New Method for Detection Ischemic Heart Cell Damage

脂肪酸结合蛋白(FABP)被认为在脂肪酸的细胞内转运和脂类代谢中起着重要的载体蛋白作用。它占可溶蛋白质的几个百分比。由于FABP的相对分子质量较低(摩尔重量为14000)，因此在缺氧性心肌细胞损伤时，FABP可能从细胞内释放出来，成为缺氧性细胞损伤的标志物。新生大鼠心肌细胞在低氧条件下孵育6h后，测定FABP和CPK的释放。根据Glatz等人的方法，使用LipidEx 1000进行样品的脱脂和脂肪酸结合的测定。LipidEx 1000是Sephadex G-25的10%(w/w)取代羟基烷氧基丙基衍生物，可在0ﾟC从蛋白质-脂肪酸复合体中去除游离脂肪酸，并根据蛋白质-脂类相互作用动力学从水溶液中去除37ﾟC下的所有脂肪酸。对于脱脂，样品(30 Mg)在LipidEx柱(1.5x7…)上进行层析More cm)，用10 mM磷酸二氢钠缓冲液(pH 7.4)在37ﾟ℃下平衡，用相同的缓冲液洗脱，所有的蛋白质都在空泡柱中回收。为了测定脂肪酸结合，样品与不同浓度的14C标记脂肪酸(amersham；Arlington Heights，IL)在聚乙烯管中在10 mm磷酸二氢钠缓冲液(pH 7.4；最终体积0.45ml)中在37℃下孵育10分钟。然后，为了去除游离脂肪酸，管在冰和冰冷LipidEx/Buffer悬浮液(1：1v/v；0.05ml)，在0ﾟC中继续孵育10min，根据离心后上清液中的放射性计算脂肪酸结合量，以pmoL/ug蛋白表示。细胞死亡率从4h开始增加，6h上升到80%，但在有氧条件下仅为8%。FABP在1h即可检测到，4h后迅速升高并达到平台期，而CPK在6h内几乎不释放。钙拮抗剂和阻断剂抑制FABP的释放，阻止细胞死亡。这些结果表明FABP是心肌细胞损伤的标志物，缺氧性细胞损伤是通过肾上腺素能受体介导的。较少

Fatty acid binding protein (FABP) is thought to play an important role as a carrier protein in intracellular transportation of fatty acids and lipid metabolism. It accounts for several percentage of soluble protein. Because the molecular weight of FABP is low (mol wt 14000), it may be released from the cells in case of the hypoxic myocyte as a maker of the hypoxic cell injury. New born rat myocytes were incubated under hypoxic treatment for 6 hrs, then the release of FABP and CPK were measured. Delipidation of samples and assay of fatty acid binding were carried out using a Lipidex 1000, according to the method of Glatz et al.. Lipidex 1000, a 10% (w/w) substituted hydroxyalkoxypropyl derivative of Sephadex G-25, removes unbound fatty acids from protein-fatty acid complexes at 0^ﾟC, and removes all fatty acids at 37^ﾟC from aqueous solutions according to protein-lipid interaction kinetics. For delipidation, samples (30mg) were subjected to chromatography on the Lipidex column (1.5 x 7 … More cm) equilibrated with 10mM sodium phosphate buffer (pH 7.4) at 37^ﾟC. The column was eluted with the same buffer and all the proteins were recovered in the void bolume. For the assay of fatty acid binding, samples were incubated with various concentrations of 14C-labeled fatty acids (Amersham ; Arlington Heights, IL) in a polyethylene tube in 10 mM sodium phosphate buffer (pH 7.4 ; final volume 0.45ml) for 10 min at 37 C. Then, to remove unbound fatty acids, the tubes were cooled on ice and ice-cold Lipidex/buffer suspension (1 : 1 v/v ; 0.05ml) was added and incubated for another 10min at 0^ﾟC. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant after centrifugation of the tubes, and was expressed as pmol/ug of protein.The cell death ratio during hypoxygenation increased since 4 h and rose to 80% at 6 h, but it was only 8% under aerobic conditions. FABP was detected at 1 h, rapidly increased and reached plateau at 4 h. On the other hand CPK release was negligible during 6h. Ca-antagonist and blocker inhibited the release of FABP and prevented the cell death. These result shows FABP is of use as a maker of myocardial cell injury, and hypoxic cell injury is mediated through adrenergic receptor. Less