The method for determination of serum lipid-free apolipoprotein concentration and its clinical application

众所周知，高密度脂蛋白颗粒大小不均一，小颗粒高密度脂蛋白比大颗粒能更有效地促进胆固醇从细胞膜外流。用双向凝胶电泳法检测小分子高密度脂蛋白，但这种方法耗时较长。采用高效液相色谱分离无脂载脂蛋白A-I，洗脱时间为51~56分钟。无脂载脂蛋白A-I的表位与高密度脂蛋白的表位相同，这是因为我们在ELISA系统中使用了载脂蛋白A-I的抗体。我们在血液中加入抗坏血酸，并用高效液相色谱法将其加入到500SOU/μ的分级溶液中。经过反复冻融试验，血清无脂载脂蛋白A-I浓度增加了2倍，即使在4℃时仍随时间变化。因此，血清样本立即进行高效液相处理或在-80℃下冷冻直到使用。冠心病患者的血清无脂载脂蛋白A-I浓度显著高于健康对照组，无论男性还是女性。这些发现表明，冠心病患者的胆固醇逆向转运发生了改变，而无脂载脂蛋白A-I的测定可能是动脉粥样硬化的一个很好的标志。

It is known that HDL are heterogeneous in particle size and small HDL particle can promote more effectively cholesterol efflux from cell membrane than large HDL particle. Small HDL are detected by two-dimentional gel electrophoresis, but this method is time consuming. Lipid-free apo A-I in this study was separated by HPLC and was elutred from 51 min to 56 min. The epitope of lipid-free apo A-I is the same as tjhat of a HDL, because we used the antibody to apo A-I in the ELISA system. We added SOU/ml of trasyrol to blood and also to 500 μ1 of fractionated solutions by HPLC. Serum lipid-free apo A-I concentration increased two times by repeated freeze and thawing test and changed with time even in the store at 4℃. Thus, serum sample was immediately treated for HPLC or freezed at -80℃ until use.Serum lipid-free apo A-I concentrations significantly increased in patients with coronary heart disease compared to healthy controls in both males and females. These findings show that reverse cholesterol transport is altered in coronary heat disease and the measurement of lipid-free apo A-I may be a good marker for atherosclerosis.