Application of supercritical fluid chromatography in the quantitative analysis of minor components (carotenes,vitamin E,sterols, and squalene) from palm oil

The application of supercritical fluid chromatography (SFC) coupled with a UV variable-wavelength detector to isolate the minor components (carotenes, vitamin E, sterols, and squalene) in crude palm oil (CPO) and the residual oil from palm-pressed fiber is reported. SFC is a good technique for the isolation and analysis of these compounds from the sources mentioned. The carotenes, vitamin E, sterols, and squalene were isolated in less than 20 min. The individual vitamin E isomers present in palm oil were also isolated into their respective components, α-tocopherol, α-tocotrienol, γ-tocopherol, γ-tocotrienol, and δ-tocotrienol. Calibration of all the minor components of palm as well as the individual components of palm vitamin E was carried out and was found to be comparable to those analyzed by other established analytical methods.     

Effects of steeping conditions during wet-milling on the retentions of tocopherols and tocotrienols in corn

Vitamin E is a natural antioxidant that plays significant roles in food preservation and disease prevention. There are eight naturally occurring vitamin E isomers (tocols): α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols. Corn oil is a major source of vitamin E. Most of the corn oil produced in the United States is a co-product of corn wet-milling. There is limited knowledge about the effects of corn wet-milling on the retention of these vitamin E isomers. A high-performance liquid chromatography method was developed for simultaneous determinations of tocols in steeped corn samples. Effects of steeping conditions (steeping time and SO₂ concentration) on retention of tocols in corn were investigated. α-Tocopherol, γ-tocopherol, α-tocotrienol, and γ-tocotrienol are the predominant vitamin E isomers in the corn variety used in the study. Steeping conditions had little effect on the concentration of α-tocopherol and α-tocotrienol. However, a higher concentration of SO₂ and a shorter steeping time gave a slightly higher γ-tocotrienol content and lower γ-tocopherol content. Corn kernels steeped in a vitamin C solution had a much higher concentration of the tocols than those steeped in SO₂ solution.     

Recovered oil from palm-pressed fiber: A good source of natural carotenoids,vitamin E,and sterols

Recovered fiber from pressed palm fruits, which is normally burned as fuel to provide energy for the palm oil mills, has now been found to be a rich source of carotenoids, vitamin E (tocopherol and tocotrienols), and sterols. Residual oil (5–6% on dry basis) extracted from palm press fibers contains a significant quantity of carotenoids (4000–6000 ppm), vitamin E (2400–3500 ppm), and sterols (4500–8500 ppm). The major identified carotenoids are α-carotene (19.5%), β-carotene (31.0%), lycopene (14.1%), and phytoene (11.9%). In terms of vitamin E, α-tocopherol constitutes about 61% of the total vitamin E present, the rest being tocotrienols (α-, γ-, and δ-). The major sterols present are β-sitosterol (47%), campesterol (24%), and stigmasterol (15%). The oil extracted from palm-pressed fiber is contaminated with about 30% of palm kernel oil. The quality of this fiber oil is slightly lower than that of crude palm oil in terms of the content of free fatty acids, peroxide value, and anisidine value.     

Composition and thermal profile of crude palm oil and its products

Gas-liquid chromatography and high-performance liquid chromatography (HPLC) were used to determine fatty acids and triglyceride (TG) compositions of crude palm oil (CPO), refined, bleached, and deodorized (RBD) palm oil, RBD palm olein, and RBD palm stearin, while their thermal profiles were analyzed by differential scanning calorimeter (DSC). The HPLC chromatograms showed that the TG composition of CPO and RBD palm oil were quite similar. The results showed that CPO, RBD palm oil, RBD olein, and superolein consist mainly of monosaturated and disaturated TG while RBD palm stearin consists mainly of disaturated and trisaturated TG. In DSC cooling thermograms the peaks of triunsaturated, monosaturated and disaturated TG were found at the range of −48.62 to −60.36, −25.89 to −29.19, and −11.22 to −1.69°C, respectively, while trisaturated TG were found between 13.72 and 27.64°C. The heating thermograms of CPO indicated the presence of polymorphs β₂′, α, β₂′, and β₁. The peak of CPO was found at 4.78°C. However, after refining, the peak shifted to 6.25°C and became smaller but more apparent as indicated by RBD palm oil thermograms. The heating and cooling thermograms of the RBD palm stearin were characterized by a sharp, high-melting point (high-T) peak temperature and a short and wide low-melting point (low-T) peak temperature, indicating the presence of occluded olein. However, for RBD palm olein, there was only an exothermic low-T peak temperature. The DSC thermograms expressed the thermal behavior of various palm oil and its products quite well, and the profiles can be used as guidelines for fractionation of CPO or RBD palm oil.     

