The Measurement of Glycosylated Hemoglobin in Man and Animals by Aminophenylboronic Acid Affinity Chromatography

Abstract

Glycosylated hemoglobin (GHb) was estimated in normal and diabetic human, rat, and dog hemolysate by m-aminophenylboronic acid (PBA) affinity chromatography and the results compared with the values determined using two ion-exchange (ION-E) methods and a colorimetric thiobarbituric acid (TBA) method. There was a good correlation between the values estimated by PBA and both ION-E chromatography methods for the human samples (r = 0.83, P < 0.0002, r = 0.86, P < 0.002). In diabetic rat and dog hemolysates, PBA chromatography demonstrated higher glycosylated hemoglobin than in normal hemolysate. In both species, there was an excellent correlation between the PBA-estimated GHb and plasma glucose levels (rat r = 0.79, P < 0.001; dog r = 0.67, P < 0.003). The IONE and TBA methods were not as effective in separating diabetic from normal samples and correlated less well with plasma glucose levels. PBA chromatography relies on the interaction of mamino phenylboronate with the hydroxyl groups of the glucose residues attached to hemoglobin. It is not affected by intra- or interspecies variations in the hemoglobin moiety and should be adaptable for measurement of GHb in a number of laboratory animals and in patients with hemoglobinopathy. It is not affected significantly by temperature and may offer advantages over the ION-E method in the routine determination of human glycosylated hemoglobin.