To determine the levels of carotenoids, retinoids, and tocopherols in normal and cataractous human lenses.

Alpha-Tocopherol and gamma-tocopherol are the two major forms of vitamin E in human plasma and the primary lipid soluble antioxidants. The dietary intake of gamma-tocopherol is generally higher than that of alpha-tocopherol. However, alpha-tocopherol plasma levels are about four fold higher than those of gamma-tocopherol. Among other factors, a preferential cellular uptake of gamma-tocopherol over alpha-tocopherol could contribute to the observed higher plasma alpha-tocopherol levels. In this investigation, we studied the uptake and depletion of both alpha-tocopherol and gamma-tocopherol (separately and together) in cultured RAW 264.7 macrophages. Similar studies were performed with alpha-tocopheryl quinone and gamma-tocopheryl quinone, which are oxidation products of tocopherols.

Vitamin E includes 8 natural compounds (4 tocopherols, 4 tocotrienols) with potential neuroprotective activity. α-Tocopherol has mainly been investigated in relation to cognitive impairment. We examined the relation of all plasma vitamin E forms and markers of vitamin E damage (α-tocopherylquinone, 5-nitro-γ-tocopherol) to mild cognitive impairment (MCI) and Alzheimer's disease (AD). Within the AddNeuroMed-Project, plasma tocopherols, tocotrienols, α-tocopherylquinone, and 5-nitro-γ-tocopherol were assessed in 168 AD cases, 166 MCI, and 187 cognitively normal (CN) people. Compared with cognitively normal subjects, AD and MCI had lower levels of total tocopherols, total tocotrienols, and total vitamin E. In multivariable-polytomous-logistic regression analysis, both MCI and AD cases had 85% lower odds to be in the highest tertile of total tocopherols and total vitamin E, and they were, respectively, 92% and 94% less likely to be in the highest tertile of total tocotrienols than the lowest tertile. Further, both disorders were associated with increased vitamin E damage. Low plasma tocopherols and tocotrienols levels are associated with increased odds of MCI and AD.

Lipopolysaccharide (LPS) induces host inflammatory responses and tissue injury and has been implicated in the pathogenesis of various age-related diseases such as acute respiratory distress syndrome, vascular diseases, and periodontal disease. Antioxidants, particularly vitamin E, have been shown to suppress oxidative stress induced by LPS, but the previous studies with different vitamin E isoforms gave inconsistent results. In the present study, the protective effects of α- and γ-tocopherols and α- and γ-tocotrienols on the oxidative stress induced by LPS against human lung carcinoma A549 cells were studied. They suppressed intracellular reactive oxygen formation, lipid peroxidation, induction of inflammatory mediator cytokines, and cell death. Tocopherols were incorporated into cultured cells much slower than tocotrienols but could suppress LPS-induced oxidative stress at much lower intracellular concentration than tocotrienols. Considering the bioavailability, it was concluded that α-tocopherol may exhibit the highest protective capacity among the vitamin E isoforms against LPS-induced oxidative stress.

Molecular imprinting is a technology that facilitates the production of artificial receptors toward compounds of interest. The molecularly imprinted polymers act as artificial antibodies, artificial receptors, or artificial enzymes with the added benefit over their biological counterparts of being highly durable. In this study, we prepared molecularly imprinted polymers for the purpose of binding specifically to tocopherol (vitamin E) and its derivative, tocopherol acetate. Binding of the imprinted polymers to the template was found to be two times greater than that of the control, non-imprinted polymers, when using only 10 mg of polymers. Optimization of the rebinding solvent indicated that ethanol-water at a molar ratio of 6:4 (v/v) was the best solvent system as it enhanced the rebinding performance of the imprinted polymers toward both tocopherol and tocopherol acetate with a binding capacity of approximately 2 mg/g of polymer. Furthermore, imprinted nanospheres against tocopherol was successfully prepared by precipitation polymerization with ethanol-water at a molar ratio of 8:2 (v/v) as the optimal rebinding solvent. Computer simulation was also performed to provide mechanistic insights on the binding mode of template-monomer complexes. Such polymers show high potential for industrial and medical applications, particularly for selective separation of tocopherol and derivatives.

