What's Wrong With Vitamin E?
By Karin Granstrom Jordan, M.D.

If you asked a group of scientists to name an antioxidant, most would point to vitamin E as the classic example of a compound that inhibits dangerous free radicals.

While numerous studies show that vitamin E suppresses free radicals, there is evidence that commercial vitamin E supplements do not provide adequate antioxidant protection.

Most vitamin E supplements consist primarily of alpha tocopherol. Recent studies indicate that a lot more than alpha tocopherol is needed to protect against degenerative disease.

To obtain optimal health benefits from vitamin E, a mixture of tocopherols (alpha, beta, delta, and gamma) and tocotrienols (alpha, beta, delta, and gamma) are required. Some of the functions of these vitamin E fractions are similar while others are completely different. When taken together, these various forms of vitamin E work synergistically as a team to provide maximum benefits.

In this article, we discuss scientific findings supporting the value of the full spectrum of vitamin E that includes the tocopherols and tocotrienols.

The Tocotrienols

In 1995, Life Extension added a small amount of tocotrienols to a Coenzyme Q10 supplement used by most Life Extension Foundation members. Evidence at that time showed that tocotrienols could help protect against free radical-induced disease.

More recent research shows that tocotrienols may be the most important members of the vitamin E family. In an animal model of aging, tocotrienols extended lifespan by 19% while reducing protein carbonylation, a particularly toxic oxidation process indicative of aging.[1] Not only have tocotrienols demonstrated a superior antioxidant effect compared to alpha tocopherol (40-60 times more effective), but in a clinical study they have been shown to reverse carotid stenosis (narrowing of the carotid artery due to atherosclerosis), thus reducing the risk of stroke.

Tocotrienols have also been shown to reduce the level of LDL (the “bad” form of cholesterol) and apolipoprotein B, both of which are important risk factors for atherosclerosis and cardiovascular disease. Furthermore, tocotrienols have been shown to inhibit the growth of cancer cells. While tocotrienols are found in high concentration in palm oil and rice bran, palm-derived tocotrienols are better supported by research.

The difference in effect between tocopherols and tocotrienols is believed to be caused by a subtle difference in molecular structure. Tocotrienols have an isoprenoid instead of a phytyl side chain. Double bonds in the isoprenoid side chain allow tocotrienols to move freely and more efficiently within cell membranes than tocopherols, giving tocotrienols greater ability to catch and fight free radicals. This greater mobility also allows tocotrienols to recycle more quickly than alpha-tocopherol.

Alpha versus Gamma Tocopherol

Several large studies have shown great benefits of vitamin E intake in reducing cardiovascular disease and death from heart attacks, while others have failed to show similar results.[2-8] This discrepancy may well be due to the fact that only alpha-tocopherol was studied in isolation, while gamma-tocopherol and toco-trienols were not considered.

This may also explain why vitamin E as found in food is more effective than alpha-tocopherol supplements in reducing death from cardiovascular disease.[9-10] Food provides a broader spectrum of the vitamin E family than conventional supplements. For example, vitamin E in the typical American diet contains considerably more gamma-tocopherol than alpha-tocopherol[11] in contrast to supplements that generally contain only alpha-tocopherol, or insignificant amounts of gamma-tocopherol, tocotrienols and other members of the vitamin E family.

Moreover, studies indicate that high dose alpha-tocopherol supplementation considerably decreases the absorption of gamma-tocopherol and reduces the effects of tocotrienols. One group of scientists observed that when human volunteers (age 30-60) were given 1,200 IU of synthetic alpha-tocopherol daily for 8 weeks, plasma gamma-tocopherol decreased in all subjects to 30-50% of initial values.[12] This is another indication of the importance of a balanced
vitamin E intake.

A Swedish study found that patients with coronary heart disease had lower levels of gamma tocopherol and a higher alpha-to-gamma ratio than healthy age-matched subjects.[13]

While alpha-tocopherol has long been known as an important antioxidant, research has now shown that the complete vitamin E team is much more effective. The different vitamin E forms have complementary effects as free radical scavengers. Together they can fight a wider spectrum of free radicals than alpha-tocopherol alone.

One research group found that gamma-tocopherol is significantly more effective than alpha-tocopherol in inhibiting the powerful and harmful oxidizing agent peroxynitrite.[14] While alpha-tocopherol can to some extent inhibit free radical generation, gamma-tocopherol is able to trap and remove existing free radicals as well as highly toxic compounds such as peroxynitrite.[15] Gamma tocopherol can, therefore, protect cells against the mutagenic and carcinogenic effects of the very damaging reactive nitrogen species (See the antioxidant section).

Tocotrienols and Lifespan Extension

Recent experimental research confirms the connection between tocotrienols, reduced oxidative damage, and increased life span. Palm-derived tocotrienols were chosen for a study of the aging process at the Life Science Research Center in Japan.[16] The study was conducted on a model organism commonly used in anti-aging research, the nematode known as C. elegans (Caenorhabditis elegans). This species of worm is widely used in basic life science research due to the fact that is has genetic sequences similar to humans.

The study demonstrated that tocotrienols, but not alpha-tocopherol, significantly extended the average life span of the organisms. Nematodes exposed to a tocotrienol enriched (80ug/ml) growth medium lived 19% longer than the control group. A lower concentration (8ug/ml) of tocotrienols extended their average lifespan by 9%. When alpha-tocopherol was tested instead of tocotrienols it had no effect on lifespan. The study also examined carbonylated proteins, which are destructive products of protein oxidation that accumulate during aging in both nematodes and humans. In humans about a third of proteins become carbonylated in the latter third of life, leading to serious degenerative changes in the structures and regulatory systems of the body, including for example the wrinkling of skin. (For further discussion of protein carbonylation, see Carnosine article on page 24 in the January 2001 issue of Life Extension magazine).

Protein carbonyl accumulation in the nematodes was a mirror image of their survival curve, increasing from 1.1nmol/mg protein in young animals to 2.8nmol/mg in “old age” (15 days). In the nematodes treated with tocotrienols protein carbonyls rose about half as much during the course of aging, to only 1.9nmol/mg at age 15 days.

Ultraviolet (UV B) irradiation of the nematodes shortened their average life span by 12%. However, when tocotrienols were added to the medium prior to irradiation, the irradiated nematodes lived as long as the non-irradiated control group. Interestingly, their lifespan increased even more when tocotrienols were added soon after irradiation, and exceeded that of the non-irradiated group, indicating that tocotrienols are more than chain-breaking antioxidants, and are, in fact, capable of repairing damage that has already occurred. Alpha-tocopherol did not lend significant protection from irradiation.

Tocotrienols and Cardiovascular Disease

One of the most striking discoveries in tocotrienol research is their ability to clear atherosclerotic blockage (stenosis) in the carotid artery, giving them the potential to significantly reduce the risk of stroke. Stroke often occurs when atherosclerotic deposits travel upstream and cut off the blood supply to part of the brain.

Tocotrienols show promise as a natural and safe alternative to risky surgery for this condition because of their ability to reverse carotid stenosis, not merely stop its progression. This was demonstrated in a clinical trial testing the effect of tocotrienols on carotid atherosclerosis.[17] The results of this 18-month trial were remarkable.

Fifty patients with carotid stenosis were randomly assigned to receive either 160 mg daily of palm tocotrienols (gamma and alpha forms) with 64 mg of alpha-tocopherol in palm oil, or palm oil only as a placebo. After 6 months the dosage in the treatment group was increased to 240 mg of tocotrienols with 96 mg of alpha-tocopherol.

At the end of the study, ultrasound scans of the carotid artery demonstrated that none of the patients in the control group had improved during the trial, while ten showed a worsening of their condition (increased stenosis). In the tocotrienol group, however, atherosclerosis was reduced and blood flow to the brain improved in 7 of 25 patients, while the condition had worsened in only two patients. No adverse side effects were reported in either group.

Tocotrienols and statin drugs such as lovastatin both lower cholesterol by suppressing the activity of the enzyme HMG-CoA reductase, although through different mechanisms. The statins are thought to affect the enzyme through competitive inhibition, while the tocotrienols accelerate enzyme degradation and decrease the efficiency of mRNA translation of the enzyme.[18] This difference in mechanism is believed to be a reason for the absence of adverse side effects with tocotrienols, contrary to the common side effects of the statin drugs.

