Conti M, Gelfi C, Bosisio A B, Righetti P G
Faculty of Sciences, Department of Cell Biology, University of Calabria, Arcavacata di Rende (Cosenza), Italy.
Electrophoresis. 1996 Oct;17(10):1590-6. doi: 10.1002/elps.1150171017.
A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), 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 polyacrylamide 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 Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.
报道了一种基于毛细管等电聚焦（IEF）评估成人红细胞糖化血红蛋白的精确且可重复的方法。为了在成人血红蛋白（Hb A）与其糖化形式（Hb A1c）之间获得基线分辨率，这两种物质的等电点差值极小，<0.03 pH单位，采用了以下步骤：聚焦混合物由5% pH 6 - 8的两性电解质、0.5% pH 3 - 10的Pharmalyte、3%短链液体聚丙烯酰胺以及两种“分离剂”的等摩尔混合物（0.33 M β-丙氨酸和0.33 M 6-氨基己酸）组成。最后两种化合物使两种血红蛋白等电点区域的pH梯度变平，从而实现完全分离。此外，血红蛋白样品不是脉冲加载，而是均匀分布在背景电解质中。如果消除所有非缓冲离子，可获得更长的毛细管使用寿命；因此，作为阴极电解液，使用50 mM赖氨酸（pH 9.7），作为阳极电解液，采用50 mM乙酸（pH 3.5）。通过毛细管等电聚焦获得的Hb A1c百分比与临床化学中一种标准区带电泳方法（即海伦娜REP糖凝胶系统）获得的数据高度一致（±6%）。
