Electron transport between cytochrome c and alpha tocopherol.

Using liposomes we have demonstrated an electron transfer between tocopherol (vitamin E) and cytochrome c. Reduced cytochrome c protects vitamin E from oxidation induced either directly by ultraviolet light or indirectly by soybean lipoxygenase-catalyzed oxidation of arachidonic acid. Oxidized cytochrome c is reduced by tocopherol and tocopherol homologues (chromanols) resulting in accumulation of tocopheroxyl radicals which we detected by ESR. The peak height of the ESR spectrum of tocopheroxyl radicals (which is proportional to the amount of radical present) is proportional to the ratio of reduced to oxidized cytochrome c. In mitochondrial membranes succinate-cytochrome c reduction is inhibited by antimycin A. Addition of exogenous chromanols facilitates a by-pass of the antimycin A blocked electron pathway, and succinate-dependent cytochrome c reductase activity is restored. Cytochrome c may act as a water-soluble complement to the lipid-soluble ubiquinol in regenerating mitochondrial tocopherol from tocopheroxyl radical.     

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.     

Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone.

Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady-state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate-reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha-tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical.     

Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.     

Mitochondria and microsomal membranes have a free radical reductase activity that prevents chromanoxyl radical accumulation

Enzyme-dependent mechanisms which prevent accumulation of chromanoxyl radicals derived from the vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydroxycromane (PMC), were characterized in rat liver microsomal and mitochondrial membranes. The free radical oxidation product of PMC (chromanoxyl radical) was generated in membranes using either photochemical (uv light) or enzymatic (lipoxygenase and arachidonic acid) methods and detected by ESR. Substrates (NADH or NADPH) prevented accumulation of chromanoxyl radicals until the substrate was fully consumed. In microsomes, reduced glutathione increased the efficacy of NADPH in preventing the accumulation of the chromanoxyl radical, but was without effect in the absence of NADPH. Ascorbate also prevented accumulation of the chromanoxyl radical. It is concluded that rat liver microsomes and mitochondria have both enzymatic and non-enzymatic mechanisms for reducing chromanoxyl radicals.     

Vitamin E and its function in membranes

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.     

Radical Exchange Reactions between Vitamin E, Vitamin C and Phospholipids in Autoxidizing Polyunsaturated Lipids

Antioxidant reactions of mixtures of vitamin E, vitamin C and phospholipids in autoxidizing lipids at 90°C have been studied by ESR spectroscopy. When the phospholipid contained a tertiary amine (e.g. phosphatidylcholine), the vitamin C and the vitamin E radicals were successively observed as these two vitamins were sequentially oxidised during lipid oxidation. In the presence of the primary amine contained in phosphatidylserine, the vitamin E oxidation was delayed for a few hours. In this case neither the vitamin C, nor the vitamin E radicals but a nitroxide radical derived from the phospholipid was observed. Similar results to those obtained with PS were obtained in the presence of either phospha-tidylethanolamine or soybean lecithin. The participation in the radical reactions of phospholipids possessing a primary amine can therefore explain the synergistic effect of these phospholipids in a mixture of vitamins E and C.     

Antioxidant effects of ubiquinones in microsomes and mitochondria are mediated by tocopherol recycling

Ubiquinones and tocopherols (vitamin E) are intrinsic lipid components which have a stabilizing function in many membranes attributed to their antioxidant activity. The antioxidant effects of tocopherols are due to direct radical scavenging. Although ubiquinones also exert antioxidant properties the specific molecular mechanisms of their antioxidant activity may be due to: (i) direct reaction with lipid radicals or (ii) interaction with chromanoxyl radicals resulting in regeneration of vitamin E. Lipid peroxidation results have now shown that tocopherols are much stronger membrane antioxidants than naturally occurring ubiquinols (ubiquinones). Thus direct radical scavenging effects of ubiquinols (ubiquinones) might be negligible in the presence of comparable or higher concentrations of tocopherols. In support of this our ESR findings show that ubiquinones synergistically enhance enzymic NADH- and NADPH-dependent recycling of tocopherols by electron transport in mitochondria and microsomes. If ubiquinols were direct radical scavengers their consumption would be expected. Further proving our conclusion HPLC measurements demonstrated that ubiquinone-dependent sparing of tocopherols was not accompanied by ubiquinone consumption.     

