Growth-inhibitory effects of vitamin E succinate on retrovirus-transformed tumor cells in vitro

Vitamin E succinate inhibited proliferation of C4#1 cells, an established avian retrovirus [reticuloendotheliosis virus (REV)]-transformed immature lymphoid tumor cell line, in a dose-dependent manner. The cytostatic effects of vitamin E succinate were reversible in that treated cells regained their ability to divide after vitamin E succinate removal. Possible mechanism(s) for the antiproliferative actions of vitamin E succinate were investigated. Analyses of C4#1 cell surface membrane antigen profiles and morphology indicated that vitamin E succinate was not inducing differentiation of the tumor cells to a more mature, differentiated, nonproliferative state. Five antioxidants, including a synthetic analogue of vitamin E, Trolox, as well as the active vitamin form, DL-alpha-tocopherol, were incapable of inhibiting C4#1 tumor cell growth, indicating that a mechanism of action other than or in addition to functions as an antioxidant may be operating. Cell cycle analyses suggested that C4#1 tumor cells treated with vitamin E succinate were blocked in the G0G1/early S phases of the cell cycle. Tumor growth arrested by vitamin E succinate did not affect the expression of the REV-encoded oncogene, v-rel, at either the RNA or protein level. These studies demonstrated that vitamin E, in the form of vitamin E succinate, inhibited the growth of retrovirus-transformed tumor cells in vitro and suggested that the antiproliferative effects of vitamin E succinate did not involve antioxidant properties but rather, as yet, unidentified mechanisms leading to cell cycle blockage.     

Vitamin E kinetics and the function of tocopherol regulatory proteins.

Plasma and tissue alpha-tocopherol concentrations are remarkably stable, which suggests that they are regulated. alpha-Tocopherol transfer protein, tocopherol-associated protein, and tocopherol-binding protein bind alpha-tocopherol. These proteins might function as tocopherol regulatory proteins, although only tocopherol transfer protein has been shown to influence plasma and tissue alpha-tocopherol concentrations. Tissue alpha-tocopherol concentrations likely depend on tocopherol regulatory protein function and tissue lipid content, vitamin E uptake and efflux, oxidative stress, and interactions between vitamin E and other antioxidants. Pharmacokinetic models often divide tissues into rapidly perfused, slowly perfused, and very slowly perfused compartments. Tissue vitamin E concentrations might equilibrate more rapidly in tissues with greater perfusion, greater vitamin E uptake, increased amounts or activities of tocopherol regulatory protein, and lower lipid contents. The rate at which tissue concentrations approach equilibrium, however, does not predict the final equilibrium concentrations because of redistribution among tissues. Redistribution of vitamin E to adipose tissue from other tissues may be significant. Intracellular trafficking of vitamin E might occur in conjunction with membrane recycling because membrane constituents rapidly recycle between the plasma membrane and intracellular endocytic compartments. Thus, tocopherol regulatory proteins may modulate rather than directly regulate vitamin E tissue distribution and intracellular trafficking.     

Relationship between red blood cell and liver tocopherol concentration after administration and depletion of vitamin E

As the assessment of the nutritional status of vitamin E, the validity of red blood cell (RBC) tocopherol concentration was examined in relation to tocopherol concentration in the liver and in its subcellular fractions. When 10 mg/kg of dl-alpha-tocopherol was injected intramuscularly to vitamin E deficient rats, plasma tocopherol reached a maximum level earlier than did RBC tocopherol and liver tocopherol. However, as the correlation in tocopherol concentrations between RBC and plasma and between RBC and tissue homogenate or its subcellular fractions was examined with respect to all the values examines during the study courses, a moderately close correlation was observed between RBC and liver tissue and between RBC and the subcellular fractions while a lack of correlation was found between RBC and plasma. When rats sufficient in vitamin E were depleted for 8 weeks by a vitamin E deficient diet, tocopherol concentrations decreased in a similar pattern in the plasma, RBC, and liver and its subcellular fractions. In this case, a very close correlation in the tocopherol concentrations was observed between RBC and other subjects, i.e., the plasma, liver homogenate and its subcellular fractions.     

Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 [PEG 1000]) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. [14C]TPGS (synthesized from [14C]PEG 1000) was taken up by Caco-2 cells but [14C]PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the [14C]TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule.     

