Protein crosslinking by the Maillard reaction: Dicarbonyl-derived imidazolium crosslinks in aging and diabetes

alpha-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 a SD, 14.5 +/- 1.8 and 141 +/- 18.4 pmol per milligram of protein, P < 0.05). Brunescent lenses had the highest levels of imidazolium crosslinks (GOLD, 18.36 +/- 2.5; and MOLD, 179.2 +/- 32.3 pmol per milligram of protein, P < 0.05). The GOLD and MOLD levels were higher in diabetic plasma proteins when compared to that of normal (GOLD, 17.5 +/- 6.34 pmol per milligram of protein vs 43.5 +/- 15.96 pmol per milligram of protein; and MOLD, 172.5 +/- 32.53 pmol per milligram of protein vs 273 +/- 62.67 pmol per milligram of protein, P < 0.05), GOLD and MOLD are significant in terms of tissue damage in aging and diabetes because they represent protein crosslinking by compounds that are major precursors of AGEs. Our method can be used for quantification of imidazolium crosslinks in tissue proteins to assess alpha-dicarbonyl-mediated protein damage in vivo. (C) 1999 Academic Press.