Lipid-soluble Chain-breaking Antioxidant Research Articles

On ‘Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes?’ by Graham W. Burton, Anne Joyce, Keith U. Ingold

Application of a time-tested quantitative method of measuring peroxyl radical production in conjunction with the determination of the stoichiometry of the reaction of peroxyl radicals with α-tocopherol has permitted the conclusion that α-tocopherol is the major lipid-soluble chain-breaking antioxidant in human plasma and red cell membranes.

The alpha‐tocopherol transfer protein regulates vitamin E transport across placental cell monolayers (758.1)

Oxidative stress is thought to cause/ play critical causative role in the etiology of pregnancy disorders such as pre‐eclampsia, intrauterine growth restriction and repeated miscarriages. Vitamin E is a major lipid soluble chain breaking antioxidant that is essential for fetal development. The alpha‐tocopherol transfer protein (TTP) is the only specific regulator of vitamin E homeostasis in vertebrates, which is expressed in liver, brain and placenta. We hypothesized that mother‐fetus transport of vitamin E across the placenta is mediated and regulated by TTP. To test this hypothesis, we used the human choriocarcinoma cell‐line BeWo cultured as monolayers on transwells. Monolayer formation was confirmed by histological evaluation and electric resistance measurements. Trans‐epithelial resistance ranged between 90‐120 Ohms, indicating functional tight junctions throughout the monolayer. Anti‐ E cadherin staining indicated the manolayers converted to a syncytiotrophoblast layer. Media containing serum‐complexed [14C]‐α tocopherol was delivered to the apical side of the cells for 48 hrs and the replaced with serum free media. Cells that express TTP displayed significant (ca. 2‐fold) increase in the extent of tocopherol secretion by the monolayer. These findings show that placental TTP is necessary for trans‐placental transport of vitamin E. The functional consequences of heritable mutations in the TTPA gene on trans‐placental vitamin E transport are being addressed in ongoing experiments.

α-Tocopherol incorporation in mitochondria and microsomes upon supranutritional vitamin E supplementation

Vitamin E (α-tocopherol) is a major lipid-soluble chain-breaking antioxidant in humans and mammals and plays an important role in normal development and physiology. The localization of α-tocopherol within the highly unsaturated phospholipid bilayer of cell membranes provides a means of controlling lipid oxidation at the initiation site. Mitochondria are the site for major oxidative processes and are important in fat oxidation and energy production, but a side effect is leakage of reactive oxygen species. Thus, incorporation of α-tocopherol and other antioxidants into mitochondria and other cellular compartments is important in order to maintain oxidative stability of the membrane-bound lipids and prevent damage from the reactive oxygen species. Many studies regarding mitochondrial disease and dysfunction have been performed in relation to deficiency of vitamin E and other antioxidants, whereas relatively sparse information is available regarding the eventual beneficial effects of antioxidant-enriched mitochondria in terms of health and function. This may be due to the fact that only little scientific information is available concerning the effect of supranutritional supplementation with antioxidants on their incorporation into mitochondria and other cellular membranes. The purpose of this review is therefore to briefly summarize experimental data performed with dietary vitamin E treatments in relation to the deposition of α-tocopherol in mitochondria and microsomes.

α-Tocopherol is an effective Phase II enzyme inducer: protective effects on acrolein-induced oxidative stress and mitochondrial dysfunction in human retinal pigment epithelial cells

Vitamin E has long been identified as a major lipid-soluble chain-breaking antioxidant in mammals. α-Tocopherol is a vitamin E component and the major form in the human body. We propose that, besides its direct chain-breaking antioxidant activity, α-tocopherol may exert an indirect antioxidant activity by enhancing the cell's antioxidant system as a Phase II enzyme inducer. We investigated α-tocopherol's inducing effect on Phase II enzymes and its protective effect on acrolein-induced toxicity in a human retinal pigment epithelial (RPE) cell line, ARPE-19. Acrolein, a major component of cigarette smoke and also a product of lipid peroxidation, at 75 μmol/L over 24 h, caused significant loss of ARPE-19 cell viability, increased oxidative damage, decreased antioxidant defense, inactivation of the Keap1/Nrf2 pathway, and mitochondrial dysfunction. ARPE-19 cells have been used as a model of smoking- and age-related macular degeneration. Pretreatment with α-tocopherol activated the Keap1/Nrf2 pathway by increasing Nrf2 expression and inducing its translocation to the nucleus. Consequently, the expression and/or activity of the following Phase II enzymes increased: glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase 1, heme-oxygenase 1, glutathione S-transferase and superoxide dismutase; total antioxidant capacity and glutathione also increased. This antioxidant defense enhancement protected ARPE-19 cells from an acrolein-induced decrease in cell viability, lowered reactive oxygen species and protein oxidation levels, and improved mitochondrial function. These results suggest that α-tocopherol protects ARPE-19 cells from acrolein-induced cellular toxicity, not only as a chain-breaking antioxidant, but also as a Phase II enzyme inducer.

