Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO^-) and superoxide (HO₂^•/O₂^•-). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely ^•OH and ^•NO₂. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with ^•NO₂ and ONOO^-. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where ^•OH and ^•NO₂ are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.