Antioxidant activity analysis by liposomal membrane system and application to anesthetics

Antioxidant activity was analyzed using the liposomal membrane system for lipid peroxidation. Diphenyl-1-pyrenylphosphine incorporated into lipid-bilayer liposomes, which were prepared with unsaturated phospholipids and cholesterol, was converted to the corresponding fluorescent phosphine oxide by the reaction with lipid hydroperoxides. Under the optimal conditions, the liposomes were pretreated with tested compounds. Thereafter, they were reacted with oxidants: 1 - 50 microM peroxynitrite, 1.0 mM hydrogen peroxide or 0.4 mM t-butylhydroperoxide, followed by fluorometric analysis. The antioxidant activity was determined by comparing the obtained fluorescence intensities with controls. The proposed method was successfully applicable to various anesthetics (10 and 100 microM for each) and reference antioxidants: alpha-tocopherol (1.0 and 2.5 microM), quercetin (1.0 and 5.0 microM) and (-)-epigallocatechin-3-gallate (1.0 and 5.0 microM) with intraand inter-assay C.V.s of 0.2 - 7.0%. In analyses of general and local anesthetics, propofol was the most active, and showed a concentration of 6.8 microM to produce 50% inhibition against the peroxynitrite-induced lipid peroxidation of cell model membranes, supporting its potential benefit in clinical use.