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Competing Interests: Declaration of competing interest The author has no competing interests to declare.

Significance: Protection from oxygen, a diradical, became a necessity with the evolution of photosynthetic organisms about 2.7 billion years. α-Tocopherol plays an essential, protective role in organisms from plants to people. An overview of human conditions that result in severe vitamin E (α-tocopherol) deficiency is provided. Recent Advances: α-Tocopherol has a critical role in the oxygen protection system by stopping lipid peroxidation, its induced damage, and cellular death by ferroptosis. Recent findings in bacteria and plants support the concept of why lipid peroxidation is so dangerous to life and why the family of tocochromanols are essential for aerobic organisms and for plants. Critical Issues: The hypothesis that prevention of the propagation of lipid peroxidation is the basis for the α-tocopherol requirement in vertebrates is proposed and further that its absence dysregulates energy metabolism, one-carbon metabolism, and thiol homeostasis. By recruiting intermediate metabolites from adjacent pathways to sustain effective lipid hydroperoxide elimination, α-tocopherol function is linked not only to NADPH metabolism and its formation through the pentose phosphate pathway via glucose metabolism but also to sulfur-containing amino acid metabolism and to one-carbon metabolism. Future Directions: Evidence from humans, animals, and plants supports the hypothesis, but future studies are needed to assess the genetic sensors that detect lipid peroxidation and cause the ensuing metabolic dysregulation. Antioxid. Redox Signal. 38, 775-791.

α-Tocopherol (α-T) is a required dietary nutrient for humans and thus is a vitamin. This narrative review focuses on vitamin E structures, functions, biological determinants and its deficiency symptoms in humans. The mechanisms for the preferential α-T tissue enrichment in the human body include the α-T transfer protein (TTPA) and the preferential metabolism of non-α-T forms. Potential new α-T biomarkers, pharmacokinetic data, and whether there are better approaches to evaluate and set the α-T dietary requirement are discussed. Finally, the possible role of α-T supplements in delay of chronic diseases and the evaluation of vitamin E safety are considered., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This review discusses why the embryo requires vitamin E (VitE) and shows that its lack causes metabolic dysregulation and impacts morphological changes at very early stages in development, which occur prior to when a woman knows she is pregnant. VitE halts the chain reactions of lipid peroxidation (LPO). Metabolomic analyses indicate that thiols become depleted in E- embryos because LPO generates products that require compensation using limited amino acids and methyl donors that are also developmentally relevant. Thus, VitE protects metabolic networks and the integrated gene expression networks that control development. VitE is critical especially for neurodevelopment, which is dependent on trafficking by the α-tocopherol transfer protein (TTPa). VitE-deficient (E-) zebrafish embryos initially appear normal, but by 12 and 24 h post-fertilization (hpf) E- embryos are developmentally abnormal with expression of pax2a and sox10 mis-localized in the midbrain-hindbrain boundary, neural crest cells and throughout the spinal neurons. These patterning defects indicate cells that are especially in need of VitE-protection. They precede obvious morphological abnormalities (cranial-facial malformation, pericardial edema, yolksac edema, skewed body-axis) and impaired behavioral responses to locomotor activity tests. The TTPA gene (ttpa) is expressed at the leading edges of the brain ventricle border. Ttpa knockdown using morpholinos is 100% lethal by 24 hpf, while E- embryo brains are often over- or under-inflated at 24 hpf. Further, E- embryos prior to 24 hpf have increased expression of genes involved in glycolysis and the pentose phosphate pathway, and decreased expression of genes involved in anabolic pathways and transcription. Combined data from both gene expression and the metabolome in E- embryos at 24 hpf suggest that the activity of the mechanistic Target of Rapamycin (mTOR) signaling pathway is decreased, which may impact both metabolism and neurodevelopment. Further evaluation of VitE deficiency in neurogenesis and its subsequent impact on learning and behavior is needed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vitamin A, acting through its metabolite, all- trans -retinoic acid, is a potent transcriptional regulator affecting expression levels of hundreds of genes through retinoic acid response elements present within these genes. However, the literature is replete with claims that consider vitamin A to be an antioxidant vitamin, like vitamins C and E. This apparent contradiction in the understanding of how vitamin A acts mechanistically within the body is a major focus of this review. Vitamin E, which is generally understood to act as a lipophilic antioxidant protecting polyunsaturated fatty acids present in membranes, is often proposed to be a transcriptional regulator. The evaluation of this claim is another focus of the review. We conclude that vitamin A is an indirect antioxidant, whose indirect function is to transcriptionally regulate a number of genes involved in mediating the body's canonical antioxidant responses. Vitamin E, in addition to being a direct antioxidant, prevents the increase of peroxidized lipids that alter both metabolic pathways and gene expression profiles within tissues and cells. However, there is little compelling evidence that vitamin E has a direct transcriptional mechanism like that of vitamin A. Thus, we propose that the term antioxidant not be applied to vitamin A, and we discourage the use of the term transcriptional mediator when discussing vitamin E.

Vitamin E, discovered in 1922, is essential for pregnant rats to carry their babies to term. However, 100 years later, the molecular mechanisms for the vitamin E requirement during embryogenesis remain unknown. Vitamin E's role during pregnancy has been difficult to study and thus, a vitamin E-deficient (E-) zebrafish embryo model was developed. Vitamin E deficiency in zebrafish embryos initiates lipid peroxidation, depletes a specific phospholipid (DHA-phosphatidyl choline), causes secondary deficiencies of choline, betaine and critical thiols (such as glutathione), and dysregulates energy metabolism. Vitamin E deficiency not only distorts the carefully programmed development of the nervous system, but it leads to defects in several developing organs. Both the α-tocopherol transfer protein and vitamin E are necessary for embryonic development, neurogenesis and cognition in this model and likely in human embryos. Elucidation of the control mechanisms for the cellular and metabolic pathways involved in the molecular dysregulation caused by vitamin E deficiency will lead to important insights into abnormal neurogenesis and embryonic malformations.

