Reaction of peroxynitrite and uric acid studied by ESR spin trapping and mass spectrometry: Free radical formation and product identification

Uric acid is the most abundant antioxidant in plasma. However, under conditions of elevated uric acid levels and oxidative stress, it becomes a pro-oxidant and causes endothelial dysfunction. It is hypothesized that the generation of reactive intermediates such as free radicals from the reaction of peroxynitrite and other oxidants mediate the pathological pro-oxidant properties of uric acid. Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. Our research is focused on the identification of free radical metabolites of uric acid formed from its reaction with peroxynitrite. Our experimental approach included the electron spin resonance (ESR) spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4. Using PBN (N-tert-butyl-alpha-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a(N) = 15.6 G; and a(H) = 3.6 G, corresponded to two carbon-based radicals. Further structural identifications of the PBN-radical adducts were carried out using liquid chromatography-mass spectrometry (LC-MS). After comparison with the control reactions, we could identify two molecules, corresponding to the fragment ions of m/z 352 and 223, respectively. The PBN-triuretcarbonyl radical adduct was characterized for m/z 352 and the latter was identified as a PBN-aminocarbonyl radical adduct.;The pH dependence study of the reaction between uric acid and peroxynitrite revealed the formation of hydrogen adduct at high pH and could be observed even without urate. Its formation was proposed to undergo the inverted spin trapping mechanism, in which the spin trap was initially oxidized rather than the antioxidant substrate, and followed by electron transfer.;We extended our studies to investigate the effect of methyl substitution at various nitrogen positions on product and radical formation. No ESR signal was observed when conducting the reactions with N-7 methylated uric acids in phosphate buffer pH 7.4. Moreover, the reactions were purposely conducted in methanol to trap the reaction intermediate. Various products have been identified by LC-MS, and those products indicated that a common intermediate in various urate oxidation conditions, dehydroisouric acid, was formed.