Formation of protein bound lysine-derived galactosyl and glucosyl pyrroles in heated model systems

Residues of lysine-substituted AGEs (advanced glycosylation end-products) arising from the Maillard reaction and containing the carbon backbone of lactose or maltose, thus deriving from 1-desoxy-ketose degradation, were produced when casein was heated in the presence of the corresponding U14C labelled disaccharides. The enzymatic hydrolysates of the washed casein were purified by SPE and submitted to HPLC on a C18 column flushed with diluted acetic acid. A specific chromatographic peak (lambda max, 288.1 nm; MW, 416.2 Da) with a different retention time was obtained for each disaccharide reacted. On the basis of the value of the specific radioactivity, the two compounds appeared to contain the whole carbon backbone of the parent sugar. Analyses by MS/MS and NMR performed on the same two compounds extracted at preparative scale from lysine-lactose and lysine-maltose model systems allowed the structure assignment of 6-[2-acetyl-3-(beta-D-galactopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid and 6-[2-acetyl-3-(alpha-D-glucopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid, respectively. Both compounds submitted to enzymatic deglycosylation by specific alpha- or beta-glucosidases produced the lysine-derived acetyl pyrrole 6-(2-acetyl-3-hydroxy-1-pyrrolyl) 2-amino hexanoic acid (lambda max, 288.1 nm; MW, 254.1 Da). Galactosyl- and glucosyl-isomaltoles, extracted from the lysine-containing systems, identified with the reference molecules and heated in the presence of lysine under slightly alkaline conditions, gave the expected lysine-derived glycosyl pyrroles as identified above. The HPLC conditions were optimized by adjusting the composition of the eluting solvent and temperature of the column to achieve the best separation and identification of the AGEs in mixtures such as foods with possible interfering molecules like Trp and lysyl pyrrole aldehyde. Because of the reported presence of the two precursors isomaltol glycosydes in some foods, the corresponding lysine-derived glycosyl pyrroles can occur as both protein bound and in free form.