HMG-CoA reductase is the enzyme that permits the body to synthesize its own cholesterol from a precursor called mevalonate. The mevalonate pathway is also of great importance in regulating cell growth and proliferation. The ability of tocotrienols to inhibit this pathway, therefore, enables them to inhibit cancer growth (see more in the cancer section).

Some studies have demonstrated a significant reduction of both total and LDL cholesterol with tocotrienols administered to patients with high serum lipids. In a double blind, crossover study on 25 patients with high cholesterol levels, the patients in the treatment group were given 4 capsules daily of 50 mg tocotrienols mixed with palm oil, while the control group received only corn oil. At the end of the 8-week trial period, total cholesterol and LDL cholesterol had decreased significantly (15% and 8%) in the 15 subjects given the palm tocotrienols. There was no change in the control group.[19]

Total cholesterol and LDL-cholesterol were reduced even more (17 % and 24 % respectively) when tocotrienols were added to a low fat, low cholesterol diet and alcohol-free regimen in another double-blind, longer-lasting trial (12 weeks).[20] Other important cardiovascular risk factors were reduced by tocotrienols. Apoli-poprotein B and lipoprotein(a), strong predictors of cardiovascular disease[21-23], as well as thromboxane B2 and platelet factor 4 were all significantly lowered in the tocotrienol-treated group (15%, 17%, 31% and 14% respectively).

Thromboxane B2 contributes to cardiovascular disease through proinflammatory activities and platelet aggregation. It is formed from pro-inflammatory prostaglandins through the function of the enzyme cyclooxygenase (COX-2), which is known to be involved in the development of both inflammatory and neoplastic (cancerous) disease. The significant reduction (31%) of thromboxane B2 in this tocotrienol study is interesting, suggesting possible similarities with gamma-tocopherol,which is known to be a COX-2 inhibitor.[24]

What is a Free Radical? Free radicals are the products of oxidative reactions in the body. They are highly reactive compounds that take electrons from other molecules to stabilize themselves. In this process of electron "theft", a new free radical is created, namely, the molecule from which the electron was taken. That new free radical then practices "theft" on another nearby molecule, and a chain-reaction cycle of cell destruction begins. It is important to realize that oxidation is a normal part of life as are free radicals. Oxidation is what enables us to get and use energy from our food. When free radicals are produced in excess, however, they are so damaging that the body maintains a sophisticated antioxidant system to hold them in check. However, when the body's prolonged exposure to oxidative factors causes an excessive output of free radicals that exceeds the body's ability to neutralize them (technically called "oxidative stress"), the body is put in an increasingly vulnerable position due to accelerated cell destruction. Antioxidants are substances that neutralize free radicals.

While both alpha and gamma-tocopherol have been shown to reduce platelet aggregation and delay thrombus formation, gamma-tocopherol was shown to be significantly more potent in a study on rats.[25]

Tocotrienols were studied in combination with the statin drug lovastatin in another study. The 28 patients with elevated cholesterol levels in this double blind, cross-over clinical trial were placed on the American Heart Association Step-1 diet before beginning the treatment. After 35 days on the diet, they were given low doses of lovastatin, tocotrienols and alpha-tocopherol (and combinations of these agents) in stages of 35 days each, while staying on the diet. The combination of lovastatin (10mg) and palm tocotrienols (50mg) had a lipid-lowering effect of 20-25%, while tocotrienols or lovastatin alone in the same dosages reduced LDL-cholesterol 18% and 15% respectively. No side effects were reported during the study. It is important to note that dosages of cholesterol-lowering drugs should not be reduced on the basis of this preliminary study.[26]

Supplementation with gamma-tocotrienol alone, or in combination with alpha-tocopherol, to rats fed a diet rich in fat for 6 weeks, showed a significant reduction in total and LDL cholesterol, triglycerides and reactive oxidation products, particularly hydroper-oxides.[27] The powerful antioxidant effects of tocotrienols will be discussed later in this article.

Tocotrienols and Cancer

Tocotrienols possess the ability to stimulate the killing of cancer cells selectively through programmed cell death (apoptosis) in order to reduce cancer cell proliferation,while leaving normal cells unaffected.[28]

One of the mechanisms by which tocotrienols are thought to suppress cancer is related to the isoprenoid side chain that makes them different from tocopherols. Isoprenoids are plant compounds that have suppressed the initiation, growth and progression of many types of cancer in experimental studies.[29-31] They are common in fruits and vegetables, which may explain why diets rich in these foods have consistently been shown to reduce the incidence of cancer.[32-34]

The anti-proliferative effect of isoprenoids is thought to be due to suppression of the mevalonate pathway, through which mutated ras proteins transform healthy cells into cancer cells. Mutated ras is the most common cellular defect found in human cancers. The mevalonate pathway escapes regulatory control in tumor tissue, but remains highly sensitive to regulation by tocotrienols. Tocotrienols are at least 5 times more powerful than farnesol, the body’s regulator of the mevalonate pathway.[33]

An early study demonstrated that long-term supplementation with palm-derived tocotrienols reduced the impact of carcinogens in rats.[35] After exposure to liver toxins, one group of rats was fed a diet supplemented with toco-trienols (30mg/kg food), while another group was given a standard diet. All 10 rats on the standard diet that were exposed to the liver toxins developed liver cancer during the 9-month-long study. In contrast, only one of the 6 rats supplemented with tocotrienols developed cancer nodules. Tocotrienol supplementation also significantly reduced the plasma level of GGT (gamma-glutamyl-transpeptidase), a marker enzyme for liver cancer.[35-36]

Another animal study demonstrated the effectiveness of tocotrienols in suppressing established tumors. When mice were given feed supplemented with gamma-tocotrienol (7umol/day), established melanoma tumors were significantly suppressed and the survival time of the hosts increased by 35%. Doubling of the dose did not further increase the duration of survival. Alpha-tocopherol had only a marginal effect.[37]

Tocotrienols and Breast Cancer

Interestingly, human breast cancer cells have been shown to respond very well to treatment with tocotrienols.[38-44]

While most breast cancers are believed to be estrogen dependent, some tumors, particularly postmenopausal tumors, do not depend upon estrogen for their growth. Anti-estrogen drugs, such as the widely used tamoxifen, are most effective on hormone sensitive tumors. The use of tamoxifen is also limited by the development of resistance to this drug in many patients.[45] Tocotrienols provide growth inhibition of breast cancer cells in culture that is independent of estrogen sensitivity, and have great potential to be a significant aid in the prevention and treatment of breast cancer.

A number of in vitro studies have demonstrated the effectiveness of tocotrienols as inhibitors of both estrogen receptor-positive (estrogen responsive) and estrogen receptor-negative (non-estrogen responsive) cell proliferation.

Researchers tested the effect of palm tocotrienols on three different cell lines of estrogen responsive and estrogen non-responsive human breast cancer cells (MCF7, MDA-MB-231 and ZR-75-1). They found that tocotrienols inhibited cell growth strongly in both the presence and absence of estradiol, the major estrogen in the body. The researchers also demonstrated that tocotrienols enhanced the effect of tamoxifen. The gamma- and delta-fractions of tocotrienols were most effective at inhibiting cell growth, while alpha-tocopherol was ineffective in doing so.[38-40]

Among the tocotrienols, delta-tocotrienol was shown in another study to be the most potent inducer of apoptosis (programmed cell death) in both estrogen-responsive and estrogen non-responsive human breast cancer cells, followed by gamma and alpha-tocotrienol (beta-tocotrienol was not tested). Interestingly, delta-tocotrienol is more plentiful in palm tocotrienols than in tocotrienols derived from rice. Of the natural tocopherols, only delta-tocopherol showed any apoptosis-inducing effect, although it was less than a tenth of the effect of palm and rice delta-tocotrienol.[42]

Similar results were obtained when mammary cancer cells from mice were studied.[44] While tocopherols had no inhibitory effect on cancerous cell growth, alpha, gamma, and delta-tocotrienols effectively arrested the cell cycle and triggered cell death. Highly malignant cells were most sensitive to the anti-proliferative effects of tocotrienols, whereas less aggressive pre-cancerous cells were the least sensitive.

