Protein Crosslinking by the Maillard Reaction: Dicarbonyl-Derived Imidazolium Crosslinks in Aging and Diabetes

Abstract α-Dicarbonyl compounds that arise from various metabolic pathways react with proteins to form a variety of adducts in a reaction known as the Maillard reaction. These adducts are collectively known as advanced glycation end products or AGEs. Methylglyoxal (MG) and glyoxal (GXL) are two such dicarbonyls. They react with proteins to produce lysine–lysine imidazolium crosslinking AGEs. The imidazolium crosslinks derived from MG (MOLD-methylglyoxal-lysine dimer) and GXL (GOLD-glyoxal-lysine dimer) are present in human tissue proteins. In this study, we report an HPLC method for the simultaneous quantification of GOLD and MOLD in biological specimens. The method consists of reverse-phase HPLC of acid-hydrolyzed proteins, collection of eluate-containing imidazoliums, phenylisothiocyanate derivatization, followed by a second reverse-phase HPLC. This method was linear for both the imidazolium compounds in the range of 0.5–300 pmol. The levels of GOLD and MOLD in aging lenses (20 to 80 years) were trace–8.4 pmol and 15–93 pmol per milligram of protein, respectively. Cataractous lenses showed significantly higher levels of both GOLD and MOLD (mean ± SD, 14.5 ± 1.8 and 141 ± 18.4 pmol per milligram of protein, P P P in vivo.