Assay for lipid peroxide content in mitochondria by the thiobarbituric acid reaction.

Abstract

The thiobarbituric acid (TBA) reaction method established by Yagi to determine the lipid peroxide level in serum and animal tissues was applied to isolated mitochondria with some modifications. The TBA reaction was carried out in acetic acid (pH 2.0) in the presence of 0.7 % sodium dodecyl sulfate at 95°C for 45 min. Chloroform was used to remove the lipid and protein from the reaction mixture. An aqueous layer was used for spectrophotometric measurement at 532.5 nm to determine the TBA reaction product with lipid peroxide. To evaluate the TBA assay system, the lipid peroxide content of mitochondrial suspensions exposed to ultraviolet light was examined. The lipid peroxide formed in mitochondria was proportional to the dose of ultraviolet energy. The lipid peroxide of methyl linoleate exposed to ultraviolet light was determined both with the TBA reaction and with the hydroperoxide reaction. The linear increase in TBA values following UV exposure was similar to the increase of hydroperoxide values determined iodometrically. The TBA assay was shown to be a good predictor of lipid peroxide formed in mitochondria. This assay system is useful for lipid peroxide determination in various tissues: epidermis, Ehrlich ascites tumor cells and B-16 melanoma culture cells.