Determination of ebselen-sensitive reactive oxygen metabolites (ebROM) in human serum based upon N,N'-diethyl-1,4-phenylenediamine oxidation.
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 Fe(2+)-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.
Clinical Biomarkers Laboratory, Allergy and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, United States.
Yongliang
Y
Liang
James R
JR
Roede
Sergey
S
Dikalov
Nana Gletsu
NG
Miller
Samuel C
SC
Dudley
Arshed
A
Quyyumi
Dean P
DP
Jones
D001393:Azoles / Q000097:blood*; D006801:Humans; D054833:Isoindoles; D015394:Molecular Structure; D016566:Organoselenium Compounds / Q000097:blood*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000097:blood / Q000378:metabolism*; D010655:Phenylenediamines / Q000097:blood / Q000737:chemistry / Q000378:metabolism*
D001393:Azoles; D054833:Isoindoles; D016566:Organoselenium Compounds; D010655:Phenylenediamines; C042986:ebselen; D010100:Oxygen