Analysis of hydroxy and keto cholesterols in oxidized brain synaptosomes

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as alpha-to-copherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7 alpha-hydroxy-, 7 beta-hydroxy-, and 5 alpha, 6 alpha-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of alpha-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.