title: Detection and characterization of the electron paramagnetic resonance-silent glutathionyl-5,5-dimethyl-1-pyrroline N-oxide adduct derived from redox cycling of phenoxyl radicals in model systems and HL-60 cells.
abstract: The antioxidant function of glutathione includes enzymatic reduction of hydrogen peroxide by glutathione peroxidase and nonenzymatic reduction of organic radicals and reactive oxygen species. The glutathionyl S-centered radical, formed by the nonenzymatic reduction process, is a marker of oxidative reactions proceeding by radical mechanisms. Spin-adducts of glutathionyl radicals with the spin trap DMPO, 5,5-dimethyl-1-pyrroline N-oxide, are not sufficiently stable and can be detected only under steady-state conditions. We developed a novel HPLC method for the detection of an EPR-silent DMPO adduct of glutathionyl radicals in model systems and in cells. We synthesized a sufficient quantity of the adduct for characterization by UV spectrophotometry, ionspray mass spectrometry, and 1H NMR spectroscopy. The UV absorption lambda max of the adduct, 258 nm, was indicative of a 2-(S-alkylthiyl)pyrroline N-oxide chromophore. The molecular mass of the adduct was 418 amu. No signal for the C2 proton of the DMPO-derived portion of the adduct was evident in its 1H NMR spectrum. The results were consistent with the structure 2-(S-glutathionyl)-5,5-dimethyl-1-pyrroline N-oxide (GS-DMPO nitrone). We showed that this adduct accumulated in the course of peroxidase-dependent redox cycling of phenol in the presence of glutathione and DMPO as well as in HL-60 cells exposed to a phenol/H2O2/DMPO reaction mixture. The EPR-silent GS-DMPO nitrone was readily assayed by HPLC under conditions incompatible with the detection of the GS-DMPO nitroxide by EPR. This is to our knowledge the first direct experimental evidence for the redox cycling of phenol in this bone marrow-derived cell line. The method may prove useful in the study of radical-driven oxidations of glutathione in various pathophysiological processes associated with radical mechanisms.
authors.affiliation: Department of Environmental & Occupational Health, University of Pittsburgh, Pennsylvania 15238, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Stoyanovosky
authors.forename: R
authors.initials: R
authors.lastname: Goldman
authors.forename: S S
authors.initials: SS
authors.lastname: Jonnalagadda
authors.forename: B W
authors.initials: BW
authors.lastname: Day
authors.forename: H G
authors.initials: HG
authors.lastname: Claycamp
authors.forename: V E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000032:analysis* / Q000138:chemical synthesis; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000032:analysis / Q000138:chemical synthesis; D018922:HL-60 Cells; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000032:analysis / Q000378:metabolism*; D010636:Phenols / Q000032:analysis / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels
chemical_list: D003497:Cyclic N-Oxides; D005609:Free Radicals; D010636:Phenols; D017382:Reactive Oxygen Species; D013113:Spin Labels; C099621:glutathionyl-5,5-dimethyl-1-pyrroline N-oxide; C042329:phenoxy radical; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006859:Hydrogen; D009195:Peroxidase; D005978:Glutathione