LC/MS/MS detection of uric acid oxidation metabolites to evaluate reactive oxygen and nitrogen species with application to pseudo-inflammation.

Uric acid, a water-soluble antioxidant ubiquitous in human bodily fluids at relatively high concentrations, reacts with a variety of reactive oxygen and nitrogen species. Ours and other previous studies identified allantoin, oxaluric acid, triuret, and 5--carboxyimino-6-aminopyrimidine-2,4(3)-dione as specific metabolites reactive against free radicals, singlet oxygen, peroxynitrite, and hypochlorous anion, respectively. We analyzed human plasma spiked with these products using high-performance liquid chromatography-tandem mass spectrometry. We observed recoveries of 40-110% and coefficients of variance within 7%. Samples remained stable at -80°C for at least 4 weeks, indicating the analytical method is sound. Detection of these metabolites in biological samples enables the identification of each species generated . We observed changes in the products in human blood during pseudo-inflammation induced by treatment with lipopolysaccharide. Levels of allantoin, oxaluric acid, triuret, and 5--carboxyimino-6-aminopyrimidine-2,4(3)-dione increased after addition of lipopolysaccharide. The formation of singlet oxygen was confirmed by increased formation of Trp-derived -hydroxide ([2,3a,8a]-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid) (-WOH). We believe our method will aid development of strategies to treat oxidative stress-associated diseases.