New Method for Detection Ischemic Heart Cell Damage

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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