Summary In a model reaction, we selected as an AGE target the dipeptide Nɛ-Z-arg-lys. The proximity of the arginine and lysine residues promotes stable intramolecular crosslik formation. Incubation of Nɛ-Z-arg-lys with 10 equivalents of glucose in 0.2 M phosphate buffer (pH 7.4) at 37°C for five weeks produces at least 25 distinct reaction products upon fractionation of this mixture by HPLC. Each fraction was isolated, concentrated, and analysed for reactivity with a polyclonal anti-AGE antibody (RU) that has been shown previsouly to recognize a class of AGEs that increase as a consequence of kyperglycemia and which are inhibited from forming in human subjects by treatment with aminoguanidine. The products present within one fraction (1.5% yield) were found to block antibody binding in a dose-dependent manner. Further purification of this fraction by PHLC revealed the presence of one major immunoreactive compound (0.6% yield). Characterization of this adduct by UV, ESMS and 1-H-NMR spectra revealed the presence of a intramolecular arg-lys-imidszole crosslink. This crosslink is non-fluorescent and acidlabile and may represent an important class of immunoreactive AGE-crosslink that forms in vivo.
