抄録
Glycosylation of human hemoglobin has been calimed, as well as it was reported that the level of glycosylated hemoglobin was elevated in the individuals in poor diabetic control.
Structual studies in human hemoglobins have shown that hemoglobin is a tetrameric protein consisting of two alpha and two beta subunits, which exists in two allosteric forms, one called T and the other did R form. In the physiological condition hemoglobin in the deoxy state is the T form with which heme-iron exists in high spin, on the other hand in the oxy one is the R form with which heme-iron in low spin. Between these two forms there are differences in physical properties so that those are able to identify from each other by a certain technique such as visible or ultraviolet spectroscopy.
Many organic phosphates are known as effective allosteric effector site binding substances, also many of which, for a example, inositol hexaphosphate (IHP) binds so tightly to the binding site that the conformation of the tetrameric protein of hemoglobin causes a shift from the R to the T allosteric form. This reaction may be failed in glycosylated hemoglobins, since these substances (i. e. IHP) binding to glycosylated hemoglobin can be blocked by the glucose residue covalently attached to the N-terminus of the beta chains. The relative amount of glycosylated hemoglobins present in the blood sample can be determined from the absorption spectrums taken before and after addition of a prefered allosteric effector site binding substance.
We evaluated the A-gent from ABBOTT KK, the kit for to determine the concentration of glycosylated hemoglobins consists imidazole buffer with potassium ferricyanide and a detergent, IHP solution and three calibrators. The potassium ferricyanide oxidizes the hemoglobin, and a detergent lyses the red blood cells to release homoglobins. The imidazole coordinates with the heme-iron shifting equilibrium allosteric isomers to the R form. Addition of IHP which reacts with the allosteric binding site of major (non-glycosylated) hemoglobin, causes a shift of the equilibrium of allosteric isomers to the T form.
The absorption spectrums at 560nm and 633nm are measured before and after addition of IHP to the treated sample. The concentration of glycosylated hemoglobin in the specimens may be given from the standard curve of three calibrators.
For this study we utilized ABBOTT-VP automated analyzer and the specimens from both diabetic and non-diabetic subjects. Three specimens were tested from different classes to carry out the precision study, their mean values were 632, 7.92 and 12.13% glycosylated hemoglobin and±S. D.(or±C. V.) values were respectively 0.39 (6.10%), 0.32 (3.98%) and 1.03 (8.51%). The regression statics for 40 clinical specimens between the values obtained by the proposed method and those by HPLC method gave the correlation coefficient, 0.659 and the linear regression appeared its slope, 1.7 and Y-intercept, 6.7.
This method is able to be automated and may has the advantages in simple and rapidly performance of the glycosylated hemoglobin test.
