Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study.

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical (()OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with ()OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary ()OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and ()OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary ()OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the ()OH sources in pathological processes such as Parkinson's disease.