In Part I of this article, evidence from a wide range of peer-reviewed research, including in vitro and in vivo studies, clinical and randomized controlled trials, epidemiological studies, meta-analyses and reviews, was presented. The case was made that while decreased chronic disease incidence is clearly associated with generous dietary intake of vitamin E (mixed tocopherols and tocotrienols), supplementation with high-dose α-tocopherol alone, whether natural (RRR) or synthetic (all-rac), may promote potentially harmful imbalances in inflammatory and detoxification pathways. Both outcomes—the consistently positive effects of a diet rich in vitamin E and the inconsistent outcomes of studies using α-tocopherol—result from the fact that each of the 8 isomers that collectively constitute vitamin E has unique actions that complement, support and balance one another. Part II of this article reviews recent research on both the unique and interactive nature of the vitamin E isomers’ therapeutic actions, and the ratios in which the tocopherols and tocotrienols have been found to be most bioavailable and effective.
Part II: Vitamin E in Action
Therapeutic effects of vitamin E isomers
All natural vitamin E compounds have antioxidant activity, but evolution has tasked this 8-isomer family of inter-related tocopherols and tocotrienols with significantly more than just serving as chain-breaking inhibitors of lipid peroxidation. In addition to their antioxidant actions within LDL and HDL cholesterol, the vitamin E isomers reside in lipid bilayers where they not only act as antioxidants, but affect cell signaling, membrane-resident enzymes and other membrane-dependent processes. Their therapeutic activities include:
Aging is associated with an increase in oxidative stress and a decline in immune function.
One key marker of lowered immunity in the elderly is a decrease in interleukin-2 (IL-2), a cytokine important for the clonal expansion of T cells, which play an important role in the specific immune response. Pro-inflammatory prostaglandin E2 (PGE2) suppresses IL-2 production and lymphocyte proliferation; α-tocopherol, in amounts that support rather than overwhelm normal physiology, inhibits production of PGE2.
Short term supplementation of healthy elderly people with α-tocopherol (800 mg/day for 4 weeks) has been shown to significantly decrease PGE2 production and increase (restore) IL-2 production. A 6-month trial also found that α-tocopherol (200 mg/day) significantly increased the delayed type hypersensitivity response to various antigens.1
In a long-term (2 year) study, however, α-tocopherol (200 mg/day) was associated with an increase in duration and severity of respiratory tract infections in well-nourished non-institutionalized elderly individuals.2
One reason for this apparent disparity may be that the majority of Americans (approximately 90%) do not consume even the RDI—a mere 15 mg/day—for vitamin E.3 Thus, most elderly Americans not taking supplemental vitamin E are likely to be vitamin E-deficient. Over the short term, high dose α-tocopherol supplementation would help replete normal physiological levels of this isomer, while a longer period of high-dose α-tocopherol supplementation would be more likely to result in immune-suppressing imbalances related to the effects of high levels of α-tocopherol on Phase I detoxification enzymes and suppression of γ-tocopherol.
Immune function also declines with age in animals. Similar to humans, PGE2 is elevated in macrophages isolated from old mice (24 months) compared to young mice (6 months). α-Tocopherol appears to decrease PGE2 production in macrophages from old mice by inhibiting the increased cyclooxygenase-2 (COX-2) activity in these cells. Inhibition of the age-related increase of PGE2 may trace back to α-tocopherol’s inhibition of lipid peroxidation by reactive oxygen species (ROS), which is thought to contribute to the enhanced COX-2 expression seen with age.
