A sandwich enzyme linked immuno-sorbent assay for the determination of rat heart fatty acid-binding protein using the streptavidin-biotin system. Application to tissue and effluent samples from normoxic rat heart perfusion

Abstract

An enzyme linked immuno-sorbent assay (ELISA) of the sandwich type for the determination of heart-type fatty acid-binding protein (H-FABPc) was developed, making use of the streptavidin-biotin system. The assay turned out to be virtually disturbance insensitive and showed a detection limit for H-FABPc of 0.2 micrograms/l with an intra- and inter-assay variation of 5% and 14%, respectively. The H-FABPc content of adult rat heart muscle was found to be 0.740 +/- 0.120 mg/g wet weight. The H-FABPc content of a number of skeletal muscles varied from 0.013 to 0.303 mg/g wet weight and was related to the content of type I muscle fibers of these tissues, suggesting a role for H-FABPc in intracellular fatty acid metabolism. The assay was further applied to study the release of H-FABPc from isolated rat heart during normoxic Langendorff perfusion, as compared to that of lactate dehydrogenase (LDH), into fluid derived from the right ventricular cavity (Qrv) and that from the interstitial space (Qi). Total release of H-FABPc per 15 min amounted to 0.015 +/- 0.010% but that of LDH to 0.080 +/- 0.040% of their total tissue content. Furthermore, for both H-FABPc and LDH 80% was released into Qi, which only accounted for 1-2% of total flow. These findings suggest that during normoxic perfusion of rat heart H-FABPc, and LDH are released from different cellular compartments and that the bulk amount of released intracellular proteins is transported via the lymph instead of being directly released into the bloodstream.