Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid

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Abstract
A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis‐parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37°C, with 5 μM for 60′ giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37°C while being examined in an Epics Elite flow cytometer equipped with a 325nm HeCd laser, and parinaric acid fluorescence at 405nm was measured over time. Addition of the oxidant tert‐butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells mrith dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant α‐tocopherol, 200 μM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without α‐tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis‐parinaric acid staining and could be excited by the latter using resonance energy transfer. Because these two probes show a degree of organelle specificity, they can be used to measure the loss of parinaric acid due to lipid peroxidation at defined subcellular sites. Although our own interest in the method is to examine the actions of redox cycling anticancer drugs and the integrity of host antioxidant defences as a possible mechanism of drug resistance, it appears to be a versatile technique for investigating an important process of cell injury that is difficult to study using standard biochemical assays.