Vitamin E status in patients on parenteral nutrition receiving Intralipid

Low vitamin E levels have been found to be a frequent side effect in patients on total parenteral nutrition (TPN). In the present study, the vitamin E content of fat emulsions (Intralipid) was measured and the influence of the intravenously administered lipid emulsion on plasma vitamin E levels was investigated. The majority of vitamin E was provided in the beta + gamma-tocopherol fractions (68.7% of total tocopherol). Vitamin E levels in patients were significantly lower (p less than 0.05) as compared to age- and sex-matched normal controls. Although sufficient amounts of vitamin E (16.9 +/- 0.8 IU daily) were infused, according to RDA requirements, E-plasma levels decreased even further during the course of TPN. It was concluded that high amounts of biologically less active tocopherol isomers are not sufficient to maintain vitamin E plasma levels. Supplemental sources of alpha-tocopherol are needed and more attention should be paid to the different tocopherol isomers in the quantitation of the daily allowances of vitamin E.     

Absorption of water-miscible forms of vitamin E in a patient with cholestasis and in thoracic duct-cannulated rats

Oral administration of vitamin E (100 mg tocopherol X kg-1 X day-1) as tocopheryl polyethylene glycol 1000 succinate (TPGS) to a child with congenital hepatic cholestasis (unresponsive to oral administration of dl-alpha-tocopheryl acetate) promoted an increase of tocopherol in plasma and adipose tissue while tocopheryl acetate emulsified with medium chain triglycerides and polysorbate 80 (MCT-E) did not. alpha-Tocopherol absorption, quantitated in thoracic duct-cannulated rats receiving intraduodenal infusions of soybean oil and saline, was similar for TPGS, MCT-E, and dl-alpha-tocopheryl acetate; gamma-tocopherol absorption from soybean oil was not affected by the presence of the supplemental alpha-tocopherol. Following bile duct ligation in one rat, TPGS promoted the absorption of alpha-tocopherol while absorption of gamma-tocopherol from soybean oil was decreased 30 fold, demonstrating that TPGS, which forms a micellar solution, delivers alpha-tocopherol through the unstirred water layer to enterocytes, while free tocopherol (alpha or gamma) absorption requires the presence of bile salts.     

Altered lipoprotein metabolism in spontaneous vitamin E deficiency of owl monkeys

Certain owl monkeys (AOT) develop spontaneous hemolytic anemia that responds to vitamin E. The anemia is associated with red blood cell lipid peroxidation and altered red blood cell membrane lipid composition. To investigate these changes, plasma lipid and lipoprotein profiles were characterized in anemic, anemia-susceptible, and anemia-resistant AOT. The plasma vitamin E and vitamin A concentrations were assessed as an index of fat absorption and the effect of corn oil supplementation and vitamin E-selenium injection were measured. Anemia-susceptible AOT had depressed plasma levels of vitamin E and A and an altered lipoprotein metabolism characterized by elevated ratios of low/high density lipoprotein cholesterol and free to esterified cholesterol in these lipoproteins. Vitamin E-selenium injection in anemia-susceptible AOT increased the plasma vitamin E, and vitamin E and corn oil supplements reduced the high density lipoprotein free to esterified cholesterol ratio. The data suggest that the AOT suffer from fat malabsorption and that the consequences (including tocopherol deficiency) result in altered cholesterol metabolism.     

Comparison of a vitamin E-rich diet and supplemental vitamin E on measures of vitamin E status and lipoprotein profile

OBJECTIVE: To determine whether dietary modification rather than use of supplements can raise indices of vitamin E status to potentially cardioprotective levels. DESIGN: Eight week randomised controlled trial with parallel treatments to compare increased use of vitamin E-rich foods, supplementation with 200 IU of vitamin E, and a placebo. SETTING: Dunedin, New Zealand. SUBJECTS: Ninety subjects were recruited, of whom 82 non-smoking, free-living individuals aged 22-72 y with plasma cholesterol <7.5 mmol/l completed the trial. MAIN OUTCOME MEASURES: Dietary intakes, plasma alpha tocopherol, plasma alpha tocopherol/cholesterol ratio and lipoprotein cholesterol. RESULTS: Consumption of an additional 12 mg of vitamin E (alpha tocopherol equivalents) from dietary sources was primarily achieved through the replacement of saturated fat-rich foods with unsaturated fats rich in vitamin E, nuts and vegetables. This resulted in a 3.4 micromol/l increase in plasma alpha tocopherol at week 6 (95% CI 1.6-5.3), and 0.9 micromol/mmol in plasma alpha tocopherol/cholesterol at weeks 4 and 6 (95% CI 0.3-1.4 and 0.4-1.4, respectively) when compared with the placebo group. In the supplement group, plasma alpha tocopherol and plasma alpha tocopherol/cholesterol were significantly increased within 2 weeks and remained so throughout the 8 week intervention. CONCLUSION: Increasing dietary vitamin E intake can increase plasma alpha tocopherol levels, although factors other than dietary intake are also important determinants. The extent of dietary modification required to achieve potentially cardioprotective levels of plasma alpha tocopherol is difficult in practice. SPONSORSHIP: The study was supported through the Otago Medical Research Foundation Laurenson Award.     

