Evaluation of a colorimetric method for determination of glycosylated hemoglobin

Abstract

We evaluated a colorimetric assay for glycosylated hemoglobin to determine the effects of several variables - oxalic acid concentration, extraneous glucose, hemoglobin concentration, hydrolysis interval, and 5-hydroxyl-methylfurfural destruction - and the precision. The interference seen when the blood glucose concentration exceeds 2.0 g/L (11 mmol/L) can be eliminated by washing the erythrocytes with 9 g/L saline. The accuracy of this assay is not influenced by hemoglobin concentrations from 80 to 150 g/L. The background nonspecific color, although substantial, is similar from sample to sample. After a 5-h hydrolysis at 100°C, about 85% of the hexose is released, and the analytical recovery of 5-hydroxymethylfurfural is constant over a wide range of glycosylated hemoglobin concentrations. The 5th to 95th percentile reference interval for a population of 65 nondiabetic individuals was 4.6 to 6.1 mol per 100 mol of total hemoglobin. The range of values for a population of 85 diabetic patients was 6.9 to 20.4 mol per 100 mol.