No abstract available

Tocopherols and tocotrienols, collectively known as vitamin E, have received a great deal of attention because of their interesting biological activities. In the present study, we reexamined and improved previous methods of sample preparation and the conditions of high-performance liquid chromatography for more accurate quantification of tocopherols, tocotrienols and their major chain-degradation metabolites. For the analysis of serum tocopherols/tocotrienols, we reconfirmed our method of mixing serum with ethanol followed by hexane extraction. For the analysis of tissue samples, we improved our methods by extracting tocopherols/tocotrienols directly from tissue homogenate with hexane. For the analysis of total amounts (conjugated and unconjugated forms) of side-chain degradation metabolites, the samples need to be deconjugated by incubating with β-glucuronidase and sulfatase; serum samples can be directly used for the incubation, whereas for tissue homogenates a pre-deproteination step is needed. The present methods are sensitive, convenient and are suitable for the determination of different forms of vitamin E and their metabolites in animal and human studies. Results from the analysis of serum, liver, kidney, lung and urine samples from mice that had been treated with mixtures of tocotrienols and tocopherols are presented as examples.

Regular consumption of fruits and vegetables, which is related to high plasma levels of lipid-soluble micronutrients such as carotenoids and tocopherols, is linked to lower incidences of various age-related diseases. Differences in lipid-soluble micronutrient blood concentrations seem to be associated with age. Our retrospective analysis included men and women aged 22-37 and 60-85 years from the Berlin Aging Study II. Participants with simultaneously available plasma samples and dietary data were included (n = 1973). Differences between young and old groups were found for plasma lycopene, α-carotene, α-tocopherol, β-cryptoxanthin (only in women), and γ-tocopherol (only in men). β-Carotene, retinol and lutein/zeaxanthin did not differ between young and old participants regardless of the sex. We found significant associations for lycopene, α-carotene (both inverse), α-tocopherol, γ-tocopherol, and β-carotene (all positive) with age. Adjusting for BMI, smoking status, season, cholesterol and dietary intake confirmed these associations, except for β-carotene. These micronutrients are important antioxidants and associated with lower incidence of age-related diseases, therefore it is important to understand the underlying mechanisms in order to implement dietary strategies for the prevention of age-related diseases. To explain the lower lycopene and α-carotene concentration in older subjects, bioavailability studies in older participants are necessary.

Vitamin E is a mixture of eight compounds alpha, beta, gamma, delta- tocopherols and alpha, beta, gamma, delta- tocotrienols. Their individual role in cellular transport as antioxidants and in metabolic pathways has been highlighted in the present work. All the eight compounds have been docked with the respective metabolizing enzymes (alpha-tocopherol transfer protein (ATTP), alpha-tocopherol associated protein (TAP), P-glycoprotein (P-gly) and human serum albumin (HSA)) to understand molecular interactions for pharmacokinetics. These have been structurally aligned against the four human phospholipids in order to reveal their individual role in chylomicron formation and hence the mechanism of cellular transport. The study of their binding with their metabolizing enzymes provides insight to the comparative antioxidant activity of each of these isomers.

Ectotherm animals including insects are known to undergo seasonal restructuring of the cell membranes in order to keep their functionality and/or protect their structural integrity at low body temperatures. Studies on insects so far focused either on fatty acids or on composition of molecular species in major phospholipid classes. Here we extend the scope of analysis and bring results on seasonal changes in minor phospholipid classes, lysophospholipids (LPLs), free fatty acids, phytosterols and tocopherols in heteropteran insect, Pyrrhocoris apterus. We found that muscle tissue contains unusually high amounts of LPLs. Muscle and fat body tissues also contain high amounts of β-sitosterol and campesterol, two phytosterols derived from plant food, while only small amounts of cholesterol are present. In addition, two isomers (γ and δ) of tocopherol (vitamin E) are present in quantities comparable to, or even higher than phytosterols in both tissues. Distinct seasonal patterns of sterol and tocopherol concentrations were observed showing a minimum in reproductively active bugs in summer and a maximum in diapausing, cold-acclimated bugs in winter. Possible adaptive meanings of such changes are discussed including: preventing the unregulated transition of membrane lipids from functional liquid crystalline phase to non-functional gel phase; decreasing the rates of ion/solute leakage; silencing the activities of membrane bound enzymes and receptors; and counteracting the higher risk of oxidative damage to PUFA in winter membranes.