In further studies, the combination of α-tocopherol and δ-tocopherol was found to inhibit PGE2 in macrophages isolated from old mice more potently than α-tocopherol alone. In addition, splenocyte IL-2 production was unaffected by α-tocopherol but was increased by γ- and δ-tocopherol. These results clearly indicate that the different tocopherol isomers exert different, yet complementary immunostimulatory effects.1
α-Tocopherol exerts beneficial opposing effects on key inflammation-regulating enzymes
Treatment of human aortic endothelial cells with physiological concentrations of α-tocopherol has been shown to dose-dependently stimulate the production of anti-inflammatory prostaglandin I2 (a.k.a. prostacyclin) and the vasodilator form of PGE2 via induction of phospholipase A2 synthesis, while, at the same time, significantly inhibiting pro-inflammatory COX activity. These results indicate that α-tocopherol improves endothelial function by exerting opposite effects on phospholipase and cyclooxygenase,
the two key enzymes involved in the conversion of essential fatty acids (eicosanoids) into prostanoids.1
It is interesting to note that α-tocopherol inhibits (pro-inflammatory) PGE2 production in macrophages, but enhances (anti-inflammatory) PGE2 production in endothelial cells, a phenomenon similar to α-tocopherol’s effects on nitric oxide (NO). α-Tocopherol reduces NO production in splenocytes and macrophages, but increases NO production in endothelial cells. Endothelium-derived NO helps to maintain normal cardiovascular function, whereas macrophage-derived NO is a pro-inflammatory signal in pathophysiological changes in the cardiovascular system. The analogous oppositional effects of α-tocopherol on PGE2 production in endothelial cells and macrophages are also protective since endothelium-derived PGE2 helps maintain vasodilatation, whereas macrophage-derived PGE2 is pro-inflammatory.4
Tocopherols target kinases to modulate cell signaling
The anti-inflammatory and immunostimulatory effects of the tocopherols are related, not only to their antioxidant activity, but to their modulation of cell signaling via their effects on kinases.1
Protein kinase C inhibited by α-tocopherol
The first experimental evidence that vitamin E isomers modulate enzymes involved in signal transduction came from studies of protein kinase C (PKC) activity. PKC was best inhibited by α-tocopherol; the other tocopherols were less potent despite having equal antioxidant activity, an outcome that first prompted researchers’ recognition that PKC is inhibited by non-antioxidant vitamin-E-mediated mechanisms.5
In vascular smooth muscle cells, α-tocopherol inhibits PKC activity by enhancing the activity of protein phosphatase type 2A (PP2A), which inhibits PKC auto-phosphorylation, thus decreasing PKC activity. Elevation of PP2A levels by α-tocopherol is also associated with a decrease in ERK1/2 phosphorylation, and a decrease in NFκB DNA-binding activity, both of which are key players in inflammatory pathways.1
NFκB controls the expression of a variety of genes involved in the inflammatory response and vascular smooth muscle cell (VSMC) proliferation. Studies carried out on human macrophages treated with α-tocopherol and activated afterwards have shown a 43% reduction in NFκB activation as compared to control cells. Both γ-tocopherol and δ-tocopherol exhibit similar VSMC anti-proliferative effects related to NFκB inhibition.6 7 8
Inhibition of PKC or ERK1/2 also leads to inhibition of COX-2 upregulation and pro-inflammatory PGE2 production. PKC regulates activation and transcription of a large number of proteins involved in the inflammatory process, including the production of IL-1b, expression of COX, and formation of O2- by NADPH oxidase. PKC is also a well-established anti-apoptotic factor expressed in several tumor types.9
PKC inhibition by α-tocopherol correlates with the reduction of cell proliferation, an observation that has been noted in many cell types including vascular smooth muscle cells, monocytes/macrophages, neutrophils, fibroblasts, mesangial cells and various cancer cell lines.5
α-Tocopherol’s inhibition of PKC activity is also associated with a significant reduction in platelet aggregation. Platelets express the endothelial form eNOS and generate NO. Platelet-derived NO plays an important role in the regulation of platelet aggregation and secretion. Inhibition of platelet eNOS is a key factor in the abnormal platelet activation encountered in different pathological situations (e.g., hypertension and diabetes).10
Platelets supplemented with α-tocopherol (both in vitro and in vivo) release ~50% more NO and produce 74% less superoxide radical than unsupplemented platelets. These beneficial effects result from α-tocopherol’s inhibition of the PKC-dependent phosphorylation of the endothelial form of nitric oxide synthase (eNOS), thus preventing a reduction in eNOS’ catalytic activity.1
RRR (natural) α-Tocopherol more effectively inhibits protein kinase C
Research comparing inhibition of protein kinase C by the natural RRR form of α-tocopherol, versus all-racemic (synthetic) α-tocopherol, shows that the former is twice as potent as the latter.9
Mixed tocopherols inhibit platelet aggregation more effectively than α-tocopherol alone
In research comparing the effects of mixed tocopherols versus α-tocopherol alone, adenosine diphosphate (ADP)-induced platelet aggregation decreased significantly in healthy people given γ-tocopherol-enriched vitamin E (100 mg γ-tocopherol, 40 mg, δ-tocopherol, and 20 mg α-tocopherol per day), but not in those receiving pure α-tocopherol alone (100 mg/day) or in controls.11 This 5:2:1 ratio among the tocopherols is that seen in vitamin E-rich foods and appears to be the most effective (further discussion in the concluding section of this article).
