Klenk D C, Hermanson G T, Krohn R I, Fujimoto E K, Mallia A K, Smith P K, England J D, Wiedmeyer H M, Little R R, Goldstein D E
Clin Chem. 1982 Oct;28(10):2088-94.
An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.
将一种用于测定糖化血红蛋白的亲和色谱法（《分析快报》14: 649 - 661, 1981）与硫代巴比妥酸比色法（I）（《临床化学》27: 669 - 672, 1981）以及离子交换液相色谱法（II）（《糖尿病》29: 623 - 628, 1980）进行比较。亲和法与II的相关性为0.98，亲和法与I的相关性为0.97（n = 51）。非糖尿病个体标本的批内变异系数为1.9%，糖尿病个体标本的批内变异系数为1.0%。各自的批间变异系数分别为3.4%和2.4%。红细胞在等渗盐水中（37℃）孵育5小时未能去除“不稳定”葡萄糖加合物，导致II的平均误差为13.1%，I的平均误差为5.4%，亲和法的平均误差为1.6%。在18至27℃之间，温度每升高1.0℃，亲和色谱法测得的糖化血红蛋白降低0.1 - 0.2%。将亲和程序中使用的洗涤缓冲液的pH从7.75变化到8.25（最适pH 8.0），一个糖尿病标本的糖化血红蛋白净变化为0.5%。使用亲和法，我们测定了124名明显健康个体的糖化血红蛋白参考区间为5.3%至7.5%（平均值6.36%，标准差0.55%）。通过II进行再色谱分析以及对亲和分离得到的馏分进行等电聚焦分析发现，存在大量未被II检测到的糖化血红蛋白。亲和法为目前使用的方法提供了一种快速、简单、精确且准确的替代方法，并且很大程度上不受许多常见干扰因素的影响。
