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 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 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 was evident in its H-1 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. (C) 1996 Academic Press, Inc.