Tocotrienols were found to be far more effective than alpha-tocopherol in inhibiting breast cancer cell growth.[41] The tocotrienol concentration needed was less than 1/20 of alpha-tocopherol in estrogen responsive cells and less than 1/10 in cells unresponsive to estrogen. Tocotrienols in combination with tamoxifen were more inhibitory than either compound alone in both estrogen responsive and non-responsive breast cancer cells. The authors pointed out that the synergism between tamoxifen and tocotrienols may allow for the use of lower doses of tamoxifen, and reduce its risk of adverse side effects. It is important to note that further studies are needed before tocotrienols can be used safely in combination with any cancer therapy.

Gamma Tocopherol and Prostate Cancer

While alpha-tocopherol has proven to be effective in inhibiting the growth of prostate cancer cells, gamma-tocopherol has been found to be more effective. In a study comparing the inhibitory effect of synthetic alpha-tocopherol and natural gamma-tocopherol on prostate cancer cell growth, it was demonstrated that gamma-tocopherol inhibited cell growth at concentrations 1,000 times lower then synthetic alpha-tocopherol.[46]

One recent study explored the association of alpha-tocopherol, gamma-tocopherol and selenium with prostate cancer. Blood samples were examined from 117 men who had developed prostate cancer and from 233 matched controls. Higher levels of gamma-tocopherol were associated with significantly lower prostate cancer risk. Men in the highest quintile of gamma-tocotrienol levels had a five-fold reduction in the risk of developing prostate cancer compared to men in the lowest quintile. Significant protection by high levels of selenium and alpha-tocopherol was observed only when gamma-tocopherol concentrations were high.[47]

The Super Antioxidant

Much of the broad involvement of vitamin E in human metabolism is due to its role as the body’s primary lipid-soluble antioxidant. Tocopherols and tocotrienols are part of the body’s highly effective defense system, without which life as we know it could not exist. This defense system consists of a network of antioxidants, interacting with and supporting each other. Antioxidants such as vitamin C, coenzyme Q10 and glutathione are needed for effective recycling of tocopherols and tocotrienols.

What is a Free Radical?

Free radicals are the products of oxidative reactions in the body. They are highly reactive compounds that take electrons from other molecules to stabilize themselves. In this process of electron “theft”, a new free radical is created, namely, the molecule from which the electron was taken. That new free radical then practices “theft” on another nearby molecule, and a chain-reaction cycle of cell destruction begins.

It is important to realize that oxidation is a normal part of life as are free radicals. Oxidation is what enables us to get and use energy from our food. When free radicals are produced in excess, however, they are so damaging that the body maintains a sophisticated antioxidant system to hold them in check.

However, when the body’s prolonged exposure to oxidative factors causes an excessive output of free radicals that exceeds the body’s ability to neutralize them (technically called “oxidative stress”), the body is put in an increasingly vulnerable position due to accelerated cell destruction.

Antioxidants are substances that neutralize free radicals.

The unique power of both tocopherols and tocotrienols is their ability to break the chain reaction of lipid peroxidation by neutralizing peroxyl radicals to prevent the spread of free radical damage in cell membranes. Tocotrienols are more potent scavengers of the peroxyl radical than alpha-tocopherol and, as we shall see below, provide far better protection against lipid peroxidation. Peroxidation of fatty acids (lipids) in cell membranes has a great impact on both cellular structure and function. Peroxidation of LDL-cholesterol, for example, is known to be the first step in the development of atherosclerosis.[48-49]

Lipid peroxidation is destructive, because lipids are an essential part of cell membranes, hormones and nerve tissue. The damage itself initiates a chain reaction of free radical generation. Vulnerable polyunsaturated fatty acids generate peroxyl radicals, which not only damage lipids, but also damage important proteins responsible for most daily functions in humans.

The efficiency of the various vitamin E members is not equal, however. While gamma-tocopherol is a more effective antioxidant than alpha-tocopherol, particularly in reducing damage from nitrogen radicals[50-51], tocotrienols have proven to be even more powerful than tocopherols. The greater antioxidant effect of delta-tocotrienol compared to alpha-tocopherol is thought to be due to its molecular structure, more uniform distribution in cell membranes, greater recycling activity, and more effective collision with free radicals.[52]

In one study, the efficacy of alpha-tocotrienol was 40 times higher than alpha-tocopherol in protecting rat liver microsomal membranes against lipid peroxidation and 6.5 times higher in protecting cytochrome P-450 against oxidative damage.[53] Cytochrome P-450 is a system of enzymes that play a central role in the detoxification of both exogenous (such as drugs and pesticides) and endogenous (such as hormones) compounds and in the synthesis of steroid hormones and bile acids in the liver.

A follow-up study demonstrated that tocotrienols protect against injury from ischemia and reperfusion (interruption and resumption of blood flow) in isolated rat hearts[54]. A mixture of tocotrienols (55%) and tocopherols (45%) from palm oil was used in this study.

Following 40 minutes of ischemia, alpha-tocotrienol was more active in free radical scavenging than alpha-tocopherol and was preferentially consumed. The recycling efficiency of alpha-tocotrienol was also higher than alpha-tocopherol, which may be one reason for its significantly higher physiological activity under oxidative stress.

An in vitro rat brain study[55] confirmed the superiority of tocotrienols as inhibitors of lipid peroxidation. The study also demonstrated that tocotrienols at low dosage can inhibit protein oxidation in brain mitochondria. Palm tocotrienols were significantly more effective than alpha-tocopherol in this study. Gamma-tocotrienol had the strongest inhibitory effect, while alpha- and delta-tocotrienols were less effective. These results suggest that palm tocotrienols may be helpful in preventing neurodegenerative disorders caused by oxidative stress. Clinical studies are eagerly awaited.

Another study on rat liver microsomes demonstrated the ability of palm tocotrienols to protect cell membranes from oxidative damage. Gamma-tocotrienol again was the most effective. At the low concentration of 5 uM, palm tocotrienols significantly inhibited protein oxidation (37%) and lipid peroxidation (27-30%).[56]

Nitrogen radicals, originating from nitric oxide (NO), cause severe damage to the body. Nitric oxide is an important signaling molecule produced in many tissues, including the blood vessel lining (endothelium) and the brain. It regulates a diverse range of physiological processes. When superoxide and NO combine, however, one of the most toxic radicals in the human body, peroxynitrite, is formed.

Gamma-tocopherol and gam-ma-tocotrienol are the vitamin E isoforms that have been found most effective in reducing damage from nitrogen radicals. In contrast to alpha-tocopherol, gamma-tocopherol has the ability to scavenge nitrogen dioxide without forming toxic nitrogen products, and was found to be a more effective inhibitor of cancerous transformation of cells.[57] Gamma-tocopherol is also significantly more effective than alpha-tocopherol in inhibiting peroxynitrite-induced lipid peroxidation. Another team of scientists demonstrated that gamma-tocopherol is required to remove peroxynitrite-derived toxic products, despite the antioxidant action of alpha-tocopherol.[51] This is an important discovery as peroxynitrite is one of the major damaging oxidants produced in humans. Its formation is particularly associated with ischemic injuries, inflammation and neurodegenerative disorders. The authors suggest that the presence of both tocopherols may be required in vivo for optimal protection against nitrogen radicals.

Indirect support for this argument can be found in a study showing that plasma levels of gamma-tocopherol (but not alpha-tocopherol) rapidly increase when long-term smokers stop smoking. This suggests that mainly gamma-tocopherol is consumed in combating free radicals produced by smoking. High doses of alpha-tocopherol have also been shown to displace gamma-tocopherol in plasma and other tissues.[58]

Tocotrienols and Hypertension

An important factor in hypertension and congestive heart failure is the body’s pool of extra- cellular fluid. Scientists have for decades searched for the hormone in the body that controls the release of excess water and thereby reduces high blood pressure. In 1996 a compound with this effect was isolated, LLU-alpha, which proved to be a metabolite of gamma-tocopherol.[59] Last year animal studies indicated that LLU-alpha also is produced from gamma-tocotrienol.[60-61]

Hypertension has also been associated with elevated lipid peroxide levels (see the antioxidant section) both in animals and humans. In a study of tocotrienols in spontaneously hypertensive rats[62], it was demonstrated that treatment with gamma-tocotrienol prevented the development of age-related hypertension by scavenging free radicals and enhancing the body’s enzymatic antioxidant defense system. Tocotrienols reduced lipid peroxidation in blood vessels and significantly increased the activity of the antioxidant superoxide dismutase (SOD).

