A Simplified Assay of Hemoglobin AIc in Diabetic Patients by Use of Isoelectric Focusing and Quantitative Microdensitometry

Abstract

Isoelectric focusing (IEF) of human erythrocyte hemolysates on poly aery la mide slab gels over a pH gradient of 6 to 8 provides sufficient resolution of hemoglobin Ale (HbAIc) from other hemoglobin components (AIa, AIb, AII, S, and F) to allow quantification by high-resolution microdensitometry. Twice-chromatographed HbAIc alone and in admixtures with normal and diabetic hemolysates were employed to verify the identification and quantification of the Aie component. Relative concentration was determined as a per cent of the total hemoglobin absorption at 556 nm on acid-fixed, unstained gels. A total of 60 patient samples were examined by IEF, and, for 35 samples, both column chromatography and IEF determination were obtained, revealing excellent correlation between these two methods.