Measurement of glycosylated amino acids and peptides from urine of diabetic patients using affinity chromatography

An affinity chromatography system has been developed that retains glycosylated amino acids and peptides. Using this system, synthetic ¹⁴C-glycosylated lysine (reduced with NaB³H₄) was completely separated from a mixture of reduced ¹⁴C-glycosylated lysine and unmodified ³H-lysine. Amino acid analysis of the retained peak from hydrolyzed human diabetic hemoglobin previously reduced with NaB³H₄ revealed an equimolar mixture of glycosylated valine and glycosylated lysine, in agreement with previously published data obtained using other methodologies. These data demonstrate that in alkaline solution, the NaB³H₄-reducible breakdown products of nonenzymatically glycosylated proteins are adsorbed to m-aminophenyl boronic acid immobilized on Bio-Gel P-6, while nonglycosylated amino acids are not. This affinity chromatography system should facilitate the rapid evaluation of nonenzymatic glycosylation in a large number of diabetic tissues. Levels of retained compounds in urine from diabetic and normal patients were determined by measuring ninhydrin-positive material. Amino acid analysis of NaB³H₄-reduced hydrolysates of these peaks showed that glycosylated lysine was the major borohydride-reducible adduct present (67%-86%). Linear regression analysis showed that the quantity of excreted compounds in normals correlated with body weight (r = 0.63). The mean level (μmol leucine-equivalent/24 hr/kg body weight) in diabetics was over 1.5 times that found in urine from normal subjects (P < 0.005).