A review of characterization of tocotrienols from plant oils and foods

Tocotrienols, members of the vitamin E family, are natural compounds found in a number of vegetable oils, wheat germ, barley and certain types of nuts and grains. Vegetable oils provide the best sources of these vitamin E forms, particularly palm oil and rice bran oil contain higher amounts of tocotrienols. Other sources of tocotrienols include grape fruit seed oil, oats, hazelnuts, maize, olive oil, buckthorn berry, rye, flax seed oil, poppy seed oil and sunflower oil. Tocotrienols are of four types, viz. alpha (α), beta (β), gamma (γ) and delta (δ). Unlike tocopherols, tocotrienols are unsaturated and possess an isoprenoid side chain. A number of researchers have developed methods for the extraction, analysis, identification and quantification of different types of vitamin E compounds. This article constitutes an in-depth review of the chemistry and extraction of the unsaturated vitamin E derivatives, tocotrienols, from various sources using different methods. This review article lists the different techniques that are used in the characterization and purification of tocotrienols such as soxhlet and solid–liquid extractions, saponification method, chromatography (thin layer, column chromatography, gas chromatography, supercritical fluid, high performance), capillary electrochromatography and mass spectrometry. Some of the methods described were able to identify one form or type while others could analyse all the analogues of tocotrienol molecules. Hence, this article will be helpful in understanding the various methods used in the characterization of this lesser known vitamin E variant.     

生育三烯酚的生理功能及合成代谢调控

维生素E是一种重要的脂溶性抗氧化剂。天然维生素E包括α-、β-、γ-和δ-生育酚及α-、β-、γ-和δ-生育三烯酚等8种组分。生育三烯酚具有独特的降低胆固醇、抗癌和神经保护功能,这些功能是生育酚所不具备的。生育三烯酚主要由谷物等单子叶植物合成。虽然生育三烯酚成分占天然维生素E的一半,但有关生育三烯酚的研究仅占维生素E全部文献的1%。可喜的是,近年来这一领域的研究取得了较大的进展。我们就生育三烯酚在人体内的生理功能、来源、生物合成及代谢调控等方面的进展进行概述。     

Unleashing the untold and misunderstood observations on vitamin E

Paradoxically, meta-analysis of human randomized controlled trials revealed that natural but not synthetic α-tocopherol supplementation significantly increases all-cause mortality at 95% confidence interval. The root cause was that natural α-tocopherol supplementation significantly depressed bioavailability of other forms of vitamin E that have better chemo-prevention capability. Meta-analysis outcome demonstrated flaws in the understanding of vitamin E. Reinterpretation of reported data provides plausible explanations to several important observations. While α-tocopherol is almost exclusively secreted in chylomicrons, enterocytes secrete tocotrienols in both chylomicrons and small high-density lipoproteins. Vitamin E secreted in chylomicrons is discriminately repacked by α-tocopherol transfer protein into nascent very low-density lipoproteins in the liver. Circulating very low-density lipoproteins undergo delipidation to form intermediate-density lipoproteins and low-density lipoproteins. Uptake of vitamin E in intermediate-density lipoproteins and low-density lipoproteins takes place at various tissues via low-density lipoproteins receptor-mediated endocytosis. Small high-density lipoproteins can deliver tocotrienols upon maturation to peripheral tissues independent of α-tocopherol transfer protein action, and uptake of vitamin E takes place at selective tissues by scavenger receptor-mediated direct vitamin E uptake. Dual absorption pathways for tocotrienols are consistent with human and animal studies. α-Tocopherol depresses the bioavailability of α-tocotrienol and has antagonistic effect on tocotrienols in chemo-prevention against degenerative diseases. Therefore, it is an undesirable component for chemo-prevention. Future research directions should be focused on tocotrienols, preferably free from α-tocopherol, for optimum chemo-prevention and benefits to mankind.     

Anti-inflammatory properties of α- and γ-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (α, β, γ, δ) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, α-tocopherol (αT) and γ-tocopherol (γT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (γT-enriched) tocopherols seems to be more potent than supplementation with αT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with αT only and thus warrants further investigation.     

Natural forms of vitamin E: metabolism,antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy

The vitamin E family consists of four tocopherols and four tocotrienols. α-Tocopherol (αT) is the predominant form of vitamin E in tissues and its deficiency leads to ataxia in humans. However, results from many clinical studies do not support a protective role of αT in disease prevention in people with adequate nutrient status. On the other hand, recent mechanistic studies indicate that other forms of vitamin E, such as γ-tocopherol (γT), δ-tocopherol, and γ-tocotrienol, have unique antioxidant and anti-inflammatory properties that are superior to those of αT in prevention and therapy against chronic diseases. These vitamin E forms scavenge reactive nitrogen species, inhibit cyclooxygenase- and 5-lipoxygenase-catalyzed eicosanoids, and suppress proinflammatory signaling such as NF-κB and STAT3/6. Unlike αT, other vitamin E forms are significantly metabolized to carboxychromanols via cytochrome P450-initiated side-chain ω-oxidation. Long-chain carboxychromanols, especially 13′-carboxychromanols, are shown to have stronger anti-inflammatory effects than unmetabolized vitamins and may therefore contribute to the beneficial effects of vitamin E forms in vivo. Consistent with mechanistic findings, animal and human studies show that γT and tocotrienols may be useful against inflammation-associated diseases. This review focuses on non-αT forms of vitamin E with respect to their metabolism, anti-inflammatory effects and mechanisms, and in vivo efficacy in preclinical models as well as human clinical intervention studies.     

