Determination of ebselen-sensitive reactive oxygen metabolites (ebROM) in human serum based upon N,N′-diethyl-1,4-phenylenediamine oxidation

Abstract

BackgroundOxidative stress occurs through free radical- and non-radical-mediated oxidative mechanisms, but these are poorly discriminated by most assays. A convenient assay for oxidants in human serum is based upon the Fe2+-dependent decomposition of peroxides to oxidize N,N′-diethyl-1,4-phenylenediamine (DEPPD) to a stable radical cation which can be measured spectrophotometrically. MethodsWe investigated modification of the DEPPD oxidation assay to discriminate color formation due to non-radical oxidants, including hydroperoxides and endoperoxides, which are sensitive to ebselen. ResultsUse of serum, which has been pretreated with ebselen as a reference, provides a quantitative assay for non-radical, reactive oxidant species in serum, including hydroperoxides, endoperoxides and epoxides. In a set of 35 human serum samples, non-radical oxidants largely accounted for DEPPD oxidation in 86% of the samples while the remaining 14% had considerable contribution from other redox-active chemicals. ConclusionsThe simple modification in which ebselen-pretreated sample is used as a reference provides means to quantify non-radical oxidants in human serum. Application of this approach could enhance understanding of the contribution of different types of oxidative stress to disease.