Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591).

A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.

Validation of the peroxidative indicators, cis-parinaric acid and parinaroyl-phospholipids, in a model system and cultured cardiac myocytes.

cis-Parinaric acid is increasingly being used in eukaryotic cells as a very sensitive marker for the initial stages of lipid peroxidation. Despite the increased application of this probe, no extensive validation, especially in cellular systems, has been performed. cis-Parinaric acid can either be inserted freely into biomembranes or incorporated (bio)synthetically into lipids (parinaroyl-lipid). Therefore, a direct comparison was made between the peroxidative behaviour of the two parinaroyl probes and the endogenous polyunsaturated fatty acids arachidonic and linoleic acid, in both an artificial lipidic system and in cultured neonatal rat heart cells. Three different radical generating systems were used, i.e., hydrogen peroxide, cumene hydroperoxide and the thermo-labile 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The data demonstrate that the peroxidation rate of cis-parinaric acid is higher than that of the parinaroyl, arachidonoyl and linoleoyl lipids. The latter three displayed comparable peroxidation rates, showing that the peroxidative decay of parinaroyl-lipid is a good marker for the degradation of endogenous polyunsaturated fatty acids. Experimental results using the freely inserted cis-parinaric acid could potentially lead to an overestimation of the inflicted damage and should be interpreted with care. In addition, a comparison was made with the measurement of conjugated dienes and malon dialdehyde as thiobarbituric acid reactive substances. The results demonstrate that measurement of conjugated dienes and malon dialdehyde only provide information on peroxidative processes in vitro, but are not suitable for in-depth studies in cultured cells. In contrast, the use of the parinaroyl probes is a suitable, straightforward, sensitive and reproducible method for detecting the initial stages of lipid peroxidation in living cells.

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 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 DL-aminocarnitine, an inhibitor of beta-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.