Analysis of vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS

Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13′-hydroxychromanols (13′-OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13′-carboxychromanol (13′-COOH), appear to be more bioactive than tocopherols in anti-inflammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with efficacy of ≥ 89% and interday/intraday variation of 3–11% for major metabolites. To ensure accurate quantification, short-chain, long-chain, and sulfated carboxychromanols are included as external/internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with γ-tocopherol or δ-tocopherol. Although plasma levels of 13′-COOHs and 13′-OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo.     

Tocotrienols: balancing the mitochondrial crosstalk between apoptosis and autophagy

Tocotrienols are a group of natural vitamin E compounds with patent antitumoral effects, mostly based on their ability to induce apoptosis in cancer cells. In activated pancreatic stellate cells (PSCs) we have determined that tocotrienols elicit a dramatic mitochondrial destabilization followed by initiation of non-necrotic forms of programmed cell death, namely apoptosis and autophagy. PSCs are the main cell type involved in the generation of pancreatic fibrosis, and their removal is critical to limit the fibrogenic process. Noteworthy, tocotrienol death-promoting actions are exclusively directed to activated PSCs, but not to their quiescent counterparts nor to terminally differentiated acinar cells. Here, we hypothesize that the transformed phenotype of PSCs may include "activated" mitochondria, which can be used by tocotrienols to trigger autophagic and apoptotic signaling. We propose that mitochondria are the cornerstone of cell sensitivity to tocotrienols, and suggest possible mechanisms, that may be interconnected, on how tocotrienols may govern mitochondrial death pathways.     

Tocotrienols: Balancing the Mitochondrial Crosstalk Between Apoptosis and Autophagy

Tocotrienols are a group of natural vitamin E compounds with patent antitumoral effects, mostly based on their ability to induce apoptosis in cancer cells. In activated pancreatic stellate cells (PSCs) we have determined that tocotrienols elicit a dramatic mitochondrial destabilization followed by initiation of non-necrotic forms of programmed cell death, namely apoptosis and autophagy. PSCs are the main cell type involved in the generation of pancreatic fibrosis, and their removal is critical to limit the fibrogenic process. Noteworthy, tocotrienol death-promoting actions are exclusively directed to activated PSCs, but not to their quiescent counterparts nor to terminally differentiated acinar cells. Here, we hypothesize that the transformed phenotype of PSCs may include “activated” mitochondria, which can be used by tocotrienols to trigger autophagic and apoptotic signaling. We propose that mitochondria are the cornerstone of cell sensitivity to tocotrienols, and suggest possible mechanisms, that may be interconnected, on how tocotrienols may govern mitochondrial death pathways.

Addendum to:

Tocotrienols Induce Apoptosis and Autophagy in Rat Pancreatic Stellate Cells through the Mitochondrial Death Pathway

M. Rickmann, E.C. Vaquero, J.R. Malagelada and X. Molero

Gastroenterology 2007; 132:2518-32     

Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24 h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (p < .05). None of the tocotrienol doses tested affected microglia viability.     

n-Hexanal and Some Volatile Alcohols

Both α-tocopherol and a 1: 1.7 mixture of α-tocopherol and tocotrienols at a 0.2% dietary level significantly depressed the age-related increase in the systolic blood pressure of spontaneously hypertensive rats (SHRs) after 3 weeks of feeding. The aortic production of prostacyclin was increased 1.5 times both by α-tocopherol and a tocotrienol mixture, suggesting a possible relevance to their hypotensive effect. These vitamins did not influence the Δ6- and Δ5-desaturase activities of liver microsomes, but fatty acid profiles of the liver phospholipids predicted a reduction of linoleic acid desaturation. These effects were in general more clear with tocotrienols than with α-tocopherol. Platelet aggregation by 5 μM ADP remained uninfluenced. Thus, tocotrienols may have effects on various lipid parameters somewhat different from those of α-tocopherol.     

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.     

Multifaceted role of tocotrienols in cardioprotection supports their structure: function relation

Tocotrienols are a class of vitamin E which modulates several mechanisms associated with cardioprotection, anti-cancer, anti-diabetic, and neuroprotection. Unlike other Vitamin E-like compounds, tocotrienols possess inimitable properties. Quite a lot of studies have determined the cardioprotective abilities of tocotrienols and have been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein and lipoprotein plasma levels. In addition, tocotrienol has been suggested to have an antioxidant, anti-thrombotic, and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. The bioactivity exhibited is due to the structural characteristics of tocotrienols. Rich sources of tocotrienols which include rice bran, palm oil, and other edible oils exhibit protective effect against cardiovascular disorders. The conclusions drawn from the early literature that vitamin E group of compounds provides an inevitable role in cardioprotection is sustained in many more recent studies.     

