A micromethod has been developed for the quantitative determination of unesterified or total cholesterol in liver microsomes. The method, sensitive to 0.1 nmole of cholesterol, is based on the conversion of cholesterol into its acetate by use of 3H-labeled acetic anhydride of known specific radioactivity. For the determination of unesterified cholesterol, liver microsomal suspensions were extracted with methylene chloride-ethanol; for the determination of total cholesterol the microsomal suspensions were hydrolyzed and extracted with n-hexane. The lipid extract was reacted with [3H]acetic anhydride in pyridine, the [3H]acetoxycholesterol was separated by tlc and assayed by scintillation counting.
Free and esterified cholesterol in tissues have been determined by various methods (1) such as column chromatography (2), colorimetry (3-7), fluorometric methods (8), or gas-liquid chromatography (9).
The following micromethod, worked out for liver homogenates and liver microsomes, but potentially applicable to any tissue extract, utilizes the quantitative acetylation of cholesterol with [3H]acetic anhydride of known specific activity and the determination of the [3H]acetoxycholesterol formed by tic and scintillation counting.