Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 (Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl_z or cytochrome b-559.     

Role of ��-tocopherol in cellular signaling: ��-tocopherol inhibits stress-induced mitogen-activated protein kinase activation

Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.     

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T₂ seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.     

Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Acute toxicity study of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol, and γ-tocopherol in rats

Liposomes have been used for the delivery of antioxidants to different tissues and organs for the treatment of oxidative stress-induced injuries. In this study, the acute toxicity of a single dose of intravenously (i.v.) administered liposomal antioxidant formulation, containing N-acetylcysteine (NAC) with or without α-tocopherol (α-T) or γ-tocopherol (γ-T), in rats was examined. Each group consisted of 5 male and 5 female Sprague-Dawley rats, with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (660 mg/kg) and test groups receiving DPPC liposomes (660 mg/kg) entrapped with 1) NAC (200 mg/kg), 2) NAC (200 mg/kg) and α-T (83.3 mg/kg), and 3) NAC (200 mg/kg) and γ-T (71.4 mg/kg). These dose levels were determined from the dose-range-finding study and were considered to be the maximum feasible dose (MFD) levels, based on the volume of 10 mL/kg and physical properties and viscosity of the test articles that could be safely administered to rats by an i.v. injection. Two weeks after treatment (day 15), rats in the control group and three test groups exhibited no clinical signs of toxicity during the dosing period or during the 14-day post-treatment period. Weight gain and food consumption in all animals was appropriate for the age and sex of animals. Clinical pathology findings (e.g., hematology, coagulation, clinical chemistry, and urinalysis) were unremarkable in all rats and in all groups. In conclusion, the results of this study showed no treatment-related toxicity in rats at the MFD level by a single bolus i.v. administration.     

γ-tocopherol is less effective than α-tocopherol in preventing oxidant-induced sister chromatid exchanges in Chinese hamster V79 cells

Although α-tocopherol (α-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, γ-tocopherol (γ-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of γ-TOC and a high α-TOC:γ-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the γ-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 μM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of γ-TOC (319.8 ± 66.2 ng/10⁶ cells) into V79 cells compared to α-TOC (66.9 ± 6.4 ng/10⁶ cells). γ-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, α-TOC acetate was partially protective against both genotoxins, whereas α-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of γ-TOC into V79 cells, α-TOC but not γ-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.     

α-tocopherol β-oxidation localized to rat liver mitochondria

Approximately 40% of Americans take dietary supplements, including vitamin E (α-tocopherol). Unlike other fat-soluble vitamins, α-tocopherol is not accumulated to toxic levels. Rather tissue levels are tightly regulated, in part via increased hepatic metabolism and excretion that could, theoretically, alter metabolism of drugs, environmental toxins, and other nutrients. To date, in vivo subcellular location(s) of α-tocopherol metabolism have not been identified. The proposed pathway of α-tocopherol metabolism proceeds via ω-hydroxylation to 13′-OH-α-tocopherol, followed by successive rounds of β-oxidation to form α-CEHC. To test the hypothesis that α-tocopherol ω-hydroxylation occurs in microsomes while β-oxidation occurs in peroxisomes, rats received daily injections of vehicle, 10 mg α-tocopherol, or 10 mg trolox/100 g body wt for 3 days, and then microsomes, mitochondria, and peroxisomes were isolated from liver homogenates. Homogenate α-tocopherol levels increased 16-fold in α-tocopherol-injected rats, while remaining unchanged in trolox- or vehicle-injected rats. Total α-tocopherol recovered in the three subcellular fractions represented 93 ± 4% of homogenate α-tocopherol levels. In α-tocopherol-injected rats, microsome α-tocopherol levels increased 28-fold, while mitochondria and peroxisome levels increased 8- and 3-fold, respectively, indicating greater partitioning of α-tocopherol to the microsomes with increasing liver α-tocopherol. In α-tocopherol-injected rats, microsome 13′-OH-α-tocopherol levels increased 24-fold compared to controls, and were 7-fold greater than 13′-OH-α-tocopherol levels in peroxisome and mitochondrial fractions of α-tocopherol-injected rats. An unexpected finding was that α-CEHC, the end product of α-tocopherol metabolism, was found almost exclusively in mitochondria. These data are the first to indicate a mitochondrial role in α-tocopherol metabolism.     