The radical scavenging effect of tocotrienols may affect blood pressure in other ways than through reduced lipid peroxidation. Earlier studies showed that free radicals can inactivate nitric oxide (NO) to impair vasodilatation, which leads to an increase of peripheral resistance and blood pressure. We look forward to further research in this area.

The Need for Full Spectrum Vitamin E

Vitamin E has an excellent safety record.[63-66] However, studies of alpha-tocopherol alone, without the mix of other tocopherols and tocotrienols, has shown pro-oxidant rather than antioxidant activity in people consuming high doses (over 1000 mg).[67]

We have seen that the various vitamin E forms have their unique role in the metabolism of the human body. Research strongly suggests that we need the full spectrum of vitamin E to maximize our chances of preventing and, possibly, treating many of the diseases of aging.

References with Abstracts: What's Wrong with Vitamin E

[1-10] [11-20] [21-30] [31-40] [41-50] [51-60] [61-67]

1. J Gerontol A Biol Sci Med Sci 2000 Jun;55(6):B280-5
Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans.
Adachi H, Ishii N.
Life Science Research Center, Lion Corporation, Kanagawa, Japan. hadachi@lion.co.jp

To assess the efficiency of tocotrienols against oxidative damage, we have demonstrated in a model-system nematode, Caenorhabditis elegans, that tocotrienol administration reduced the accumulation of protein carbonyl (a good indicator of oxidative damage during aging) and consequently extended the mean life span (LS), but not the maximum LS. Conversely, alpha-tocopherol acetate did not affect these parameters. As a way to evaluate the protective ability of tocotrienols against oxidative stress, the life spans of animals administrated tocotrienols before or after exposure to ultraviolet B-induced oxidative stress were measured. Ultraviolet B irradiation shortened the mean LS of animals, whereas preadministration of tocotrienols recovered the mean LS to that of unirradiated animals. Interestingly, postadministration also extended the mean LS more than that of unirradiated animals, and administration through the LS conferred greater protection. Thus, the administration of tocotrienols to animals results in a reduction of oxidative stress risks. These data indicated that tocotrienols merit further investigation as possible agents for antiaging and oxidative stress prevention. In addition, they suggest that C. elegans will continue to provide provocative clues into the mechanisms of aging.

2. N Engl J Med 1993 May 20;328(20):1450-6
Vitamin E consumption and the risk of coronary heart disease in men.
Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC.
Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115.

BACKGROUND. The oxidative modification of low-density lipoproteins increases their incorporation into the arterial intima, an essential step in atherogenesis. Although dietary antioxidants, such as vitamin C, carotene, and vitamin E, have been hypothesized to prevent coronary heart disease, prospective epidemiologic data are sparse. METHODS. In 1986, 39,910 U.S. male health professionals 40 to 75 years of age who were free of diagnosed coronary heart disease, diabetes, and hypercholesterolemia completed detailed dietary questionnaires that assessed their usual intake of vitamin C, carotene, and vitamin E in addition to other nutrients. During four years of follow-up, we documented 667 cases of coronary disease. RESULTS. After controlling for age and several coronary risk factors, we observed a lower risk of coronary disease among men with higher intakes of vitamin E (P for trend = 0.003). For men consuming more than 60 IU per day of vitamin E, the multivariate relative risk was 0.64 (95 percent confidence interval, 0.49 to 0.83) as compared with those consuming less than 7.5 IU per day. As compared with men who did not take vitamin E supplements, men who took at least 100 IU per day for at least two years had a multivariate relative risk of coronary disease of 0.63 (95 percent confidence interval, 0.47 to 0.84). Carotene intake was not associated with a lower risk of coronary disease among those who had never smoked, but it was inversely associated with the risk among current smokers (relative risk, 0.30; 95 percent confidence interval, 0.11 to 0.82) and former smokers (relative risk, 0.60; 95 percent confidence interval, 0.38 to 0.94). In contrast, a high intake of vitamin C was not associated with a lower risk of coronary disease. CONCLUSIONS. These data do not prove a causal relation, but they provide evidence of an association between a high intake of vitamin E and a lower risk of coronary heart disease in men. Public policy recommendations with regard to the use of vitamin E supplements should await the results of additional studies.

3. N Engl J Med 1993 May 20;328(20):1444-9
Vitamin E consumption and the risk of coronary disease in women.
Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC.
Channing Laboratory, Boston, MA 02115.

BACKGROUND. Interest in the antioxidant vitamin E as a possible protective nutrient against coronary disease has intensified with the recognition that oxidized low-density lipoprotein may be involved in atherogenesis. METHODS. In 1980, 87,245 female nurses 34 to 59 years of age who were free of diagnosed cardiovascular disease and cancer completed dietary questionnaires that assessed their consumption of a wide range of nutrients, including vitamin E. During follow-up of up to eight years (679,485 person-years) that was 97 percent complete, we documented 552 cases of major coronary disease (437 nonfatal myocardial infarctions and 115 deaths due to coronary disease). RESULTS. As compared with women in the lowest fifth of the cohort with respect to vitamin E intake, those in the top fifth had a relative risk of major coronary disease of
0.66 (95 percent confidence interval, 0.50 to 0.87) after adjustment for age and smoking. Further adjustment for a variety of other coronary risk factors and nutrients, including other antioxidants, had little effect on the results. Most of the variability in intake and reduction in risk was attributable to vitamin E consumed as supplements. Women who took vitamin E supplements for short periods had little apparent benefit, but those who took them for more than two years had a relative risk of major coronary disease of 0.59 (95 percent confidence interval, 0.38 to 0.91) after adjustment for age, smoking status, risk factors for coronary disease, and use of other antioxidant nutrients (including multi-vitamins). CONCLUSIONS. Although these prospective data do not prove a cause-and-effect relation, they suggest that among middle-aged women the use of vitamin E supplements is associated with a reduced risk of coronary heart disease. Randomized trials of vitamin E in the primary and secondary prevention of coronary disease are being conducted; public policy recommendations about the widespread use of vitamin E should await the results of these trials.

4. Lancet 1996 Mar 23;347(9004):781-6
Randomised controlled trial of vitamin E in patients with coronary disease:
Cambridge Heart Antioxidant Study (CHAOS)
Stephens NG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ.
Department of Medicine, Cambridge University.

BACKGROUND: Vitamin E (alpha-tocopherol) is thought to have a role in prevention of atherosclerosis, through inhibition of oxidation of low-density lipoprotein. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of alpha-tocopherol and lower rates of ischaemic heart disease. We tested the hypothesis that treatment with a high dose of alpha-tocopherol would reduce subsequent risk of myocardial infarction (MI) and cardiovascular death in patients with established ischaemic heart disease. METHODS: In this double-blind, placebo-controlled study with stratified randomisation, 2002 patients with angiographically proven coronary atherosclerosis were enrolled and followed up for a median of 510 days (range 3-981). 1035 patients were assigned alpha-tocopherol (capsules containing 800 IU daily for first 546 patients; 400 IU daily for remainder); 967 received identical placebo capsules. The primary endpoints were a combination of cardiovascular death and non-fatal MI as well as non-fatal MI alone. FINDINGS: Plasma alpha-tocopherol concentrations (measured in subsets of patients) rose in the actively treated group (from baseline mean 34.2 micromol/L to 51.1 micromol/L with 400 IU daily and 64.5 micromol/L with 800 IU daily) but did not change in the placebo group. Alpha-tocopherol treatment significantly reduced the risk of the primary trial endpoint of cardiovascular death and non-fatal MI (41 vs 64 events; relative risk 0.53 [95% Cl 0.34-0.83; p=0.005). The beneficial effects on this composite endpoint were due to a significant reduction in the risk of non-fatal MI (14 vs 41; 0.23 [0.11-0.47]; p=0.005); however, there was a non-significant excess of cardiovascular deaths in the alpha-tocopherol group (27 vs 23; 1.18 [0.62-2.27]; p=0.61). All-cause mortality was 36 of 1035 alpha-tocopherol-treated patients and 27 of 967 placebo recipients. INTERPRETATION: We conclude that in patients with angiographically proven symptomatic coronary atherosclerosis, alpha-tocopherol treatment substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment. The effect of alpha-tocopherol treatment on cardiovascular deaths requires further study.