γ-tocotrienol as a hypocholesterolemic and antioxidant agent in rats fed atherogenic diets

This study was designed to determine whether incorporation of γ-tocotrienol or α-tocopherol in an atherogenic diet would reduce the concentration of plasma cholesterol, triglycerides and fatty acid peroxides, and attenuate platelet aggregability in rats. For six weeks, male Wistar rats (n=90) were fed AIN76A semisynthetic test diets containing cholesterol (2% by weight), providing fat as partially hydrogenated soybean oil (20% by weight), menhaden oil (20%) or corn oil (2%). Feeding the ration with menhaden oil resulted in the highest concentrations of plasma cholesterol, low and very low density lipoprotein cholesterol, triglycerides, thiobarbituric acid reactive substances and fatty acid hydroperoxides. Consumption of the ration containing γ-tocotrienol (50 μ/kg) and α-tocopherol (500 mg/kg) for six weeks led to decreased plasma lipid concentrations. Plasma cholesterol, low and very low density lipoprotein cholesterol, and triglycerides each decreased significantly (P<0.001). Plasma thiobarbituric acid reactive substances decreased significantly (P<0.01), as did the fatty acid hydroperoxides (P<0.05), when the diet contained both chromanols. Supplementation with γ-tocotrienol resulted in similar, though quantitatively smaller, decrements in these plasma values. Plasma α-tocopherol concentrations were lowest in rats fed menhaden oil without either chromanol. Though plasma α-tocopherol did not rise with γ-tocotrienol supplementation at 50 mg/kg, γ-tocotrienol at 100 mg/kg of ration spared plasma α-tocopherol, which rose from 0.60±0.2 to 1.34±0.4 mg/dL (P<0.05). The highest concentration of α-tocopherol was measured in plasma of animals fed a ration supplemented with α-tocopherol at 500 mg/kg. In response to added collagen, the partially hydrogenated soybean oil diet without supplementary cholesterol led to reduced platelet aggregation as compared with the cholesterol-supplemented diet. However, γ-tocotrienol at a level of 50 mg/kg in the cholesterol-supplemented diet did not significantly reduce platelet aggregation. Platelets from animals fed the menhaden oil diet released less adenosine triphosphate than the ones from any other diet group. The data suggest that the combination of γ-tocotrienol and α-tocopherol, as present in palm oil distillates, deserves further evaluation as a potential hypolipemic agent in hyperlipemic humans at atherogenic risk.     

Tissue Distribution of α- and γ-Tocotrienol and γ-Tocopherol in Rats and Interference with Their Accumulation by α-Tocopherol

The aim of this study was to evaluate tissue distribution of vitamin E isoforms such as α- and γ-tocotrienol and γ-tocopherol and interference with their tissue accumulation by α-tocopherol. Rats were fed a diet containing a tocotrienol mixture or γ-tocopherol with or without α-tocopherol, or were administered by gavage an emulsion containing tocotrienol mixture or γ-tocopherol with or without α-tocopherol. There were high levels of α-tocotrienol in the adipose tissue and adrenal gland, γ-tocotrienol in the adipose tissue, and γ-tocopherol in the adrenal gland of rats fed tocotrienol mixture or γ-tocopherol for 7 weeks. Dietary α-tocopherol decreased the α-tocotrienol and γ-tocopherol but not γ-tocotrienol concentrations in tissues. In the oral administration study, both tocopherol and tocotrienol quickly accumulated in the adrenal gland; however, their accumulation in adipose tissue was slow. In contrast to the dietary intake, α-tocopherol, which has the highest affinity for α-tocopherol transfer protein (αTTP), inhibited uptake of γ-tocotrienol to tissues including adipose tissue after oral administration, suggesting that the affinities of tocopherol and tocotrienol for αTTP in the liver were the critical determinants of their uptake to peripheral tissues. Vitamin E deficiency for 4 weeks depleted tocopherol and tocotrienol stores in the liver but not in adipose tissue. These results indicate that dietary vitamin E slowly accumulates in adipose tissue but the levels are kept without degradation. The property of adipose tissue as vitamin E store causes adipose tissue-specific accumulation of dietary tocotrienol.     