Electronic-cigarette, or vaping, product use-associated lung injury (EVALI) is a syndrome of acute respiratory failure characterized by monocytic and neutrophilic alveolar inflammation. Epidemiological and clinical evidence suggests a role of vitamin E acetate (VEA) in the development of EVALI, yet it remains unclear whether VEA has direct pulmonary toxicity. To test the hypotheses that aerosolized VEA causes lung injury in mice and directly injures human alveolar epithelial cells, we exposed adult mice and primary human alveolar epithelial type II (AT II) cells to an aerosol of VEA generated by a device designed for vaping oils. Outcome measures in mice included lung edema, BAL analysis, histology, and inflammatory cytokines; in vitro outcomes included cell death, cytokine release, cellular uptake of VEA, and gene-expression analysis. Comparison exposures in both models included the popular nicotine-containing JUUL aerosol. We discovered that VEA caused dose-dependent increases in lung water and BAL protein compared with control and JUUL-exposed mice in association with increased BAL neutrophils, oil-laden macrophages, multinucleated giant cells, and inflammatory cytokines. VEA aerosol was also toxic to AT II cells, causing increased cell death and the release of monocyte and neutrophil chemokines. VEA was directly absorbed by AT II cells, resulting in the differential gene expression of several inflammatory biological pathways. Given the epidemiological and clinical characteristics of the EVALI outbreak, these results suggest that VEA plays an important causal role.

Vitamin E (VitE) deficiency results in embryonic lethality. Knockdown of the gene ttpa encoding for the VitE regulatory protein [α-tocopherol transfer protein (α-TTP)] in zebrafish embryos causes death within 24 h post-fertilization (hpf). To test the hypothesis that VitE, not just α-TTP, is necessary for nervous system development, adult 5D strain zebrafish, fed either VitE sufficient (E+) or deficient (E-) diets, were spawned to obtain E+ and E- embryos, which were subjected to RNA in situ hybridization and RT-qPCR. Ttpa was expressed ubiquitously in embryos up to 12 hpf. Early gastrulation (6 hpf) assessed by goosecoid expression was unaffected by VitE status. By 24 hpf, embryos expressed ttpa in brain ventricle borders, which showed abnormal closure in E- embryos. They also displayed disrupted patterns of paired box 2a (pax2a) and SRY-box transcription factor 10 (sox10) expression in the midbrain-hindbrain boundary, spinal cord and dorsal root ganglia. In E- embryos, the collagen sheath notochord markers (col2a1a and col9a2) appeared bent. Severe developmental errors in E- embryos were characterized by improper nervous system patterning of the usually carefully programmed transcriptional signals. Histological analysis also showed developmental defects in the formation of the fore-, mid- and hindbrain and somites of E- embryos at 24 hpf. Ttpa expression profile was not altered by the VitE status demonstrating that VitE itself, and not ttpa, is required for development of the brain and peripheral nervous system in this vertebrate embryo model.

BACKGROUNDWe hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls.METHODSCustom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies.RESULTSIn healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3-4 hours; d3- and d6-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d6-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration.CONCLUSIONSThe unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins.TRIAL REGISTRATIONClinicalTrials.gov, NCT00862433.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.

Background: Determining the human vitamin E [α-tocopherol (α-T)] requirement is difficult, and novel approaches to assess α-T absorption and trafficking are needed., Objective: We hypothesized that the dual-isotope technique, using 2 deuterium-labeled [intravenous (IV) d6- and oral d3-] α-T, would be effective in determining α-T fractional absorption. Further, defined liquid meal (DLM) fat or fasting would modulate α-T fractional absorption and lipoprotein transport., Methods: A 3-phase cr ossover design was used. At 0 h, participants received IV d6-α-T and consumed d3-α-T with a 600-kcal DLM (40% or 0% fat) followed by controlled meals or by the 0% fat DLM, a 12-h fast, and then controlled meals. Blood samples and fecal samples were collected at intervals and analyzed by LC-MS. Pharmacokinetic parameters were calculated from plasma tracer concentrations and enrichments. Fractional absorption was calculated from d3- to d6-α-T areas under the curve, from a novel mathematical model, and from the balance method (oral d3-α-T minus fecal d3-α-T excreted)., Results: Estimated α-T fractional absorption during the 40% fat intervention was 55% ± 3% (mean ± SEM; n = 10), which was 9% less than during the 0% fat intervention (64% ± 3%, n = 10; P < 0.02). Fasting had no apparent effect (56% ± 3%, n = 7), except it slowed plasma oral d3-α-T appearance. Both balance data and model outcomes confirmed that the DLM fat did not potentiate d3-α-T absorption. During the IV emulsion clearance, HDL rapidly acquired d6-α-T (21 ± 2 nmol/L plasma per minute). During the first 8 h postdosing, triglyceride-rich lipoproteins (TRLs) were preferentially d3-α-T enriched relative to LDL or HDL, showing the TRL precursor role., Conclusions: Quantitatively, α-T absorption is not limited by fat absence or by fasting. However, α-T leaves the intestine by a process that is prolonged during fasting and potentiated by eating, suggesting that α-T absorption is highly dependent on chylomicron assembly processes. This trial was registered at clinicaltrials.gov as NCT00862433., (Copyright © American Society for Nutrition 2019.)

Vitamin E, as all-rac α-tocopheryl-acetate (TAc), has a bioavailability of only 5.4% in swine and, therefore, is a poor vitamin E source. Tocopheryl-phosphate (TP) has been used successfully as a vitamin E source around 1940 but it was subsequently replaced by TAc as it was easier to manufacture. Recently, it has been proposed as an in vivo intermediate in vitamin E metabolism with possibly gene-regulatory functions. TP may be more bioavailable than TAc as intestinal hydrolysis and emulsification are not required. The objective of this work was to compare the bioavailability of TAc and TP in swine. Piglets (18.6 ± 0.6 kg) fitted with jugular catheters received a single test meal (350 g) containing either deuterated (trimethyl-d9) TAc or TP (75 IU/kg body weight, n = 8 per treatment). Twelve serial blood samples were obtained starting premeal until 78 h postmeal for analysis of deuterated T and TP using LC MS/MS. Results were standardized by dividing them by the dose per kg body weight and were subsequently modeled with a multicompartment model. T from TAc had a slow appearance rate (0.040 ± 0.014 h-1) and rapid disappearance rate (0.438 ± 0.160 h-1) with a plateau value of 0.414 ± 0.129 µM/(µmol/kg BW). TP appeared faster in plasma (0.119 ± 0.058 h-1, P = 0.01) while the elimination rate was similar (0.396 ± 0.098 h-1, P = 0.51). The plateau value of TP was only numerically higher (0.758 ± 0.778 µM/(µmol/kg BW), P = 0.34). TP was quickly converted to T; its appearance rate was 0.026 ± 0.009 h-1, slower than the appearance rate of T from TAc (P = 0.01), whereas the elimination rate was 0.220 ± 0.062 h-1, slower than that of T from TAc (P = 0.00). The conversion of TP to T may have been incomplete, as its plateau value was only 0.315 ± 0.109 µM/(µmol/kg BW). The area under the curve, expressed relative to area under the curve for T from TAc, was 34.5% for TP and 107.3% for T from TP. These data confirm that TP is more quickly absorbed than T from TAc. TP is also converted to T and thus a functional precursor of T. Nevertheless, as a source of T, TP failed to offer a clear advantage over TAc in bioavailability.

