1993 Volume 39 Issue Supplement Pages S9-S22
We studied the location of α -tocopherol (α -Toc) in the liposome membranes, and the dynamics of its radical scavenging and recycling by ascorbic acid. The quenching efficiency of α -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. α -Toc was oxidized by positively charged Fe3+ more slowly in DMPC liposomes negatively charged with dicetylphosphate (DCP) (1st order rate constant, 1.41×10-3 sec-1) than in negatively charged SDS micelles (7.14×10-1 sec-1). Assuming that 100% of the OH-groups of α -Toc are at the membrane surface of the SDS micelles, as the oxidation rate of α -Toc in liposomes is 0.32μM sec-1, which is about 150 times slower than that in micelles (49.3μM sec-1), only 0.65% of the OH-groups of α -Toc are probably present at the membrane surface of the liposomes. The fluorescence of α -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 α -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. α -Toc inhibited AMVN-induced lipid peroxidation and AsA extended its inhibition period, but glutathione (GSH) did not affect this inhibition period. Oxidation of α -Toc in association with inhibition of lipid peroxidation was suppressed completely in the presence of negatively charged AsA and slightly in the presence of neutrally charged GSH, although AsA and GSH could not penetrate into these liposome membranes. These findings suggest that the resulting reversible oxidized product, probably the α -Toc cation, moves to the membrane surface, where α -Toc is regenerated by AsA.
