Analytical Sciences

Antioxidant Activity Analysis by Liposomal Membrane System and Application to Anesthetics

Hironori TSUCHIYA,* Takahiro UENO,** Maki MIZOGAMI,** and Ko TAKAKURA**

* Department of Dental Basic Education, Asahi University School of Dentistry, 1851 Hozumi, Mizuho, Gifu 501-0296, Japan
** Department of Anesthesiology, Asahi University School of Dentistry, 1851 Hozumi, Mizuho, Gifu 501-0296, Japan

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 µM 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 µM for each) and reference antioxidants: α-tocopherol (1.0 and 2.5 µM), quercetin (1.0 and 5.0 µM) and (-)-epigallocatechin-3-gallate (1.0 and 5.0 µM) 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 µM to produce 50% inhibition against the peroxynitrite-induced lipid peroxidation of cell model membranes, supporting its potential benefit in clinical use.

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