Development of dual stage solvent fractionation as an approach to concentrate carotenoids from crude palm oil with high recovery: The potential use of molecular simulation as a pre-screening tool

Crude palm oil (CPO) is highly abundant in carotenoids. Previous findings found that dry fractionation can concentrate carotenoids from CPO but resulted in a significant loss of carotenoids. Therefore, the present study aimed to utilize solvent fractionation, which offers a better separation efficiency, to concentrate carotenoids from CPO with improved recovery. Computational study revealed a high binding affinity of phytonutrient towards unsaturated triacylglycerols (TAGs) species in olein fraction due to similar polarity. This prediction was further verified with evidence showing strong, positive correlation between the iodine value and carotenoids concentrations of fractionated oil. The difference in binding affinity of saturated and unsaturated TAG towards different solvents can be used as a guide for screening and selection of solvent suitable for recovery of phytonutrient during solvent fractionation. Subsequently, a lab-scale single- stage fractionation study disclosed that crystallization temperature of 15°C, oil to acetone ratio of 1:5 (w/v) for 4 h under agitation at 100 rpm produced olein with the highest carotenoid concentration (637 ppm) and recovery (94%). Subsequent double-stage fractionation successfully concentrated the carotenoids up to 125% with a recovery of >93%. Conclusively, solvent fractionation provides an effective way to concentrate valuable carotenoids from CPO while minimizing the lost.     

Cytochrome P450-Dependent Metabolism of Vitamin E Isoforms is a Critical Determinant of Their Tissue Concentrations in Rats

The aim of this study was to clarify the contribution of cytochrome P450 (CYP)-dependent metabolism of vitamin E isoforms to their tissue concentrations. We studied the effect of ketoconazole, a potent inhibitor of CYP-dependent vitamin E metabolism in cultured cells, on vitamin E concentration in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 weeks were administered by oral gavage a vitamin E-free emulsion, an emulsion containing α-tocopherol, γ-tocopherol or a tocotrienol mixture with or without ketoconazole. α-Tocopherol was detected in the serum and various tissues of the vitamin E-deficient rats, but γ-tocopherol, α- and γ-tocotrienol were not detected. Ketoconazole decreased urinary excretion of 2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychroman after α-tocopherol or a tocotrienol mixture administration, and that of 2,7,8-trimethyl-2(2′-carboxyethyl)-6-hydroxychroman (γ-CEHC) after γ-tocopherol or a tocotrienol mixture administration. The γ-tocopherol, α- and γ-tocotrienol concentrations in the serum and various tissues at 24 h after their administration were elevated by ketoconazole, while the α-tocopherol concentration was not affected. The γ-tocopherol or γ-tocotrienol concentration in the jejunum at 3 h after each administration was also elevated by ketoconazole. In addition, significant amount of γ-CEHC was in the jejunum at 3 h after γ-tocopherol or γ-tocotrienol administration, and ketoconazole inhibited γ-tocopherol metabolism to γ-CEHC in the jejunum. These results showed that CYP-dependent metabolism of γ-tocopherol and tocotrienol is a critical determinant of their concentrations in the serum and tissues. The data also suggest that some amount of dietary vitamin E isoform is metabolized by a CYP-mediated pathway in the intestine during absorption.     

