Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited

Uri Samuni,Sara Goldstein,Amram Samuni

doi:10.3390/antiox11010040

Abstract

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.

Highlights

Superoxide (HO2 /O2 − ) is formed during normal metabolism, as well as in pathophysiological processes through the action of various drugs, poisons and radiation [1].Superoxide is a moderately reactive radical that oxidizes relatively few biological compounds [2]
There are some reactions of superoxide that contribute to its toxicity and are potentially deleterious including its recombination with NO forming peroxynitrite (ONOOH/ONOO− ), which is implicated in pathophysiology of various diseases including acute and chronic inflammatory processes, sepsis, ischemia-reperfusion, and neurodegenerative disorders [3,4]
The present study demonstrates that cyclic hydroxylamines (RNO-H) do not react directly with peroxynitrite, but rather indirectly with the radicals formed during its self-decomposition (Scheme 1)

Summary

Introduction

Superoxide (HO2 /O2 − ) is formed during normal metabolism, as well as in pathophysiological processes through the action of various drugs, poisons and radiation [1]. Superoxide is a moderately reactive radical that oxidizes relatively few biological compounds [2]. There are some reactions of superoxide that contribute to its toxicity and are potentially deleterious including its recombination with NO forming peroxynitrite (ONOOH/ONOO− ), which is implicated in pathophysiology of various diseases including acute and chronic inflammatory processes, sepsis, ischemia-reperfusion, and neurodegenerative disorders [3,4]

Methods

Results

Conclusion