PKC activation in platelets was markedly decreased in both tocopherol-treated groups, yet mixed tocopherols were much more effective in preventing platelet aggregation. Why? Oxidation of tetrahydrobiopterin by peroxynitrite in oxidant-stressed endothelium compromises nitric oxide synthase (eNOS) activity while amplifying superoxide production; this mechanism contributes prominently to the endothelial dysfunction that characterizes many common clinical disorders. Peroxynitrite is a reactive nitrogen species (RNS), which γ-tocopherol traps much more efficiently than α-tocopherol. As an effective peroxynitrite scavenger, γ-tocopherol can protect tetrahydrobiopterin and preserve eNOS activity.12
α-tocopherol effective against ROS, γ-tocopherol against RNS
Despite α-tocopherol’s action as an antioxidant against ROS, when lipids are exposed to peroxynitrite, nitrogen dioxide and other nitrating species, γ-tocopherol is required to effectively remove the resulting RNS. Excess generation of RNS is associated with chronic inflammation-related diseases including cardiovascular disease (CVD), cancer, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Chronic inflammation induced by phagocytes, a major contributor to cancer and other chronic degenerative diseases, is also largely due to peroxynitrite, which is formed during phagocyte activation. γ-tocopherol, but not α-tocopherol, is able to act as a trap for peroxynitrite and other membrane-soluble electrophilic nitrogen oxides and electrophilic mutagens, forming stable carbon-centered adducts through its unsubstituted nucleophilic 5-position, which is already filled in α-tocopherol.13
Because not only is γ-tocopherol responsible for scavenging RNS, but large doses of α-tocopherol displace γ-tocopherol in plasma and other tissues, the current wisdom of vitamin E supplementation with α-tocopherol alone may produce an imbalance that contributes to cancer and heart disease.13 Adding support to this hypothesis, a recent clinical trial of individuals suffering from coronary heart disease showed decreased serum levels of γ-tocopherol, but not α-tocopherol.14
γ-tocopherol may be more protective than α-tocopherol against CVD
Plasma concentrations of γ-tocopherol, but not α-tocopherol, have been found to be lower in CVD patients than in healthy control subjects in several studies.15 14 16
A cross sectional study of Swedish and Lithuanian middle-aged men, found that plasma γ-tocopherol concentrations were twice as high in the Swedish men, who had a 25% lower incidence of CVD-related mortality.17 In a case control study to test whether fat-soluble antioxidants can play a role against the occurrence of myocardial infarction, subjects with high plasma levels of γ-tocopherol were found to have a 70% lower risk for MI than those with low levels.18 19 The inverse correlations seen in these studies were not found for α-tocopherol.
As noted in Part I of this article, high-dose α-tocopherol has been found to reduce γ-tocopherol levels by 30-70%.1 Additionally, compared to α-tocopherol, δ-tocotrienol exerts the highest inhibitory action on monocytic cell adherence. Similarly, compared to α-tocopherol, α-tocotrienol displays a more profound inhibitory effect on adhesion molecule expression (ICAM-1, E-selectin, VCAM-1) and monocytic cell adherence induced by TNFα.6 Clearly, mixed tocopherols and tocotrienols should be the preferred form of vitamin E supplementation for individuals with cardiovascular disease.
Tocopherols also inhibit other kinases, including protein kinase B (a.k.a. AKT)
The tocopherols, including γ-, α- , and δ-tocopherol have been shown to strongly inhibit mast cell proliferation via inhibition of protein kinase B (a.k.a. AKT) phosphorylation. (Mast cells are involved in the immediate hypersensitivity response during allergic reactions and in the specific immune defense against certain parasites and bacteria.)
Specifically, the tocopherols inhibit the phosphatidylinositol 3-kinase (PI3K)-phosphoinositide-dependent kinase pathway, which in turn, activates AKT.1 5 19
By inhibiting AKT, tocopherols inhibit not only mast cell proliferation, but the AKT-mediated activation of the transcription factor NFκB. NFκB acts as a master switch for the upregulation of inflammatory cytokines, adhesion molecules and other inflammatory gene products produced in response to inflammatory stimuli. Activation of NFκB induced by TNF-α, and NFκB DNA binding induced by lipopolysaccharide (which stimulates formation of the pro-inflammatory cytokine PGE2), have been shown to be dose-dependently inhibited by α-tocopherol. Among the tocotrienols, γ-tocotrienol has also been shown to inhibit AKT phosphorylation, causing a reduction in NFκB activity induced by carcinogens, growth factors, and several inflammatory stimuli.1 5
Since AKT has recently been identified as a player in so many tumorigenic activities that a recent paper suggests AKT activation alone might be sufficient to induce cancer, the combined inhibitory effects of these vitamin E isomers on AKT phosphorylation may also have cancer-preventive effects.20
Tocopherols upregulate PPAR expression and activity
Peroxisome proliferator-activated receptors (PPAR) are a family of transcription factors induced by various ligands that act as anti-inflammatory agents by interfering with inflammatory signaling cascades, including the NFκB pathway.
