The first two decades of vitamin E.

This presentation reviews highlights of the first 20 years (1922-1942) of vitamin E. It begins with background information leading to identification of an antisterility factor for rats of both sexes and its acceptance into the vitamin family as vitamin E (1925). Research of the next 12 years revealed a multiplicity of deficiency manifestations: embryonic mortality, testis degeneration, encephalomalacia and exudative diathesis in the chick, and nutritional muscular dystrophy in avian and mammalian species. Toward the close of this period came the isolation of vitamin E from natural sources, determination of its empirical formula, and introduction of the designation alpha-tocopherol for vitamin E (1936). Within the next two years the structural formula of alpha-tocopherol was elucidated, its chemical synthesis accomplished, and its production from natural plant oils by molecular distillation was well established. The existence of other tocopherols with lesser degrees of biological activity became recognized. Also, the concurrent development of a chemical method for determining the vitamin E content of alpha-tocopherol in foods, body tissues and body fluids, which replaced the very laborious bioassay procedure, greatly facilitated later advances in knowledge of the distribution and nature of vitamin E.     

High dietary intake of alpha-tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of three high levels of dietary intake of alpha-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg alpha-tocopherol kg-1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg alpha-tocopherol acetate kg-1) for 15, 30 or 45 days. Growth, serum alpha-tocopherol levels, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between alpha-tocopherol acetate intake and serum alpha-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg-1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg-1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head-kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg-1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800 mg kg-1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg-1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.     

Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

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.     

Effect of High Doses of Dietary Vitamin E on the Concentrations of Vitamin E in Several Brain Regions, Plasma, Liver, and Adipose Tissue of Rats

The object of this study was to assess the influence of high levels of dietary vitamin E on vitamin E concentrations in specific areas of the brain. Four-week-old male rats were fed vitamin E-deficient, control, and high-vitamin E (1,000 IU/kg) diets for 4 months. Concentrations of alpha-tocopherol in serum, adipose tissue, liver, cerebrum, cerebellum, and striatum were determined by liquid chromatography with fluorescence detection. In the high-vitamin E group, alpha-tocopherol concentrations in cerebrum, cerebellum, and striatum increased uniformly to 1.4-fold of values in controls; serum, adipose tissue, and liver attained even higher concentrations: 2.2-, 2.2-, and 4.6-fold, respectively, of control values. As observed before, brain levels of alpha-tocopherol were somewhat resistant to vitamin E deficiency, in contrast to the peripheral tissues.     

Plasma concentrations of vitamin E in six species of bustard (Gruiformes: Otididae)

Vitamin E (measured as alpha-tocopherol) and cholesterol concentrations were determined in plasma samples collected from 86 clinically healthy captive adult bustards of six species and 23 captive juveniles (6-12 mo old) of two of these species. Adult houbara bustards (Chlamydotis undulata macqueenii) had higher plasma alpha-tocopherol concentrations than juveniles (adult: mean +/- SE, 11.07 +/- 0.41 micrograms/ml, n = 32; juvenile: 6.33 +/- 0.48, n = 12) and higher alpha-tocopherol: cholesterol ratios (adult: 6.09 +/- 0.44, n = 12; juvenile: 2.94 +/- 0.22, n = 11). No age difference was evident for kori bustard (Ardeotis kori) plasma alpha-tocopherol concentrations (adult: 4.43 +/- 0.42, n = 21; juvenile: 4.46 +/- 0.26, n = 11) or alpha-tocopherol: cholesterol ratios (adult: 3.67 +/- 0.44, n = 20; juvenile: 3.71 +/- 0.36, n = 11). Adult houbara bustards had significantly higher (P < 0.01) alpha-tocopherol concentrations compared with adult rufous-crested (Eupodotis ruficrista; 6.64 +/- 0.33, n = 19) and white-bellied (Eupodotis senegalensis; 7.75 +/- 0.81, n = 8) bustards, but similar alpha-tocopherol: cholesterol ratios (rufous-crested: 5.56 +/- 0.32, n = 18; white-bellied: 5.83 +/- 0.43, n = 8). Juvenile houbara bustards had higher plasma alpha-tocopherol concentrations than juvenile kori bustards but similar alpha-tocopherol:cholesterol ratios. Adult houbara bustard plasma alpha-tocopherol levels and alpha-tocopherol:cholesterol ratios did not differ significantly between sexes. The vitamin E status of adult bustards appeared to be influenced by environmental conditions that varied due to species-specific husbandry regimens, but no clear relationship was seen with dietary vitamin E levels. Juvenile bustards did not have higher vitamin E levels than adults, despite being maintained on four-fold dietary vitamin E concentrations and in similar environmental conditions. This paper presents the first published data for plasma vitamin E concentrations in bustards. The plasma alpha-tocopherol and cholesterol concentrations and alpha-tocopherol:cholesterol ratios of captive bustards were similar to those previously reported for omnivorous avian species. Further research is required to determine which components of the identified environmental conditions affect bustard vitamin E status and to confirm whether differences exist between species independent of the variation in their management regimes.     

