Determination of microsomal cholesterol by an isotope derivative method

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