Investigation of the heterogeneity of hemoglobin by cation-exchange chromatography on Bio-REX 70

The use of Bio-Rex 70 cation-exchange resin for chromatography of normal and diabetic hemoglobin provides a reproducible pattern of the "fast components". Particular attention to the choice of sample preparation, pH of elution, and the increase of ionic strength by sodium chloride linear gradients results in the separation of Hb-A1b into two components and in the isolation of a new component eluting between Hb-A1c and Hb-A0. Experiments with [3H]glucose and the colorimetric test (thiobarbituric acid) normally used to determine the extent of non-enzymatic glycosylation, as well as an increase of this component in diabetic samples compared with normoglycemic ones and a significant linear correlation with Hb-A1c, indicate that this component should be a part of the hemoglobins glucosylated on the epsilon-NH2 group of the lysines of both chains and/or the hemoglobin glucosylated on the alpha-NH2 of the valine of the alpha-chain. We propose to call this component Hb-A1x, pending confirmation of its identity. Normally Hb-A1x accounts for about 3% of Hb-A, but up to 5-7% of glucosylated hemoglobins should be confined to the early part of Hb-A0. In diabetics, the percentage of Hb-A1x rises to 4-5% and that of the other glucosylated hemoglobins increases to 12-16%.