Chromatographic and colorimetric detection of glycosylated hemoglobins: A comparative analysis of two different methods

Abstract

The measurement of glycosylated hemoglobins in diabetic patients represents a new approach to the problem of the long-term glycemie control. Chromatographic procedures are usually employed to separate the glycosylated components of hemoglobin; we performed a comparative analysis of two different methods: Chromatographic and colorimetric. Chromatographic separation on small ion-exchange resin columns gives high precision (coefficient of variation = 1.7%) over the whole range of normal and diabetic values of percentage of glycosylated hemoglobin (3.2–15.9%). An alternative technique is the measurement of 5-hydroxymethylfurfural released by oxalic acid hydrolysis of the hexoses bound to hemoglobin, as proposed by Fluckiger and Winterhalter (Fluckiger, R. and Winterhalter, K.H. (1976) FEES Lett. 71, 356–360). Normal values, expressed as 5-hydroxymethylfurfural absorbance per 10 g of total hemoglobin, range from 133 to 235, with mean ± S.D. = 189 ± 26; while diabetic patients show a range from 220 to 443 and mean ± S.D. = 318 ± 65. This last method gives satisfactory precision over the entire range of values examined (coefficient of variation = 4.2%) and has proved simple and inexpensive. The correlation between the two methods is very high ( n = 20; r = 0.98; p < 0.001) with a regression line, y = 25. Ix + 40.3. The storage of the hemolysates at −20°C for up to 70 days for the colorimetric method and up to 260 for the Chromatographic procedure does not decrease the precision of either technique.