Real Time Detection of Reactions Between Radicals of Lycopene and Tocopherol Homologues

Laser flash photolysis of lycopene in homogeneous chloroform solution together with tocopherol homolopes results in rapid formation of the lycopene radical cation and slower formation of tocopheroxyl radicals. Time-resolved detection by absorption spectroscopy of decay of the lycopene radical cation, of formation of the tocopheroxyl radicals, and of bleaching of lycopene has shown that a-tocopherol is able to reduce the lycopene radical cation and thereby partially regenerate lycopene on a ms timescale. In contrast, lycopene is able to reduce the δ-tocopheroxyl radical, whereas an equilibrium exists between the lycopene radical cation and β- or γ-tocopherol. The relative stability of these antioxidant radicals is hence: a-tocopheroxyl > lycopene radical cation ≈ β-tocopheroxyl - γ-tocopheroxyl > S-toco-pheroxyl.     

Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine

Oxidations of soybean phosphatidylcholine liposomes in an aqueous dispersion initiated by free radicals generated initially either in the aqueous phase or in the lipid phase were efficiently suppressed by vitamin E in the membranes. Vitamin E was consumed linearly with time and, when the inhibition period was over the oxidation proceeded rapidly at a rate similar to that in the absence of vitamin E. L-Cysteine was also effective by itself in scavenging radicals in the aqueous region, but it was consumed more rapidly than vitamin E. On the other hand, cysteine could not scavenge the radicals efficiently in a lipid region. Nevertheless, when vitamin E was incorporated into liposomes, the addition of cysteine in the aqueous phase prolonged the inhibition period and it reduced the rate of decay of vitamin E markedly even when the radicals were generated initially in the lipid bilayer. Furthermore, it was found by an electron spin resonance study that chromanoxyl radical disappeared quite rapidly when it was mixed with cysteine and that the spin adduct of cysteine radical was observed in the presence of alpha-(4-pyridyl-N-oxide)-N-tert-butyl nitrone. It was concluded that L-cysteine located in an aqueous region could regenerate vitamin E by reacting with vitamin E radical formed in a lipid region and show a synergistic antioxidant effect, although its efficiency of vitamin E regeneration was lower than that by vitamin C.     

Interaction of vitamin C and vitamin E during free radical stress in plasma: An ESR study

To study the interaction of the antioxidant vitamins C and E in a biological system, we used electron spin resonance (ESR) spectroscopy to make serial measurement of ascorbate tocopheroxyl free radicals in plasma subjected to continuous free radical-mediated oxidative stress. Upon initiation of a continuous oxidative stress, we observed an immediate increase in the concentration of ascorbate radical, which reached a peak, and then steadily declined. Only after the virtual disappearance of the ascorbate radical did we observe the appearance of the tocopheroxyl radical. These data are consistent with the hypothesis that ascorbate is the terminal small-molecule antioxidant in biological systems. This is the first experimental demonstration that the predicted thermodynamic hierarchy of ascorbate, -tocopherol, and their free radicals holds in a biological system containing endogenous levels of these antioxidant vitamins.     

Active oxygen chemistry within the liposomal bilayer. Part III: Locating Vitamin E, ubiquinol and ubiquinone and their derivatives in the lipid bilayer

We have previously shown that the location and orientation of compounds intercalated within the lipid bilayer can be qualitatively determined using an NMR chemical shift-polarity correlation. We describe herein the results of our application of this method to analogs of Vitamin E, ubiquinol and ubiquinone. The results indicate that tocopherol--and presumably the corresponding tocopheroxyl radical--reside adjacent to the interface, and can, therefore, abstract a hydrogen atom from ascorbic acid. On the other hand, the decaprenyl substituted ubiquinol and ubiquinone lie substantially deeper within the lipid membrane. Yet, contrary to the prevailing literature, their location is far from being the same. Ubiquinone-10 is situated above the long-chain fatty acid "slab". Ubiquinol-10 dwells well within the lipid slab, presumably out of "striking range" of Vitamin C. Nevertheless, ubiquinol can act as an antioxidant by reducing C- or O-centered lipid radicals or by recycling the lipid-resident tocopheroxyl radical.     

