Chemiluminescent determination of cholesterol hydroperoxides in human erythrocyte membrane

A method for separating, detecting, and quantifying cholesterol hydroperoxide (Ch-OOH) based on extraction, purification by solid-phase extraction cartridge, high-performance liquid chromatography with chemiluminescent detection (HPLC-CL), and liquid chromatography-mass spectrometry has been developed for human erythrocyte membrane. We prepared standard compounds of the cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (Ch 5alpha-OOH, Ch 7alpha-OOH, and Ch 7beta-OOH). An octyl silica column with methanol/water/acetonitrile 89:9:2 (by vol) as eluent was used to determine Ch-OOH. HPLC-CL that incorporated cytochrome c and luminol as the post-column luminescent reagent was used. We also investigated the optimal assay conditions and how to prevent formation of artifact Ch-OOH. Analysis of erythrocyte membranes from seven healthy volunteers identified Ch 7alpha-OOH and Ch 7beta-OOH, but not Ch 5alpha-OOH, as commonly occurring components. The respective mean concentrations of Ch 7alpha-OOH and Ch 7beta-OOH were 2.5+/-1.6 and 5.4+/-3.5 pmol/mL blood.