Quantification of Superoxide Radicals and Peroxynitrite in Vascular Cells Using Oxidation of Sterically Hindered Hydroxylamines and Electron Spin Resonance

Abstract

The reactions of two hydroxylamines, 1-hydroxy-3-carboxy-pyrrolidine (CP-H) and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H), with superoxide radicals and peroxynitrite were studied. In these reactions corresponding stable nitroxyl radicals 3-carboxy-proxyl (CP) and 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidinoxyl (TEMPONE) are formed and the amount of them can be quantified by electron spin resonance (ESR). It was found that CP-H and TEMPONE-H provide almost the same efficacy in assaying peroxynitrite by ESRin vitroat pH 7.4. The formation of superoxide radicals in suspensions of cells was discriminated from that of peroxynitrite using superoxide dismutase or dimethyl sulfoxide as competitive reagents. The stability of the radicals CP and TEMPONE in the presence of ascorbate or thiols was studiedin vitro.The reduction rate of CP by ascorbate was 66-fold slower than the rate of reduction of TEMPONE. Therefore, the quantification of the formation of superoxide radicals and of peroxynitrite is much less affected by ascorbic acid when CP-H, but not TEMPONE-H, is used. Both TEMPONE-H and CP-H were used to determine the formation rates of superoxide radicals and peroxynitrite in suspensions of cultured aortic smooth muscle cells and endothelial cells, in washedex vivoplatelets, and in blood treated with glycerol trinitrate (GTN) as an NO donor. It was shown that both the acute addition of GTN (0.5 mM) to vascular cells and the incubation of smooth muscle or endothelial cells in culture with 0.1 mM GTN for 24 h enhance significantly the formation of reactive oxygen species in cells. The rates of of superoxide radical formation were increased at least in two times and peroxynitrite was detected. Hydroxylamines TEMPONE-H and CP-H can be used as nontoxic compounds in ESR assay capable of quantifying the formation of superoxide radicals and peroxynitrite in suspensions of cells and in the whole blood with high sensitivity.