The aim of this study was to investigate the most important oxidation products of α-tocotrienol (α-T3) along with other tocochromanols in lipid matrices and tocotrienol-rich foods. For this purpose, an efficient molecular distillation procedure was developed for the extraction of analytes, and α-T3-spiked and thermally oxidized natural lipids (lard and wheat germ oil) and α-T3-rich foods (wholemeal rye bread and oil from dried brewer’s spent grain) were investigated through HPLC-DAD-F. The following α-T3 oxidation products were extractable from lipid matrices along with tocochromanols: α-tocotrienolquinone (α-T3Q), α-tocotrienolquinone-4a,5-epoxide (α-T3Q-4a,5-E), α-tocotrienolquinone-7,8-epoxide (α-T3Q-7,8-E), 7-formyl-β-tocotrienol (7-FβT3), and 5-formyl-γ-tocotrienol (5-FγT3). Recovery rates were as high as 88% and enrichment factors up to 124. The proposed method allows the investigation of α-T3Q, α-T3Q-4a,5-E, α-T3Q-7,8-E, 7-FβT3, and 5-FγT3 in small quantities (<0.78 μg/g) in lipid matrices, which is necessary for the investigation and analysis of the formation kinetics of these oxidation products in fat, oils, and tocotrienol-rich foods.
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Vitamin E is a family of naturally occurring and structurally related lipophilic antioxidants, one of which, α-tocopherol (α-TOH), selectively accumulates in vertebrate tissues. The ω-hydroxylase cytochrome P450-4F2 (CYP4F2) is the only human enzyme shown to metabolize vitamin E. Using cDNA cloning, cell culture expression, and activity assays, we identified Cyp4f14 as a functional murine ortholog of CYP4F2. We then investigated the effect of Cyp4f14 deletion on vitamin E metabolism and status in vivo. Cyp4f14-null mice exhibited substrate-specific reductions in liver microsomal vitamin E-ω-hydroxylase activity ranging from 93% (γ-TOH) to 48% (γ-tocotrienol). In vivo data obtained from metabolic cage studies showed whole-body reductions in metabolism of γ-TOH of 90% and of 68% for δ- and α-TOH. This metabolic deficit in Cyp4f14(-/-) mice was partially offset by increased fecal excretion of nonmetabolized tocopherols and of novel ω-1- and ω-2-hydroxytocopherols. 12′-OH-γ-TOH represented 41% of whole-body production of γ-TOH metabolites in Cyp4f14(-/-) mice fed a soybean oil diet. Despite these counterbalancing mechanisms, Cyp4f14-null mice fed this diet for 6 weeks hyper-accumulated γ-TOH (2-fold increase over wild-type littermates) in all tissues and appeared normal. We conclude that CYP4F14 is the major but not the only vitamin E-ω-hydroxylase in mice. Its disruption significantly impairs whole-body vitamin E metabolism and alters the widely conserved phenotype of preferential tissue deposition of α-TOH. This model animal and its derivatives will be valuable in determining the biological actions of specific tocopherols and tocotrienols in vivo.
Vitamin E has been suggested to modulate age-associated changes by altering the redox balance resulting in altered gene and/or protein expression. Here we have utilized proteomics to determine whether such regulation in protein expression occurs in human lymphocytes from two different age groups stressed with H₂O₂ and then treated with vitamin E in the form of tocotrienol-rich fraction (TRF). In this study, lymphocytes obtained from young (30-49 years old) and old (>50 years old) volunteers were first challenged with 1 mM H₂O₂. They were then treated by exposure to 50, 100 and 200 μg/ml TRF. Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF (matrix-assisted laser desorption/ionization time-of-flight/time-of-flight) tandem mass spectrometry was then performed on whole-cell protein extracts to identify proteins that have changed in expression. A total of 24 proteins were found to be affected by H₂O₂ and/or TRF treatment. These included proteins that were related to metabolism, antioxidants, structural proteins, protein degradation and signal transduction. Of particular interest was the regulation of a number of proteins involved in stress response–peroxiredoxin-2, peroxiredoxin-3 and peroxiredoxin-6-all of which were shown to be down-regulated with H₂O₂ exposure. The effect was reversed following TRF treatment. The expression of peroxiredoxin-2 and peroxiredoxin-6 was confirmed by quantitative reverse transcriptase polymerase chain reaction. These results suggested that TRF directly influenced the expression dynamics of the peroxiredoxin-2, thus improving the cells ability to resist damage caused by oxidative stress.
Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The α-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. γ-tocopherol methyltransferase (γ-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of γ- and δ-isomers to α- and β-isomers. In present study, the Arabidopsis γ-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the α-/γ-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the γ-isomers were converted to α-isomers, especially the γ- and δ-tocotrienol levels were dramatically decreased. As a result, the α-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the α-tocotrienol content. The results showed that the α-/γ-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on α-tocopherol level, which may reflect the fact that γ-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign γ-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world’s most important food crops.
The aim of this work was to compare the intestinal absorption kinetics and bioavailability of γ-tocotrienol (γ-T3) and α-tocopherol (α-Tph) administered separately as oil solutions to rats invivo. Also, to explain the significant difference in both compounds oral bioavailability bycomparing their: 1) release profiles using the dynamic in-vitro lipolysis model, 2) intestinalpermeability, and 3) carrier-mediated uptake by Niemann-Pick C1-like 1 (NPC1L1) transporterwere examined. Absolute bioavailability studies were conducted after oral administration of γ-T3 or α-Tph prepared in corn oil to rats. In-situ rat intestinal perfusion with ezetimibe (NPC1L1 inhibitor) was performed to compare intestinal permeability. In-vitro interaction kinetics with NPC1L1 was examined in NPC1L1 transfected cells. While the in-vitro release studies demonstrated significantly higher release rate of γ-T3 in the aqueous phase, the oral bioavailability of α-Tph (36%) was significantly higher than γ-T3 (9%). Consequent in-situ studies revealed significantly higher intestinal permeability for α-Tph compared to γ-T3 in rats. Moreover, NPC1L1 kinetic studies demonstrated higher Vmax and Km values for α-Tph compared to γ-T3. Collectively, these results indicate that intestinal permeability is the main contributing factor for higher bioavailability of α-Tph. Also, these results emphasize the potentially important role of intestinal permeability in the bioavailability of γ-T3 suggesting that enhancing its permeability would increase its oral bioavailability.
BACKGROUND: Today, few known plant species provide both an essential oil (EO) and a vegetable oil (VO). Seed and husk of two Aframomum species were investigated and compared in terms of EO, fatty acids, tocopherols, and tocotrienols.
RESULTS: EO yield reaches 15.3 g kg(-1) in the seeds and 3.2 g kg(-1) in the husks, while VO yield is 180.0 g kg(-1) in the seeds and 25.0 g kg(-1) in the husks. β-Pinene, 1,8-cineol, α-selinene, terpine-4-ol, linalool, myrtenal and β-caryophyllene are the major compounds of seed and husk EO. Fatty acid analysis of two Aframomum species shows that oleic, linoleic, and palmitic acids were the major compounds of VO. Total sterol contents reached 4.3 g kg(-1) in seed VO and 8.5 g kg(-1) in husk VO. An appreciable amount of tocopherols (0.52 g kg(-1) ) was found in seed VO.
CONCLUSION: The seed and husk oil of A. stipulatum and A. giganteum fruits are rich sources of many bioactive constituents such as fatty acids, sterols, tocopherols and tocotrienols. These tropical wild fruits can be considered as new Aroma Tincto Oleo Crops (ATOC) resources that contain both EOs and VOs.
Vitamin E and coenzyme Q10 (CoQ10) have antioxidant effects that may benefit cardiovascular health. Meta-analyses of randomized controlled trials have not shown a protective effect of supplementation with the vitamin E isomer α-tocopherol on the risk of acute myocardial infarction (AMI), but data on other isomers and CoQ10 are limited. Our objective was to examine the association of the plasma concentrations of vitamin E isomers (α-, γ-, and δ-tocopherol and α-, γ-, and δ-tocotrienol) and CoQ10 (ubiquinol and ubiquinone) with the incidence of AMI. We conducted a nested case-control study with 233 cases of incident AMI and 466 matched controls selected from the Singapore Chinese Health Study, aged 45-74 y at the time of recruitment and free of cardiovascular disease at the time of blood collection. We used conditional logistic regression to examine the association between vitamin E and CoQ10 and the risk of AMI adjusted for other risk factors. In the basic model, higher δ-tocopherol and ubiquinone concentrations were significantly associated with a higher risk of AMI, whereas there were no significant associations for the other vitamin E and CoQ10 isomers. After adjusting for lifestyle and other risk factors, only the association between δ-tocopherol and AMI risk remained significant [OR = 3.09 (95% CI: 1.53, 6.25) highest vs. lowest quintile; P-trend = 0.028]. We did not observe an inverse association between plasma concentrations of vitamin E isomers or CoQ10 and risk of AMI in Singapore Chinese. In contrast, plasma δ-tocopherol concentrations were associated with a higher risk of AMI. Our findings do not support a role of higher vitamin E or CoQ10 intakes in the prevention of AMI.