Prolonged α-Tocopherol Deficiency Decreases Oxidative Stress and Unmasks α-Tocopherol-dependent Regulation of Mitochondrial Function in the Brain

Vitamin E is the major lipid-soluble chain-breaking antioxidant in mammals and plays an important role in normal development and physiology. Deficiency (whether dietary or genetic) results in primarily nervous system pathology, including cerebellar neurodegeneration and progressive ataxia (abnormal gait). However, despite the widely acknowledged antioxidant properties of vitamin E, only a few studies have directly correlated levels of reactive oxygen species with vitamin E availability in animal models. We explored the relationship between vitamin E and reactive oxygen species in two mouse models of vitamin E deficiency: dietary deficiency and a genetic model (tocopherol transfer protein, Ttp-/- mice). Both groups of mice developed nearly complete depletion of alpha-tocopherol (the major tocopherol in vitamin E) in most organs, but not in the brain, which was relatively resistant to loss of alpha-tocopherol. F4-neuroprostanes, an index of lipid peroxidation, were unexpectedly lower in brains of deficient mice compared with controls. In vivo oxidation of dihydroethidium by superoxide radical was also significantly lower in brains of deficient animals. Superoxide production by brain mitochondria isolated from vitamin E-deficient and Ttp-/- mice, measured by electron paramagnetic resonance spectroscopy, demonstrated a biphasic dependence on exogenously added alpha-tocopherol. At low concentrations, alpha-tocopherol enhanced superoxide flux from mitochondria, a response that was reversed at higher concentrations. Here we propose a mechanism, supported by molecular modeling, to explain decreased superoxide production during alpha-tocopherol deficiency and speculate that this could be a beneficial response under conditions of alpha-tocopherol deficiency.

Possible neuroprotective effects of lecithin and α‐tocopherol alone or in combination against ischemia/reperfusion insult in rat brain

A close correlation exists between ischemia/reperfusion (I/R)-induced insult and the release of free radicals. Lecithin is a polyunsaturated phosphatidylcholine that corresponds to the phosphatidylcholine molecule. Phosphatidylcholines are high-energy functional and structural elements of all biologic membranes. alpha-Tocopherol is the major lipid-soluble chain-breaking antioxidant in the body tissues and effectively protects against neuronal damage. Therefore, we studied the effect of lecithin (300 mg/kg, p.o., 14 days) and alpha-tocopherol (200 mg/kg, p.o., 14 days), alone or in combination, on the brain redox state during I/R. Adult male Wistar rats were subjected to global ischemia by the occlusion of the two carotid arteries 24 h after the last dose of drug administration. Reperfusion was carried out 1 h after induction of ischemia and lasted for another hour. Brain lipid peroxides (MDA) and glutathione (GSH) contents, as well as superoxide dismutase (SOD) and catalase (CAT) activities were assessed. The results showed that I/R elevated brain lipid peroxides content which was accompanied by a reduction in both antioxidant enzyme activities, however, brain GSH level remained unaltered. Lecithin, alpha-tocopherol and their combination restored MDA content, as well as CAT activity with a slight tendency to normalize SOD activity. We conclude that lecithin has a possible neuroprotective effect partly through its antioxidant action which is comparable to that of alpha-tocopherol.

Vitamin E and Cardiovascular Disease

SummaryInterest in the use of antioxidants for the treatment of human disease, and in the role of dietary antioxidants in the prevention of disease development, has been sustained for at least two decades. Several anti-oxidant protective mechanisms exist and constitute a primary defensive system including enzymatic defences (glutathione peroxidase and superoxide dismutase, which depend on the presence of ions such as selenium, zinc, copper, and manganese) and naturally occurring vita-mins such as vitamin E, vitamin A, beta-carotene, and vitamin C. The most important natural antioxidants are vitamin E (in the form of α-, β-, γ-, and δ-tocopherols), beta-carotene, vitamin C and selenium (fundamental constituent of glutathione-peroxidase, i.e., an enzyme with antioxidant function). The first two are lipophilic substances whilst ascorbic acid is hydrophilic. Each antioxidant has a different important mechanism of action since oxidative damage can be caused by lipid- or water-soluble molecules. Lipid-soluble antioxidants are likely to be very important in preventing the peroxidation of low-density lipo-proteins (LDL) and this action could be paramount in the prevention of atherosclerosis. On the other hand, water-soluble antioxidants could be useful where a water-soluble oxidative stress occurs (e.g., inflammation). As lipophilic molecules, vitamin E and beta-carotene are incorporated into the LDL particle. Vitamin E is the main lipid-soluble chain-breaking antioxidant in plasma and tissues and converts the peroxyl-free radical to hydroperoxide, a less reactive radical. It acts as a first-line anti-oxidative defence of LDL particles, protecting unsatu-rated fatty acids from peroxidation. Beta-carotene is a carotenoid (precursor of vitamin A, pro-vitamin) that acts as scavenger of oxidising radicals such as singlet oxygen and is a second-line antioxidative defence of LDL cholesterol. Vitamin C (ascorbic acid) can react with singlet oxygen, superoxide, hydroxyl radicals, and is the first line of antioxi-dative defence in water-soluble compartments. In addition, it plays an important role in regenerating reduced -tocopherol.

