Research Institute for Children, Children's Hospital, New Orleans, LA 70118, USA.
Anal Biochem. 2012 May 15;424(2):149-55. doi: 10.1016/j.ab.2012.02.011. Epub 2012 Feb 25.
Glucose spontaneously reacts with hemoglobin amino groups to produce unstable Schiff base complexes that can dissociate or rearrange to form stable Amadori products. We used dynamic capillary isoelectric focusing and boronate affinity chromatography to assess the formation and dissociation of unstable hemoglobin complexes in vitro. Formation was studied by incubating erythrocytes at 37°C for up to 24h in phosphate-buffered saline (PBS) supplemented with 0 to 55.6 mmol/L glucose. Dissociation was studied by incubating glucose-loaded erythrocytes in PBS without glucose. Dynamic capillary isoelectric focusing separated hemoglobin A1c into two subfractions identified as A1c1 and A1c2. The A1c1 subfraction contained both stable and unstable hemoglobin complexes. The A1c2 subfraction contained only unstable hemoglobin complexes. Both subfractions quantitatively increased in the presence of glucose and decreased in its absence. Rates of increase and decrease were faster and time to equilibrium was shorter for A1c2 (4 h) compared with A1c1 (20 h). Unstable hemoglobin complexes did not bind to boronate affinity columns but instead eluted intact in A1c1 and A1c2 subfractions from nonglycated affinity fractions. Cyanoborohydride reduction confirmed the presence of Schiff base complexes. Evidence of multiple unstable hemoglobin complexes with different rates of glycation suggests that new models are needed to describe nonenzymatic hemoglobin glycation.
葡萄糖会自发地与血红蛋白的氨基反应，生成不稳定的希夫碱复合物，这些复合物可以解离或重排，形成稳定的艾考糊精产物。我们使用动态毛细管等电聚焦和硼酸亲和层析技术来评估体外不稳定血红蛋白复合物的形成和解离。通过在磷酸盐缓冲盐水（PBS）中孵育红细胞，在 37°C 下孵育长达 24 小时，并补充 0 至 55.6mmol/L 的葡萄糖，研究其形成过程。通过在没有葡萄糖的 PBS 中孵育载葡萄糖的红细胞，研究其解离过程。动态毛细管等电聚焦将血红蛋白 A1c 分离成两个亚组分，分别鉴定为 A1c1 和 A1c2。A1c1 亚组分含有稳定和不稳定的血红蛋白复合物。A1c2 亚组分仅含有不稳定的血红蛋白复合物。在存在葡萄糖的情况下，这两种亚组分均定量增加，在没有葡萄糖的情况下则减少。与 A1c1（20 小时）相比，A1c2（4 小时）的增加和减少速度更快，达到平衡的时间更短。不稳定的血红蛋白复合物不会与硼酸亲和柱结合，但会在 A1c1 和 A1c2 亚组分中完整洗脱，而不会从非糖化亲和级分中洗脱。氰基硼氢化钠还原证实了希夫碱复合物的存在。存在具有不同糖化速率的多种不稳定血红蛋白复合物的证据表明，需要新的模型来描述非酶促血红蛋白糖化。