Tocopherol Succinate Reference Standard (Control 981) of National Institute of Health Sciences

The raw material of tocopherol succinate was tested for preparation of the "Tocopherol Succinate Reference Standard (Control 981)". The analytical data obtained were: infrared spectrum same as that of the Tocopherol Succinate Reference Standard (Control 8510); specific absorbance, E(1%)1 cm (286 nm) = 40.7; thin-layer chromatography, no impurities detected until 50.0 microgram; high-performance liquid chromatography (HPLC),three impurities detected and amount of tocopherol succinate estimated to be 98.2%, loss on drying, 0.19%, assay by HPLC, 101.7%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 981).     

Pharmacokinetic disposition in sheep of various vitamin E preparations given orally or intravenously

1. Increases in plasma tocopherol concentrations were compared in sheep after a single oral administration of (per kg body-weight): 67 mg D- and 91 mg DL-epimers of alpha-tocopherol, and 74 mg D- and 100 mg DL-epimers of alpha-tocopheryl acetate, or intravenous administration of DL-alpha-tocopherol and DL-epimers of DL-alpha-tocopheryl acetate. 2. The results showed that biological availability was higher after D-alpha-tocopherol dosing than after the other forms. Intravenous administration of D-alpha-tocopherol acetate was a more effective way of dosing in sheep than equivalent intravenous amounts of DL-alpha-tocopheryl acetate or DL-alpha-tocopherol.     

Tocopherol Succinate Reference Standard (Control 021) of National Institute of Health Sciences

The raw material of tocopherol succinate was tested for the preparation of the "Tocopherol Succinate Reference Standard (Control 021)". Analytical data obtained were as follows: infrared spectrum, same as that of the Tocopherol Succinate Reference Standard (Control 981); specific absorbance, E1% 1 cm (286 nm) = 40.7; thin-layer chromatography, no impurities were detected until 50.0 micrograms; high-performance liquid chromatography (HPLC), four impurities were detected and the amount of tocopherol succinate was estimated to be 99.0%; loss on drying, 0.11%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 021).     

Changes in vitamin E concentrations in human plasma and platelets with age

It has been postulated that vitamin E compounds (tocopherols) can retard the aging process. Therefore, changes in concentrations of tocopherols in human plasma and platelets with aging were studied. Fasting blood samples were obtained from 48 healthy male volunteers aged 24-91 years. Concentrations of the tocopherols in plasma and platelets were determined by a liquid chromatographic method. Alpha and gamma tocopherols were the major forms of vitamin E found in both plasma and platelets. The concentrations of alpha and total tocopherols in plasma did not change significantly with age. However, the plasma gamma tocopherol and the platelet alpha, gamma, and total tocopherol concentrations decreased significantly with age. The platelet to plasma ratios of tocopherol concentrations also decreased with age.     

Changes in vitamin E concentrations in human plasma and platelets with age.

It has been postulated that vitamin E compounds (tocopherols) can retard the aging process. Therefore, changes in concentrations of tocopherols in human plasma and platelets with aging were studied. Fasting blood samples were obtained from 48 healthy male volunteers aged 24-91 years. Concentrations of the tocopherols in plasma and platelets were determined by a liquid chromatographic method. Alpha and gamma tocopherols were the major forms of vitamin E found in both plasma and platelets. The concentrations of alpha and total tocopherols in plasma did not change significantly with age. However, the plasma gamma tocopherol and the platelet alpha, gamma, and total tocopherol concentrations decreased significantly with age. The platelet to plasma ratios of tocopherol concentrations also decreased with age.     

