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

Yongliang Liang, James R. Roede, Sergey Dikalov, Nana Gletsu Miller, Samuel C. Dudley, Arshed Quyyumi, Dean P. Jones

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Background Oxidative 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. Methods We 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. Results Use 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. Conclusions The 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.

Original languageEnglish (US)
Pages (from-to)1-6
Number of pages6
JournalClinica Chimica Acta
StatePublished - Dec 24 2012

Bibliographical note

Funding Information:
This research was supported by NIH grants ES011195 and ES009047 , RR00039 , DK067167 , DK075745 and the Woodruff Foundation .

Publisher Copyright:
© 2012 Elsevier B.V.


  • Cardiovascular disease
  • Endoperoxides
  • Epoxides
  • Hydroperoxides
  • Oxidative stress
  • Peroxides


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