α-Tocopherol has been shown to upregulate PPARγ in rat hepatocytes almost as much as comparable concentrations of the PPARγ ligand drug, troglitazone, which is structurally similar to α-tocopherol. Troglitazone (a.k.a. Rezulin, Resulin or Romozin), was an anti-diabetic and anti-inflammatory drug of the class of the thiazolidinediones, but was withdrawn from the U.S. market in 2000 because it increased risk of drug-induced hepatitis. α-Tocopherol does not have these negative effects.1
In human cells, all four natural tocopherol homologues have been shown to increase PPARγ mRNA levels 24 hours post-treatment. The strongest transcriptional activity is produced by γ-tocopherol, which increased PPARγ mRNA levels ~ 3-fold, followed by α-tocopherol, which produced ~1.5 to 2-fold increase.1
Tocopherols and their CEHC metabolites inhibit COX and LOX activity1
Tocopherols and their CEHC metabolites also exert anti-inflammatory actions at the post-transcriptional level, by inhibiting either cyclooxygenase (COX) or 5-lipoxygenase (LOX) activity.
COX-1, the constitutive form of the enzyme, performs the ‘housekeeping’ function of synthesizing prostaglandins that regulate normal cellular activities. COX-2, the inducible form, is produced in response to endotoxins and cytokines in many immune cells including macrophages and monocytes, and in epithelial cells. COX-2 is upregulated under most inflammatory conditions and is the primary enzyme responsible for the formation of pro-inflammatory PGE2. 5-LOX is the rate-limiting enzyme involved in the formation of the pro-inflammatory leukotriene B4 (LTB4). Because of the central roles played by both PGE2 and LTB4 in inflammation, COX-2 and 5-LOX have been identified as key targets for drug therapy in inflammatory diseases.
γ-Tocopherol and its water-soluble metabolite γ-CEHC have been shown to significantly reduce PGE2 synthesis in challenged macrophages and human lung epithelial cells at physiologically relevant concentrations; α-tocopherol was much less potent than γ-tocopherol in reducing PGE2 formation in these cells. In carrageenan-induced inflammation in male Wistar rats, γ-tocopherol was shown to inhibit pro-inflammatory PGE2 and LTB4, decrease TNFα, and significantly attenuate the drop off in food consumption in these animals caused by inflammation-associated discomfort.
These anti-inflammatory effects of γ-tocopherol and γ-CEHC appear to be rooted in their inhibition of COX-2 activity and are independent of their antioxidant activity. In other research, additional vitamin E isomers have also been found to inhibit COX-2-catalyzed formation of PGE2, e.g., in one study, δ-tocopherol was an even stronger COX-2 inhibitor than γ-tocopherol.
Although some studies have found an inverse correlation between high plasma levels of α-tocopherol and the formation of 5-LOX pathway products such as LTB4 by human leukocytes, γ-Tocopherol is a stronger inhibitor of COX and possibly LOX than α-tocopherol. Furthermore, as noted above, γ-tocopherol traps reactive nitrogen species (RNS) much more efficiently than α-tocopherol, although α-tocopherol is generally considered to be more potent than γ-tocopherol as a chain-breaking antioxidant for inhibiting lipid peroxidation due to ROS.
Mixed tocopherols more effective than single isomers against oxidative stress and inflammation
Clinical trials involving hemodialysis patients and subjects with metabolic syndrome (MetS), two conditions involving significant oxidative stress and inflammation, have shown that supplementation with γ-tocopherol or mixed tocopherols results in much better outcomes than supplementation with α-tocopherol alone.
In hemodialysis patients, mixed RRR tocopherols (60% γ-tocopherol, 28% δ-tocopherol, 18% α-tocopherol), but not α-tocopherol (300 mg/day) for 14 days, significantly reduced levels of the prototypic marker of inflammation, high sensitivity C reactive protein (hsCRP).21
In MetS subjects, in a randomized, placebo-controlled double-blind trial in which patients were given either α-tocopherol (800 mg/day), γ-tocopherol (800 mg/day), a combination of both (400 mg of each/day) or placebo for 6 weeks, only the combination produced significant differences (a median reduction of 15% in hsCRP) compared to placebo.
