A novel colorimetric method for determination of glycated protein based on 2-keto-glucose release with hydrazine

We tried to measure glycated proteins by a novel method based on colorimetry of 2-keto-glucose which is released from the glycated protein (ketoamine) on heating with hydrazine. Reaction conditions were optimized with glycated human serum albumin (glc HSA) as a model compound. Ketoamine reacted quantitatively with hydrazine on heating at 100 degrees C for 0.5 h, followed by heating with phenylhydrazine at 60 degrees C for 1 h. Glucose interference with the assay was eliminated by preincubation of the sample with glucose oxidase at 37 degrees C for 0.5 h. Time courses for the coloration of glc HSA and human serum showed a profile similar to that of N-p-tolyl-D-isoglucosamine under optimized reaction conditions. The lower limit for the assay of glc HSA was 0.7 microM. The serum level of glycated proteins measured by the present method correlated well with that (fructoamine value, microM) measured by the conventional method (nitroblue tetrazolium-reducing method) (r = 0.92, n = 35). In conclusion, the present method is a novel, highly sensitive and reliable one for measuring glycated proteins in biological samples.