Effect of α-tocopherol tissue levels on beef quality

To evaluate meat quality of beef with different α-tocopherol tissue levels, 55 feedlot steers were fed a barley-based finisher diet with four vitamin E supplementation levels (0, 350, 700 and 1400 IU DL-α-tocopheryl acetate/animal per day) for 120 days. Although the increase in oxidation levels overtime was much smaller (P < 0.001) in the high-medium and high groups, α-tocopherol tissue levels did not affect (P > 0.05) pH, proximate analysis, drip and cooking losses, and shear force of steaks. No effect of α-tocopherol tissue levels was found in retail evaluation of steaks after a short ageing time of 6 days, but with 21 days of ageing, a delay in formation of metmyoglobin (P = 0.008) was observed in steaks with higher tissue levels of α-tocopherol. Similar results were found for ground beef (25% fat) prepared from 6-day aged meat. Thus, higher α-tocopherol tissue levels protect ground beef and long-aged steaks from discolouration and lipid oxidation.     

Serum α-tocopherol and γ-tocopherol concentrations and prostate cancer risk in the PLCO Screening Trial: a nested case-control study

Background

Vitamin E compounds exhibit prostate cancer preventive properties experimentally, but serologic investigations of tocopherols, and randomized controlled trials of supplementation in particular, have been inconsistent. Many studies suggest protective effects among smokers and for aggressive prostate cancer, however.

Methods

We conducted a nested case-control study of serum α-tocopherol and γ-tocopherol and prostate cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, with 680 prostate cancer cases and 824 frequency-matched controls. Multivariate-adjusted, conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CIs) for tocopherol quintiles.

Results

Serum α-tocopherol and γ-tocopherol were inversely correlated (r = −0.24, p<0.0001). Higher serum α-tocopherol was associated with significantly lower prostate cancer risk (OR for the highest vs. lowest quintile = 0.63, 95% CI 0.44–0.92, p-trend 0.05). By contrast, risk was non-significantly elevated among men with higher γ-tocopherol concentrations (OR for the highest vs. lowest quintile = 1.35, 95% CI 0.92–1.97, p-trend 0.41). The inverse association between prostate cancer and α-tocopherol was restricted to current and recently former smokers, but was only slightly stronger for aggressive disease. By contrast, the increased risk for higher γ-tocopherol was more pronounced for less aggressive cancers.

Conclusions

Our findings indicate higher α-tocopherol status is associated with decreased risk of developing prostate cancer, particularly among smokers. Although two recent controlled trials did not substantiate an earlier finding of lower prostate cancer incidence and mortality in response to supplementation with a relatively low dose of α-tocopherol, higher α-tocopherol status may be beneficial with respect to prostate cancer risk among smokers. Determining what stage of prostate cancer development is impacted by vitamin E, the underlying mechanisms, and how smoking modifies the association, is needed for a more complete understanding of the vitamin E-prostate cancer relation.     

Gas chromatography of retinol and α-tocopherol without derivatization

An improved gas chromatographic method for the analysis of retinol and α-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D₂, D₃, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identifies were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.     

Specific binding sites for d-α-tocopherol on human erythrocytes

Since vitamin E deficiency is associated with increased susceptibility of erythrocytes to hemolysis, we investigated the presence of tocopherol binding sites in human red blood cells. Erythrocytes were found to have specific binding sites for d-α-[³H]tocopherol with properties of receptors. Kinetic studies of binding demonstrated two binding sites: one with high affinity (equilibrium association constant K_a = 2.6·10⁷ M^?1), low capacity (7600 sites/cell) and the second with low affinity (K_a = 1.24·10⁶ M^?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature.     

Serum α-tocopherol and selenium in belgian infants and children

Previous studies showed low selenium (Se) concentrations in Belgian children. Serum α-tocopherol, retinol, total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, selenium (Se), and thiobarbituric acid-reactive substances were examined. In order to obtain further information on the Se status in Belgian children, Se, α-tocopherol, retinol, and lipid concentrations were examined and signs of peroxidative lipid damage were evaluated in a subgroup. The study was performed in 524 children (0–14 yr old) during vaccination campaigns. Three age groups were analyzed: 0–1, 1–4, and 4–14 yr. In 87 of them, where sufficient amounts of serum were available, analysis of thiobarbituric acid-reactive substances was done. Infants have high serum α-tocopherol concentrations: (23.2 μmol/L [median and interquartile range: 18.6–30.2]) and low Se concentrations (0.37 mol/L [0.27–0.47]). Se concentrations rise significantly during the first 4 yr (p < 0.0001) (Mann-Whitney U-test, tied p-values): 0.70 μmol/L (0.59–0.82); in the 4–14 yr olds, it was 0.75 μmol/L (0.67–0.86). These values remain low compared to results coming from other parts of the world. α-Tocopherol concentrations decrease significantly after infancy (p < 0.0001). The ratio α-tocopherol/total cholesterol is higher in infants. This is induced by the high vitamin E content of infant formulas. Signs of serum lipid peroxidation could not be detected by analysis of serum malondialdehyde concentrations. High α-tocopherol concentrations, as those observed in infant serum lipids, could be one of the protective mechanisms from the peroxidative lipid damages, sometimes observed in a low-Se status.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.