In addition, levels of nitrotyrosine (a marker of cytotoxicity) in MetS subjects’ urine were significantly decreased following γ-tocopherol supplementation, either alone or in combination with α-tocopherol, while α-tocopherol alone failed to decrease urinary nitrotyrosine.21
Anti-Cancer Actions of γ-Tocopherol, γ-CEHC
Preferential uptake of γ‐rather than α-tocopherol has been noted in a number of different cancer cell lines, and γ-tocopherol, but not α-tocopherol, exhibits anti-proliferative and pro-apoptotic effects on a number of cancerous but not normal cell types.22 6
γ-Tocopherol and its metabolic product, γ-carboxyethyl hydroxychroman (γ-CEHC), have been found to be effective inhibitors of human breast, prostate and colon cancer cell proliferation. γ-Tocopherol has been shown induce human breast cancer cells to undergo apoptosis in cell culture and to be more effective than α-tocopherol in inhibiting proliferation and inducing apoptosis of human prostate cells in cell culture. γ-Tocopherol, but not α-tocopherol, has been shown to induce apoptosis in human colon cancer cells, and to do so without damage to normal colon cells.22 23 Among the vitamin E isomers, γ‐tocopherol, δ‐tocopherol, and all four tocotrienols, but not α-tocopherol, have been shown to selectively induce cancer cells to undergo apoptosis.22
In a study involving more than 20,000 men, patients who developed prostate cancer had significantly lower blood levels of γ-tocopherol than men who did not, and the higher the blood level of γ-tocopherol, the lower the risk of prostate cancer. Men with the highest levels were 80% less likely to develop prostate cancer, compared to men with the lowest levels.24
In addition to offering significant protective effects against prostate cancer, γ-tocopherol appears to be integral to the anti-cancer actions of selenium and α-tocopherol. A nested case-control study examined the association of α-tocopherol, γ-tocopherol, and selenium with the incidence of prostate cancer. Not only did men in the highest quintile of plasma γ-tocopherol concentrations have a 5-fold reduction in the risk of prostate cancer compared to those in the lowest quintile, but significant protective effects of high concentrations of selenium and α-tocopherol were only observed when γ-tocopherol concentrations were high.25 8
As noted in Part I of this article, γ-tocopherol is preferentially metabolized into γ-CEHC, and the latter compound has been observed to exert much stronger anti-proliferative activity than α-CEHC in prostate cancer cells and rhabdomyosarcoma cells. CEHCs are as effective as their vitamin E isomer precursors in producing these anti-proliferative effects, which have been shown to occur through inhibition of cyclin signaling with subsequent arrest of the cell cycle.26 γ-CEHC may also have blood pressure-lowering effects as it has been shown to be a natriuretic factor that promotes sodium excretion and contributes to the regulation of the extracellular fluid volume.27 19
Furthermore, tocotrienols, which are more extensively metabolized to CEHCs than the tocopherols, have been found to be more effective anti-proliferative agents than the tocopherols in various cancer cell lines. This adds support to the hypothesis that the metabolic transformation of vitamin E isomers into CEHCs is not merely a step in their elimination from the body, but provides compounds with specific protective functions.28
Tocotrienols’ antioxidant anti-cancer actions
Recent in vitro and in vivo studies suggest that tocotrienols, but not α-tocopherol, may play a role in breast cancer prevention by preventing E2 (17 β-estradiol) epoxidation. E2 can be activated by epoxide-forming oxidants resulting in the ability to bind to DNA and form DNA adducts, which can cause mutation leading to carcinogenesis. Research evaluating and comparing the ability of α-tocopherol, α-tocotrienol, γ-tocotrienol and δ-tocotrienol in preventing the formation of E2 epoxide and the initiation of E2 epoxide-induced breast cancer found all three tocotrienols were more potent antioxidants and more effective than α-tocopherol.29
Why this dramatic difference between α-tocopherol and tocotrienols in preventing the oxidative formation of E2 epoxide? The researchers conducting these studies believe the polyunsaturated phytol side chain in tocotrienols is responsible. The 8 naturally occurring forms of vitamin E differ in number and position of methyl groups on the chroman head (α, β, γ, δ) and the presence or absence of double bonds in the phytyl tail (saturated = tocopherols; unsaturated = tocotrienols). Since α-tocopherol and α-tocotrienol share the same chromanol ring, the more potent antioxidant effects of α-tocotrienol and the other tocotrienols must be due to the difference in their isoprenoid tails. Tocotrienols have an isoprenoid tail with three unsaturation points instead of the saturated phytol tail seen in tocopherols.29 30
Based on this finding, it appears that as a family, vitamin E provides two anti-oxidative functional groups, its chromanol ring and its polyunsaturated phytol side chain. Since tocotrienols have both of these anti-oxidant options while tocopherols have only one, this fundamental structural difference may explain why the tocotrienols are the more potent antioxidants.29 Some researchers have also suggested that the presence of unsaturations in their side chain render tocotrienols much more flexible than tocopherols, allowing for more efficient penetration into tissues that have saturated fatty layers such as the brain and liver.31
Tocotrienols’ cell signaling anti-cancer actions31
The NFκB pathway plays a central role in tumorigenesis. γ-Tocotrienol inhibits the NFκB activation pathway, leading to down-regulation of various gene products and potentiation of apoptosis. γ-Tocotrienol has been shown to completely abolish NFκB activation induced by a wide range of agonists including tumor necrosis factor, phorbol myristate acetate, okadaic acid, lipopolysaccharide, cigarette smoke, interleukin-1beta, and epidermal growth factor. Even constitutive NFκB activation expressed by certain tumor cells is abrogated by γ-tocotrienol.