Tocotrienols from palm oil: Electron spin resonance spectra of tocotrienoxyl radicals

The major vitamin E components present in palm oil, viz. α-tocopherol, α⁻, ψ-and δ-tocotrienols, have been isolated and their structures verified by the NMR spectra of their acetate and succinate derivatives. Oxidation of γ-and δ-tocotrienols with alkaline K₃Fe(CN)₆ gave isolable dimeric species, which were studied by¹³C NMR. Free radicals generated from the monomeric and dimeric tocotrienols were investigated using ESR spectroscopy. The distinction between antioxidant activity and antioxidant capacity of vitamin E isomers is discussed.     

Tocotrienols Suppress Proinflammatory Markers and Cyclooxygenase-2 Expression in RAW264.7 Macrophages

Tocotrienols are powerful chain breaking antioxidant. Moreover, they are now known to exhibit various non-antioxidant properties such as anti-cancer, neuroprotective and hypocholesterolemic functions. This study was undertaken to investigate the anti-inflammatory effects of tocotrienol-rich fraction (TRF) and individual tocotrienol isoforms namely δ-, γ-, and α-tocotrienol on lipopolysaccharide-stimulated RAW264.7 macrophages. The widely studied vitamin E form, α-tocopherol, was used as comparison. Stimulation of RAW264.7 with lipopolysaccharide induced the release of various inflammatory markers. 10 μg/ml of TRF and all tocotrienol isoforms significantly inhibited the production of interleukin-6 and nitric oxide. However, only α-tocotrienol demonstrated a significant effect in lowering tumor necrosis factor-α production. Besides, TRF and all tocotrienol isoforms except γ-tocotrienol reduced prostaglandin E₂ release. It was accompanied by the down-regulation of cyclooxygenase-2 gene expression by all vitamin E forms except α-tocopherol. Collectively, the data suggested that tocotrienols are better anti-inflammatory agents than α-tocopherol and the most effective form is δ-tocotrienol.     

Effect of sesaminol on plasma and tissue alpha-tocopherol and alpha-tocotrienol concentrations in rats fed a vitamin E concentrate rich in tocotrienols

We have shown that sesame lignans added to rat diet resulted in significantly greater plasma and tissue concentrations of α- and γ-tocopherol concentrations in supplemented rats than in rats without supplementation. In the present studies we examined whether sesaminol, a sesame lignan, enhances tocotrienol concentrations in plasma and tissues of rats fed diets containing a tocotrienol-rich fraction of palm oil (T-mix). In Ex-periment 1, effects of sesaminol on tocotrienol concentrations in plasma, liver, and kidney were evaluated in rats fed diets containing 20 mg/kg of T-mix (20T) and 50 mg/kg of T-mix (50T) with or without 0.1% sesaminol. Although the T-mix contained 23% α-tocopherol, 22% α-tocotrienol, and 34% γ-tocotrienol, α-tocopherol constituted most or all of the vitamin E in plasma and tissue (from 97% in kidney to 100% in plasma), with no or very little α-tocotrienol and no γ-tocotrienol at all. Addition of sesaminol to the T-mix resulted in significantly higher plasma, liver, and kidney α-tocopherol concentrations compared to values for T-mix alone. Further, T-mix with sesaminol resulted in significantly higher α-tocotrienol concentrations in kidney, although the concentration was very low. In Experiment 2, we examined whether sesaminol caused enhanced absorption of α-tocopherol and α-tocotrienol in a dosage regimen supplying T-mix and sesaminol on alternating days and observed significantly higher levels of α-tocopherol and α-tocotrienol in rats fed sesaminol, even without simultaneous intake, compared to those in rats without sesaminol. In Experiment 3, α-tocopherol was supplied to the stomach with and without sesaminol, and α-tocopherol concentrations in the lymph fluid were measured, α-Tocopherol concentrations were not different between groups. These results indicated that sesaminol produced markedly higher α-tocopherol concentrations in plasma and tissue and significantly greater α-tocotrienol concentrations in kidney and various other tissues, but the concentrations of α-tocotrienol were extremely low compared to those of α-tocopherol (Exps. 1 and 2). However, the sesaminol-induced increases of α-tocopherol and α-tocotrienol concentrations in plasma and tissue were not caused by their enhanced absorption since sesaminol did not enhance their absorption.     

Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status

Potential antiproliferative effects of tocotrienols, the major vitamin E component in palm oil, were investigated on the growth of both estrogen-responsive (ER+) MCF7 human breast cancer cells and estrogen-unresponsive (ER-) MDA-MD-231 human breast cancer cells, and effects were compared with those of α-tocopherol (αT). The tocotrienol-rich fraction (TRF) of palm oil inhibited growth of MCF7 cells in both the presence and absence of estradiol with a nonlinear dose-response but such that complete suppression of growth was achieved at 8 μg/mL. MDA-MB-231 cells were also inhibited by TRF but with a linear dose-response such that 20 μg/mL TRF was needed for complete growth suppression. Separation of the TRF into individual tocotrienols revealed that all fractions could inhibit growth of both ER+ and ER- cells and of ER+ cells in both the presence and absence of estradiol. However, the γ- and δ-fractions were the most inhibitory. Complete inhibition of MCF7 cell growth was achieved at 6 μg/mL of γ-tocotrienol/δ-tocotrienol (γT₃/δT₃) in the absence of estradiol and 10μm/mL of δT₃ in the presence of estradiol, whereas complete suppression of MDA-MB-231 cell growth was not achieved even at concentrations of 10μg/mL of δT₃. By contrast to these inhibitory effects of tocotrienols, αT had no inhibitory effect on MCF7 cell growth in either the presence or the absence of estradiol, nor on MDA-MB-231 cell growth. These results confirm studies using other sublines of human breast cancer cells and demonstrate that tocotrienols can exert direct inhibitory effects on the growth of breast cancer cells. In searching for the mechanism of inhibition, studies of the effects of TRF on estrogen-regulated pS2 gene expression in MCF7 cells showed that tocotrienols do not act via an estrogen receptor-mediated pathway and must therefore act differently from estrogen antagonists. Furthermore, tocotrienols did not increase levels of growth-inhibitory insulin-like growth factor binding proteins (IGFBP) in MCF7 cells, implying also a different mechanism from that proposed for retinoic acid inhibition of estrogen-responsive breast cancer cell growth. Inhibition of the growth of breast cancer cells by tocotrienols could have important clinical implications not only because tocotrienols are able to inhibit the growth of both ER+ and ER- phenotypes but also because ER+ cells could be growth-inhibited in the presence as well as in the absence of estradiol. Future clinical applications of TRF could come from potential growth suppression of ER+ breast cancer cells otherwise resistant to growth inhibition by antiestrogens and retinoic acid.     

Effect of roasting temperature and time on the chemical composition of rice germ oil

Compositional changes of rice germ oils prepared at different roasting temperatures (160–180°C) and times (5–15 min) from rice germ were evaluated and compared with those of unroasted rice germ oil. The color development and phosphorus content of oils increased significantly as roasting temperature and time increased, whereas the FA compositions of rice germ oils did not change with roasting temperature and time. Four phospholipid classes, i.e., PE, PI, PA and PC, were identified. PE had the lowest stability under roasting conditions. There were no significant differences in γ-oryzanol levels of rice germ oils prepared at different roasting temperatures and times. Four tocopherol isomers (α−, β−, γ−, and δ-tocopherol) and three tocotrienol isomers (α−, γ−, and δ-tocotrienol) were identified, but no β-tocotrienol was detectable. The content of α− and γ−tocopherol in rice germ oil gradually increased as roasting temperature and time increased.     

α-tocopherol concentrations of the nervous system and selected tissues of adult dogs fed three levels of vitamin E

