DETERMINATION OF THE AUTOXIDATION PRODUCTS FROM FREE OR TOTAL CHOLESTEROL - A NEW MULTISTEP ENRICHMENT METHODOLOGY INCLUDING THE ENZYMATIC RELEASE OF ESTERIFIED CHOLESTEROL

A new method for the determination of the autoxidation products from free cholesterol, viz., the isomeric 5,6-epoxycholestanols, the C-7 oxidized cholesterol, 20 α-hydroxycholesterol, 25-hydroxycholesterol, and cholestanetriol, has been developed. The method is also useful in combination with an enzymatic method to release esterified cholesterol to facilitate total cholesterol oxide determination. For analysis of total cholesterol oxides, the lipids were dispersed in phosphate buffer by using bovine serum albumin as a detergent and incubated with triacylglycerol lipase under nitrogen atmosphere. The phospholipids and the released fatty acids were removed by column chromatography. The remaining lipids were dispersed again and incubated with cholesterol ester hydrolase under nitrogen atmosphere. Cholesterol oxides were isolated in three enrichment steps. The isolated cholesterol oxides were separated as trimethylsilyl ether derivatives on a capillary methyl silicone column. © 1990, American Chemical Society. All rights reserved.