ascorbic-acid and tocopherylquinone

Results from in vivo studies of the capacity of vitamin C to spare and/or recycle vitamin E are equivocal. While some in vitro and membrane models reveal an interaction between vitamins C and E, the characterization of this relationship in biologically relevant systems is lacking. Thus, we investigated this relationship using hepatocytes isolated from 3- to 6-month-old male Sprague-Dawley rats. Cells were incubated for 18-20 h in medium supplemented with 0.1-4 mM ascorbic acid. The loss of alpha-tocopherol and the formation of its primary oxidized metabolite, alpha-tocopherolquinone, was determined by HPLC. Levels of alpha-tocopherol in hepatocytes incubated without ascorbic acid declined from 390 to 35 pmol/mg protein; hepatocyte ascorbic acid levels declined from 9 to 0.5 nmol/mg protein. alpha-Tocopherolquinone was undetectable in freshly isolated hepatocytes but following incubation in ascorbate-free medium reached 10 pmol/mg protein. The formation of alpha-tocopherolquinone was not detected in hepatocytes incubated with ascorbic acid. Dehydroascorbic acid (DHA) levels represented 10-20% of the total ascorbate content in freshly isolated hepatocytes but after 3 h incubation the proportion of DHA increased to 50%; after 18-20 h incubation DHA was undetectable. Hepatocytes incubated with 1.0, 2.0, 2.5, or 4.0 mM ascorbic acid lost significantly less alpha-tocopherol (62, 69, 67, and 56%, respectively) than unsupplemented controls (90%). Twelve percent of the alpha-tocopherol lost from hepatocytes during incubation was detected in the medium of cells incubated with ascorbic acid, but vitamin E was undetectable in the medium of cells incubated without ascorbic acid. These results demonstrate an interaction between vitamins C and E in cell culture and are not inconsistent with a potential recycling of oxidized alpha-tocopherol by ascorbic acid.

Topics: Animals; Ascorbic Acid; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Glutathione; Liver; Male; Rats; Rats, Sprague-Dawley; Time Factors; Vitamin E

A kinetic study of the regeneration reaction of vitamin E (tocopherol) with eight biological hydroquinones (HQs) (ubiquinol-10 (Q10H2 1); ubiquinol-0 (Q0H2 2); vitamin K1 HQ (VK1H2 3); vitamin K3 HQ (VK3H2 4); alpha-, beta-, and gamma-tocopherol-HQs (alpha-, beta-, and gamma-TQH2 5-7); and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) in solution was performed. The second-order rate constants (k4) for the reaction of HQs 1-8 with alpha-tocopheroxyl and 5,7-diisopropyltocopheroxyl radicals in ethanol, benzene, and isopropyl alcohol/water (5:1, v/v) solutions were measured with a stopped-flow spectrophotometer. The order of magnitude of k4 values obtained for HQs is VK1H2 > VK3H2 > alpha-TQH2 > beta-TQH2 approximately gamma-TQH2 approximately TMQH2 > Q10H2 > Q0H2, being independent of the kinds of tocopheroxyl radicals and the polarity of the solvents. The log of the k4 values obtained for HQs was found to correlate with their peak oxidation potentials. Comparing the k2 value (2.68 x 10(6) M-1 s-1 obtained for the reaction of alpha-tocopheroxyl with vitamin C (sodium ascorbate) with those (k4 = 2.54 x 10(5) and 8.15 x 10(5) M-1 s-1) obtained for the reaction of alpha-tocopheroxyl with Q10H2 and alpha-TQH2 in isopropyl alcohol/water mixtures, the former is approximately 11 and 3 times as reactive as the latter, respectively. On the other hand, the k2 value obtained for sodium ascorbate is smaller than the k4 values obtained for VK1H2 and VK3H2. These results suggest that mixtures of vitamin E and these HQs (as well as those of vitamins E and C) may function synergistically as antioxidants in various tissues and mitochondria.

