Microanalysis of Tissue Cholesterol Using a Fluorometric Enzymatic Method

Abstract

A simple fluoroenzymatic method is presented for the microdetermination of tissue cholesterol.The method is based on the same principle as a conventional enzymatic cholesterol determination except that homovanillic acid, a fluorogenic reagent, was used as the substrate for the hydrogen peroxide-peroxidase system, and Triton X-100 was added to the enzymatic reagent solution for solubilization of cholesterol and activation of cholesteryl ester hydrolase and cholesterol oxidase.Rat liver, aorta, serum and high-density lipoprotein fraction samples were used to compare this fluoroenzymatic method with the standard colorimetric method.Results of the two methods were Similar.The advantages of the fluoroenzymatic assay for free and esterified cholesterol include speed, easy procedure and high sensitivity.Using this method, as little as 0.25 μg of cholesterol can be determined.This. method should prove very useful for the quantification of cholesterol in limited amounts of tissue such as in biopsies.