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. 2002 May 15;364(Pt 1):15–24. doi: 10.1042/bj3640015

Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence.

Naila Ahmed 1, Paul J Thornalley 1
PMCID: PMC1222540  PMID: 11988071

Abstract

Glycation of proteins leads to the formation of advanced glycation endproducts (AGEs) of diverse molecular structure and biological function. Serum albumin derivatives modified to minimal and high extents by methylglyoxal and glucose in vitro have been used in many studies as model AGE proteins. The early and advanced glycation adduct contents of these proteins were investigated using the 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) chromatographic assay of enzymic hydrolysates. AGEs derived from methylglyoxal, glyoxal and 3-deoxyglucosone, the hydroimidazolones N(delta)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), N(delta)-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) and N(delta)-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine (3DG-H1), bis(lysyl)imidazolium cross-links methylglyoxal-derived lysine dimer (MOLD), glyoxal-derived lysine dimer (GOLD), 3-deoxyglucosone-derived lysine dimer (DOLD), monolysyl adducts N(epsilon)-(1-carboxyethyl)lysine (CEL), N(epsilon)-carboxymethyl-lysine (CML) and pyrraline, other AGEs, N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)ornithine (THP), argpyrimidine and pentosidine, and fructosyl-lysine were determined. AGEs with intrinsic fluorescence (argpyrimidine and pentosidine) were assayed without derivatization. Human serum albumin (HSA) glycated minimally by methylglyoxal in vitro contained mainly MG-H1 with minor amounts of THP and argpyrimidine. Similar AGEs were found in prothrombin glycated minimally by methylglyoxal and in N(alpha)-t-butyloxycarbonyl-arginine incubated with methylglyoxal. HSA glycated highly by methylglyoxal contained mainly argpyrimidine, MG-H1 and THP, with minor amounts of CEL and MOLD. HSA glycated minimally by glucose in vitro contained mainly fructosyl-lysine and CML, with minor amounts of THP, MG-H1, G-H1, 3DG-H1, argpyrimidine and DOLD. HSA glycated highly by glucose contained these AGEs and pyrraline, and very high amounts ( approximately 8 mol/mol of protein) of fructosyl-lysine. Most AGEs in albumin glycated minimally by methylglyoxal and glucose were identified. Significant proportions of arginine and lysine-derived AGEs in albumin modified highly by methylglyoxal, and lysine-derived AGEs in albumin modified highly by glucose, remain to be identified.

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Selected References

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