Model studies of the maillard reaction of arg-lys with D-glucose

Possible routes leading from Amadori product precursors to glucose-derived protein crosslinks has been suggested by model studies examining the fate o f the Amadori products in vitro. For instance, the Amadori product can unde rgo dehydration to give 1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione) , which has been isolated by trapping with aminoguanidine. In a model react ion, we selected as an AGE target the dipeptide N-alpha-Z-arg-lys. The prox imity of the arginine and lysine residues to each other promotes stable int ramolecular crosslink formation. Incubation of N-alpha-Z-arg-lys with IO eq uivalents of glucose in 0.2 M phosphate buffer (pH 7.4) at 37 degrees C for five weeks produces at least 25 distinct reaction products upon fractionat ion of this mixture by HPLC. Each fraction was isolated, concentrated, and analyzed for its reactivity with a polyclonal anti-AGE antibody (RU) that h as been shown previously to recognize a class of AGEs that increase as a co nsequence of hyperglycemia and which are inhibited from forming in human su bjects by treatment with the advanced glycation inhibitor aminoguanidine. T he products present within one fraction (1.5% yield) were found to block an tibody binding in a dose-dependent fashion. Further purification of this fr action by HPLC revealed the presence of one major (0.6% yield) immunoreacti ve compound. Characterization of this adduct by UV, ESMS and H-1-NMR spectr a revealed the presence of intramolecular arg-lys-imidazole crosslink. This crosslink is non-fluorescent and acid labile and may represent an importan t class of immunoreactive AGE-crosslinks that form in vivo.