The reaction of glyoxylic acid with lysine chemically protected on the α- amine group studied using mass spectrometry

Abstract

Glyoxylic acid (GA) is a human metabolite potentially involved in glycation processes and is considered a precursor of Nε-carboxymethyl lysine (CML). Different mechanisms of GA reaction with chemically protected at α-amine group or unprotected lysine have been reported, depending on the applied conditions. The objective of this study was to shed new light on the reaction that occurs at physiological pH, using mass spectrometry and some complementary tools. In the first approach, six GA species were found in water, methanol and in their mixtures with relative distribution depending on the solvent composition. The product of GA reaction with Z-Lys (Nα-(carbobenzyloxy)-L-lysine) was Z-CML and two participating GA species were assigned to those preferentially formed in methanol. The reaction mixture in HEPES buffer, pH 7.2, was analyzed by high resolution MS at different times over 100 days. Protonated molecules [M+H]+, respective sodium adducts ([M+Na]+), changes of their abundances over time and MS/MS data were used to assign the reaction intermediates. Additional experiments using liquid chromatography with MS detection were performed for confirmation of key intermediates. At physiological pH, Z-CML could be detected in the reaction mixture after 40 days. A plausible reaction mechanism is proposed, which involves formation of adducts between GA species and -amine group of Z-Lys, followed either by two decarboxylation steps and oxidation of aldehyde- to carboxylic group or by decarboxylation and hydrolytic degradation. A slow formation of Z-CML was attributed to the accumulation of intermediates directly preceding the final product in the proposed reaction scheme, indicating that oxidative conditions would favor Z-CML production, in agreement with well-known accelerated formation of AGEs under oxidative stress.