Glioblastoma is associated with poor prognosis with a median survival of one year. High doses of ionizing radiation is the only established exogenous risk factor. To explore new potential biological risk factors for glioblastoma, we investigated alterations in metabolite concentrations in pre-diagnosed serum samples from glioblastoma patients diagnosed up to 22 years after sample collection, and undiseased controls. The study points out a latent biomarker for future glioblastoma consisting of nine metabolites (γ-tocopherol, α-tocopherol, erythritol, erythronic acid, myo-inositol, cystine, 2-keto-L-gluconic acid, hypoxanthine and xanthine) involved in antioxidant metabolism. We detected significantly higher serum concentrations of α-tocopherol (p=0.0018) and γ-tocopherol (p=0.0009) in future glioblastoma cases. Compared to their matched controls, the cases showed a significant average fold increase of α- and γ-tocopherol levels: 1.2 for α-T (p=0.018) and 1.6 for γ-T (p=0.003). These tocopherol levels were associated with a glioblastoma odds ratio of 1.7 (α-T, 95% CI:1.0-3.0) and 2.1 (γ-T, 95% CI:1.2-3.8). Our exploratory metabolomics study detected elevated serum levels of a panel of molecules with antioxidant properties as well as oxidative stress generated compounds. Additional studies are necessary to confirm the association between the observed serum metabolite pattern and future glioblastoma development.

A fast HPLC method with fluorescence detector (FD) was developed for the determination of three tocopherols (TOCs) in milk samples from Modicana cattle breed. The ultrasound-assisted procedure was optimized for the extraction of TOCs prior to HPLC/FD analysis, reducing sample preparation time and allowing a fast quantification of α-tocopherol, δ-tocopherol and γ tocopherol. The optimized ultrasonic extraction combines an efficient and simple saponification at room temperature and a rapid HPLC quantification of TOCs in milk. The precision of the full analytical procedure was satisfactory and the recoveries at three spiked levels were between 95.3% and 87.8%. The linear correlations were evaluated (R2 > 0.99) and the relative standard deviation (RSD) values for intra-day and inter-day tests at three spiked levels were below 1% for the retention time and below 5.20% for the area at low level spiking. The proposed procedure, reducing the experimental complexity, allowed accurate extraction and detection of three TOCs in milk samples from Modicana cattle breed.

Tocopherols (vitamin E) play essential roles in human health because of their antioxidant activity, and plant-derived oils are the richest sources of tocopherols in the human diet. Although soybean (Glycine max) is one of the main sources of plant-derived oil and tocopherol in the world, the relationship between tocopherol and oil in soybean seeds remains unclear. Here, we focus on dissecting tocopherol metabolism with the long-term goal of increasing α-tocopherol content and soybean oil quality. We first collected tocopherol and fatty acid profiles in a soybean population (>800 soybean accessions) and found that tocopherol content increased during soybean domestication. A strong positive correlation between tocopherol and oil content was also detected. Five tocopherol pathway-related loci were identified using a metabolite genome-wide association study strategy. Genetic variations in three tocopherol pathway genes were responsible for total tocopherol content and composition in the soybean population through effects on enzyme activity, mainly caused by non-conserved amino acid substitution or changes in gene transcription level. Moreover, the fatty acid regulatory transcription factor GmZF351 directly activated tocopherol pathway gene expression, increasing both fatty acid and tocopherol contents in soybean seeds. Our study reveals the functional differentiation of tocopherol pathway genes in soybean populations and provides a framework for development of new soybean varieties with high α-tocopherol content and oil quality in seeds.