Effect of exercise training on tissue vitamin E and ubiquinone content

Endurance exercise training led to an adaptive increase in the ubiquinone content and cytochrome c reductase activity of red quadriceps and soleus muscles and adipose tissues, but not of cardiac or white quadriceps muscle. These findings are consistent with the well-known positive adaptation of skeletal muscle mitochondria to endurance training. However, there was no concomitant increase in the vitamin E content of tissues, which showed an increase in mitochondrial content. Since ubiquinone is located in the mitochondrial inner membrane and the major pool of vitamin E is also associated with mitochondrial membranes, the results suggest that training causes a substantial decrease in vitamin E concentration in the proliferating muscle mitochondrial membranes, thus depleting muscle mitochondria of their major lipid antioxidant. Since vitamin E is the major cellular, lipid-soluble, chain-breaking antioxidant, these findings indicate increased free radical reactions in the tissues of exercising animals.     

Effect of RoseOx on peroxidation of rat mitochondrial lipids.

The effect of the RoseOx drug on rat liver mitochondrial lipids free-radical oxidation in vivo was studied. During the period of 14 days 50 mG/kg per body weight of RoseOx was added to the diet of normal rats each day. Free radical oxidation in liver mitochondrial fraction was determined by the help of a chemiluminescence method. Four kinetic The RoseOx addition to the usual diet led for free radical oxidation braking in mitochondrial fraction of liver as was shown. The RoseOx antioxidizing effect was stipulated by availability of carnosic acid as a supplement. One of the mechanism of the caronosic acid antioxidizing action could be its participation in LFRO reactions breaking by its OH-groups. Carnosic acid contains OH-groups in its molecule as well as a vitamin E for example. So, the mechanism of carnosic acid antioxidizing action is probably similar to vitamin E action in lipid free radical oxidation reactions.     

Olive oil supplemented with vitamin E affects mitochondrial coenzyme Q levels in liver of rats after an oxidative stress induced by adriamycin.

In this study we have evaluated the supplementation of olive oil with vitamin E on coenzyme Q concentration and lipid peroxidation in rat liver mitochondrial membranes. Four groups of rats were fed on virgin olive, olive plus 200 mg/kg of vitamin E or sunflower oils as lipid dietary source. To provoke an oxidative stress rats were administered intraperitoneally 10 mg/kg/day of adriamycin the last two days of the experiment. Animals fed on olive oil plus vitamin E had significantly higher coenzyme Q and vitamin E levels but a lower mitochondrial hydroperoxide concentration than rats fed on olive oil. Retinol levels were not affected, by either different diets or adriamycin treatment. In conclusion, an increase in coenzyme Q and alpha-tocopherol in these membranes can be a basis for protection against oxidation and improvement in antioxidant capacity.     

Synergistic interaction between vitamin E and the bile pigments bilirubin and biliverdin

The oxidation of soybean phosphatidylcholine (PC) liposomes initiated with a lipid-soluble azo compound within the liposomal membranes has been studied in the absence and presence of membrane-bound vitamin E and water-soluble bile pigments. In the absence of vitamin E, lipid peroxidation proceeded linearly and without delay. Low micromolar amounts of bilirubin ditaurine (BR-DT, a model compound of conjugated bilirubin) or biliverdin (BV) inhibited the oxidation of PC significantly and in a concentration-dependent way. In contrast, neither taurine, ascorbic acid nor reduced glutathione inhibited significantly under these conditions. Both bile pigments were consumed during their protective action. Vitamin E incorporated into the liposomal membranes suppressed the oxidation initially almost completely, thereby producing an induction period. In the combined presence of vitamin E and either of the two bile pigments at 10 microM each, this induction period was increased by at least 200%. In contrast, when 10 microM vitamin E was combined with an equimolar concentration of reduced glutathione, the induction period increased by only about 30%. BR-DT and BV both spared the consumption of vitamin E during the oxidation of PC liposomes. These results demonstrate that conjugated bilirubin and BV located in the aqueous phase can directly scavenge lipid radicals to some extent. Furthermore, both bile pigments can act synergistically with membrane-bound vitamin E to prevent lipid peroxidation initiated in the lipid phase, most likely through regeneration of the vitamin from its chromanoxyl radical.     

