LC/MS/MS detection of uric acid oxidation metabolites to evaluate reactive oxygen and nitrogen species in vivo 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-N-carboxyimino-6-aminopyrimidine-2,4(3H)-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 in vivo. 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-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione increased after addition of lipopolysaccharide. The formation of singlet oxygen was confirmed by increased formation of Trp-derived cis-hydroxide ([2S,3aR,8aR]-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid) (cis-WOH). We believe our method will aid development of strategies to treat oxidative stress-associated diseases.