A new colorimetric method for the estimation of glycosylated hemoglobin

A new colorimetric method, based on the phenol sulphuric acid reaction of carbohydrates, is described for the determination of glycosylated hemoglobin. Hemolyzates were treated with 1 mol/1 oxalic acid in 2 mol/1 HCl for 4 h at 100°C, the protein was precipitated with trichloroacetic acid, and the free sugars and hydroxymethyl furfural in the protein free supernatant were treated with phenol and sulphuric acid to form the color. The new method is compared to the thiobarbituric acid method and the ion-exchange chromatographic method for the estimation of glycosylated hemoglobin in normals and diabetics. The increase in glycosylated hemoglobin in diabetic patients as estimated by the phenol-sulphuric acid method was more significant ( P < 0.001) than the increase observed by the thiobarbituric acid method ( P < 0.01). The correlation between the phenol-sulphuric acid method and the column method was better ( r = 0.91) than the correlation between the thiobarbituric acid method and the column method ( r = 0.84). No significant correlation between fasting and postprandial blood sugar level and glycosylated hemoglobin level as determined by the two colorimetric methods was observed in diabetic patients.