ESR and HPLC analysis of the interaction of hydroxyl radical with DMSO: Rapid reduction and quantification of POBN and PBN nitroxides

The low stability of hydroxyl radical (OH.)-derived nitroxides is a limitin g factor for direct spin-trapping of OH. in biological systems. The latter experimental difficulty is partly solved with the introduction of dimethyl sulfoxide (DMSO) into the studied systems. Hydroxyl radical oxidizes DMSO t o methyl radical, which forms relatively stable nitroxides. The results of the present work provide evidence that in alpha-(4-pyridyl-1-oxide)-N-tert- butylnitrone (POBN) and alpha-phenyl-N-tert-butylnitrone (PBN) spin-trappin g experiments aimed to detect methyl radical in biological systems, the nit roxides formed can be reduced to their ESR-"silent" hydroxylamine derivativ es. The nitroxides and their hydroxylamine derivatives were successfully an alyzed by HPLC with electrochemical (EC) and UV detection. The lowest limit s of UV and EC detection of POBN/CH3 hydroxylamine was evaluated to be in t he micro- and nanomolar range, respectively. In parallel ESR and HPLC-EC an alysis of the metabolism of menadione by either HepG2 cells or isolated rat hepatocytes in the presence of DMSO, the HPLC-EC method has proven to be m ore sensitive in detecting the production of methyl radical. The use of the HPLC-EC detection of POBN/CH3 and PBN/CH3 is expected to be advantageous i n detection of hydroxyl radical in biological systems in the presence of DM SO.