A sensitive electrochemical method for quantitative hydroperoxide determination.

We report a general assay for hydroperoxides that is simple, selective, and sensitive. The assay is based on the reduction of hydroperoxides by glutathione (GSH) catalyzed by GSH peroxidase. Stoichiometric amounts of oxidized glutathione (GSSG) are produced that are separated from GSH by HPLC. GSSG eluting from the column is quantitated with a coulometric detector operating in the oxidizing mode (E = 0.82 V vs Pd). Picomole amounts of GSSG can be measured and related to the hydroperoxide concentration in the incubation mixture. GSH peroxidase has broad substrate specificity to many different hydroperoxides. Therefore, this method allows the determination of the total hydroperoxide concentration in the reaction mixture. For analysis of peroxidized phospholipids, phospholipase A2 is included in the reaction to release fatty acid hydroperoxides from the 2-position of the glycerol moiety. The presence of hydroperoxide is verified by addition of sodium borohydride or stannous chloride to sample extracts of biological fluids before analysis. The applicability of this method was tested by examination of human plasma from normal individuals for hydroperoxide levels.