5. JAMA 2001 Mar 7;285(9):1178-82
Effects of vitamin E on lipid peroxidation in healthy persons.
Meagher EA, Barry OP, Lawson JA, Rokach J, FitzGerald GA.
Center for Experimental Therapeutics, 811 Biomedical Research Bldg II/III,
University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, USA.

CONTEXT: Oxidative stress may play a role in the development or exacerbation of many common diseases. However, results of prospective controlled trials of the effects of antioxidants such as vitamin E are contradictory. OBJECTIVE: To assess the effects of supplemental vitamin E on lipid peroxidation in vivo in healthy adults. DESIGN: Randomized, double-blind, placebo-controlled trial conducted March 1999 to June 2000. SETTING: A general clinical research center in a tertiary referral academic medical center. PARTICIPANTS: Thirty healthy men and women aged 18 to 60 years. INTERVENTIONS: Participants were randomly assigned to receive placebo or alpha-tocopherol dosages of 200, 400, 800, 1200, or 2000 IU/d for 8 weeks (n = 5 in each group), followed by an 8-week washout period. MAIN OUTCOME MEASURES: Three indices of lipid peroxidation, urinary 4-hydroxynonenal (4-HNE) and 2 isoprostanes, iPF(2alpha)-III and iPF(2alpha)-VI, measured by gas chromatography/mass spectrometry and compared among the 6 groups at baseline, 2, 4, 6, and 8 weeks, and 1, 3, and 8 weeks after discontinuation. RESULTS: Circulating vitamin E levels increased in a dose-dependent manner during the study. No significant effect of vitamin E on levels of urinary 4-HNE or either isoprostane was observed. Mean (SEM) baseline vs week 8 levels of iPF(2alpha)-III were 154 (20.1) vs 168 (22.3) pg/mg of creatinine for subjects taking placebo; 165 (19.6) vs 234 (30.1) pg/mg for those taking 200 IU/d of vitamin E; and 195 (26.7) vs 213 (40.6) pg/mg for subjects taking 2000 IU/d. Corresponding iPF(2alpha)-VI levels were 1.43 (0.6) vs 1.62 (0.4) ng/mg of
creatinine for subjects taking placebo; 1.64 (0.3) vs 1.24 (0.8) ng/mg for those taking 200 IU/d of vitamin E; and 1.83 (0.3) vs 1.94 (0.9) ng/mg for those taking 2000 IU/d. Baseline vs week 8 levels of 4-HNE were 0.5 (0.04) vs 0.4 (0.05) ng/mg of creatinine for subjects taking placebo; 0.4 (0.06) vs 0.5 (0.02) ng/mg with 200 IU/d of vitamin E; and 0.2 (0.02) vs 0.2 (0.1) ng/mg with 2000 IU/d. CONCLUSIONS: Our results question the rationale for vitamin E supplementation in healthy individuals. Specific quantitative indices of oxidative stress in vivo should be considered as entry criteria and for dose selection in clinical trials of antioxidant drugs and vitamins in human disease.

6. JAMA 1995 Jun 21;273(23):1849-54
Serial coronary angiographic evidence that antioxidant vitamin intake reduces progression of coronary artery atherosclerosis.
Hodis HN, Mack WJ, LaBree L, Cashin-Hemphill L, Sevanian A, Johnson R, Azen SP.
Atherosclerosis Research Unit, University of Southern California School of Medicine, Los Angeles 90033, USA.

OBJECTIVE--To explore the association of supplementary and dietary vitamin E and C intake with the progression of coronary artery disease. DESIGN--A subgroup analysis of the on-trial antioxidant vitamin intake database acquired in the Cholesterol Lowering Atherosclerosis Study, a randomized, placebo-controlled, serial angiographic clinical trial evaluating the risk and benefit of colestipol-niacin on coronary artery disease progression. SETTING--Community-and university-based cardiac catheterization laboratories. SUBJECTS--A total of 156 men aged 40 to 59 years with previous coronary artery bypass graft surgery. INTERVENTION--Supplementary and dietary vitamin E and C intake (nonrandomized) in association with cholesterol-lowering diet and either colestipol-niacin or placebo (randomized). OUTCOME--Change per subject in the percentage of vessel diameter obstructed because of stenosis (%S) determined by quantitative coronary angiography after 2 years of randomized therapy on all lesions, mild/moderate lesions (< 50%S), and severe lesions (> or = 50%S). RESULTS--Overall, subjects with supplementary vitamin E intake of 100 IU per day or greater demonstrated less coronary artery lesion progression than did subjects with supplementary vitamin E intake less than 100 IU per day for all lesions (P = .04) and for mild/moderate lesions (P = .01). Within the drug group, benefit of supplementary vitamin E intake was found for all lesions (P = .02) and mild/moderate lesions
(P = .01). Within the placebo group, benefit of supplementary vitamin E intake was not found. No benefit was found for use of supplementary vitamin C exclusively or in conjunction with supplementary vitamin E, use of multivitamins, or increased dietary intake of vitamin E or vitamin C. CONCLUSIONS--These results indicate an association between supplementary vitamin E intake and angiographically demonstrated reduction in coronary artery lesion progression. Verification from carefully designed, randomized, serial arterial imaging end point trials is needed.

7. Eur Heart J 2001 Jan;22(2):103-4
Clinical, public health, and research implications of the Heart Outcomes
Prevention Evaluation (HOPE) Study.
Yusuf S.

8. Am J Clin Nutr 1996 Mar;63(3):377-85
Inverse relation between the concentration of low-density-lipoprotein vitamin E and severity of coronary artery disease.
Regnstrom J, Nilsson J, Moldeus P, Strom K, Bavenholm P, Tornvall P, Hamsten A.
Department of Medicine, the King Gustaf V Research Institute, Karolinska Hospital, Stockholm, Sweden.

Oxidation of low-density lipoprotein (LDL) is believed to play an important role in atherogenesis, and antioxidant vitamins are thought to protect against coronary artery disease (CAD). We investigated whether the vitamin E concentrations in serum and LDL were associated with the severity of CAD as assessed by a semiquantitative scoring system in which coronary angiograms are analyzed for the number and size of distinct stenotic lesions (global stenosis score). The study group consisted of 64 consecutive male survivors of myocardial infarction aged < 45 y. Lipid-adjusted serum and LDL vitamin E concentrations were significantly lower in the patients than in 35 age-matched male control subjects, whereas the absolute serum and LDL vitamin E concentrations did not differ significantly. No associations were found between the serum concentration or lipid-adjusted serum values of vitamin E and the stenosis score. In contrast, significant inverse correlation was found between the LDL vitamin E concentration, whether adjusted to the lipid (r=-0.477,P<0.001) or protein (r=-0.375, P<0.01) content of LDL, and the global coronary stenosis score. We conclude that a low LDL vitamin E concentration might play a role in the development of stenoses in coronary arteries and may contribute to clinically manifest CAD.

9. N Engl J Med 1996 May 2;334(18):1156-62
Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women.
Kushi LH, Folsom AR, Prineas RJ, Mink PJ, Wu Y, Bostick RM.
Division of Epidemiology, University of Minnesota School of Public Health,
Minneapolis 55454-1015, USA.

