Comparison of a colorimetric assay for glycosylated hemoglobin with ion-exchange chromatography

Abstract

Because levels of glycosylated hemoglobin (GHb) are increased in diabetes and reflect the previous metabolic control, clinicians and clinical investigators are finding increasing applications for measurements of GHb in diabetic patients. We report the characterization of a colorimetrie assay procedure for GHb and compare its performance with that of a commonly used assay by ion-exchange chromatography. Although results of GHb determination by both methods correlate highly ( r = 0.943, P < 0.001), the two procedures estimate different glycosylated fractions. The colorimetrie procedure is nonstoichiometric, requiring careful standardization of assay conditions, including the concentration of total hemoglobin in the assayed aliquot, to achieve precision and permit comparison of results. We characterized the effect of storage of hemolysates or packed erythrocytes on the subsequent determination of GHb by both methods. Determinations of GHb by the colorimetrie method, but not by column chromatography, are reproducible on hemoly-sates or packed erythrocytes stored frozen for at least 5 mo. A unique advantage of the colorimetrie procedure is the capability to estimate GHb levels when variant hemoglobins, including fetal and sickle hemoglobins, are present.