Topics: Ascorbic Acid; Free Radicals; Hydroquinones; In Vitro Techniques; Kinetics; Lipid Peroxides; Oxidation-Reduction; Solutions; Ubiquinone; Vitamin E; Vitamin K

An earlier report from this laboratory showed that tocopherol in human platelets is oxidized when the platelets are incubated in vitro in Tyrode medium with arachidonate (or other oxidants). Arachidonate is a more potent oxidizing agent in 50 mM potassium phosphate buffer at pH 7.4 with 0.1 mM ethylenediaminetetraacetic acid (EDTA) than in Tyrode medium. Forty to fifty percent of total platelet tocopherol was oxidized upon incubation with 40-50 microM arachidonate in the phosphate-buffered medium. The tocopherol oxidation took place within 15 min after the addition of arachidonate. Preincubation of platelets with ascorbate blocked the oxidation of tocopherol. This is one of the first direct in vitro demonstrations of the vitamin E-sparing action of vitamin C in media containing biological cellular material. Other compounds which blocked the oxidation of platelet tocopherol were ascorbyl palmitate, propyl gallate, butylated hydroxytoluene, hydroquinone and glutathione. If ascorbate or glutathione was added after the tocopherol was oxidized to the quinone there was no reversal of the oxidation.

Topics: Adult; Antioxidants; Arachidonic Acid; Arachidonic Acids; Ascorbic Acid; Blood Platelets; Butylated Hydroxytoluene; Chromatography, High Pressure Liquid; Fasting; Glutathione; Humans; In Vitro Techniques; Male; Middle Aged; Oxidation-Reduction; Time Factors; Vitamin E

The effects of chronic ethanol feeding on hepatic lipid peroxidation, ascorbic acid, glutathione and vitamin E levels were investigated in rats fed low or adequate amounts of dietary vitamin E. Hepatic lipid peroxidation was significantly increased after chronic ethanol feeding in rats receiving a low-vitamin E diet, indicating that dietary vitamin E is an important determinant of hepatic lipid peroxidation induced by chronic ethanol feeding. No significant change was observed in hepatic non-heme iron content, but hepatic content of ascorbic acid and glutathione was increased by ethanol feeding. Both low dietary vitamin E and ethanol feeding significantly reduced hepatic alpha-tocopherol content, and the lowest hepatic alpha-tocopherol was found in rats receiving a combination of low vitamin E and ethanol. Plasma alpha-tocopherol was elevated after ethanol feeding, probably because of the associated hyperlipemia. Both the ratio of plasma alpha-tocopherol/plasma lipid and the red blood cell alpha-tocopherol were reduced by ethanol feeding. Furthermore, ethanol feeding caused a marked increase of hepatic alpha-tocopheryl quinone, a metabolite of alpha-tocopherol by free radical reactions. Ethanol feeding caused little changes of alpha-tocopherol and alpha-tocopheryl quinone content in mitochondria, whereas a striking increase in alpha-tocopheryl quinone was observed in microsomes. These data suggest that ethanol feeding causes a marked alteration of vitamin E metabolism in the liver and that the combination of ethanol with a low-vitamin E intake results in a decrease of hepatic alpha-tocopherol content which renders the liver more susceptible to free radical attack.

Topics: Animals; Antioxidants; Ascorbic Acid; Drug Administration Schedule; Ethanol; Free Radicals; Glutathione; Lipid Peroxidation; Liver; Male; Microsomes, Liver; Mitochondria, Liver; Rats; Rats, Inbred Strains; Vitamin E

The antioxidant effect of alpha-tocopherolquinone and alpha-tocopherolhydroquinone was studied in liposomes and rat liver submitochondrial particles. Both alpha-tocopherolquinone and alpha-tocopherolhydroquinone inhibit lipid peroxidation induced by ascorbate/Fe2+ in liposomes and by cumene hydroperoxide in submitochondrial particles. Alpha-tocopherolhydroquinone is much more effective than alpha-tocopherolquinone in inhibiting lipid peroxidation. Submitochondrial particles, depleted of ubiquinones and reincorporated with alpha-tocopherolquinone, are protected from lipid peroxidation only in the presence of succinate. Alpha-tocopherolquinone cannot replace endogenous ubiquinones in the respiratory chain function, nevertheless it can be reduced by the mitochondrial respiratory chain substrates, presumably through the reduced ubiquinones.

Topics: alpha-Tocopherol; Animals; Ascorbic Acid; Benzene Derivatives; Cattle; Electron Transport; Ferrous Compounds; Lipid Peroxides; Liposomes; Malondialdehyde; Mitochondria, Liver; Oxidation-Reduction; Rats; Submitochondrial Particles; Succinates; Succinic Acid; Ubiquinone; Vitamin E