ESR and HPLC-EC 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 limiting 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 to 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-trapping experiments aimed to detect methyl radical in biological systems, the nitroxides formed can be reduced to their ESR-"silent" hydroxylamine derivatives. The nitroxides and their hydroxylamine derivatives were successfully analyzed by HPLC with electrochemical (EC) and UV detection. The lowest limits of UV and EC detection of POBN/CH3 hydroxylamine was evaluated to be in the micro- and nanomolar range, respectively. In parallel ESR and HPLC-EC analysis 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 more 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 in detection of hydroxyl radical in biological systems in the presence of DMSO.
Stoyanovsky
D A
DA
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Melnikov
Z
Z
Cederbaum
A I
AI
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003497:Cyclic N-Oxides; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D017665:Hydroxyl Radical / Q000737:chemistry*; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry*; D010084:Oxidation-Reduction; D011725:Pyridines; D051381:Rats; D017207:Rats, Sprague-Dawley; D013113:Spin Labels