Zebrafish (Danio rerio) are a recognized model for studying the pathogenesis of cognitive deficits and the mechanisms underlying behavioral impairments, including the consequences of increased oxidative stress within the brain. The lipophilic antioxidant vitamin E (α-tocopherol; VitE) has an established role in neurological health and cognitive function, but the biological rationale for this action remains unknown. In the present study, we investigated behavioral perturbations due to chronic VitE deficiency in adult zebrafish fed from 45 days to 18-months of age diets that were either VitE-deficient (E-) or VitE-sufficient (E+). We hypothesized that E- zebrafish would display cognitive impairments associated with elevated lipid peroxidation and metabolic disruptions in the brain. Quantified VitE levels at 18-months in E- brains (5.7 ± 0.1 nmol/g tissue) were ~20-times lower than in E+ (122.8 ± 1.1; n = 10/group). Using assays of both associative (avoidance conditioning) and non-associative (habituation) learning, we found E- vs E+ fish were learning impaired. These functional deficits occurred concomitantly with the following observations in adult E- brains: decreased concentrations of and increased peroxidation of polyunsaturated fatty acids (especially docosahexaenoic acid, DHA), altered brain phospholipid and lysophospholipid composition, as well as perturbed energy (glucose/ketone), phosphatidylcholine and choline/methyl-donor metabolism. Collectively, these data suggest that chronic VitE deficiency leads to neurological dysfunction through multiple mechanisms that become dysregulated secondary to VitE deficiency. Apparently, the E- animals alter their metabolism to compensate for the VitE deficiency, but these compensatory mechanisms are insufficient to maintain cognitive function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Vitamin E (α-tocopherol, VitE) was discovered in 1922 for its role in preventing embryonic mortality. We investigated the underlying mechanisms causing lethality using targeted metabolomics analyses of zebrafish VitE-deficient embryos over five days of development, which coincided with their increased morbidity and mortality. VitE deficiency resulted in peroxidation of docosahexaenoic acid (DHA), depleting DHA-containing phospholipids, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation also increased NADPH oxidation, which depleted glucose by shunting it to the pentose phosphate pathway. VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. Thus, embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis with lethal outcomes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We hypothesized that brains from vitamin E-deficient (E-) zebrafish (Danio rerio) would undergo increased lipid peroxidation because they contain highly polyunsaturated fatty acids, thus susceptible lipids could be identified. Brains from zebrafish fed for 9 months defined diets without (E-) or with (E+) added vitamin E (500 mg RRR-α-tocopheryl acetate per kilogram diet) were studied. Using an untargeted approach, 1-hexadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine [DHA-PC 38:6, PC 16:0/22:6]was the lipid that showed the most significant and greatest fold-differences between groups. DHA-PC concentrations were approximately 1/3 lower in E- (4.3 ± 0.6 mg/g) compared with E+ brains (6.5 ± 0.9 mg/g, mean ± SEM, n = 10 per group, P = 0.04). Using lipidomics, 155 lipids in brain extracts were identified. Only four phospholipids (PLs) were different (P < 0.05) between groups; they were lower in E- brains and contained DHA with DHA-PC 38:6 at the highest abundances. Moreover, hydroxy-DHA-PC 38:6 was increased in E- brains (P = 0.0341) supporting the hypothesis of DHA peroxidation. More striking was the depletion in E- brains of nearly 60% of 19 different lysophospholipids (lysoPLs) (combined P = 0.0003), which are critical for membrane PL remodeling. Thus, E- brains contained fewer DHA-PLs, more hydroxy-DHA-PCs, and fewer lysoPLs, suggesting that lipid peroxidation depletes membrane DHA-PC and homeostatic mechanisms to repair the damage resulting in lysoPL depletion., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Vitamin E is an essential human nutrient that was first isolated from wheat. Emmer wheat, the cereal of Old World agriculture and a precursor to durum wheat, grows wild in the Fertile Crescent. Evolution Canyon, Israel, provides a microsite that models effects of contrasting environments. The north-facing and south-facing slopes exhibit low and high stress environments, respectively. Wild emmer wheat seeds were collected from both slopes and seed tocochromanol contents measured to test the hypothesis that high stress alters emmer wheat seed tocol-omics. Seeds from high stress areas contained more total vitamin E (108±15 nmol/g) than seeds from low stress environments (80±17 nmol/g, P=.0004). Vitamin E profiles within samples from these different environments revealed significant differences in isoform concentrations. Within each region, β- plus γ-tocotrienols represented the highest concentration of wheat tocotrienols (high stress, P<.0001; low stress, P<.0001), while α-tocopherol represented the highest concentration of the tocopherols (high stress, P=.0002; low stress, P<.0001). Percentages of both δ-tocotrienol and δ-tocopherol increased in high stress conditions. Changes under higher stress apparently are due to increased pathway flux toward more tocotrienol production. The production of more δ-isoforms suggests increased flow through a divergent path controlled by the VTE1 gene. Hence, stress conditions alter plant responses such that vitamin E profiles are changed, likely an attempt to provide additional antioxidant activity to promote seed viability and longevity.