Colchicine and Vinblastine Prevent the Piperonyl Butoxide-Induced Increase in Rat Biliary Output of α-Tocopherol

α-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the α-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, ip injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary α-tocopherol output increased to 132% of pretreatment measurements. Hepatic α-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic α-tocopherol and the increase in biliary output of α-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of α-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic α-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.

Ultraviolet light-induced generation of vitamin E radicals and their recycling. A possible photosensitizing effect of vitamin E in skin.

Vitamin E (alpha-tocopherol) is the major lipid-soluble chain-breaking antioxidant of membranes. Its UV-absorbance spectrum (lambda max 295 nm) extends well into the solar spectrum. We hypothesize that in skin alpha-tocopherol may absorb solar UV light and generate tocopheroxyl radicals. Reduction of tocopheroxyl radicals by other antioxidants (e.g. ascorbate, thiols) will regenerate (recycle) vitamin E at the expense of their own depletion. Hence, vitamin E in skin may act in two conflicting manners upon solar illumination: in addition to its antioxidant function as a peroxyl radical scavenger, it may act as an endogenous photosensitizer, enhancing light-induced oxidative damage. To test this hypothesis, we have illuminated various systems (methanol-buffer dispersions, liposomes and skin homogenates) containing alpha-tocopherol or its homologue with a shorter 6-carbon side chain, chromanol-alpha-C6 with UV light closely matching solar UV light, in the presence or absence of endogenous or exogenous reductants. We found that: (i) alpha-tocopheroxyl (chromanoxyl) radicals are directly generated by solar UV light in model systems (methanol-water dispersions, liposomes) and in skin homogenates; (ii) reducing antioxidants (ascorbate, ascorbate+dihydrolipoic acid) can donate electrons to alpha-tocopheroxyl (chromanoxyl) radicals providing for vitamin E (chromanol-alpha-C6) recycling; (iii) recycling of UV-induced alpha-tocopheroxyl radicals depletes endogenous antioxidant pools (accelerates ascorbate oxidation); (iv) beta-carotene, a non-reducing antioxidant, is not active in alpha-tocopherol recycling, and its UV-dependent depletion is unaffected by vitamin E.

Function of vitamin E in physical exercise: a review

Even though vitamin E may not improve physical achievements in sports competitions, as shown in several swimming experiments, it is important for the health of skeletal muscle: in its role as the major lipid-soluble chainbreaking antioxidant in lipid cell membranes, vitamin E protects muscle tissue in aerobic exercise, in which oxygen metabolism and, consequently, free radical production are greatly accelerated. Animal studies in several laboratories have shown that endurance exercise results in the same type of oxidative muscle damage as does vitamin E deficiency: there is an increase in the peroxidation products pentane and malondialdehyde and in enzymes leaked from muscles to plasma. Oxidative tissue damage in vitamin-E deficient animals is exacerbated by endurance training and, conversely, it is reduced by high-dose vitamin E supplementation; also, preliminary studies in humans have demonstrated antioxidant protection by high-dose vitamin E supplementation. After endurance exercise leakage of enzymes into the plasma and output of pentane in the breath were significantly reduced. During a high-altitude expedition in the Himalayas, protection was shown to be significantly better in the supplemented group than in the placebo group, as determined by anaerobic threshold and pentane exhalation.

Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol

d-Alpha-tocopherol (2R, 4′R,8′R-Alpha-tocopherol) and d-alpha-tocotrienol are two main vitamin E constituents having the same aromatic chromanol “head” but differing in their hydrocarbon “tail”: tocopherol with a saturated and toctrienol with an unsaturated isoprenoid chain. d-Alpha-tocopherol has the highest vitamin E activity, while d-alpha-tocotrienol manifests only about 30% of this activity. Since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes, we studied alpha-tocotrienol as compared to alpha-tocopherol under conditions which are important for their antioxidant function. d-Alpha-tocotrienol possesses 40–60 times higher antioxidant activity against (Fe 2+ + ascorbate)- and (Fe 2+ + NADPH)-induced lipid peroxidation in rat liver microsomal membranes and 6.5 times better protection of cytochrome P-450 against oxidative damage than d-alpha-tocopherol. To clarify the mechanisms responsible for the much higher antioxidant potency of d-alpha-tocotrienol compared to d-alpha-tocopherol, ESR studies were performed of recycling efficiency of the chromanols from their chromanoxyl radicals, 1H-NMR measurements of lipid molecular mobility in liposomes containing chromanols, and fluorescence measurements which reveal the uniformity of distribution (clusterizations) of chromanols in the lipid bilayer. From the results, we concluded that this higher antioxidant potency of d-alpha-tocotrienol is due to the combined effects of three properties exhibited by d-alpha-tocotrienol as compared to d-alpha-tocopherol: (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membraned bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. The data presented show that there is a considerable discrepancy between the relative in vitro antioxidant activity of d-alpha-tocotrienol with the conventional bioassays of their vitamin activity.

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.