Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes

• Published in: Chemical research in toxicology 2015-08, Vol.28 (8), p.1556-1566
• Main Authors: Patrício, Eliziane S, Prado, Fernanda M, da Silva, Railmara P, Carvalho, Larissa A. C, Prates, Marcus V. C, Dadamos, Tony, Bertotti, Mauro, Di Mascio, Paolo, Kettle, Anthony J, Meotti, Flavia C
• Format: Article
• Language: English
• Subjects: Chromatography, Liquid; Glutathione - chemistry; Hydrogen Peroxide - chemistry; Kinetics; Light; Molecular Structure; Oxidation-Reduction; Oxidative Stress; Peroxides - chemistry; Spectrometry, Mass, Electrospray Ionization; Uric Acid - analogs & derivatives; Uric Acid - chemistry; Uric Acid - metabolism
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/acs.chemrestox.5b00132

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS3 ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 103 M–1 cm–1. This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10–4 s–1 and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than −0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M–1 s–1. In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.