It is estimated that >90% of Americans do not consume sufficient dietary vitamin E, as α-tocopherol, to meet estimated average requirements. What are the adverse consequences of inadequate dietary α-tocopherol intakes? This review discusses health aspects where inadequate vitamin E status is detrimental and additional vitamin E has reversed the symptoms. In general, plasma α-tocopherol concentrations <12 μmol/L are associated with increased infection, anemia, stunting of growth, and poor outcomes during pregnancy for both the infant and the mother. When low dietary amounts of α-tocopherol are consumed, tissue α-tocopherol needs exceed amounts available, leading to increased damage to target tissues. Seemingly, adequacy of human vitamin E status cannot be assessed from circulating α-tocopherol concentrations, but inadequacy can be determined from “low” values. Circulating α-tocopherol concentrations are very difficult to interpret because, as a person ages, plasma lipid concentrations also increase and these elevations in lipids increase the plasma carriers for α-tocopherol, leading to higher circulating α-tocopherol concentrations. However, abnormal lipoprotein metabolism does not necessarily increase α-tocopherol delivery to tissues. Additional biomarkers of inadequate vitamin E status are needed. Urinary excretion of the vitamin E metabolite α-carboxy-ethyl-hydroxychromanol may fulfill this biomarker role, but it has not been widely studied with regard to vitamin E status in humans or with regard to health benefits. This review evaluated the information available on the adverse consequences of inadequate α-tocopherol status and provides suggestions for avenues for research.

The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin E-deficient parental diet (E-) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E- diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E- embryos at 48h post-fertilization (hpf), embryos were collected from each group at 36hpf. Lab embryos differentially expressed (p<0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E- embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E+ diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E- embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vitamin E improved liver histology in children and adults with NAFLD who participated in TONIC and PIVENS clinical trials, but with significant inter-individual variability in its efficacy. Cytochrome P450 4F2 (CYP4F2) is the major enzyme metabolizing Vit E, with two common genetic variants (V433M, rs2108622 and W12G, rs3093105) found to alter its activity. We investigated the relationship between CYP4F2 genotypes, α-tocopherol levels and histological improvement in these two trials. V433M and W12G variants were genotyped in TONIC (n = 155) and PIVENS (n = 213) DNA samples. The relationships between CYP4F2 genotypes, plasma α-tocopherol levels at baseline and weeks 48 (w48) and 96 (w96) and histological end points (overall improvement in liver histology and resolution of NASH) were investigated. As a result, the V433M genotype was significantly associated with baseline plasma α-tocopherol in the TONIC trial (p = 0.004), but not in PIVENS. Among those receiving Vit E treatment, CYP4F2 V433M genotype was associated with significantly decreased plasma α-tocopherol levels at w48 (p = 0.003 for PIVENS and p = 0.026 for TONIC) but not at w96. The w96 α-tocopherol level was significantly associated with resolution of NASH (p = 0.006) and overall histology improvement (p = 0.021)in the PIVENS, but not in the TONIC trial. There was no significant association between CYP4F2 genotypes and histological end points in either trial. Our study suggested the a moderate role of CYP4F2 polymorphisms in affecting the pharmacokinetics of Vit E as a therapeutic agent. In addition, there may be age-dependent relationship between CYP4F2 genetic variability and Vit E pharmacokinetics in NAFLD.

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) is ubiquitous in plants and functions in the tyrosine catabolic pathway, resulting in the formation of homogentisate. Homogentisate is the aromatic precursor of all plastoquinones and tocochromanols, including tocopherols and tocotrienols. Soybean (Glycine max (L.) Merr.) has been genetically modified to express the gene avhppd-03 that encodes the protein AvHPPD-03 derived from oat (Avena sativa L.). The AvHPPD-03 isozyme has an inherent reduced binding affinity for mesotrione, a herbicide that inhibits the wild-type soybean HPPD enzyme. Expression of avhppd-03 in soybean plants confers a mesotrione-tolerant phenotype. Seeds from three different avhppd-03-expressing soybean events were quantitatively assessed for content of eight vitamin E isoforms. Although increased levels of two tocopherol isoforms were identified for each of the three soybean events, they were within, or not substantially different from, the ranges of these isoforms found in nontransgenic soybean varieties. The increases of these tocopherols in the avhppd-03-expressing soybean events may have a slight benefit with regard to vitamin E nutrition but, given the commercial processing of soybeans, are unlikely to have a material impact on human nutrition with regard to vitamin E concentrations in soybean oil.

The purpose of this study was to determine the system-wide consequences of deficiencies in two essential micronutrients, vitamins E and C, on the proteome using zebrafish (Danio rerio) as one of the few vertebrate models that similar to humans cannot synthesize vitamin C. We describe a label-free proteomics workflow to detect changes in protein abundance estimates dependent on vitamin regimes. We used ion-mobility-enhanced data-independent tandem mass spectrometry to determine differential regulation of proteins in response to low dietary levels of vitamin C with or without vitamin E. The detection limit of the method was as low as 20 amol, and the dynamic range was five orders of magnitude for the protein-level estimates. On the basis of the quantitative changes obtained, we built a network of protein interactions that reflect the whole organism's response to vitamin C deficiency. The proteomics-driven study revealed that in vitamin-E-deficient fish, vitamin C deficiency is associated with induction of stress response, astrogliosis, and a shift from glycolysis to glutaminolysis as an alternative mechanism to satisfy cellular energy requirements.

The liver is at the nexus of the regulation of lipoprotein uptake, synthesis, and secretion, and it is the site of xenobiotic detoxification by cytochrome P450 oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III). These two major liver systems control vitamin E status. The mechanisms for the preference for α-tocopherol relative to the eight naturally occurring vitamin E forms largely depend upon the liver and include both a preferential secretion of α-tocopherol from the liver into the plasma for its transport in circulating lipoproteins for subsequent uptake by tissues, as well as the preferential hepatic metabolism of non-α-tocopherol forms. These mechanisms are the focus of this review.

Scope: The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4)., Methods and Results: In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 μmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters., Conclusion: Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hepatic α-tocopherol transfer protein (TTP) is required for optimal α-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP's significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.

