Determination of ebselen-sensitive reactive oxygen metabolites (ebROM) in human serum based upon N,N′-diethyl-1,4-phenylenediamine oxidation
Highlights
►We modified a simple assay in order to distinguish non-radical from radical species. ►Human serum was pretreated with ebselen to determine non-radical involvement. ►Non-radical species largely account for derum-mediated DEPPD oxidation. ►This assay can enhance understanding of contribution of different oxidants to disease.
Introduction
Circumstantial evidence indicates that oxidative stress is a component of many chronic and age-related diseases, yet evidence from large-scale, double-blind interventional trials with the free radical-scavenging antioxidants, vitamins C and E, provides little support for involvement of free radical mechanisms [1]. An alternative hypothesis is that the quantitatively important oxidative reactions contributing to disease involve non-radical oxidant species, such as peroxides, which disrupt redox signaling and control mechanisms [2]. However, distinction between radical and non-radical mechanisms of injury is technically challenging, especially when both free radicals and non-radical oxidants are present in a biologic system.
Another difficulty concerns the nature of oxidants relevant to disease risk. Although much of the oxidative stress literature focuses on highly reactive oxygen species (ROS) which are short-lived in biologic systems, many studies show that there are relatively stable oxidants which persist in human serum even after months of storage at − 80 °C. The latter have been termed “reactive oxygen metabolites,” for which we use the generic term “ROM” in the present manuscript. ROM in serum can be operationally defined by a reaction with N,N’-diethyl-para-phenylenediamine (DEPPD) in the presence of Fe2+, which produces a stable radical cation (Fig. 1A) easily measured using a spectrophotometer because it is a red chromophore [3].
This reaction is the basis for the popular d-ROMs assay (Diacron International, Italy; www.diacrom.com) [3] and similar FORT (Free Oxygen Radicals Test; INCOMAT Mediznische, Glashutten, Germany). These tests are simple, relatively inexpensive and have been widely used to measure oxidative stress in human serum [4], [5], [6], [7], [8], [9], [10], [11]. Importantly, the results of studies from different research groups suggest that these tests could be useful to assess disease risk. Such use is limited by uncertainty about the chemical nature of ROM.
The ROM test is standardized relative to tert-butylhydroperoxide or H2O2, giving the impression that the test is measuring H2O2 and organic peroxides in serum. However, H2O2 and many hydroperoxides are relatively unstable and would not be expected in serum at concentrations indicated by the DEPPD reaction. Moreover, background chromophores as well as other oxidative reactions, e.g., ceruloplasmin activity [3] can contribute to the spectral change. Use of an EDTA-containing blank can correct for background chromophores; however, EDTA inhibits both peroxide-dependent color formation and ceruloplasmin activity so that an EDTA control does not discriminate factors contributing to the DEPPD oxidation by serum.
Antioxidants, such as butylated hydroxytoluene, added to serum could block DEPPD oxidation, but would not discriminate between the types of oxidants present in the sample. In the present study, we examined whether ebselen, a selenium-containing chemical which reduces peroxides in the presence of thiols [12], could be used to improve the specificity of the DEPPD reaction for peroxides in human serum. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2 H)one (Fig. 1B), catalyzes thiol-dependent reduction of peroxides by a non-radical mechanism [12], [13]. Although earlier literature suggested that ebselen has properties such as free radical quenching [12], this activity was subsequently shown to have little effect [14]. Sequential reduction of ebselen by glutathione produces a selenol which is the direct reductant for the peroxides [13].
The present research shows that ebselen pretreatment of serum largely eliminates the serum-dependent oxidation of DEPPD. Studies with a purified endoperoxide and two epoxides show that, in addition to previously characterized oxidation of DEPPD by hydroperoxides, both of these oxidants support the DEPPD oxidation reaction and are eliminated by pretreatment with ebselen. Use of an imidazole N-oxide to trap radicals provided further evidence that the radical products are derived from non-radical species. Thus, use of an ebselen-pretreated serum sample as a reference for the DEPPD-dependent spectrophotometric assay provides means to improve specificity for non-radical oxidants in serum. This simple, yet novel, approach provides means to discriminate ebselen-dependent and ebselen-independent oxidative mechanisms that contribute to the DEPPD signal and potentially can be useful for population studies to test for distinct associations of disease risk with non-radical and radical mechanisms.
Section snippets
Materials
N,N-diethyl-1,4-phenylenediamine (DEPPD), tert-butylhydroperoxide (tBH), FeSO4, bathocuproine disulfonate (BCDS), glutathione peroxidase (bovine erythrocyte, Gpx), catalase, microsomal epoxide hydrolase (human, EH), rhodococcus epoxide hydrolase (EHr), styrene oxide, and 16,17-epoxy-21-acetoxy-pregnenolone were from Sigma-Aldrich (St Louis, MO). Prostaglandin H2 (PGH2) was from Cayman Chemical (Ann Arbor, MI). Peroxiredoxin-1 (human) was purchased from Lab Frontier (Seoul, South Korea). Ebselen
DEPPD-dependent color formation with human serum
Previous research showed that hydroperoxides in the presence of Fe2+ oxidize DEPPD and that this reaction is blocked by EDTA, a metal ion chelator [3]. We initially performed experiments to adapt the principles of the previous research to a 96-well plate format. In this development, we observed that DEPPD oxidation occurred with serum and tert-butylhydroperoxide (tBH) and that both were inhibited by EDTA (Fig. 2A). Time course studies showed that two phases of oxidation were present, one which
Discussion
Much of oxidative stress research has been focused on free radical mechanisms yet large-scale interventional trials with moderately high intakes of two of the best characterized free radical scavenging antioxidants, vitamins C and E, provided little evidence for benefit [1]. An alternative, non-radical hypothesis for oxidative stress has been proposed based upon the evidence that free radicals represent only a small fraction of the total oxidant load [2]. In this hypothesis, non-radical
Acknowledgements
This research was supported by NIH grants ES011195 and ES009047, RR00039, DK067167, DK075745 and the Woodruff Foundation.
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