QUANTITATION OF GLYCATED HEMOGLOBINS IN HUMAN ADULT-BLOOD BY CAPILLARY ISOELECTRIC-FOCUSING

A precise and reproducible method for assessment of glycated hemoglobi n in human adult red blood cells is reported, based on capillary Isoel ectric focusing (IEF). in order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A(1c)), two species which differ by minute Delta pI values, <0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamid e and an equimolar mixture of two ''separators'', 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid, The last two compounds flatten the pH gradient in the pI region of the two Kbs, thus allowing full separatio n. Additionally, the Hb samples, instead of being pulse-loaded, are un iformly distributed in the background electrolyte. A longer capillary life-time is obtained if ail nonbuffering ions are eliminated; thus, a s catholyte, 50 mM Lys (PH 9.7) is utilized and as anolyte 50 mM aceti c acid (pH 3.5) is adopted. The percentages of Hb A(1c), as obtained b y capillary IEF; are in good agreement (+/-6%) with data obtained by o ne of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.