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

The antioxidant function of glutathione includes enzymatic reduction o f hydrogen peroxide by glutathione peroxidase and nonenzymatic reducti on 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-a dducts of glutathionyl radicals with the spin trap DMPO, 5,5-dimethyl- 1-pyrroline N-oxide, are not sufficiently stable and can be detected o nly under steady-state conditions, We developed a novel HPLC method fo r 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 mas s spectrometry, and H-1 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 C-2 proton of the DMPO-derived portion of the adduct w as evident in its H-1 NMR spectrum, The results were consistent with t he structure 2-(S-glutathionyl)-5,5-dimethyl-1-pyrroline N-oxide (GS-D MPO nitrone), We showed that this adduct accumulated in the course of peroxidase-dependent redox cycling of phenol in the presence of glutat hione 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-DMP O nitroxide by EPR, This is to our knowledge the first direct experime ntal evidence for the redox cycling of phenol in this bone marrow-deri ved cell line, The method may prove useful in the study of radical-dri ven oxidations of glutathione in various pathophysiological processes associated with radical mechanisms. (C) 1996 Academic Press, Inc.