The cytotoxicity of vitamin E is both vitamer- and cell-specific and involves a selectable trait

During a study of the effect of vitamin E in activated mouse macrophages, we observed a reduction in the viability of cells treated with various forms of vitamin E. We show in this report that some tocopherols (both gamma- and delta-tocopherol) are cytotoxic to some but not all cell types. Mouse macrophages were especially sensitive (40 micromol/L), whereas human hepatocytes and bovine endothelial cells were almost completely refractory (90 micromol/L). The fully methylated tocopherol, alpha-tocopherol (alpha-Toc), was not cytotoxic in any cell type tested. The cytotoxicity observed with delta-tocopherol (delta-Toc) was associated with 2 markers of apoptosis. Vitamer-specific cytotoxicity was not due to differences in cellular uptake/accumulation because both alpha-Toc and delta-Toc accumulated equally in any cell type tested. In contrast, the cell-specific cytotoxicity was related in part to uptake/accumulation of the tocopherols. Macrophages accumulated nearly 5 times more tocopherol compared with hepatocytes cultured under similar conditions. To address the hypothesis that uptake accounted for the cell-specific sensitivity, we developed a macrophage "subtype" that was markedly resistant (>150 micromol/L) to delta-Toc. Under many different cell culture conditions (including human serum) uptake/accumulation of tocopherols was reduced in this subtype by approximately 50%. Further selection and evaluation of this phenotype, however, demonstrated no cytotoxicity even when cellular levels were elevated. Our results show that undermethylated tocopherols are cytotoxic to macrophages and that there are independent and selectable processes that determine cellular tocopherol uptake/accumulation and delta-Toc cytotoxicity.     

Effect of high vitamin A or tocopherol intake on hepatic lipid metabolism and intestinal absorption and secretion of lipids and bile acids in the chick

The effect of high dietary vitamin A or tocopherol, or both, on lipid absorption and secretion in the gastrointestinal tract and on serum and hepatic lipids was determined in the chick. High dietary vitamin A increased secretion of cholesterol, bile acids, phospholipids and fatty acids to the duodenum, and tocopherol enhanced cholesterol and bile acid secretion to the duodenum. No differences in over-all absorption were observed. Serum cholesterol was depressed by vitamin A and hepatic lipids were enhanced, except phosphatidyl choline which was depressed. Tocopherol increased hepatic triglycerides and cholesterol. In vitro fatty acid synthesis from lactate by liver homogenates was enhanced by vitamin E and cholesterogenesis enhanced by both vitamin A and tocopherol. delta 9 Desaturation was also enhanced by vitamin E. It is concluded that both vitamin A and tocopherol affect the hepatic synthesis and catabolism of cholesterol and that vitamin E also influences fatty acid metabolism.     

Vitamin E supplementation decreases basal levels of F(2)-isoprostanes and prostaglandin f(2alpha) in rats

Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of aging. We investigated the effects of supplementation with vitamin E on lipid peroxidation in rats. Both free radical-induced nonenzymatic- and cyclooxygenase-catalyzed enzymatic lipid peroxidation were investigated by measuring the levels of F(2)-isoprostanes (8-iso-PGF(2alpha)) and PGF(2alpha)-metabolite (15-K-DH-PGF(2alpha)), respectively, in blood, urine and liver. Samples were collected from control rats (n = 6) and from rats supplemented with vitamin E in the diet for 3 wk (n = 8, 20 g/kg diet of DL-alpha-tocopherol hydrogen succinate). Plasma alpha-tocopherol concentration and antioxidative capacity were greater in the vitamin E-supplemented rats than in the control rats (17.9 +/- 1.7 vs. 50.4 +/- 10.4 micromol/L, P < 0.001 and 181 +/- 6 vs. 275 +/- 27 micromol/L trolox equivalents, P < 0.001). Urine 8-iso-PGF(2alpha) tended to be lower in the vitamin E-supplemented rats (0.72 +/- 0.40 vs. 0.34 +/- 0.19 nmol/mmol creatinine, P = 0.056). Urine 15-K-DH-PGF(2alpha) was lower due to vitamin E supplementation (0.97 +/- 0.38 vs. 0.56 +/- 0. 21 nmol/mmol creatinine, P < 0.05), as was liver-free 8-iso-PGF(2alpha) concentration (0.47 +/- 0.11 vs. 0.18 +/- 0.04 nmol/g, P < 0.001). Supplementation with vitamin E did not affect plasma 8-iso-PGF(2alpha) or 15-K-DH-PGF(2alpha) concentrations, liver total 8-iso-PGF(2alpha) or plasma malondialdehyde levels. Thus, vitamin E supplementation reduced urine basal levels of biomarkers of both nonenzymatic and enzymatic lipid peroxidation. In liver, vitamin E reduced the basal level of free 8-iso-PGF(2alpha) but not total 8-iso-PGF(2alpha).     