The mechanistic properties of two dietary antioxidants that are required by humans, vitamins C and E, are discussed relative to their biological effects. Vitamin C (ascorbic acid) is an essential cofactor for α-ketoglutarate-dependent dioxygenases. Examples are prolyl hydroxylases, which play a role in the biosynthesis of collagen and in down-regulation of hypoxia-inducible factor (HIF)-1, a transcription factor that regulates many genes responsible for tumor growth, energy metabolism, and neutrophil function and apoptosis. Vitamin C-dependent inhibition of the HIF pathway may provide alternative or additional approaches for controlling tumor progression, infections, and inflammation. Vitamin E (α-tocopherol) functions as an essential lipid-soluble antioxidant, scavenging hydroperoxyl radicals in a lipid milieu. Human symptoms of vitamin E deficiency suggest that its antioxidant properties play a major role in protecting erythrocyte membranes and nervous tissues. As an antioxidant, vitamin C provides protection against oxidative stress-induced cellular damage by scavenging of reactive oxygen species, by vitamin E-dependent neutralization of lipid hydroperoxyl radicals, and by protecting proteins from alkylation by electrophilic lipid peroxidation products. These bioactivities bear relevance to inflammatory disorders. Vitamin C also plays a role in the function of endothelial nitric oxide synthase (eNOS) by recycling the eNOS cofactor, tetrahydrobiopterin, which is relevant to arterial elasticity and blood pressure regulation. Evidence from plants supports a role for vitamin C in the formation of covalent adducts with electrophilic secondary metabolites. Mechanism-based effects of vitamin C and E supplementation on biomarkers and on clinical outcomes from randomized, placebo-controlled trials are emphasized in this review., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: : The purpose of this study was to identify the influence of vitamin E and C supplementation on inflammatory cytokines and the association between reciprocally regulated cytokines after anterior cruciate ligament surgery., Design: : A double-blind, placebo-controlled study was conducted in men undergoing anterior cruciate ligament surgery who were randomly assigned to one of two groups (n = 10/group): (1) antioxidant (vitamins E and C) or (2) matching placebos starting ∼2 wks before (baseline) and concluding 3 mos after surgery. Plasma inflammatory cytokines were measured in fasting blood draw samples before and after anterior cruciate ligament surgery., Results: : Plasma interleukin (IL) 1β concentrations were double at 3 mos after surgery compared with baseline. Plasma IL-1β increased to a greater (P < 0.05) extent relative to IL-4 in the placebo group (mean ± SE slope, 18.87 ± 0.68; r = 0.97) than in the antioxidant group (mean ± SE slope, 4.84 ± 0.42; r = 0.89). Similarly, the relative increase in IL-1β to IL-2 was greater (P < 0.05) in the placebo (mean ± SE slope, 2.70 ± 0.21) than in the antioxidant (mean ± SE slope, 1.08 ± 0.23) group., Conclusions: : Vitamins E and C were ineffective in ameliorating the increases in IL-1β but altered associations between reciprocally regulated cytokines after anterior cruciate ligament surgery.

Background: Chronic ethanol self-administration induces oxidative stress and exacerbates lipid peroxidation. α-Tocopherol is a potent lipid antioxidant and vitamin that is dependent upon lipoprotein transport for tissue delivery., Methods: To evaluate the extent to which vitamin E status is deranged by excessive alcohol consumption, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg/d for 19 months, n = 11) were compared with nondrinkers (n = 5, control)., Results: Three alcohol-drinking animals developed hyperlipidemia with plasma triglyceride levels (1.8 ± 0.9 mM) double those of normolipidemic (NL) drinkers (0.6 ± 0.2) and controls (0.6 ± 0.3, p < 0.05); elevated plasma cholesterol (3.6 ± 0.5 mM) compared with NL drinkers (2.3 ± 0.2, p < 0.05) and controls (2.9 ± 0.3); and lower plasma α-tocopherol per triglycerides (14 ± 6 mmol/mol) than controls (27 ± 8) and NL drinkers (23 ± 6, p < 0.05). Hyperlipidemic monkey liver α-tocopherol (47 ± 15 nmol/g) was lower than NL drinkers (65 ± 13) and controls (70 ± 15, p = 0.080), as was adipose α-tocopherol (84 ± 37 nmol/g) compared with controls (224 ± 118) and NL drinkers (285 ± 234, p < 0.05). Plasma apolipoprotein (apo) CIII increased compared to baseline at both 12 and 19 months in the normolipidemic (p = 0.0016 and p = 0.0028, respectively) and in the hyperlipidemic drinkers (p < 0.05 and p < 0.05, respectively). Plasma apo H concentrations at 19 months were elevated hyperlipidemics (p < 0.05) relative to concentrations in control animals. C-reactive protein (CRP), a marker of inflammation, was increased compared to baseline at both the 12- and 19-month time points in the normolipidemic (p = 0.005 and p = 0.0153, respectively) and hyperlipidemic drinkers (p = 0.016 and p = 0.0201, respectively)., Conclusion: A subset of alcohol-drinking monkeys showed a predisposition to alcohol-induced hyperlipidemia. The defect in lipid metabolism resulted in lower plasma α-tocopherol per triglycerides and depleted adipose tissue α-tocopherol, and thus decreased vitamin E status., (Copyright © 2010 by the Research Society on Alcoholism.)

Background: The transfer of vitamin E across the placenta is limited, but no data exist on the concentrations of vitamin E metabolites carboxyethyl hydroxychromans (α- and γ-CEHCs) in the fetal circulation., Objective: We measured α- and γ-CEHC concentrations in maternal and umbilical cord blood pairs and examined their relations to circulating vitamin E (α- and γ-tocopherol) and maternal dietary vitamin E intake., Design: Healthy, pregnant women were enrolled from Oregon Health and Science University's obstetric clinic (<22 wk gestation), and at least one fasting blood sample and a previous day's 24-h diet recall were collected during their pregnancy (n = 19). Umbilical cord blood samples were obtained at the time of delivery and were analyzed for α- and γ-tocopherol, α- and γ-CEHC, and total lipid concentrations., Results: Mean (±SD) concentrations of umbilical cord blood α-CEHC (30.2 ± 28.9 nmol/L) and γ-CEHC (104.5 ± 61.3 nmol/L) were not significantly different from maternal concentrations (P = 0.07 and 0.08, respectively), but metabolite:tocopherol ratios were significantly higher in cord blood (P < 0.01 and 0.001, respectively). Maternal α-tocopherol:total lipids ratios were correlated with cord blood α-CEHCs (r = 0.67, P = 0.004), and higher vitamin E intakes were associated with higher cord blood α-CEHC concentrations (r = 0.75, P < 0.003)., Conclusion: Higher maternal intake of vitamin E during pregnancy may result in increased metabolite concentrations in the fetal circulation, suggesting increased maternal or fetal liver metabolism of vitamin E. This trial was registered at clinicaltrials.gov as NCT00632476.

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more alpha- and gamma-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.

