A Simple and Rapid Method for The Determination of Total Glycosylated Hemoglobins (HbAIa+b+b+c)

Abstract

A simple and rapid method for the measurement of total glycosylated hemoglobins (HbAIa+b+c) is described. The washed erythrocytes were lysed by adding distilled water and toluene. The lysate was incubated with O.1 volume of a 10-times concentrated solution of phosphate cyanide buffer (Buffer I) overnight at 4°C. Bio-Rex 70 cation exchanger equilibrated with Buffrt I was poured into a small column (6 cm×1.2 cm) to a packed height of approximately 4 cm. Then, O.1 ml of the lysate was layered onto the surface of the resin and 8 ml of the eluate was collected. This fraction (F1) contained HbAia+b+c.Subsequently, Buffer I was replaced by high phosphate buffer (Buffer II) and further eluate (8 ml) was collected. This fraction (F2) contained all the other components of HbA in the lysate. The absorbance of Fl and of F2 diluted to 1: 8 with Buffer II was measured at 415 nm, and the percentage HbAIa+b+c was calculated as follows:
Excellent separation between Fl and F2 was obtained with the present small scaled column. The intra-assay variability of HbAIa+b+c, was evaluated by duplicate determinations in sera obtained from 5 normal subjects and 5 diabetic patients. There was no significant difference in HbAia+bas determined duplicate assays by the paired t test (0.4<p (t≥0.755)<0.5). Moreover, where measurements were repeated in lysate stored for up to 32 days at 4°C, the coefficient of variation was 3.8-11.5%. These results indicated that the present method was reproducible and HbA in the lysate remained stable on storing at 4°C for at least 32 days. The procedure thus took 2 hr to complete manually and more than 20 blood samples could be analyzed by one person ina day. The %HbAIa+b, +c., determined by the present method ranged from 7.0% to 7.9% in 9 normal subjects with no family history of diabetes mellitus (mean ± SD=7.4±0.3%). In 29 patients with diabetes mellitus, the %HbAia, b+c ranged from 7.2% to 17.9%. The average (11.8 ±2.8%) was significantly higher than that in normal subject (p<0.001). These findings suggest that our method may be useful as a routine clinical test for monitoring blood glucose in diabetic patients.