Alpha-tocopheryl phosphate: a novel, natural form of vitamin E

We have detected alpha-tocopheryl phosphate in biological tissues including liver and adipose tissue, as well as in a variety of foods, suggesting a ubiquitous presence in animal and plant tissue. Alpha-tocopheryl phosphate is a water-soluble molecule that is resistant to both acid and alkaline hydrolysis, making it undetectable using standard assays for vitamin E. A new method was therefore developed to allow the extraction of both alpha-tocopheryl phosphate and alpha-tocopherol from a single specimen. We used ESMS to detect endogenous alpha-tocopheryl phosphate in biological samples that also contained alpha-tocopherol. Due to the significance of these findings, further proof was required to unequivocally demonstrate the presence of endogenous alpha-tocopheryl phosphate in biological samples. Four independent methods of analysis were examined: HPLC, LCMS, LCMS/MS, and GCMS. Alpha-tocopherol phosphate was identified in all instances by comparison between standard alpha-tocopheryl phosphate and extracts of biological tissues. The results show that alpha-tocopheryl phosphate is a natural form of vitamin E. The discovery of endogenous alpha-tocopheryl phosphate has implications for the expanding knowledge of the roles of alpha-tocopherol in biological systems.     

Alpha-tocopherol is secreted from rat liver in very low density lipoproteins

Three separate studies were carried out to test the hypothesis that rat liver secretes vitamin E (alpha-tocopherol) within very low density lipoproteins (VLDL). i) When the clearance of plasma chylomicrons (CM) and VLDL was blocked by the administration of Triton WR-1339, alpha-tocopherol concentrations increased linearly with time in both classes of triacylglycerol-rich lipoproteins, although accumulation rates within VLDL exceeded those within CM. For fasted rats, appearance of alpha-tocopherol in VLDL persisted at slightly reduced rates. alpha-Tocopherol and triglycerides in the VLDL fraction responded to Triton WR-1339 administration by coordinate increases. In contrast to the situation in serum, alpha-tocopherol concentrations decreased in the liver following injection of Triton. ii) In order to inhibit the secretion of hepatic lipoproteins containing apolipoprotein B (apoB), rats were fed a diet containing orotic acid. This resulted in a reduction of apoB and alpha-tocopherol concentrations in serum and VLDL, whereas the vitamin E content of liver was increased. iii) In primary cultures of hepatocytes, alpha-tocopherol was secreted into the culture media predominantly within VLDL. We, therefore, conclude that the liver secretes alpha-tocopherol within VLDL and in this way contributes to the maintenance of serum vitamin E concentrations.     

Three series of high molecular weight alkanoates found in Amazonian plants

Electron impact mass spectra were measured by high temperature high resolution gas chromatography-mass spectrometry (HT-HRGC-MS) for three homologous series of high molecular weight compounds present in the Amazonian plants Marupá (Simaruba amara) and Brazil nut (Bertholettia excelsa). Based on their mass spectra, the compounds were identified as three wax ester series of alpha-tocopherol (vitamin E), beta-tocopherol and phytol (2,6,10,14-tetramethylhexadec-14-en-16-ol). The interpretations are supported by high resolution mass spectrometry and GC retention indices of authentic standards.     