Interleukin (IL)-10 is an anti-inflammatory cytokine that suppresses pro-inflammatory cytokines. We previously demonstrated that supplementation with vitamins E and C ameliorated the increase in IL-10 immediately following anterior cruciate ligament (ACL) surgery in the absence of other cytokine perturbations. Since both oxidative stress and insulin-like growth factor-1 (IGF-1) can modulate IL-10 concentrations, the mechanisms for these changes warranted further investigation. Our objective was to evaluate the mechanism for the IL-10 decrease following ACL surgery. This study consisted of randomized, double-blind, placebo-controlled experimental design. Subjects were randomly assigned to daily supplementation with either: (i) antioxidants (AO; vitamins E [alpha-tocopherol] and C [ascorbic acid]; n = 10); or (ii) matching placebos (PL; n = 10). Supplementation started approximately 2 weeks prior to surgery (baseline) and concluded 3 months after surgery. Subjects provided six fasting blood samples at: (i) baseline; (ii) immediately pre-surgery (Pre); (iii) 90 min; (iv) 72 h; (v) 7 days; and (vi) 3 months post-surgery. alpha-Tocopherol, ascorbic acid, F(2)-isoprostane and IGF-1 concentrations were measured in each blood sample. At 90 min relative to other times, plasma F(2)-isoprostane concentrations were significantly (P < 0.05) elevated in both groups, while at 90 min IGF-1 was significantly (P < 0.05) lower in the AO compared to the PL group. The changes in IGF-1 at 90 min relative to baseline were correlated (P < 0.0001) with the changes in IL-10. The decrease in IL-10 observed in the AO group is likely dependent on the decrease IGF-1 since lipid peroxidation was unchanged between the two groups.

Oxidants are involved in the pathogenesis of many disorders caused by burn and smoke inhalation; alpha- and gamma-tocopherols are major tissue antioxidants, and their depletion should reflect oxidant injury. To determine whether plasma and tissue vitamin E levels would thus be depleted in severe burn, prepared sheep were randomly divided into the following groups: non-injured, burn- and smoke-exposed, burned only and smoke-exposed only. All were resuscitated with Ringer's lactate solution, mechanically ventilated and sacrificed at various time intervals. Immediately following injury plasma, lung, trachea, heart and liver tocopherols/lipids were measured and found to be significantly depleted except in the heart. Reduction of tissue gamma-tocopherol appeared earlier than reduction of alpha-tocopherol. Thus animals receiving combined burn and inhalation injury underwent marked oxidative stress, suggesting that vitamin E might be depleted also in humans with burn and smoke inhalation injury, and that appropriate supplementation should be evaluated.

The mechanisms by which vitamin E interferes with vitamin K activity, especially blood clotting, are not known, but hypothetically this interference may involve metabolic pathways. Phylloquinone (K(1)) must be converted to menaquinone (MK-4, the most potent extrahepatic tissue vitamin K) by truncation of the K(1) side chain and replacement with geranylgeranyl. Possible mechanisms for the vitamin E and K interaction include: 1) vitamin E competes for the yet undiscovered enzyme that truncates the K(1) side chain; 2) vitamin E competes with K(1) for the hypothetical cytochrome P450 enzyme that omega-hydroxylates the K(1) side chain, thereby preventing its beta-oxidation and its removal for MK-4 formation; or 3) vitamin E increases xenobiotic pathways that increase hepatic metabolism and excretion of all vitamin K forms. Currently, the pathway for K(1) conversion to MK-4 is unknown, the process for regulating vitamin K metabolism to urinary excretion products is unknown, and why vitamin E supplements have such a dramatic effect, causing bleeding in some individuals and not in others, remains a mystery.

Purpose of Review: Vitamin E benefits in human health and chronic disease prevention are evaluated with respect to established alpha-tocopherol functions during vitamin E deficiency, adequacy, and excess., Recent Findings: Baseline vitamin E status of the 29 092 Finnish men participating in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention study showed that the men in the highest compared with the lowest quintile of serum alpha-tocopherol had significantly lower incidences of total and cause-specific mortality. New findings from the Women's Health Study support a role for vitamin E supplements in decreasing the risk for sudden death from cardiovascular disease and from thromboembolism. We speculate that a potential mechanism may involve vitamin E interference in vitamin K activation., Summary: alpha-Tocopherol acts as a peroxyl and alkoxyl radical scavenger in lipid environments, and thus it prevents lipid peroxidation in lipoproteins and membranes, especially nervous tissues. Decreased chronic disease incidence is associated with lifelong generous dietary vitamin E intakes, but more than 90% of Americans do not consume the recommended dietary amounts (15 mg/day). Vitamin E supplements can have beneficial effects on health beyond those from dietary amounts, perhaps because pharmacologic levels also upregulate hepatic xenobiotic pathways.

Purpose: The effects of vitamin E supplementation on the cholesterol levels of hypercholesterolemic patients receiving statin therapy were studied., Methods: In this prospective, single-blind, placebo-controlled, randomized trial, patients who were currently taking either lovastatin or simvastatin for a primary diagnosis of hypercholesterolemia were given placebo for two weeks and then randomized to receive a supplement of either 400 IU of vitamin E or matching placebo after dinner for eight weeks, followed by a two-week washout period., Results: Vitamin E supplementation increased plasma alpha-tocopherol concentrations approximately 1.6-fold and increased excretion of its urinary metabolite 4-fold significantly from week 2 to week 6 (p < 0.001 for both comparisons). During the eight-week supplementation period, no statistically significant differences in any lipoprotein cholesterol fraction were detected between groups; however, a 6% decrease in high-density-lipoprotein (HDL) cholesterol was detected within the vitamin E group from week 2 to week 6 (p < 0.05), but the decrease was not sufficient to change the cardiac risk ratio. Neither cytochrome P-450 isoenzyme (CYP) 3A (as measured by hydroxylation of urinary cortisol) nor cholesteryl ester transfer protein (CETP) activity was significantly altered during the study., Conclusion: Vitamin E supplementation did not affect total or low-density-lipoprotein cholesterol levels in hypercholesterolemic patients receiving lovastatin or simvastatin. A small but significant decrease in HDL cholesterol levels was observed in the group that received vitamin E supplementation during the supplementation period, but this decrease was no longer significantly different from the placebo group's levels two weeks postsupplementation. The decrease in HDL cholesterol levels did not appear to be related to either CYP3A or CETP.

