Preksha Meditation & Human Health: 5.3.3 Estimation of HDL Cholesterol in Serum (Castelli, 1977)

(a) Principle

Phosphotungstste/Mg²⁺ precipitates chylomicrons, LDL and VLDL fractions. High Density Lipoprotein (HDL) fraction remains unaffected in supernatant. Cholesterol content of HDL fraction is assayed using AutoZyme Cholesterol.

(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

HDL separation

Prewarm at room temperature the required amount of Precipitating Reagent and AutoZyme Cholesterol working solution before use and perform the assay as following -

Pipette 0.5 mL of both Serum and HDL-Precipitating reagent mix thoroughly and centrifuge at 4000 r.p.m. for 10 minutes in a common laboratory centrifuge to obtain a clear supernatant.

Pipette into clean, dry tubes labeled Blank (B), Standard (s) and Test (T), 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

$\mathrm{Concentration\; (mg\; \%)}=\frac{\mathrm{Absorbance\; of\; sample}}{\mathrm{Absorbance\; of\; standard}} \mathrm{X\; 100}$