Influence of vitamin E on gastric mucosal injury induced by Helicobacter pylori infection

We investigated the effect of vitamin E on gastric mucosal injury induced by Helicobacter pylori (H. pylori) infection. Male Mongolian gerbils were divided into 4 groups (normal group without H. pylori infection, vitamin E-deficient, -sufficient and -supplemented groups with H. pylori infection). Following oral inoculation with H. pylori (ATCC43504 2 x 10(8) CFU), animals were fed diets alpha-tocopherol 2 mg/100 g diet in the normal and vitamin E-sufficient groups and alpha-tocopherol 0.1 mg/100 g and 50 mg/100 g in the vitamin E-deficient and -supplemented groups, respectively, for 24 weeks. Chronic gastritis was detected in all gerbils inoculated H. pylori. Gastric ulcer was detected in 2 of 7 gerbils only in the vitamin E-deficient group. In the vitamin E-deficient group, myeloperoxidase activity and mouse keratinocyte derived chemokine (KC) in gastric mucosa was significantly higher than in the vitamin E supplemented group. Subsequently, in an in vitro study expression of CD11b/CD18 on neutrophils was enhanced by H. pylori water extract. This effect was suppressed in a dose dependent manner by the addition of alpha-tocopherol. These results suggest that vitamin E has a protective effect on gastric mucosal injury induced by H. pylori infection in gerbils, through the inhibition of accumulation of activated neutrophils.     

Burn and smoke inhalation injury in sheep depletes vitamin E: kinetic studies using deuterated tocopherols

To test the hypothesis that burn and smoke injury will deplete tissue alpha-tocopherol and cause its faster plasma disappearance, deuterium-labeled vitamin E was administered to sheep exposed to both surface skin burn and smoke insufflation, which cause injuries similar to those of human victims of fire accidents. Two different protocols were used: (1) deuterated vitamin E was administered orally with food at time 0 (just before injury) or (2) the labeled vitamin E was administered orally with food the day before injury. The animals, which had been operatively prepared seven days before, were anesthetized and then received both 40% body surface area third-degree burn and 48 breaths of cotton smoke or sham injuries. All were resuscitated with Ringer's lactate solution (4 ml/kg/% BSA burn/24 h) and mechanically ventilated. Blood samples were collected at various times after vitamin E dosing. In both studies the depletion of plasma alpha-tocopherol was faster in the injured sheep. The sheep given deuterated vitamin E 24 h before injury had similar maximum alpha-tocopherol concentrations at similar times. The exponential rates of alpha-tocopherol disappearance were 1.5 times greater and half-lives were 12 h shorter (p < 0.05) in the injured sheep. In separate studies, various tissues were obtained from sheep that were sacrificed from 4 to 48 h after injury. The liver alpha-tocopherol concentrations in sheep killed at various times after injury seem to show a linear decrease at a rate of 0.1 nmol alpha-tocopherol/g liver per hour, suggesting that the liver is supplying alpha-tocopherol to maintain the plasma and lung alpha-tocopherol concentrations, but that this injury is so severe the liver is unable to maintain lung alpha-tocopherol concentrations. These findings suggest that alpha-tocopherol should be administered to burn patients to prevent vitamin E depletion and to protect against oxidative stress from burn injury.     

Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants

Vitamin E analogs such as alpha-tocopherol and alpha-tocotrienol have been shown to reduce endothelial expression of adhesion molecules. The reactivity of alpha-tocopherol and alpha-tocotrienol in inhibiting lipid peroxidation in vitro was essentially identical but the inhibition of adhesion of THP-1 cells, a monocytic-"like" cell line, to endothelial cells differs substantially. To determine the mechanism underlying this response, human umbilical vein endothelial cells (HUVECs) were assessed for their ability to accumulate vitamin E analogs. alpha-Tocotrienol accumulated in HUVECs to levels approximately 10-fold greater than that of alpha-tocopherol. The decrease in expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of THP-1 cells to HUVECs by alpha-tocopherol and alpha-tocotrienol was also determined. Both alpha-tocopherol and alpha-tocotrienol suppressed VCAM-1 expression and adhesion of THP-1 cells to HUVECs in a concentration-dependent manner. The efficacy of tocotrienol for reduction of VCAM-1 expression and adhesion of THP-1 cells to HUVECs was also 10-fold higher than that of tocopherol. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells clearly correlated with their intracellular concentrations. The data demonstrated that, in assessing the biological responses of antioxidants, intracellular accumulation and metabolism were additional important factors that must be considered.     