Why tocotrienols work better: insights into the in vitro anti-cancer mechanism of vitamin E

The selective constraint of liver uptake and the sustained metabolism of tocotrienols (T3) demonstrate the need for a prompt detoxification of this class of lipophilic vitamers, and thus the potential for cytotoxic effects in hepatic and extra-hepatic tissues. Hypomethylated (γ and δ) forms of T3 show the highest in vitro and in vivo metabolism and are also the most potent natural xenobiotics of the entire vitamin E family of compounds. These stimulate a stress response with the induction of detoxification and antioxidant genes. Depending on the intensity of this response, these genes may confer cell protection or alternatively they stimulate a senescence-like phenotype with cell cycle inhibition or even mitochondrial toxicity and apoptosis. In cancer cells, the uptake rate and thus the cell content of these vitamers is again higher for the hypomethylated forms, and it is the critical factor that drives the dichotomy between protection and toxicity responses to different T3 forms and doses. These aspects suggest the potential for marked biological activity of hypomethylated “highly metabolized” T3 that may result in cytoprotection and cancer prevention or even chemotherapeutic effects. Cytotoxicity and metabolism of hypomethylated T3 have been extensively investigated in vitro using different cell model systems that will be discussed in this review paper as regard molecular mechanisms and possible relevance in cancer therapy.     

A naturally occurring mixture of tocotrienols inhibits the growth of human prostate tumor,associated with epigenetic modifications of cyclin-dependent kinase inhibitors p21 and p27

Tocotrienols, members of the vitamin E family, have three unsaturated bonds in their side chains. Recently, it has been suggested that the biological effects of tocotrienols may differ from that of tocopherols. Several in vitro studies have shown that tocotrienols have stronger anticancer effects than tocopherols. VCaP cell line used in this study is from a vertebral bone metastasis from a patient with prostate cancer. Eight-week-old male NCr(−/−) nude mice were subcutaneously injected with VCaP-luc cells in matrigel and then administered a tocotrienol mixture for 8 weeks. The tocotrienol mixture inhibited the growth of human prostate tumor xenografts in a dose-dependent manner. The concentrations of tocotrienols and their metabolites were significantly increased in treatment groups. Tocotrienols inhibited prostate tumor growth by suppressing cell proliferation, which was associated with the induction of the cyclin-dependent kinase (CDK) inhibitors p21 and p27. In addition, tocotrienol treatment was associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27 and with decreased expression of histone deacetylases. Tocotrienols inhibited human prostate tumor growth, associated with up-regulation of the CDK inhibitors p21 and p27. Elevated expression of p21 and p27 could be partly due to the suppressed expression of HDACs.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of γ-tocopherol and γ-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Effects of Late Administration of Pentoxifylline and Tocotrienols in an Image-Guided Rat Model of Localized Heart Irradiation

Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.     

Serum α-tocopherol and γ-tocopherol concentrations and prostate cancer risk in the PLCO Screening Trial: a nested case-control study

Background

Vitamin E compounds exhibit prostate cancer preventive properties experimentally, but serologic investigations of tocopherols, and randomized controlled trials of supplementation in particular, have been inconsistent. Many studies suggest protective effects among smokers and for aggressive prostate cancer, however.

Methods

We conducted a nested case-control study of serum α-tocopherol and γ-tocopherol and prostate cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, with 680 prostate cancer cases and 824 frequency-matched controls. Multivariate-adjusted, conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CIs) for tocopherol quintiles.

Results

Serum α-tocopherol and γ-tocopherol were inversely correlated (r = −0.24, p<0.0001). Higher serum α-tocopherol was associated with significantly lower prostate cancer risk (OR for the highest vs. lowest quintile = 0.63, 95% CI 0.44–0.92, p-trend 0.05). By contrast, risk was non-significantly elevated among men with higher γ-tocopherol concentrations (OR for the highest vs. lowest quintile = 1.35, 95% CI 0.92–1.97, p-trend 0.41). The inverse association between prostate cancer and α-tocopherol was restricted to current and recently former smokers, but was only slightly stronger for aggressive disease. By contrast, the increased risk for higher γ-tocopherol was more pronounced for less aggressive cancers.

Conclusions

Our findings indicate higher α-tocopherol status is associated with decreased risk of developing prostate cancer, particularly among smokers. Although two recent controlled trials did not substantiate an earlier finding of lower prostate cancer incidence and mortality in response to supplementation with a relatively low dose of α-tocopherol, higher α-tocopherol status may be beneficial with respect to prostate cancer risk among smokers. Determining what stage of prostate cancer development is impacted by vitamin E, the underlying mechanisms, and how smoking modifies the association, is needed for a more complete understanding of the vitamin E-prostate cancer relation.