γ-Tocotrienol induces poly (ADP-ribose) polymerase (PARP) cleavage activating caspase-3, an enzyme that plays a key role in programmed cell death.
Tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death in breast cancer cells. When MCF-7 breast cancer cells were injected into athymic nude mice, feeding large amounts (1 mg/d) of a tocotrienol-rich fraction (TRF) of palm oil for 20 weeks delayed the onset, incidence, and size of tumors. At autopsy, tumor tissue was excised and cDNA array analysis showed that 30 genes were significantly affected by TRF. Ten genes were down-regulated and 20 genes up-regulated with respect to untreated animals.
Even low levels of tocotrienols induce human breast cancer cells in culture to undergo cell death by apoptosis. In rank order, the most effective vitamin E isomers for inhibition of colony formation of human breast cancer cells are δ‐, γ‐, and α-tocotrienols; and δ‐ and γ-tocopherols;-α-tocopherol was ineffective.22
In an animal model of prostate cancer, mice were injected with prostate cancer cells. When the tumors were about 5 mm in diameter, test animals were subcutaneously injected with 400 mg/kg γ-tocotrienol and irradiated 24 h later at the site of the tumor with a dose of 12 Gy (60) cobalt. Tumor size was reduced by almost 40%, but only in tocotrienol-treated and irradiated mice.
The growth-inhibitory and apoptotic effects of TRF has been tested on normal human prostate epithelial cells, virally transformed normal human prostate epithelial cells, and human prostate cancer cells. TRF selectively inhibits the growth of cancer cells, but not of normal cells, and produces significant apoptosis of cancer cells, but not normal cells.
In colon cancer cells, the pathway involved in TRF-induced apoptosis has been well characterized. In RKO cells, a poorly differentiated colon carcinoma cell line commonly used as an in vitro model for human colon carcinoma, TRF alters the Bax/Bcl2 ratio in favor of apoptosis, which triggers activation of caspase-3, an enzyme that plays a key role in programmed cell death.
Since the discovery that telomerase is repressed in most normal human somatic cells but strongly expressed in most human tumors, telomerase has become a target for therapeutic agents to combat human cancer. Tocotrienols inhibit telomerase activity in human colorectal adenocarcinoma cells in a time- and dose-dependent manner. δ-Tocotrienol demonstrated the highest inhibitory activity. Tocotrienols also inhibited protein kinase C activity, which caused down-regulation of human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. (Although α-tocopherol inhibits PKC, it does not share tocotrienols’ telomerase-inhibiting effect.)
Tocotrienols’ inhibit HMG-CoA reductase activity
Micromolar amounts of tocotrienols suppress the activity of HMG-CoA reductase, the hepatic enzyme responsible for cholesterol synthesis via a post-transcriptional mechanism. α-Tocopherol, however, induces HMGCoA activity.31
After a study conducted in hypercholesterolemic pigs fed supplements of the tocotrienol-rich fraction of palm oil showed a 44% decrease in total serum cholesterol, a 60% decrease in LDL-cholesterol, and significant decreases in platelet aggregation and levels of apolipoprotein B (-26%), thromboxane-B2 (-41%), and platelet factor 4 (-29%), these promising results were tested in humans. Twenty-five hypercholesterolemic human subjects were enrolled in a double-blind, crossover, 8-week study comparing the effects of a tocotrienol-rich fraction (TRF) of palm oil (200 mg/day) with corn oil (300 mg/day) on serum lipids.31
Serum total cholesterol (-15%), LDL cholesterol (-8%), Apo B (-10%), thromboxane (-25%), platelet activating factor 4 (-16%), and glucose (-12%) decreased significantly only in the 15 subjects given palm oil during the initial four weeks. In 7 subjects, serum total cholesterol decreased 31% during the four-week period in which they were given 200 mg γ-tocotrienol/day.32 31
In a follow up study, a second group of 16 subjects received 200 mg/day of γ-tocotrienol for 4 weeks. The cholesterol-suppressive results were equivalent to that of the mixture of tocotrienols (220 mg) used in the prior study. Cholesterol levels of subjects in the follow up study decreased by 13% during the 4-week trial, while apolipoprotein A (the lipid binding protein that transports HDL in the bloodstream) and HDL cholesterol levels were unaffected. 33 31 Interestingly, other research showed that when the tocotrienol-rich mixture included α-tocopherol in amounts higher than 15%, it lessened the tocotrienols’ cholesterol-lowering effects.34
The same researchers then looked at the effect of combining tocotrienols isolated from rice bran with lovastatin. Twenty-eight hypercholesterolemic subjects on the American Heart Association (AHA) Step-1 diet were given a tocotrienol preparation alone or in combination with lovastatin. While tocotrienols or lovastatin plus AHA Step-1 diet effectively lowered serum total cholesterol (14%, 13%) and LDL-cholesterol (18%, 15%), respectively, the combination significantly reduced cholesterol by 20–25%, while also producing an average increase of 49.5% in the HDL/LDL ratio. No adverse side effects were reported by any of the subjects throughout the 25-week study.35