All of the naturally occurring vitamin E forms, as well as those of synthetic all-rac-alpha-tocopherol, have relatively similar antioxidant properties, so why does the body prefer alpha-tocopherol as its unique form of vitamin E? We propose the hypothesis that all of the observations concerning the in vivo mechanism of action of alpha-tocopherol result from its role as a potent lipid-soluble antioxidant. The purpose of this review then is to describe the evidence for alpha-tocopherol's in vivo function and to make the claim that alpha-tocopherol's major vitamin function, if not only function, is that of a peroxyl radical scavenger. The importance of this function is to maintain the integrity of long-chain polyunsaturated fatty acids in the membranes of cells and thus maintain their bioactivity. That is to say that these bioactive lipids are important signaling molecules and that changes in their amounts, or in their loss due to oxidation, are the key cellular events that are responded to by cells. The various signaling pathways that have been described by others to be under alpha-tocopherol regulation appear rather to be dependent on the oxidative stress of the cell or tissue under question. Moreover, it seems unlikely that these pathways are specifically under the control of alpha-tocopherol given that various antioxidants other than alpha-tocopherol and various oxidative stressors can manipulate their responses. Thus, virtually all of the variation and scope of vitamin E's biological activity can be seen and understood in the light of protection of polyunsaturated fatty acids and the membrane qualities (fluidity, phase separation, and lipid domains) that polyunsaturated fatty acids bring about.

Heart disease is the number one cause of death in the United States and has long been recognized to be multifactorial. A growing body of evidence suggests that not only free radical-mediated reactions but also inflammatory responses play major roles in atherogenesis. Vitamin E has both antioxidant and antiinflammatory properties and is the most widely studied vitamin in clinical trials and thus will be the primary example used in this review. Clinical trials of vitamin E efficacy, in hindsight, have been overly optimistic in their expectation that a vitamin could reverse poor dietary habits and a sedentary lifestyle as well as provide benefit beyond that of pharmaceutical agents in treating heart disease. However, it is also apparent that most Americans do not consume dietary amounts adequate to meet established vitamin E requirements. In response to oxidative stressors, vitamin E can decrease biomarkers of lipid peroxidation, is itself killed, and requires optimal vitamin C status to function most effectively. Thus, adequate vitamin E intakes are clearly needed, but what is adequate for what function has yet to be defined. It is noteworthy that in most trials, biomarkers were not used nor were oxidative stress and lipid peroxidation markers used or plasma vitamin E concentrations measured.

Dietary and supplemental vitamin E is absorbed and delivered to the liver, but of the various antioxidants with vitamin E activity, only alpha-tocopherol is preferentially recognized by the alpha-tocopherol transfer protein (alpha-TTP) and is transferred to plasma, while the other vitamin E forms (e.g., gamma-tocopherol or tocotrienols) are removed from the circulation. Hepatic alpha-TTP is required to maintain plasma and tissue alpha-tocopherol concentrations. The liver is the master regulator of the body's vitamin E levels in that it not only controls alpha-tocopherol concentrations, but also appears to be the major site of vitamin E metabolism and excretion. Vitamin Es are metabolized similarly to xenobiotics; they are initially omega-oxidized by cytochrome P450s, undergo several rounds of beta-oxidation, and then are conjugated and excreted. As a result of these various mechanisms, liver alpha-tocopherol and other vitamin E concentrations are closely regulated; thus, any potential adverse vitamin E effects are limited.

The term vitamin E is used to describe eight lipophilic, naturally occurring compounds that include four tocopherols and four tocotrienols designated as alpha-, beta-, gamma-, and delta-. The most well-known function of vitamin E is that of a chain-breaking antioxidant that prevents the cyclic propagation of lipid peroxidation. Despite its antioxidant function, dietary vitamin E requirements in humans are limited only to alpha-tocopherol because the other forms of vitamin E are poorly recognized by the hepatic alpha-tocopherol transfer protein (TTP), and they are not converted to alpha-tocopherol by humans. In attempts to gain a better understanding of vitamin E's health benefits, the molecular regulatory mechanisms of vitamin E have received increased attention. Examples of these mechanisms include: (1) the role of the hepatic alpha-TTP in preferentially secreting alpha-tocopherol into the plasma, (2) phase I and phase II metabolism of vitamin E and the potential impact for drug-vitamin E interactions, and (3) the regulation of biliary excretion of vitamin E by ATP-binding cassette protein(s). It is expected that the continued studies of these regulatory pathways will provide new insights into vitamin E function from which additional human health benefits will evolve.

Metabolites of α- and γ-tocopherol, 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (α-CEHC) and 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC), respectively, are produced in the liver and have been measured in biological fluids and tissue. Compared to α-tocopherol concentrations, metabolite concentrations are as much as a factor of a thousand lower, requiring extremely sensitive methodology to attain accurate measurements. This unit presents a protocol for CEHC extraction from biological samples, and describes very specific and sensitive HPLC/MS analysis.

During endurance exercise, oxygen consumption by the skeletal muscle can increase 100-200 times. We previously found that during an ultramarathon race (50 km, forest trail through hilly terrain) compared with a day of rest, vitamin E disappeared faster (as measured using 2H-labelled alpha-tocopherol) and lipid peroxidation increased. Therefore, we hypothesized that prior supplementation with antioxidants (vitamins E and C) would decrease oxidative stress during distance running and, therefore, decrease lipid peroxidation and inflammation, decrease DNA damage, decrease muscle damage and/or improve recovery. To test these hypotheses, we carried out a randomized, double-blind study in runners (n 11 females, 11 males) who were participants in an annual ultramarathon race. We found that supplementation with both vitamins E and C only prevented increases in lipid peroxidation, but had no apparent effect on DNA damage, inflammation or muscle damage. These results suggest that the mechanism of oxidative damage is operating independently of the inflammatory and muscle damage responses.

