Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E-enriched rat liver mitochondria and microsomes

Substantial loading of rat liver mitochondrial and microsomal membranes with D-alpha-tocopherol was achieved by dietary supplementation with no adverse effects of this loading being apparent, e.g. on treadmill exercise endurance. The tocopheroxyl radical was readily detected by ESR in the enriched microsomes and mitochondria. Continuous enzymatic oxidation with horseradish peroxidase and a hydrophilic phenol, to favor selective oxidation of tocopherol without the involvement of lipid peroxidation, allowed the tocopheroxyl radical to be observed for up to 1 h in liposomes of dioleoylphosphatidylcholine and for about 15 min in the subcellular membranes. Total alpha-tocopherol decreased throughout this period, but a significant residual fraction remained after all the ESR signal of tocopheroxyl had disappeared. Decay kinetics of the tocopheroxyl radical ESR signal produced by a burst of intense UV irradiation consisted of a rapid initial phase and a slower exponential decay. A more narrow and more persistent ESR signal, not yet chemically identified, was observed after the tocopheroxyl radical had disappeared under prolonged oxidation. Ascorbic acid prevented formation of the tocopheroxyl radical until the ascorbyl radical ESR signal had decayed, whereas uric acid, up to saturating concentration in phosphate buffer, had no effect.