A further study confirmed that 100 mg/day of rice bran-derived tocotrienols produced maximum decreases of serum total cholesterol (-20%), LDL-cholesterol (-25%), apolipoprotein B (carrier for LDL) (-14%) and triglycerides (-12%) compared with the baseline values in hypercholesterolemic subjects. Researchers concluded that a dose of 100 mg/day of tocotrienols derived from rice bran plus the AHA Step-1 diet could control the risk of coronary heart disease in hypercholesterolemic humans.36
How do tocotrienols lower cholesterol? The tocotrienols, primarily γ-tocotrienol, accelerate, via a post-transcriptional process, the rate at which HMGCoA reductase is degraded by 2.35-fold compared to control.37 Unlike statin drugs, which competitively inhibit and thus block HMG-CoA reductase activity (and therefore the production of other mevalonate-derived products, including ubiquinone [Coenzyme Q10]), tocotrienols simply increase the rate at which HMG-CoA reductase is degraded, thus allowing for normal, albeit shortened duration of the enzyme’s activity (and therefore some production of CoQ10).
Not only do tocotrienols, mainly the γ-isoform, significantly increase the rate at which HMG-CoA reductase is degraded, they also activate the conversion of LDL to HDL through the interphase VLDL–VDL, and finally, γ-tocotrienol or mixed tocotrienols increase the number of HDL, which then interact with LDL to reduce the concentration of LDL in the plasma.38
Tocotrienols’ neuroprotective actions31
Protection against glutamate toxicity and lipoxygenase activation via inhibition of c-Src kinase
Glutamate toxicity is a major contributor to neurodegeneration. It includes excitotoxicity and an oxidative stress component. Nanomolar concentrations of α-tocotrienol, not α-tocopherol, provide complete neuroprotection not only to neural cell lines, but also to primary cortical neurons, and not only against glutamate toxicity, but also against other insults such as homocysteic acid, glutathione deficiency, and linoleic acid (the omega-6 fatty acid precursor to arachidonic acid)-induced oxidative stress. Studies have revealed that at micromolar concentrations, tocotrienols protect neural cells via their antioxidant capabilities, while at nanomolar concentrations, tocotrienols regulate specific neurodegenerative signaling processes.31
Glutamate markedly enhances c-Src kinase activity. c-Src and the structurally related members of the Src family are associated with cell membranes and transduce signals involved in the death pathway from transmembrane receptors to the cell interior. c-Src is heavily expressed in the brain and in human neural tissues, and rapid c-Src activation plays a central role in executing neurodegeneration. Treatment with nanomolar amounts of a-tocotrienol completely prevents glutamate-induced death even in active c-Src kinase over-expressing cells.31
Lowered glutathione (GSH) levels, which can be induced by a variety of neurotoxins including glutamate, is an early marker of neurotoxicity. α-Tocotrienol strongly protects primary cortical neurons against a number of GSH-lowering neurotoxins, enabling the neurons to survive even in the face of GSH loss.
c-Src also catalyzes the phosphorylation of 12-lipoxygenase (12-Lox), which is greatly upregulated in glutamate-challenged neurons. By inhibiting c-Src activation, α-tocotrienol also prevents activation of 12-Lox, suppressing arachidonic acid metabolism. Neurons and the brain are rich in arachidonic acid, which is released in massive amounts from cell membranes in response to brain ischemia or trauma and can be metabolized into neurotoxic compounds.
The lipoxygenase pathway is the primary metabolic pathway of arachidonic acid in the brain. A decrease in GSH triggers the activation of neuronal 12-Lox, whose metabolism of arachidonic acid leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The 12-Lox metabolite 12-HPETE, specifically, has been shown to cause neuron death. 12-Lox has been also implicated in the pathogenesis of Alzheimer’s, and inhibition of 12-Lox has been shown to protect cortical neurons from β-amyloid induced toxicity. In sum, by inhibiting c-Src, α-tocotrienol prevents 12-Lox activation, lessens arachidonic acid metabolism, and promotes neuronal survival under GSH-depleted conditions.30
Tocotrienol-dependent inhibition of c-Src is beneficial not only for protecting neurons, but cardiac myocytes, since c-Src mediates post-ischemic cardiac injury and dysfunction. Tocotrienol supplementation has been shown to inhibit c-Src phosphorylation in cardiac myocytes and protect the heart.39
High c-Src is also tightly associated with carcinogenesis. Although no research has yet been published, it seems reasonable to postulate that tocotrienols’ c-Src inhibitory properties may also provide anti-cancer benefits.