The effect of long-term selenium and vitamin E-enriched diet on the content of lipid peroxides and cholesterol in rats

The effect of long-term diets enriched with natural antioxidants was studied on Wistar rats with average initial body weight 150 g. After enrichment of the diet with selenium (0.1 ppm of sodium selenite per 100 g of diet), with vitamin E (6 mg of alpha-tocopherol per 100 g of diet) and selenium and vitamin E together the following results were obtained: diets enriched with selenium or vitamin E given for 12 months reduced the production of lipid peroxides in the liver and serum of the rats. On the other hand, addition of both antioxidants to the diet had no effect on lipid peroxide levels in the animals. Diet enrichment for 12 and 18 months with selenium or vitamin E had no effect on the levels of total cholesterol and HDL cholesterol. The obtained results suggest that selenium and alpha-tocopherol exert an inhibitory action on the processes of ageing in the experimental animal model.     

Synthesis and analysis of conjugates of the major vitamin E metabolite,alpha-CEHC

Glucuronide and sulphate conjugates of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the major metabolite of alpha-tocopherol (vitamin E), have been synthesized and used for the first direct analysis of conjugated urinary vitamin E metabolites. The metabolites of vitamin E (alpha-tocopherol) could be useful as markers of the function(s) of vitamin E in vivo. A number of methods have been described for the analysis of urinary vitamin E metabolites but these have relied on either acid or enzymatic deconjugation of the metabolites prior to analysis by high performance liquid chromatography or gas chromatography/mass spectrometry. These methods have provided useful information about the amount and types of metabolites excreted in the urine but suffer from a number of disadvantages. Deconjugation has been shown to produce artifacts as a result of the conversion of alpha-CEHC to alpha-tocopheronolactone and the efficiency of deconjugation is also difficult to assess. Methods that allow the direct measurement of the conjugated metabolites would overcome these problems and would also substantially reduce the preparation and analysis time. Here we describe the use of conjugated standards to characterize alpha-CEHC conjugates in human urine by tandem mass spectrometry (MS-MS). The future use of MS-MS to measure urinary vitamin E metabolites is also discussed.     

Rapid determination of alpha-tocopherol in sarcoplasmic reticulum membranes by reverse phase HPLC.

A simple isocratic high performance liquid chromatograph (HPLC) system is described to perform a rapid separation, identification and quantitative determination of vitamin E (alpha-tocopherol) in biological membranes. It makes use of a reverse phase C18 column with pure methanol as the mobile phase, and an ultraviolet detector which enables its quantification in the nanogram scale. This procedure was applied to lipid extracts from whole muscle homogenate and from a preparation of sarcoplasmic reticulum vesicles from skeletal muscle, where the vitamin E contents was determined.     

Influence of dietary vitamins A and E on serum alpha- and gamma-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti)

Serum retinol, retinyl palmitate, beta-carotene, cryptoxanthin, lutein, alpha-tocopherol and gamma-tocopherol were measured in 18 captive Humboldt penguins (Spheniscus humboldti) prior to and following the removal of Columbia River (CR) smelt (Thaleichthys pacificus) from the diet. Dietary vitamin A was reduced from 59.8 to 13.5 IU g-1 (dry matter basis) when CR smelt was removed from the diet. Minimal changes were noted in dietary vitamin E. Serum samples Without-CR smelt had significantly lower circulating retinol (1.19 +/- 0.09 vs. 1.94 +/- 0.08 micrograms ml-1) and retinyl palmitate (0.033 +/- 0.012 vs. 0.105 +/- 0.004 microgram ml-1) compared to samples With-CR. The Without-CR smelt diet resulted in increased serum alpha-tocopherol from 26.4 +/- 0.94 to 39.1 +/- 3.72 micrograms ml-1. More serum samples taken Without-CR smelt had detectable levels of gamma-tocopherol than those With-CR smelt. Serum lutein was higher for the samples taken Without versus With-CR smelt. Serum cryptoxanthin did not differ. beta-Carotene was not detected. Data indicate that high levels of dietary vitamin A can affect circulating levels of retinol, retinyl palmitate and vitamin E. Thus, dietary vitamin A and the interrelationship between vitamins A and E should be considered when assessing captive penguins.     

