The use of cis-parinaric acid to measure lipid peroxidation in cardiomyocytes during ischemia and reperfusion

摘要

cis-Parinaric acid (PnAc), a fluorescent, polyunsaturated fatty acid, was used to measure lipid peroxidation during simulated ischemia and reperfusion in cultured neonatal rat cardiomyocytes. PnAc was used both as free fatty acid, inserted in the membranes following cultivation of the cells, as well as constituent of the cellular complex lipids by metabolically integrating the fatty acid during growth. In the insertion experiments a pre-incubation with -aminocamitine, an inhibitor of β-oxidation, was necessary to prevent loss of fluorescent signal. Such a pre-incubation resulted in an enrichment of PnAc in the sarcolemma: In pre-treated cells 57 ± 1.3% of total inserted PnAc is present in the sarcolemma compared to 27 ± 5.7% in cells containing the integrated probe. Both methods to introduce PnAc into the cells were compared with respect to their sensitivity for an externally applied oxidative stress and thereafter lipid peroxidation during simulated ischemia and reperfusion was assayed. Going from normoxic to ischemic conditions lipid peroxidation did not increase and remained at a low level. When the ischemic cells were subsequently subjected to reperfusion (reintroduction of both oxygen and glucose), large scale lipid peroxidation was obvious. When, on the other hand, oxygen alone was reintroduced (reoxygenation) no increased lipid peroxidation was observed. These observations led to the conclusion that ischemia does not lead to an enhanced lipid peroxidation and that resumption of metabolic activity during reperfusion is necessary to induce lipid peroxidation.