THE USE OF CIS-PARINARIC ACID TO MEASURE LIPID-PEROXIDATION IN CARDIOMYOCYTES DURING ISCHEMIA AND REPERFUSION

cis-Parinaric acid (PnAc), a fluorescent, polyunsaturated fatty acid, was used to measure lipid peroxidation during simulated ischemia and r eperfusion in cultured neonatal rat cardiomyocytes. PnAc was used both as free fatty acid, inserted in the membranes following cultivation o f the cells, as well as constituent of the cellular complex lipids by metabolically integrating the fatty acid during growth. In the inserti on experiments a pre-incubation with DL-aminocarnitine, an inhibitor o f beta-oxidation, was necessary to prevent loss of fluorescent signal. Such a pre-incubation resulted in an enrichment of PnAc in the sarcol emma: In pre-treated cells 57 +/- 1.3% of total inserted PnAc is prese nt in the sarcolemma compared to 27 +/- 5.7% in cells containing the i ntegrated probe. Both methods to introduce PnAc into the cells were co mpared with respect to their sensitivity for an externally applied oxi dative stress and thereafter lipid peroxidation during simulated ische mia and reperfusion was assayed. Going from normoxic to ischemic condi tions lipid peroxidation did not increase and remained at a low level. When the ischemic cells were subsequently subjected to reperfusion (r eintroduction of both oxygen and glucose), large scale lipid peroxidat ion was obvious. When, on the other hand, oxygen alone was reintroduce d (reoxygenation) no increased lipid peroxidation was observed. These observations led to the conclusion that ischemia does not lead to an e nhanced lipid peroxidation and that resumption of metabolic activity d uring reperfusion is necessary to induce lipid peroxidation. (C) 1997 Elsevier Science B.V.