Transitory stimulation of human platelet 12-lipooxygenase by vitamin E supplementation

Human platelet lipooxygenase converts arachidonic acid to 12-hydroperoxy-eicosatetraenoic acid (12-HPETE), which is rapidly reduced by peroxidases to 12-hydroxy-eicosatetraenoic acid (12-HETE). This study was conducted to examine the effect of vitamin E supplementation on platelet 12-lipooxygenase activity. Sixteen healthy subjects were supplemented with 400 IU/day of either D-alpha-tocopherol (268 mg) or DL-alpha-tocopherol (364 mg) for 4 wk. Supplements elicited a transitory increase of lipooxygenase activity but a suppression of peroxidase activity, as indicated by increased 12-HETE production and 12-HPETE accumulation. Plasma-tocopherol concentration was double the presupplement value and remained stable during supplementation. Neither age, sex, nor isomeric form of tocopherol supplement significantly influenced the pattern of response. Results show that vitamin E exerts a differential effect on platelet lipooxygenase and peroxidase activities.     

An evaluation of nutritional status of vitamin E in pregnant women with respect to red blood cell tocopherol level

Changes in RBC tocopherol level as an index for vitamin E status were studied in relation to plasma levels of tocopherol and lipids during pregnancy. Plasma tocopherol levels increased gradually and significantly during gestation. The changes depended on changes in total lipids in plasma, of which an increase in triglycerides was most predominant. A similar increase was shown in beta-lipoprotein levels, while HDL-cholesterol did not show any significant change. The ratio of plasma tocopherol to plasma total lipids, proposed as the best index for vitamin E status by Horwitt, remained unchanged during gestation, while RBC tocopherol levels somewhat decreased during the last trimester. The result indicates that tissue tocopherol available for biological function in the biomembrane may decrease due to hyperlipemia during gestation, as reflected in RBC tocopherol change.     

Serum concentrations of alpha-tocopherol after ingestion of various vitamin E preparations

The rat-fetal-resorption test currently is used to assess the biological activity of vitamin E compounds. Previous studies in humans, however, suggest that rat assays underestimate the potency of free tocopherol relative to the acetate ester form and of RRR-alpha-tocopheryl acetate relative to all-rac-alpha-tocopheryl acetate. Therefore, we studied the serum concentrations of alpha-tocopherol after ingestion of RRR-alpha-tocopheryl acetate, all-rac-alpha-tocopheryl acetate, RRR-alpha-tocopherol, RRR-alpha-tocopheryl succinate, and RRR-alpha-tocopheryl acetate plus apple pectin by each of 20 adult human subjects. Measurements were made at 0, 8, 24, and 48 h after ingestion of 800 IU of the various preparations. The results at 24 h were representative of the differences observed. The mean increase in concentration of alpha-tocopherols (mg/g lipid) in 24 h was 71.2% after RRR-alpha-tocopherol, 63.3% after RRR-alpha-tocopherol acetate, and 41.2% after RRR-alpha-tocopherol succinate. Animal assay data do not correlate with data from studies of absorption and retention in serum of alpha-tocopherols ingested by humans.     

Vitamin E and relationships among tocopherols in human plasma, platelets, lymphocytes, and red blood cells

Plasma, RBC, platelets, and lymphocytes from human subjects on graded intakes of vitamin E were analyzed for tocopherols to determine which humoral compartment most closely followed changes in the dietary intake. Relative merits of the various blood elements to reflect changes in vitamin E intake were calculated by the sensitivity concept of Mandel and Stiehler (ie, rate of change of tocopherol levels with dose divided by the standard deviation). Sensitivities of alpha-tocopherol levels of the various blood components to vitamin E intake decreased in the order platelets greater than RBC greater than plasma lipids greater than plasma greater than lymphocytes. Changes in tocopherol levels in platelets most closely followed changing dietary intakes of vitamin E when compared with data from RBC, lymphocytes, and plasma. Furthermore, for studying the effects of diets on vitamin E status, tocopherol levels of platelets appear to be a better measure than tocopherol levels of either RBC, lymphocytes, or plasma lipids.