Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms

Nitroxide stable radicals generally serve for probing molecular motion in m embranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging, Rece ntly nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which an depleted while attenuati ng oxidative damage, nitroxides can be recycled. In many cases the antioxid ative activity of nitroxides is associated with switching between their oxi dized and reduced forms. In the present work, superoxide radicals were gene rated either radiolytically or enzymatically using hypoxanthine/xanthine ox idase, Electron paramagnetic resonance (EPR) spectrometry was used to follo w the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxida tion. The results indicate that: a) The rate constant of superoxide reactio n with cyclic hydroxylamines is pH-independent and is lower by several orde rs of magnitude than the rate constant of superoxide reaction with nitroxid es; b) The oxidation of hydroxylamine by superoxide is primarily responsibl e for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superox ide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simula tion of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxid es in vitro can be distinguished from that of common LMWA due to hydroxylam ine oxidation by superoxide, which allows nitroxide recycling and enables i ts catalytic activity. (C) 1999 Elsevier Science Inc.