BACKGROUND: The role of dietary antioxidant vitamins in preventing coronary heart disease has aroused considerable interest because of the knowledge that oxidative modification of low-density lipoprotein may promote atherosclerosis. METHODS. We studied 34,486 postmenopausal women with no cardiovascular disease who in early 1986 completed a questionnaire that assessed, among other factors, their intake of vitamins A, E, and C from food sources and supplements. During approximately seven years of follow-up (ending December 31, 1992), 242 of the women died of coronary heart disease. RESULTS. In analyses adjusted for age and dietary energy intake, vitamin E consumption appeared to be inversely associated with the risk of death from coronary heart disease. This association was particularly striking in the subgroup of 21,809 women who did not consume vitamin supplements (relative risks from lowest to highest quintile of vitamin E intake, 1.0, 0.68, 0.71, 0.42, and 0.42; P for trend 0.008). After adjustment for possible confounding variables, this inverse association remained (relative risks from lowest to highest quintile, 1.0, 0.70, 0.76, 0.32, and 0.38; P for trend, 0.004). There was little evidence that the intake of vitamin E from supplements was associated with a decreased risk of death from coronary heart disease, but the effects of high-dose supplementation and the duration of supplement use could not be definitely addressed. Intake of vitamins A and C did not appear to be associated with the risk of death form coronary heart disease. CONCLUSIONS. These results suggest that in postmenopausal women the intake of vitamin E from food is inversely associated with the risk of death from coronary heart disease and that such women can lower their risk without using vitamin supplements. By contrast, the intake of vitamins A and C was not associated with lower risks of dying from coronary disease.

10. Am J Epidemiol 1994 Jun 15;139(12):1180-9
Antioxidant vitamin intake and coronary mortality in a longitudinal population study.
Knekt P, Reunanen A, Jarvinen R, Seppanen R, Heliovaara M, Aromaa A.
Social Insurance Institution, Helsinki, Finland.

Oxidation of lipoproteins is hypothesized to promote atherosclerosis and, thus, a high intake of antioxidant nutrients may protect against coronary heart disease. The relation between the intakes of dietary carotene, vitamin C, and vitamin E and the subsequent coronary mortality was studied in a cohort of 5,133 Finnish men and women aged 30-69 years and initially free from heart disease. Food consumption was estimated by the dietary history method covering the total habitual diet during the previous year. Altogether, 244 new fatal coronary heart disease cases occurred during a mean follow-up of 14 years beginning in 1966-1972. An inverse association was observed between dietary vitamin E intake and coronary mortality in both men and women with relative risks of 0.68 (p for trend = 0.01) and 0.35 (p for trend < 0.01), respectively, between the highest and lowest tertiles of the intake. Similar associations were observed for the dietary intake of vitamin C and carotenoids among women and for the intake of important food sources of these micronutrients, i.e., of vegetables and fruits, among both men and women. The associations were not attributable to confounding by major nondietary risk factors of coronary heart disease, i.e., age, smoking, serum cholesterol, hypertension, or relative weight. The results support the hypothesis that antioxidant vitamins protect against coronary heart disease, but it cannot be excluded that foods rich in these micronutrients also contain other constituents that provide the protection.

11. Sources And Consumption Of Antioxidants In The Diet
Bieri J G
J Am Oil Chem Soc 61 (12). 1984. 1917-1918. 1984

12. J Nutr 1985 Jun;115(6):807-13
Oral alpha-tocopherol supplements decrease plasma gamma-tocopherol levels in humans.
Handelman GJ, Machlin LJ, Fitch K, Weiter JJ, Dratz EA.

In a cross-sectional survey of 86 elderly persons, it was observed that subjects with elevated plasma alpha-tocopherol levels had depressed plasma gamma-tocopherol. Tocopherols were measured by both reverse-phase and normal-phase high performance liquid chromatography (HPLC). When eight human volunteers (age range 30-60) were given 1200 IU of all-rac-alpha-tocopherol daily for 8 wk, plasma gamma-tocopherol and beta-tocopherol decreased in all subjects. After supplementation, gamma-tocopherol values were typically 30-50% of initial values, and alpha-tocopherol values were typically 200-400% of initial values. These results suggest that intestinal uptake and/or plasma transport make more efficient use of alpha-tocopherol than of gamma- or beta-tocopherol. Moreover, the results indicate that the ratio of gamma- to alpha-tocopherol in plasma would be a more satisfactory index to measure compliance in trials involving supplementation with alpha-tocopherol.

13. J Intern Med 1996 Feb;239(2):111-7
Gamma, but not alpha, tocopherol levels in serum are reduced in coronary heart disease patients.
Ohrvall M, Sundlof G, Vessby B.
Department of Geriatrics, University of Uppsala, Sweden.

OBJECTIVES. Low concentrations of alpha tocopherol are claimed to be associated with an increased prevalence of coronary heart disease. This study was undertaken to see whether measurements of serum tocopherol concentrations can contribute to discrimination between subjects with and without coronary heart disease. SETTING. All patients had been referred to the department of cardiology of the University Hospital in Uppsala, Sweden. SUBJECTS. Male patients (n = 69) below 60 years of age with coronary heart disease (CHD) and healthy age-matched reference subjects (n = 138) were compared. RESULTS. Lipid-corrected alpha tocopherol concentrations did not differ significantly between the groups, but the CHD group had a lower mean concentration of gamma tocopherol and a higher alpha/gamma ratio. In a stepwise logistic regression analysis, the LDL/HDL ratio was the best independent discriminator between the groups, followed by the proportion of palmitic acid in the cholesterol esters and the alpha/gamma tocopherol ratio. CONCLUSIONS. The lower gamma tocopherol concentration and the high ratio between alpha and gamma tocopherol in the CHD group indicate a difference in antioxidative status between CHD patients and healthy subjects. The lipid-lowering treatment of these CHD patients is far from optimal.

14. Proc Natl Acad Sci U S A 1997 Apr 1;94(7):3217-22
Gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications.
Christen S, Woodall AA, Shigenaga MK, Southwell-Keely PT, Duncan MW, Ames BN.
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720, USA.

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of .NO and O2.- during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how gamma-tocopherol (gammaT), the principal form of vitamin E in the United States diet, and alpha-tocopherol (alphaT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the .NO and O2.- generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by gammaT than alphaT. More importantly, nitration of gammaT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of approximately 50% and approximately 75%, respectively, was not affected by the presence of alphaT. These results suggest that despite
alphaT's action as an antioxidant gammaT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that gammaT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in alphaT. Because large doses of
dietary alphaT displace gammaT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily alphaT should be reconsidered.

15. Proc Natl Acad Sci U S A 1993 Mar 1;90(5):1771-5
Gamma-tocopherol detoxification of nitrogen dioxide: superiority to alpha-tocopherol.
Cooney RV, Franke AA, Harwood PJ, Hatch-Pigott V, Custer LJ, Mordan LJ.
Cancer Research Center of Hawaii, University of Hawaii, Honolulu 96813.

In the vitamin E group, alpha-tocopherol is generally considered to be the most potent antioxidant with the highest vitamin bioactivity, yet gamma-tocopherol is produced in greater amounts by many plants and is the principal tocopherol in the United States diet. This report describes a fundamental difference in the chemical reactivities of alpha-tocopherol and gamma-tocopherol with nitrogen dioxide (NO2), which leads to the formation of a nitrosating agent from alpha-tocopherol, but not from gamma-tocopherol. Nitric oxide (NO) is a major product of the reaction of gamma-tocopherol with NO2, while alpha-tocopherol reacts with NO2 to form an intermediate tocopheroxide analogue. The biological significance of gamma-tocopherol is suggested by limited epidemiological data as well as the observation that it is a more potent inhibitor than alpha-tocopherol of neoplastic transformation during the postinitiation phase in
3-methylcholanthrene-treated C3H/10T1/2 murine fibroblasts. This latter property suggests the superiority of gamma-tocopherol in a mammalian biological assay and a role for endogenous NO production in promotion of neoplastic transformation.

16. J Gerontol A Biol Sci Med Sci 2000 Jun;55(6):B280-5
Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans.
Adachi H, Ishii N.
Life Science Research Center, Lion Corporation, Kanagawa, Japan.
hadachi@lion.co.jp

To assess the efficiency of tocotrienols against oxidative damage, we have demonstrated in a model-system nematode, Caenorhabditis elegans, that tocotrienol administration reduced the accumulation of protein carbonyl (a good indicator of oxidative damage during aging) and consequently extended the mean life span (LS), but not the maximum LS. Conversely, alpha-tocopherol acetate did not affect these parameters. As a way to evaluate the protective ability of tocotrienols against oxidative stress, the life spans of animals administrated tocotrienols before or after exposure to ultraviolet B-induced oxidative stress were measured. Ultraviolet B irradiation shortened the mean LS of animals, whereas preadministration of tocotrienols recovered the mean LS to that of unirradiated animals. Interestingly, postadministration also extended the mean LS more than that of unirradiated animals, and administration through the LS conferred greater protection. Thus, the administration of tocotrienols to animals results in a reduction of oxidative stress risks. These data indicated that tocotrienols merit further investigation as possible agents for antiaging and oxidative stress prevention. In addition, they suggest that C. elegans will continue to provide provocative clues into the mechanisms of aging.