The effects of dietary vitamin E levels on tissue α-tocopherol (α−T) concentrations in different parts of the nervous system are largely unknown. Therefore, we measured the α−T contents of nervous and other tissues obtained from beagle dogs fed for two years a vitamin E-deficient diet (−E, 0.05±0.02 mg vitamin E/kg diet, n=2), a vitamin E-supplemented diet (+E, 114±14 mg/kg, n=2), or a standard chow diet (E_n, 74±6 mg/kg, n=3). Brain regions and spinal cords of +E dogs contained about double the α−T concentrations of E_n dogs, and about 10-fold those of −E dogs. The various brain regions of −E dogs, compared with E_n dogs, retained 12–18% of the α−T concentrations, with the exception of the caudal colliculus, which retained 48%. Peripheral nerve α−T concentrations in +E dogs (67 ng/mg wet weight) were nearly 5-fold higher than in E_n dogs (13.4±5.9 ng/mg) and 80-fold higher than in −E dogs (0.8 ng/mg). Within each dietary group, the lowest α−T concentrations in the central nervous system (CNS) were in the spinal cord. Peripheral nerves were the most susceptible to vitamin E repletion or depletion: in +E dogs, nerves contained higher concentrations of α−T than most brain regions; in E_n dogs, they contained similar concentrations; but in −E dogs, they contained less α−T than most brain regions. Muscles and other tissues of −E dogs retained from 1 to 10% of E_n values. The studies demonstrate that the CNS conserved α−T compared to peripheral nerves and nonnervous tissues in adult dogs, but contained lower absolute concentrations of α−T compare with most other tissues.     

Tocol-derived minor constituents in selected plant seed oils

Various crude and processed seed oils were analyzed for tocopherols (T) and tocotrienols (T₃) by reversed-phase HPLC with fluorescence detection (FL). The oils included processed canola oil, crude corn oil, crude milkweed oil, crude palm oil, crude/processed rice bran oils, crude/processed soybean oil, crude/processed sunflower oil, and related modified oil, crude/processed sunflower oil, and related modified oil varieties. The HPLC system consisted of a pentafluorophenylsilica (PFPS) column and a mobile phase of methanol and water. The results of comparative methodological studies with rice bran oils and milkweed oils indicated that the reversed-phase PEPS-HPLC method in conjunction with the use of less hazardous solvents proved to be superior and a viable alternative to the conventional normal-phase HPLC method. Unlike the traditional nonpolar octadecylsilica phase, which fails to resolve β-γ pairs of T and T₃, HPLC with the unique polar PFPS column enables separations of all compounds of interest. Except for palm oil, βT and γT were detected in all other crude oils. Although most milkweed oils contained moderale levels of βT and γT, the βT species was present in relatively low abundance in edible oils despite the observation of fairly high concentrations of γT in the latter oils. βT₃ and γT₃ were detected along with αT₃ and σT₃ only in palm and rice bran oils. Tocolderived antioxidant distribution data for zero-time processed oils provided potential utility in correlation studies of frying quality and stability. The variable distribution data for crude oils shed some light on market profitability of oilseeds with rich sources of vitamin E-related minor constituents.     

Removal of fat from Cow's milk decreases the vitamin E contents of the resulting dairy products

The present study was undertaken to determine whether decreases in fat contents result in lower vitamin E contents. Milk samples of varying fat contents (half and half, whole milk, reduced-fat milk low-fat milk, and nonfat milk) were obtained from a local dairy on six different occasions, α-locopherol was the major form of vitamin E (>85%); γ-tocopherol and α-tocotrienol were present to a lesser extent. As the fat contents of milk products decreased from 11 to 0.3%, the vitamin E contents decreased. For example, raw milk as compared to nonfat milk had both higher α-tocopherol contents (45.5+-4.6 vs. 4.5±0.5 μg/100 g; P<-0.0001) and higher total lipids ( 3.46±0.49 vs. 0.30±0.07 g/100 g; P≤0.0001). Vitamin E, cholesterol, and total lipids increased as cream was added back to nonfat milk during production. For every 1 mg cholesterol increase, there was an increase of approximately 4 μg of α-tocopherol; for every 1 g total lipids increase, the α-tocopherol content increased by 17 μg. These data demonstrate that removal of milk fat markedly decreases the vitamin E content of various milk products     

Vitamin E supplementation increases circulating vitamin E metabolites tenfold in end-stage renal disease patients

