Spicer K M, Allen R C, Hallett D, Buse M G
J Clin Invest. 1979 Jul;64(1):40-8. doi: 10.1172/JCI109461.
Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.
在体外研究了影响完整红细胞合成血红蛋白（Hb）A1c的因素。孵育后细胞裂解，通过在聚丙烯酰胺平板凝胶上进行等电聚焦分离血红蛋白，并通过微量光密度测定法定量。HbA1c随时间、葡萄糖浓度（5 - 500 mM）和孵育温度（4℃ - 37℃）增加。低温允许长时间孵育且溶血最小。在4℃时，HbA1c随时间线性增加6周；在最高葡萄糖浓度下孵育后，HbA1c占总血红蛋白的50%。胰岛素（1和0.1 mU/ml）在体外不影响HbA1c合成。除葡萄糖外，半乳糖和甘露糖而非果糖可作为HbA1c的前体。己糖激酶的良好底物（2 - 脱氧葡萄糖）和不良底物（3 - O - 甲基葡萄糖）比葡萄糖是更好的HbA1c合成前体，表明HbA1c合成不需要葡萄糖的酶促磷酸化。红细胞与（32）P - 磷酸盐孵育后的放射自显影显示放射性掺入HbA1a1和A1a2，但不掺入HbA1b、A1c或A。与乙酰水杨酸孵育期间产生的乙酰化HbA向阳极迁移至HbA1c并与其明显分离。胰岛素缺乏型糖尿病患者的红细胞在相同葡萄糖浓度下孵育时，合成HbA1c的速率与对照组相同。同样，囊性纤维化和先天性球形红细胞增多症患者的红细胞合成HbA1c的速率与对照组平行。当脐血以及HbC和镰状细胞贫血患者的红细胞在高浓度葡萄糖下孵育时，胎儿血红蛋白、HbC和镰状血红蛋白减少，而聚焦在接近相应糖基化衍生物预期等电点的血红蛋白出现的比例相应增加。