The alpha-tocopherol transfer protein (TTP) plays an important role in the regulation of plasma alpha-tocopherol concentrations. We hypothesized that hepatic TTP levels would be modulated by dietary vitamin E supplementation and/or by oxidative stress. Mice were fed either a High E (1150 mg RRR-alpha-tocopheryl acetate/kg diet) or a Low E (11.5 mg/kg diet) diet for 2 wk. High E increased plasma and liver alpha-tocopherol concentrations approximately 8- and 40-fold, respectively, compared with Low E-fed mice, whereas hepatic TTP increased approximately 20%. Hepatic TTP concentrations were unaffected by fasting (24 h) in mice fed either diet. To induce oxidative stress, chow-fed mice were exposed for 3 d to environmental tobacco smoke (ETS) for 6 h/d (total suspended particulate, 57.4 +/- 1.8 mg/m3). ETS exposure, while resulting in pulmonary and systemic oxidative stress, had no effect on hepatic alpha-tocopherol concentrations or hepatic TTP. Overall, changes in hepatic TTP concentrations were minimal in response to dietary vitamin E levels or ETS-related oxidative stress. Thus, hepatic TTP concentrations may be at sufficient levels such that they are unaffected by either modulations of dietary vitamin E or by the conditions of environmentally related oxidative stress used in the present studies.

Background: Little is known about factors that modulate dietary alpha-tocopherol bioavailability., Objectives: The study aimed to assess the efficacy of vitamin E-fortified apples as a low-fat vitamin E delivery system, the influence of fat on vitamin E absorption, and human vitamin E requirements by using plasma alpha-tocopherol kinetics at a dosage of alpha-tocopherol found in food., Design: Apples fortified with deuterium-labeled alpha-tocopheryl acetate were consumed by 5 participants at a breakfast containing 0%, 6%, or 21% kcal from fat in 3 sequential trials. The trials were separated by a 2-wk washout period. Blood samples were obtained up to 72 h, and plasma was analyzed for labeled and unlabeled alpha-tocopherol., Results: Compared with observations in the 0% fat trial, the maximum observed plasma d6-alpha-tocopherol concentrations (Cmax) and the areas under the curve increased 2- and 3-fold during the 6% and 21% fat trials, respectively. The mean (+/-SD) estimated percentage d6-alpha-tocopherol absorbed increased from 10 +/- 4% during the 0% fat trial to 20 +/- 3% and 33 +/- 5% during the 6% and 21% fat trials, respectively. The mean time to Cmax (9 +/- 2 h), fractional disappearance rates (0.022 +/- 0.003 pools/d), and half-lives (32 +/- 4 h) did not differ significantly between the trials. With the use of fractional disappearance rates and baseline plasma alpha-tocopherol concentrations, the estimated daily plasma alpha-tocopherol efflux was 13-14 mg. The estimated rate of alpha-tocopherol delivery to tissues was 5 mg/d., Conclusions: Given an estimated 33% absorption, the amount of dietary vitamin E needed daily to replace irreversible losses is

To evaluate vitamin E metabolism, a method was developed to quantitate liver alpha- and gamma-tocopherol metabolites, alpha-carboxyethyl hydroxychroman [alpha-CEHC; 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman] and gamma-CEHC [2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman], respectively. Vitamin E supraenriched livers were obtained from rats that were injected with vitamin E daily for 18 days. Liver samples (approximately 50 mg) were homogenized, homogenate CEHC-conjugates were hydrolyzed, CEHCs were extracted with ethyl ether, and then CEHCs were quantitated using liquid chromatography-mass spectrometry (LC-MS). Precision, based on intersample variability, ranged from 1% to 3%. Recovery of alpha- and gamma-CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of alpha- and gamma-CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver alpha-tocopherol, the MS peak for liver alpha-CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 +/- 0.01 to 15 +/- 2 nmol/g). Liver alpha-CEHC concentrations were correlated with serum alpha-CEHC, liver alpha-tocopherol, and serum alpha-tocopherol (P < 0.001 for each comparison). alpha-CEHC represented 0.5-1% of the liver alpha-tocopherol concentration. Thus, LC-MS can be successfully used to quantitate alpha- and gamma-CEHC in liver samples. These data suggest that in times of excess liver alpha-tocopherol, increased metabolism of alpha-tocopherol to alpha-CEHC occurs.

Vitamin E is a lipophilic chain-breaking antioxidant that prevents lipid peroxidation. Although cigarette smoke is a potent source of oxidative stress that depletes vitamin E in vitro, it is unclear whether it has a similar effect in vivo, particularly in humans. Therefore, this review will discuss the role of cigarette smoke on gamma-tocopherol (gamma-T) nitration, its effect on alpha-tocopherol (alpha-T) biokinetics in smokers, and the changes in the synthesis, plasma concentrations, and urinary excretion of the vitamin E metabolite (CEHC; carboxy-ethyl-hydroxy-chroman). Last, the possibility of CEHC as a biomarker of vitamin E status will be assessed as will the question whether smokers have increased dietary requirements of vitamin E.

A robust database shows that dietary supplements of vitamins E and C are safe for the general population. Because these nutrients supply antioxidant and other functions for homeostasis and protection against free radical damage, supplementation has been intensively studied. Because of perceived benefits, many persons consume quantities of vitamins E and C well above the recommended dietary allowances. As safety guidance, tolerable upper intake levels have been established by the Food and Nutrition Board, Institute of Medicine, at 1000 mg for vitamin E and 2000 mg for vitamin C in adults. Many clinical trials with these vitamins have involved subjects with various diseases, and no consistent pattern of adverse effects has occurred at any intake. Numerous studies of vitamin C supplementation have provided no pattern of evidence to support concerns about safety other than occasional gastrointestinal upset or mild diarrhea resulting from the osmotic effects of unabsorbed quantities of vitamin C. Evidence of bleeding effects and other potential adverse effects of high vitamin E intakes in humans is not convincing. Evidence of adverse effects of vitamin C that result from its effects on iron absorption and metabolism has not been confirmed in clinical trials. Thus, we conclude from clinical trial evidence that vitamin E supplements appear safe for most adults in amounts

Purpose of Review: Vitamin E deficiency in humans has lead to the discovery of regulatory mechanisms that control plasma alpha-tocopherol concentrations and prevent the accumulation of other molecules with vitamin E-antioxidant activity, such as gamma-tocopherol. This review describes these regulatory mechanisms., Recent Findings: alpha-tocopherol regulatory proteins have been cloned and crystallized and their mechanisms of action are under intense scrutiny. Studies of vitamin E metabolism suggest that xenobiotic metabolism may not only regulate vitamin E concentrations, but that vitamin E may regulate xenobiotic clearance pathways., Summary: Advances in our understanding of vitamin E nutrition suggest that vitamin E is a potent molecule that is closely regulated such that alpha-tocopherol is at the appropriate tissue concentrations necessary for some as yet to be described functions.