Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method

Abstract

Methods were developed for the separation and measurement of lipid and protein hydroperoxides, which can be used for biological materials. Lipids were extracted with methanol:chloroform and their hydroperoxides measured in solutions of methanol and chloroform containing 110mM perchloric acid, xylenol orange, and ferrous iron. Proteins were isolated by precipitation with 0.2M perchloric acid. The precipitates were redissolved in 6M guanidine hydrochloride and washed with chloroform, and the hydroperoxides were measured in the presence of perchloric acid, xylenol orange, and ferrous iron. Optimum conditions for hydroperoxide measurements were established and the assays were applied to oxidized human blood serum and to cultured cells.