A Simple Assay for Lipid Hydroperoxides in Serum or Plasma.

Abstract

In order to develop a simple and reliable assay method for lipid hydroperoxides in serum or plasma, we sought to determine the suitable conditions for direct application of the previously reported colorimetric method using a methylene blue derivative, 10-(N-methylcarbamoyl)-3, 7-(dimethylamino)-phenothiazine, for the measurement of lipid hydroperoxides in a sample without extraction of them with organic solvents. For such purpose, dissociation of lipid hydroperoxides from proteins by lipoprotein lipase was found to be necessary. Also, we found that holotransferrin, which oxidizes the methylene blue derivative, should be eliminated by its chelation with trimethylenetetraminehexaacetic acid. Pretreatment of the sample with ascorbate oxidase was also necessary to eliminate interference by ascorbic acid in a sample. Thus the recommended method is to mix the sample with lipoprotein lipase, trimethylenetetraminehexaacetic acid, ascorbate oxidase, and the detergent Triton X-100, then to incubate the mixture with the methylene blue derivative dissolved in the detergent in the presence of hemoglobin, and to measure the oxidized product, methylene blue. The amount of lipid hydroperoxides is calculated by use of an external standard, either linoleic acid hydroperoxide or cumene hydroperoxide.