Location, antioxidant and recycling dynamics of alpha-tocopherol in liposome membranes

We studied the location of alpha-tocopherol (alpha-Toc) in the liposome membranes, and the dynamics of its radical scavenging and recycling by ascorbic acid. The quenching efficiency of alpha-Toc fluorescence by acrylamide, a water soluble quencher with a very low capacity to penetrate through phospholipid bilayers, was very low in dimyristoyl-phosphatidylcholine (DMPC) liposomes with and without charges, but relatively high in sodium dodecylsulphate (SDS) or tetradecyl-trimethylammonium bromide (TTAB) micelles. These findings indicate the low exposure of the chromanol at the surface of the liposome membranes. alpha-Toc was oxidized by positively charged Fe3+ more slowly in DMPC liposomes negatively charged with dicetylphosphate (DCP) (1st order rate constant, 1.41 x 10(-3) sec-1) than in negatively charged SDS micelles (7.14 x 10(-1) sec-1). Assuming that 100% of the OH-groups of alpha-Toc are at the membrane surface of the SDS micelles, as the oxidation rate of alpha-Toc in liposomes is 0.32 microM sec-1, which is about 150 times slower than that in micelles (49.3 microM sec-1), only 0.65% of the OH-groups of alpha-Toc are probably present at the membrane surface of the liposomes. The fluorescence of alpha-Toc was most effectively quenched by interaction with the spin group of the probe 5-(N-oxyl-4,4'-dimethyloxazolidin-2-yl) stearic acid (5-NS), indicating that its OH-group was located in a position corresponding to an inner 5-methylene carbon under the membrane surface. Ascorbic acid (AsA) was rapidly oxidized by 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) when it was ionically trapped at the positively charged membrane surface of egg yolk phosphatidylcholine (egg PC) liposomes with stearylamine (SA), but was scarcely oxidized in negatively charged egg PC-DCP liposomes because it was present in the bulk water phase. These findings suggest that lipid peroxy-radicals move from the hydrophobic region to near the membrane surface, where they are trapped by alpha-Toc. The electron spin resonance (ESR) spectra of 5-NS and 16-NS labeled in DMPC or DMPC-DCP liposomes were not changed by the addition of AsA in the buffer solution of pH 7.0, indicating that negatively charged AsA could not penetrate into neutrally or negatively charged membranes. alpha-Toc inhibited AMVN-induced lipid peroxidation and AsA extended its inhibition period, but glutathione (GSH) did not affect this inhibition period.(ABSTRACT TRUNCATED AT 400 WORDS)