17. Lipids 1995 Dec;30(12):1179-83
Antioxidant effects of tocotrienols in patients with hyperlipidemia and carotid stenosis.
Tomeo AC, Geller M, Watkins TR, Gapor A, Bierenbaum ML.
Kenneth L. Jordan Research Group, Montclair, New Jersey 07042, USA.

Antioxidants may have a role in the prevention of atherosclerosis. In thepresent trial, we investigated the antioxidant properties of Palm Vitee, a gamma-tocotrienol-, and alpha-tocopherol enriched fraction of palm oil, in patients with carotid atherosclerosis. Serum lipids, fatty acid peroxides, platelet aggregation and carotid artery stenosis were measured over an 18-month period in fifty patients with cerebrovascular disease. Change in stenosis was measured with duplex ultrasonography. Ultrasound scans were done at six months, twelve months, and yearly thereafter. Bilateral duplex ultrasonography revealed apparent carotid atherosclerotic regression in seven and progression in two of the 25 tocotrienol patients, while none of the control group exhibited regression and ten of 25 showed progression (P < 0.002). Serum thiobarbituric acid reactive substances, an ex vivo indicator of maximal platelet peroxidation, decreased in the treatment group from 1.08 0.70 to 0.80 0.55 microM/L (P < 0.05) after 12 mon, and in the placebo group, they increased nonsignificantly from 0.99 0.80 to 1.26 0.54 microM/L. Both tocotrienol and placebo groups displayed significantly attenuated collagen-induced platelet aggregation responses (P < 0.05) as compared with entry values. Serum total cholesterol, low-density lipoprotein cholesterol, and triglyceride values remained unchanged in both groups, as did the plasma high density lipoprotein cholesterol values. These findings suggest that antioxidants, such as tocotrienols, may influence the course of carotid atherosclerosis.

18. J Biol Chem 1993 May 25;268(15):11230-8
Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Parker RA, Pearce BC, Clark RW, Gordon DA, Wright JJ.
Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.

Tocotrienols are natural farnesylated analogues of tocopherols which decrease hepatic cholesterol production and reduce plasma cholesterol levels in animals. For several cultured cell types, incubation with gamma-tocotrienol inhibited the rate of [14C]acetate but not [3H] mevalonate incorporation into cholesterol in a concentration- and time-dependent manner, with 50% inhibition at approximately 2 microM and maximum approximately 80% inhibition observed within 6 h in HepG2 cells. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase total activity and protein levels assayed by Western blot were reduced concomitantly with the decrease in cholesterol synthesis. In HepG2 cells, gamma-tocotrienol suppressed reductase despite strong blockade by inhibitors at several steps in the pathway, suggesting that isoprenoid flux is not required for the regulatory effect. HMG-CoA reductase protein synthesis rate was moderately diminished (57% of control), while the degradation rate was increased 2.4-fold versus control (t1/2 declined from 3.73 to 1.59 h) as judged by [35S] methionine pulse-chase/immunoprecipitation analysis of HepG2 cells treated with 10 microM gamma-tocotrienol. Under these conditions, the decrease in reductase protein levels greatly exceeded the minor decrease in mRNA (23 versus 76% of control, respectively), and the low density lipoprotein receptor protein was augmented. In contrast, 25-hydroxycholesterol strongly cosuppressed HMG-CoA reductase protein and mRNA levels and the low density lipoprotein receptor protein. Thus, tocotrienols influence the mevalonate pathway in mammalian cells by post-transcriptional suppression of HMG-CoA reductase, and appear to specifically modulate the intracellular mechanism for controlled degradation of the reductase protein, an activity that mirrors the actions of the putative non-sterol isoprenoid regulators derived from mevalonate.

19. Am J Clin Nutr 1991 Apr;53(4 Suppl):1021S-1026S
Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (palmvitee).
Qureshi AA, Qureshi N, Wright JJ, Shen Z, Kramer G, Gapor A, Chong YH, DeWitt G, Ong A, Peterson DM, et al.
Advanced Medical Research, Madison, WI 53719.

A double-blind, crossover, 8-wk study was conducted to compare effects of the tocotrienol-enriched fraction of palm oil (200 mg palmvitee capsules/day) with those of 300 mg corn oil/d on serum lipids of hypercholesterolemic human subjects (serum cholesterol 6.21-8.02 mmol/L). Concentrations of serum total cholesterol (-15%), LDL cholesterol (-8%), Apo B (-10%), thromboxane (-25%), platelet factor 4 (-16%), and glucose (-12%) decreased significantly only in the 15 subjects given palmvitee during the initial 4 wk. The crossover confirmed these actions of palmvitee. There was a carry over effect of palmvitee. Serum cholesterol concentrations of seven hypercholesterolemic subjects (greater than 7.84 mmol/L) decreased 31% during a 4-wk period in which they were given 200 mg gamma-tocotrienol/d. This indicates that gamma-tocotrienol may be the most potent cholesterol inhibitor in palmvitee capsules. The results of this pilot study are very encouraging.

20. Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolomic humans
Qureshi A.A.; Bradlow B.A.; Salser W.A.; Brace L.D. Dr. A.A. Qureshi, Advance Medical Research, 8251 Raymond Road, Madison, WI 53719 United States
Journal of Nutritional Biochemistry ( J. NUTR. BIOCHEM. ) ( United States ) 1997 , 8/5 (290-298)

Tocotrienols inhibit cholesterol synthesis by post-transcriptionally suppressing beta-hydroxy-beta-methylglutaryl-coenzyme A reductase activity. A double blind, 12-week study was performed to investigate the effect of a novel tocotrienol-rich fraction (TRFinf 2$D5: obtained by molecular distillation from specially processed rice bran oil) on cardiovascular disease risk factors of hypercholesterolomic human subjects (serum total cholesterol >5.69 mmol/L). After acclimation to an alcohol-free regimen (baseline) participants were assigned to the National Cholesterol Education Program (NCEP) Step-1 diet (saturated fat <19%, total fat <30% of total calories and cholesterol <7.76 mmol/L). The participants were evaluated after 4 weeks of exposure to the NCEP Step-1 diet: one group of 21 participants was continued on the NCEP Step-1 diet for 4 weeks receiving an additional 1.2 gm corn oil (placebo group) and a second group of 20 received 200 mg TRFinf 2$D5 dissolved in 1.0 gm corn oil (TRFinf 2$D5 group). Serum total cholesterol and LDL-cholesterol levels of all the participants, stable during the baseline phase of the study, decreased 5% and 8%, respectively, during the 4-week NCEP Step-1 diet. Placebo continuing on the NCEP Step-1 diet for an additional 4 weeks experienced additional but modest decreases in serum total cholesterol (2%) and LDL-cholesterol (3%), yielding significant (P<0.05) decreases when compared with the baseline values. These responses confirm the cholesterol- lowering action of a low fat, low cholesterol diet. Participants receiving TRFinf 2$D5 had 12% and 16% reductions (P<0.05) in total cholesterol and LDL- cholesterol levels during the 4-week experimental phase; during the two phases (NCEP Step-1 diet plus treatment) the serum total cholesterol and LDL- cholesterol levels of these of these participants were decreased (P<0.05) by 17% and 24%, respectively, TRFinf 2$D5-mediated decreases in Apo B(a), platelet factor 4 and thromboxane Binf 2 (15%, 17%, 14%, and 31%, respectively) were significant (P<0.05). There was no change in the levels of HDL-cholesterol and apolipoprotein A-I by this treatment. The treatment also resulted in remarkable increases in the levels of LDL-bound antioxidants, especially tocotrienols, which have substantially greater antioxidant activity than vitamin E.

