Specific quantitation by HPLC of protein (lysine) bound glucose in human serum albumin and other glycosylated proteins

Abstract

A specific and sensitive method for quantification of the fructose-lysine linkages present in non-enzymatically glycosylated albumin and other proteins is described. Protein is hydrolyzed for 18 h in 6 mol/l HCl at 95 degrees C to yield furosine (epsilon-N-(2-furoylmethyl)-L-lysine) known as a specific degradation product of fructose-lysine. Furosine is then separated on HPLC and quantified by its UV-absorbance against a prepared fructose-lysine standard. The method has been successfully used for the determination of glycosyl-albumin in diabetic patients starting from 100 microliter serum or less, as well as for various other proteins. Unlike the usually employed thiobarbituric acid assay the present procedure is truly specific for the detection of ketoamine linkages of glycosylated proteins.