the reagents in following order.

Incubate the assay mixture for 5 minutes at 37° C. After incubation, absorbance value of the colour against blank was measured at 510 nm with the help of autoanalyser.

(d) Calculation

$\mathrm{Concentration\; (mg\; \%)}=\frac{\mathrm{Absorbance\; of\; sample}}{\mathrm{Absorbance\; of\; standard}} \mathrm{X\; 200}$