Vitamin E supplementation could elevate circulating vitamin E metabolites while modulating oxidative and inflammatory status in end-stage renal failure patients undergoing hemodialysis. Plasma concentrations of carboxyethyl-hydroxychromanols (α-and γ-CEHC), ascorbic acid, α-and γ-tocopherols, E₂-isoprostanes, and inflammatory biomarkers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), ferritin, and C-reactive protein (CRP)] were measured in blood samples obtained from patients (n=11) before and after dialysis on two occasions prior to, and at 1 and 2 mon of daily vitamin E supplementation (400 IU RRR-α-tocopherol). Supplementation nearly doubled plasma α-tocopherol concentrations (from 18±0.5 to 31±1.7 μM, P<0.0001), whereas γ-tocopherol concentrations decreased (from 2.8±0.3 to 1.7±0.2 μM, P=0.001). Serum α-CEHC increased 10-fold from 68±3 to 771±175 nM (P<0.0001), and γ-CEHC increased from 837±164 to 1136±230 nM (P=0.008). Vitamin E supplementation also increased postdialysis hematocrits from 38±1% to 41±1% (P<0.001). Dietary antioxidant intakes (vitamins E and C) were low in most subjects; plasma ascorbic acid levels (88±27 μM) decreased significantly with dialysis (33±11 μM, P=0.01). Plasma Il-6, CRP, TNF-α, and free F₂-isoprostane concentrations were elevated throughout the study. There is a complex relationship between chronic inflammation and oxidative stress that is not mitigated by short-term vitamin E supplementation. Importantly, serum vitamin E metabolite concentrations that increased 10-fold within 30 d of supplementation did not increase further, suggesting routes other than urine for removal of metabolites.     

Intestinal Epithelial Cells Absorb γ-Tocotrienol Faster than α-Tocopherol

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of α-tocopherol and γ-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of γ-tocotrienol was also larger than that of α-tocopherol. To investigate the intestinal uptake of α-tocopherol and γ-tocotrienol in vivo, the mice were fed single doses of α-tocopherol or γ-tocotrienol with triolein. The γ-tocotrienol responded faster in plasma than α-tocopherol, although the maximal level of γ-tocotrienol was lower than that of α-tocopherol. This suggested that the intestinal uptake properties of administered α-tocopherol and γ-tocotrienol would characterize their plasma level transitions in mice.     

Penetration and distribution of α-tocopherol, α- or γ-tocotrienols applied individually onto murine skin

To evaluate skin penetration of various vitamin E homologs, a 5% solution of either α-tocopherol, α-tocotrienol, or γ-tocotrienol in polyethylene glycol was topically applied to SKH-1 hairless mice. After 0.5, 1, 2, or 4 h (n=four per time point and four per vitamin E homolog), the skin was washed, the animals killed, the skin rapidlly removed, frozen on dry ice, and a biopsy taken and sectioned: stratum corneum (two uppermost, 5-μm sections—SC1 and SC2), epidermis (next two 10μm sections—E1 and E2), papillary dermis (next 100μ, PD), dermis (next 400 μm, D), and subcutaneous fat (next 100 μm, SF). SC1 contained the highest vitamin E concentrations per μ thickness. To compare the distribution of the various vitamin E forms into the skin layers, the percentage of each form was expressed per its respective total. Most surprising was that the largest fraction of skin vitamin E following topical application was found in the deeper subcutaneous layers—the lowest layers, PD (40±15%) and D (36±15%), contained the major portion of the applied vitamin E forms. Although PD only represents about 16% of the total skin thickness, it contains sebaceous glands—lipid secretory organs, and, thus, may account for the vitamin E affinity for this layer. Hence, applied vitamin E penetrates rapidly through the skin, but the highest concentrations are found in the uppermost 5 microns.     

Assessment of olive oil adulteration by reversed-phase high-performance liquid chromatography/amperometric detection of tocopherols and tocotrienols

A method involving reversed-phase high-performance liquid chromatography with amperometric detection has been developed for the analysis of tocopherols and tocotrienols in vegetable oils. The sample preparation avoids saponification. Recoveries of α-tocotrienol and γ-tocotrienol in extra virgin olive oil were 97.0 and 102.0%, respectively. No tocotrienols were detected in olive, hazelnut, sunflower, and soybean oils, whether virgin or refined. However, relatively high levels of tocotrienols were found in palm and grapeseed oils. This method could detect small quantities (1–2%) of palm and grapeseed oils in olive oil or in any tocotrienol-free vegetable oil and might, therefore, help assess authenticity of vegetable oils.