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