Lipophilic antioxidants play dual key roles in edible seeds (i) as preservatives of cell integrity and seed viability by preventing the oxidation of fats, and (ii) as essential nutrients for human and animal life stock. It has been well documented that plant domestication and post-domestication evolution frequently resulted in increased seed size and palatability, and reduced seed dormancy. Nevertheless, and surprisingly, it is poorly understood how agricultural selection and cultivation affected the physiological fitness and the nutritional quality of seeds. Fabaceae have the greatest number of crop species of all plant families, and most of them are cultivated for their highly nutritious edible seeds. Here, we evaluate whether evolution of plants under cultivation has altered the integrated system formed by membranes (fatty acids) and lipophilic antioxidants (carotenoids and tocopherols), in the ten most economically important grain legumes and their closest wild relatives, i.e.: Arachis (peanut), Cicer (chickpea), Glycine (soybean), Lathyrus(vetch), Lens (lentil), Lupinus (lupin), Phaseolus (bean), Pisum (pea), Vicia (faba bean) and Vigna (cowpea).

The elevated level of circulating estradiol increases the risk of breast tumor development. To gain further insight into mechanisms involved in their actions, we investigated the molecular mechanisms of 4-hydroxyestradiol (4-OHE(2)) to initiate and/or promote abnormal cell growth, and of alpha- or gamma-tocopherol to inhibit this process. MCF-10A, human breast epithelial cells were incubated with 0.1 microM 4-OHE(2), either with or without 30 microM tocopherols for 96 h. 4-OHE(2) caused the accumulation of intracellular ROS, while cellular GSH/GSSG ratio and MnSOD protein levels were decreased, indicating that there was an oxidative burden. 4-OHE(2) treatment also changed the levels of DNA repair proteins, BRCA1 and PARP-1. gamma-Tocopherol suppressed the 4-OHE(2)-induced increases in ROS, GSH/GSSG ratio, and MnSOD protein expression, while alpha-tocopherol up-regulated BRCA1 and PARP-1 protein expression. In conclusion, 4-OHE(2) increases oxidative stress reducing the level of proteins related to DNA repair. Tocopherols suppressed oxidative stress by scavenging ROS or up-regulating DNA repair elements.

To assess the efficacy of various forms of vitamin E in protection of skin from UV-light-induced oxidative stress, vitamin E (tocotrienol-rich fraction of palm oil, TRF) was applied to mouse skin and the contents of antioxidants before and after exposure to UV-light were measured. Four polypropylene plastic rings (1 cm2) were glued onto the animals' backs, and 20 microliters 5% TRF in polyethylene glycol-400 (PEG) was applied to the skin circumscribed by two rings and 20 microliters PEG to the other two rings. After 2 h, the skin was washed and half of the sites were exposed to UV-irradiation (2.8 mW/cm2 for 29 mi: 3 MED). TRF treatment (n = 19 mice) increased mouse skin alpha-tocopherol 28 +/- 16-fold, alpha-tocotrienol 80 +/- 50-fold, gamma-tocopherol 130 +/- 108-fold, and gamma-tocotrienol 51 +/- 36-fold. A significantly higher percentage of alpha-tocopherol was present in the skin as compared with that in the applied TRF. After UV-irradiation, all vitamin E forms decreased significantly (p < .01), while a larger proportion of the vitamin E remained in PEG-treated (approximately 80%) compared with TRF-treated (approximately 40%) skin. Nonetheless, vitamin E concentrations in irradiated TRF-treated skin were significantly higher than in the nonirradiated PEG-treated (control) skin (p < .01). Thus, UV-irradiation of skin destroys its antioxidants: however, prior application of TRF to mouse skin results in preservation of vitamin E.

In the present study, we determined the effects of a γ-tocopherol-rich mixture of tocopherols (γ-TmT) on the growth and apoptosis of cultured human prostate cancer LNCaP cells. We also determined the effects of dietary γ-TmT on the formation and growth of LNCaP tumors in immunodeficient mice. In the in vitro study, we found that the activity of γ-TmT was stronger than α-tocopherol for inhibiting the growth and stimulating apoptosis in LNCaP cells. In the animal study, treatment of severe combined immunodeficient (SCID) mice with dietary γ-TmT inhibited the formation and growth of LNCaP xenograft tumors in a dose-dependent manner. Mechanistic studies showed that g-TmT administration inhibited proliferation as reflected by decreased mitosis and stimulated apoptosis as reflected by increased caspase-3 (active form) expression in LNCaP tumors. In addition, dietary administration of g-TmT increased the levels of a-, γ- and δ- tocopherol in plasma, and increased levels of γ- and δ- tocopherol were also observed in the prostate and in tumors. The present study demonstrated that g-TmT had strong anticancer activity both in vitro and in vivo. Additional studies are needed to determine the potential preventive effect of g-TmT for prostate cancer in humans.

