Characterisation of the major autoxidation products of 3-hydroxykynurenineunder physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily au toxidised to products that may be involved in protein modification and cyto toxicity. The oxidation of 3-OHKyn has been studied here with a view to cha racterising the major products as well as determining their relative rates of formation and the role that H2O2 and hydroxyl radical (HO.) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OH Kyn, was the major product formed initially. It was, however, found to be u nstable, particularly in the presence of H2O2, and degraded to other produc ts including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (D HQCA). A compound that has a structure consistent with that of hydroxyxanth ommatin (OHXan) was also formed in addition to at least two minor species t hat we were unable to identify. Hydrogen peroxide was formed rapidly upon o xidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25 degrees C to 35 degrees C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxid ation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan(.)). Hydroxyl radicals wer e also produced during 3-OHKyn autoxidation. The HO. EPR signal disappeared and the Xan(.) EPR signal increased when catalase was added to the autoxid ation mixture. The HO. did not appear to play a role in the formation of th e autoxidation products as evidenced using HO. traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation o f H2O2 and by the formation of reactive 3-OHKyn autoxidation products such as the Xan(.) and DHQCA.