Of the eight natural vitamin E congeners (α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol), the non-α-tocopherol congeners have unique biological properties that may contribute to human health. Their study in vivo has been complicated by the lack of a simple analytical method that completely resolves and sensitively detects all eight natural tocopherols and tocotrienols in biological matrices. We thus developed and validated (according to the FDA guidelines for bioanalytical method validation) the first reversed-phase liquid chromatographic method for the baseline-separation and quantification of all eight tocopherols and tocotrienols. Analytes were extracted from human plasma or mouse liver and separated on a Phenomenex Kinetex PFP column (2.6 μm, 150 × 4.6 mm) by elution with methanol:water (85:15, vol/vol) at a flow rate of 0.8 mL/min. The developed RP-LC method used a solid-core pentafluorophenyl stationary phase and achieved baseline separation of all eight vitamin E congeners within 15 min at a backpressure of 23 MPa, which is suitable for most conventional HPLC systems. The method was fast, linear, accurate, and precise with detection limits of 27-156 pg and good recoveries (82-122%) for all analytes. In conclusion, we developed and validated the first RP-LC method for baseline resolution of all eight tocopherols and tocotrienols extracted from plasma and liver, which should be useful for the quantification of individual vitamin E congeners in large epidemiological studies and randomized controlled trials.
BACKGROUND: Tocotrienols and tocopherols (tocols) are important phytochemical compounds with antioxidant activity and potential benefits for human health. Among cereals, barley is a good source of tocols. In the present study the effect of two cultivation methods, organic and conventional, on the tocol content in 12 Greek barley varieties was investigated. A validated reverse phase high-performance liquid chromatography method (RP-HPLC) with fluorescence detection (excitation at 292 nm, emission at 335 nm) was applied along with direct solvent extraction with acetonitrile at a 1:30 (w/v) sample/solvent ratio for tocol quantification.
RESULTS: The results showed statistically significant differences (P < 0.05) between the two cultivation methods (except for δ-tocopherol) as well as among varieties. In the case of organic cultivation the four homologues of tocotrienol (α-, β + γ- and δ-) increased, by 3.05-37.14% for α-tocotrienol, 15.51-41.09% for (β + γ)-tocotrienol and 30.45-196.61% for δ-tocotrienol, while those of tocopherol (α- and β + γ- but not δ-) decreased, by 5.90-36.34% for α-tocopherol and 2.84-46.49% for (β + γ)-tocopherol. A simple correlation analysis between tocols revealed a good correlation between (β + γ)-tocotrienol and δ-tocotrienol. Although there was a significant decrease in the important α-tocopherol in the varieties studied under organic cultivation, there was an overall increase in tocotrienol content.
This study investigated the effects of α-tocopherol and palm oil tocotrienol supplementations on bone fracture healing in postmenopausal osteoporosis rats. 32 female Sprague-Dawley rats were divided into four groups. The first group was sham operated (SO), while the others were ovariectomised. After 2 months, the right femora were fractured under anesthesia and fixed with K-wire. The SO and ovariectomised-control rats (OVXC) were given olive oil (vehicle), while both the alpha-tocopherol (ATF) and tocotrienol-enriched fraction (TEF) groups were given alpha-tocopherol and tocotrienol-enriched fraction, respectively, at the dose of 60 mg/kg via oral gavages 6 days per week for 8 weeks. The rats were then euthanized and the femora dissected out for bone biomechanical testing to assess their strength. The callous of the TEF group had significantly higher stress parameter than the SO and OVXC groups. Only the SO group showed significantly higher strain parameter compared to the other treatment groups. The load parameter of the OVXC and ATF groups was significantly lower than the SO group. There was no significant difference in the Young’s modulus between the groups. In conclusion, tocotrienol is better than α-tocopherol in improving the biomechanical properties of the fracture callous in postmenopausal osteoporosis rat model.