Application of affinity chromatography for separation and quantitation of glycosylated hemoglobins

The use of m-aminophenylboronic acid immobilized on agarose as the affinity matrix for the separation and quantitation of Glyco Hb has been investigated. The glyco fraction isolated from the affinity columns contained about 10% Hb A1a + b, 52% Hb A1c, and 38% Hb A0-like glyco components. The nonglyco fraction had major portions of Hb A1a + b and Hb A0 and a small amount of Hb A1c (also contained Hb F). In normals and diabetic patients, the comparison of the affinity method with the ion-exchange chromatographic, fluorometric, and colorimetric methods showed good correlation. The affinity method also showed acceptable precision and reproducibility. The presence of labile aldimine did not influence the affinity method because it seems that only the stable ketoamine was bound to the affinity gel and thus separated by this method. Moreover, this method was less sensitive to variations in column temperature and sample age. Glyco Hb levels were determined in newborn infants and in adults with various hemoglobinopathies. The results indicated that the presence of Hb F, Hb S, and Hb C did not affect the glyco Hb determinations. Thus the affinity chromatographic approach has wider application than ion-exchange chromatography and is, at the same time, much simpler and faster than the chemical methods.