Identification and Quantification of Major Maillard Cross-links in Human Serum Albumin and Lens Protein

Glycation reactions leading to protein modifications (advanced glycation end products) contribute to various pathologies associated with the general aging process and long term complications of diabetes. However, only few relevant compounds have so far been detected in vivo. We now report on the first unequivocal identification of the lysine-arginine cross-links glucosepane 5, DOGDIC 6, MODIC 7, and GODIC 8 in human material. For their accurate quantification by coupled liquid chromatography-electrospray ionization mass spectrometry,13C-labeled reference compounds were synthesized independently. Compounds 5–8 are formed via the α-dicarbonyl compoundsN6-(2,3-dihydroxy-5,6-dioxohexyl)-l-lysinate (1a,b), 3-deoxyglucosone (2), methylglyoxal (3), and glyoxal (4), respectively. The protein-bound dideoxyosone 1a,b seems to be of prime significance for cross-linking because it presumably is not detoxified by mammalian enzymes as readily as 2–4. Hence, the follow-up product glucosepane 5 was found to be the dominant compound. Up to 42.3 pmol of 5/mg of protein was identified in human serum albumin of diabetics; the level of 5 correlates markedly with the glycated hemoglobin HbA1c. In the water-insoluble fraction of lens proteins from normoglycemics, concentration of 5 ranges between 132.3 and 241.7 pmol/mg. The advanced glycoxidation end product GODIC 8 is elevated significantly in brunescent lenses, indicating enhanced oxidative stress in this material. Compounds 5–8 thus appear predestined as markers for pathophysiological processes.