Refinement of a HPLC assay of lipid hydroperoxides caused by free radical-mediated oxidation of membrane lipids : an honors thesis (HONRS 499)

Abstract

A sensitive and easy-to-use HPLC assay that measures the amount of free radicalmediated oxidation of membrane lipids is refined. A reaction that models the effects of free radical oxidation on cell membrane lipids is used. The rate of the reaction between diphenyl-l-pyrenylphosphine (DPPP) and tert-butylhydroperoxide (tBuOOH; a model compound for peroxidized lipids) is detected by measuring the amount of fluorescent DPPPO. The levels of two catalysts, pyridine and iron (III), are adjusted so that the reaction they catalyze is complete within approximately one minute, with a goal of extrapolating back to reaction conditions that would be conducive to completion within one second. Samples of the reacting solution are injected into an HPLC machine, bypassing the column in favor of a spectrofluorometer.