The combination of doxorubicin and cyclophosphamide commonly used to treat breast cancer can cause premature ovarian failure and infertility. α-Tocopherol is a potent antioxidant whereas γ-tocopherol causes apoptosis in a variety of cancer models in vitro including breast cancer. We hypothesised that the combination of doxorubicin (Dox) and 4-hydroperoxycyclophosphamide (4-Cyc) would be more cytotoxic in vitro than each agent alone, and that α-tocopherol would reduce and γ-tocopherol would augment the cytotoxicity of the combined chemotherapeutics. Human MCF-7 breast cancer and KGN ovarian cells were exposed to Dox, 4-Cyc, combined Dox and 4-Cyc, α-tocopherol, γ-tocopherol, or a combination of Dox and 4-Cyc with α-tocopherol or γ-tocopherol. Cell viability was assessed using a crystal violet assay according to four schedules: 24h exposure, 24h exposure + 24h culture in medium, 24h exposure + 48h culture in medium, or 72h continuous exposure. Supernatants from each separate KGN culture experiment (n=3) were examined using an estradiol ELISA. Dox was cytotoxic to both MCF-7 and KGN cells, but 4-Cyc only killed MCF-7 cells. γ-Tocopherol significantly decreased MCF-7 but not KGN cell viability. The combined chemotherapeutics and γ-tocopherol were more cytotoxic to MCF-7 than KGN cells, and α-tocopherol reduced the cytotoxicity of the combined chemotherapeutics towards KGN ovarian cells, but not MCF-7 cells. The addition of both γ-tocopherol and α-tocopherol to the chemotherapeutic combination of Dox and cyclophosphamide has the potential to increase in vitro chemotherapeutic efficacy against breast cancer cells whilst decreasing cytotoxicity towards ovarian granulosa cells.

The glycosylation of alpha- and delta-tocopherols using Klebsiella pneumoniae and cyclodextrin glucanotransferase (CGTase) was investigated. K. pneumoniae converted alpha- and delta-tocopherols into the corresponding beta-glucosides in 10 and 8% yield, respectively. CGTase glycosylated alpha-tocopheryl beta-glucoside to alpha-tocopheryl beta-maltoside (51%) and alpha-tocopheryl beta-maltotrioside (35%). On the other hand, delta-tocopheryl beta-glucoside was converted into the corresponding beta-maltoside (45%) and beta-maltotrioside (29%) by CGTase. The beta-glucoside of alpha-tocopherol, and beta-glucoside and beta-maltoside of delta-tocopherol showed inhibitory effects on IgE antibody production and on histamine release from rat peritoneal mast cells.

In the present study, modified extraction methods using supercritical CO2 were investigated in order to obtain high-added value compounds from rapeseed oil deodorizer distillate and comparisons were done with modified Soxhlet extraction (solvent extraction + silica). For supercritical fluid extraction (SFE), the optimal extraction parameters were temperature of 40 °C, pressure of 350 bar (for phytosterols), 400 bar (for tocopherol), 5 wt% ethanol as co-solvent, and saponification pretreatment. The optimized SFE procedure led to the recovery of three main phytosterols (50 wt % β-sitosterol, 23.91 wt % Brassicasterol, and 36.25 wt % Campesterol) and only α-tocopherol. Moreover, there was no synergistic effect with saponification pretreatment + co-solvent and the efficiency and concentration of target compounds were less than supercritical CO2 + co-solvent. Also, comparative Data showed that the efficiency of phytosterols and tocopherols was approximately three times higher (p < 0.05) in SFE relative to modified Soxhlet extraction. Furthermore, the use of ethanol (5 wt %) as co-solvent, improved phytosterols and tocopherol efficiency and purity. The SFE technique offers various advantages over the modified Soxhlet extraction technique, including increasing the solubility of tocopherols and sterols by using CO2+ co-solvent, minimized usage of toxic organic solvents and increased purity of extracted products.