α-Tocotrienol can restore fertility in α-tocopherol-deficient mice
Interestingly, in animal studies, infertility caused by α-tocopherol deficiency can be restored by tocotrienols. Vitamin E was initially discovered in rat studies when its presence in the animals’ diet was found to be essential to prevent fetal resorption. Soon afterwards, α-tocopherol was identified as the most bioactive form in rats. In humans, as well, α-tocopherol has the highest plasma concentration of the vitamin E isomers, due to the fact that tocopherol transport protein (TTP) has such a strong preference for α-tocopherol that it is often referred to as α-TTP.
Recent studies have reported that TTP-knockout female mice are infertile, even in the presence of dietary α-tocopherol, but that oral supplementation with α-tocotrienol restores fertility. This indicates that, despite the fact that TTP binds to α-tocotrienol with 8.5-fold lower affinity than that for α-tocopherol,31 not only is α-tocotrienol successfully delivered to the relevant tissues without TTP, but α-tocotrienol can functionally stand in for α-tocopherol.40
Ratio of vitamin E isomers found in nature is best absorbed, most effective
Research in which cells were pretreated with different mixtures of α-, β-, δ-, and γ-tocopherol before exposure to hydrogen peroxide found a mixture of γ-, δ-, and α-tocopherol with the ratio of 5:2:1 not only had a much better antioxidant effect than α- tocopherol alone but was better absorbed. The total uptake of tocopherol in cell membranes was significantly higher after the cells were incubated with the mixed tocopherol preparation than when the same concentration of α-tocopherol alone was used.41 This ratio of tocopherols is similar to that found in nature. For example, the tocopherol content in corn oil and soybean oil are 77% and 70% γ-tocopherol, 2% and 23% δ-tocopherol, and 14% and 7% of α-tocopherol, respectively.19
A similar ratio was seen in other research in which a mixed tocopherol preparation was found to be significantly more able to protect myocytes against hypoxia-reoxygenation injury than α-tocopherol alone. In these experiments, the mixed tocopherol preparation contained 62% γ-tocopherol, 25% δ-tocopherol, and 13% α-tocopherol, a ratio of isomers of ~6: 3: 1.42
One strategy for increasing the bioavailability of γ-tocopherol and α- and γ-tocotrienol is the inhibition of P450 xenobiotic metabolizing enzymes with sesame seeds or sesamin, the lignan found in sesame seeds. Studies show that dietary sesame seeds as well as sesamin lead to elevated levels of tissue and plasma γ-tocopherol due to the inhibition of CYP450 3A metabolism of the tocopherols and tocotrienols.22 43 44
In Palm oil, one of the most abundant natural sources of tocotrienols, the distribution of vitamin E isomers is 30% tocopherols and 70% tocotrienols.31 45 The use of moderate amounts of red palm oil in replacement of other fats in the diet of experimental animals (the equivalent of ~2 tablespoons daily in humans) has been shown to reduce blood pressure and risk of arterial thrombosis and atherosclerosis, inhibit endogenous cholesterol biosynthesis and platelet aggregation, and improve immune function.46
Like olive oil, palm oil contains predominantly oleic acid at the crucial sn2-position in its triacylglycerols, and as a result of this high monounsaturation, offers health benefits similar to those seen when olive oil is consumed in place of other fats. In addition to its high concentration of tocotrienols, red palm oil is rich in carotenes, a combination of nutrients that greatly enhances its oxidative stability and renders it a more healthful choice for cooking compared to other oils.47
Among the natural vitamin E molecules, RRR-α-tocopherol has the highest bioavailability and has been the standard against which all the others are compared. Current research investigating the potential therapeutic actions of the other vitamin E isomers, however, clearly indicates that these compounds both potentiate and balance the actions of α-tocopherol, which, in high doses, might otherwise be detrimental. Disappointments with outcomes-based studies investigating α-tocopherol are, in large part, the result of abstracting this single compound from the vitamin E family and ignoring the interplay among the eight isomers that constitute natural vitamin E. Best clinical outcomes will be achieved with vitamin E supplementation providing mixed tocopherols and tocotrienols in ratios similar to those found in nature.
Read Part I: In Defense of Vitamin E
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