New synthesis of (+/-)-alpha-CMBHC and its confirmation as a metabolite of alpha-tocopherol (vitamin E)

There is currently interest in the metabolism of the various compounds which make up the vitamin E family, especially with regards to the possible use of vitamin E metabolites as markers of oxidative stress and adequate vitamin E supply. A number of vitamin E metabolites have been described to date and we have recently developed a method to extract and quantitate a range of vitamin E metabolites in human urine. During the development of this method a new metabolite of alpha-tocopherol was identified, which we tentatively characterised as 5-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-2-methyl-pentanoic acid (alpha-CMBHC).(1) Here we describe the synthesis of alpha-CMBHC as a standard and confirm that it is a metabolite of alpha-tocopherol.     

Utilization of vitamin E

Natural (RRR) or synthetic (all-rac) forms of alpha-tocopherol are available (usually as acetate esters) for use as vitamin E supplements. In animal tests, the natural stereoisomer, RRR-alpha-tocopheryl acetate, is 1.36 times more biologically potent than all-rac-alpha-tocopheryl acetate, an equimolar mixture of eight stereoisomers [8,15,40-43]. The higher biologic activity of natural compared with synthetic vitamin E does not result from differences in antioxidant activity [2,3], but could hypothetically be explained by differences in (1) absorption, (2) plasma transport, (3) delivery to tissues, or (4) metabolism. These possibilities will be considered in this review.     

Less than adequate vitamin E status observed in a group of preschool boys and girls living in the United States

In that data were not available on the vitamin E status of young children, the aim of the study was to evaluate the vitamin E status of preschool children by three commonly used criteria: vitamin E intakes, plasma alpha-tocopherol concentrations and plasma alpha-tocopherol/total lipid ratios. Twenty-two ethnically diverse preschool children (13 males and 9 females), aged 2 to 5 years, living in Lincoln, NE, served as subjects. The subjects were in two groups: 2-3 and 4-5 years old. Energy, fat, and alpha- and gamma-tocopherol intakes of the subjects were estimated utilizing two 24-h food recalls. Plasma alpha- and gamma-tocopherol and total lipid concentrations were ascertained. No significant differences by age grouping or gender were observed for vitamin E intakes, plasma alpha-tocopherol concentrations, plasma gamma-tocopherol concentrations and plasma alpha-tocopherol/total lipid ratios of subjects. Plasma alpha-tocopherol concentrations indicative of less than adequate status (<12 micromol/L) were observed in 91% of the children, and values <7 micromol/L (proposed cutoff for pediatric populations) in 68%. Sixty-eight percent of the subjects had plasma alpha-tocopherol/total lipid values <0.8 mg/g. The majority of the 2- to 5-year-old children included in the study had less than adequate vitamin E status.     

Influence of dietary vitamin E on the intermembrane transfer of alpha-tocopherol as mediated by an alpha-tocopherol binding protein

The effect of dietary vitamin E on the intermembrane transfer of (3R)-alpha-tocopherol, a spontaneous process accelerated in the presence of an alpha-tocopherol binding protein (alpha TBP), was examined. The transfer activity of this cytosolic liver protein was assayed via in vitro transfer of (3R)-alpha-[3H]tocopherol (alpha[3H]T) from egg lecithin liposomes to human erythrocyte ghosts (EG). Male Fisher 344 rats (1 and 20 months old) were fed diets containing 0, 30, and 500 mg/kg vitamin E (dl-alpha-tocopheryl acetate) for 15 weeks. Liver cytosol fractions were assayed for alpha[3H]T transfer activity (alpha TTA). Among young rats, those fed vitamin E-deficient diets had the highest alpha TTA, 5.02 +/- 3.10 pmole alpha[3H]T/min (mean +/- SD), which was different (P less than 0.05) from the spontaneous transfer rate of 2.10 pmole/min. Neither young rats fed 30 and 500 mg/kg vitamin E diets nor any of the aged rats showed alpha TTA which differed significantly from the spontaneous transfer rate. To examine the relationship between hepatic alpha-tocopherol levels and alpha TTA, alpha-tocopherol concentration per gram of wet liver was assayed by HPLC. A steep positive slope (6.39 +/- 1.46 pmole min-1 nmole g-1) and strong correlation (r = 0.873) between hepatic alpha-tocopherol and alpha TTA were observed (P less than 0.005) among young vitamin E-deficient rats. The data indicates that alpha TTA varies directly with hepatic alpha-tocopherol concentration when total liver vitamin E stores are very low. Thus, alpha TBP-mediated transfer of alpha-tocopherol may be manifest only when vitamin E status is compromised.