Stopped-flow investigation of the reaction of vitamin C with tocopheroxyl radical in aqueous triton X-100 micellar solutions. The structure-activity relationship of the regeneration reaction of tocopherol by vitamin C

A kinetic study of the reaction between vitamin C (L-ascorbic acid, AsH2) and a tocopheroxyl radical (7-tert-butyl-5-isopropyltocopheroxyl) in Triton X-100 micellar solution has been performed using stopped-flow spectrophotometry. The second-order rate constants (k2) obtained showed notable pH dependence with a broad maximum around pH 8. For instance, the k2 values obtained were 26 M-1 S-1 at pH 3, 322 M-1 S-1 at pH 7, and 273 M-1 S-1 at pH 10. A good correlation between the rate constants and the mole fraction of ascorbate monoanion (AsH-) was observed, showing that ascorbate (AsH-) can regenerate the tocopherol from tocopheroxyl in biological systems. Furthermore, the results indicate that reduced ascorbic acid (AsH2) does not have the ability to regenerate the tocopherol in aqueous solution. On the other hand, it was found that AsH2 can reduce the tocopheroxyl to tocopherol in benzene/ethanol (2:1) mixtures, although the rate of reaction is only approximately 15% of that observed in micellar solution at pH 7.     

Vitamin D3 and 20-hydroxyecdysone -- inhibitors of free radical lipid oxidation during D-hypervitaminosis

Under D-hypovitaminosis (control) conditions the statistically reliable increase of blood serum lipids free radical oxidation was revealed in comparison with the intact animals. Administration of vitamin D3 to the animals suffering from D-hypovitaminosis leads for statistically reliable decrease of blood serum lipids free radical oxidation, while 20-hydroxyecdysone in quantity of 0.02 mg per 1 kg of the animal body weight displays the antioxidative properties. Its antioxidative effect is characterized by a statistically reliable increase of Tind chemiluminescence kinetical parameter as compared with the control. Under D-hypovitaminosis conditions in the mitochondrial membranes the products of lipids free radical oxidation--dien conjugates are accumulated. In the case of administrating to the animals suffering from D-hypovitaminosis D3 or 20-hydroxyecdysone these oxidation products are absent. 20-Hydroxyecdysone under these conditions have been revealed as inducing accumulation in the mitochondrial and microsomal membranes of the substances with lambda 225 nm.     

Coenzyme Q and vitamin E need each other as antioxidants

Summary Both vitamin E and coenzyme Q possess distinct lipoprotective antioxidant properties in biological membranes. Their combined antioxidant activity, however, is markedly synergistic when both are present together. While it is likely that vitamin E represents the initial chain-breaking antioxidant during lipid peroxidation, both fully reduced CoQH₂ (ubiquinol) and semireduced CoQH^. (ubisemiquinone) appear to efficiently recycle the resultant vitamin E phenoxyl radical back to its biologically active reduced form. We describe and support a potential kinetic mechanism whereby vitamin E and coenzyme Q interact in such a way as to usurp the prooxidant effects of O ₂ ^−. . Physical interactions of vitamin E and coenzyme Q within the environment of the membrane lipid bilayer facilitate the recycling of vitamin E by ubisemiquinone and ubiquinol. Lastly, data are linked into a catalytic cycle that serves to connect normal electron transport mechanisms within biological membranes to the maintenance of lipoprotective antioxidant mechanisms.     

Recycling of vitamin E in human low density lipoproteins.

Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2'-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.