21. N Engl J Med 1990 Nov 8;323(19):1289-98
Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B.
Brown G, Albers JJ, Fisher LD, Schaefer SM, Lin JT, Kaplan C, Zhao XQ, Bisson BD, Fitzpatrick VF, Dodge HT.
Department of Medicine, University of Washington School of Medicine, Seattle.

BACKGROUND AND METHODS. The effect of intensive lipid-lowering therapy on coronary atherosclerosis among men at high risk for cardiovascular events was assessed by quantitative arteriography. Of 146 men no more than 62 years of age who had apolipoprotein B levels greater than or equal to 125 mg per deciliter, documented coronary artery disease, and a family history of vascular disease, 120 completed the 2 1/2-year double-blind study, which included arteriography at base line and after treatment. Patients were given dietary counseling and were randomly assigned to one of three treatments: lovastatin (20 mg twice a day) and colestipol (10 g three times a day); niacin (1 g four times a day) and colestipol (10 g three times a day); or conventional therapy with placebo (or colestipol if the low-density lipoprotein [LDL] cholesterol level was elevated). RESULTS. The levels of LDL and high-density lipoprotein (HDL) cholesterol changed only slightly in the conventional-therapy group (mean changes, -7 and +5 percent, respectively), but more substantially among patients treated with lovastatin and colestipol (-46 and +15 percent) or niacin and colestipol (-32 and +43 percent). In the conventional-therapy group, 46 percent of the patients had definite lesion progression (and no regression) in at least one of nine proximal coronary segments; regression was the only change in 11 percent. By comparison, progression (as the only change) was less frequent among patients who received lovastatin and colestipol (21 percent) and those who received niacin and colestipol (25 percent), and regression was more frequent (lovastatin and colestipol, 32 percent; niacin and colestipol, 39 percent; P less than 0.005). Multivariate analysis indicated that a reduction in the level of apolipoprotein B (or LDL cholesterol) and in systolic blood pressure, and an increase in HDL cholesterol correlated independently with regression of coronary lesions. Clinical events (death, myocardial infarction, or revascularization for worsening symptoms) occurred in 10 of 52 patients assigned to conventional therapy, as compared with 3 of 46 assigned to receive lovastatin and colestipol and 2 of 48 assigned to receive niacin and colestipol (relative risk of an event during intensive treatment, 0.27; 95 percent confidence interval, 0.10 to 0.77). CONCLUSIONS. In men with coronary artery disease who were at high risk for cardiovascular events, intensive lipid-lowering therapy reduced the frequency of progression of coronary lesions, increased the frequency of regression, and reduced the incidence of cardiovascular events.

22. N Engl J Med 1983 Aug 18;309(7):385-9
Apolipoprotein A-I as a marker of angiographically assessed coronary-artery disease.
Maciejko JJ, Holmes DR, Kottke BA, Zinsmeister AR, Dinh DM, Mao SJ.

This study was designed to determine whether the plasma level of apolipoprotein A-I is a better discriminator of angiographically documented coronary-artery disease than the level of high-density-lipoprotein (HDL) cholesterol in male subjects. The level of plasma apolipoprotein A-I in 83 patients with coronary-artery disease was 96.7 4.2 mg per deciliter (mean S.E.M.), which was significantly lower (P less than 0.0001) than the level in 25 patients without coronary-artery disease (146.9 2.1 mg per deciliter). The levels of HDL cholesterol were also lower (P less than 0.0001) in patients with coronary-artery disease (31.9 1.5 mg per deciliter) than in those without it (45.9 2.3 mg per deciliter). A stepwise discriminant analysis, however, indicated the superiority of apolipoprotein A-I over HDL cholesterol in detecting coronary-artery disease. Furthermore, a linear discriminant analysis suggested that although HDL cholesterol by itself was a discriminator of coronary-artery disease, it did not provide a substantial increase in discriminatory value over that provided by apolipoprotein A-I; in contrast,
apolipoprotein A-I levels added discriminatory value to the information obtained by measuring HDL cholesterol alone. We conclude that apolipoprotein A-I by itself is more useful than HDL cholesterol for identifying patients with coronary-artery disease.

23. Br Heart J 1988 Nov;60(5):397-403
High prevalence of hypertriglyceridaemia and apolipoprotein abnormalities in coronary artery disease.
Barbir M, Wile D, Trayner I, Aber VR, Thompson GR.
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London.

Serum lipids and apolipoproteins A-I and B were measured in 174 men aged less than 60 with angiographically confirmed coronary artery disease and in 572 healthy control men. Two thirds of the patients had raised age-corrected values of fasting serum cholesterol and/or triglyceride and/or a low high density lipoprotein (HDL) cholesterol compared with the controls. Eighteen (30%) of the 61 normolipidaemic patients had a concentration of serum apolipoprotein A-I below the 5th percentile of 233 controls. In normolipidaemic patients on beta blockers the relative prevalence of serum low density lipoprotein
(LDL)-apolipoprotein B values above the 95th percentile of 339 controls was significantly increased. Discriminant function analysis showed that a raised concentration of serum triglyceride was the best discriminant between patients and controls, with raised LDL-apolipoprotein B and reduced apolipoprotein A-I coming second only to triglyceride in analyses where each was separately compared with all the lipid variables. These associations were highly significant and were independent of other influences, including beta blockade. These findings re-emphasise the importance of hypertriglyceridaemia as a risk factor and confirm that apolipoprotein abnormalities occur frequently in coronary disease, even in normolipidaemic patients.

24. Proc Natl Acad Sci U S A 2000 Oct 10;97(21):11494-9
Gamma-tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells.
Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN.
Division of Biochemistry and Molecular Biology, University of California,
Berkeley, CA 94720, USA.

Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E(2) (PGE(2)) plays a key role in inflammation and its associated diseases, such as cancer and vascular heart disease. Here we report that gamma-tocopherol (gammaT) reduced PGE(2) synthesis in both lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and IL-1beta-treated A549 human epithelial cells with an apparent IC(50) of 7.5 and 4 microM, respectively. The major metabolite of dietary gammaT, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), also exhibited an inhibitory effect, with an IC(50) of approximately 30 microM in these cells. In contrast, alpha-tocopherol at 50 microM slightly reduced (25%) PGE(2) formation in macrophages, but had no effect in epithelial cells. The inhibitory effects of gammaT and gamma-CEHC stemmed from their inhibition of COX-2 activity, rather than affecting protein expression or substrate availability, and appeared to be independent of antioxidant activity. gamma-CEHC also inhibited PGE(2) synthesis when exposed for 1 h to COX-2-preinduced cells followed by the addition of arachidonic acid (AA), whereas under similar conditions, gammaT required an 8- to 24-h incubation period to cause the inhibition. The inhibitory potency of gammaT and gamma-CEHC was diminished by an increase in AA concentration, suggesting that they might compete with AA at the active site of COX-2. We also observed a moderate reduction of nitrite accumulation and suppression of inducible nitric oxide synthase expression by gammaT in lipopolysaccharide-treated macrophages. These findings indicate that gammaT and its major metabolite possess anti-inflammatory activity and that gammaT at physiological concentrations may be important in human disease prevention.

25. J Am Coll Cardiol 1999 Oct;34(4):1208-15
Differential effects of alpha- and gamma-tocopherol on low-density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis.
Saldeen T, Li D, Mehta JL.
Department of Forensic Medicine, University of Uppsala, Sweden.

OBJECTIVES: This study was designed to examine the differential effects of alpha- and gamma-tocopherol on parameters of oxidation-antioxidation and thrombogenesis. BACKGROUND: Experimental studies have shown that antioxidants, such as vitamin E (alpha-tocopherol), improve atherosclerotic plaque stability and vasomotor function, and decrease platelet aggregation and tendency to thrombus formation. METHODS: Sprague Dawley rats were fed chow mixed with alpha- or gamma-tocopherol (100 mg/kg/day) for 10 days. A filter soaked in 29% FeCl3 was applied around the abdominal aorta to study the patterns of arterial thrombosis. The aortic blood flow was observed and continuously recorded using an ultrasonic Doppler flow probe. ADP-induced platelet aggregation, low-density lipoprotein oxidation induced by phorbol 12-myristate 13-acetate (PMA)-stimulated leukocytes, superoxide anion generation and superoxide dismutase (SOD) activity were also measured. RESULTS: Both alpha- and gamma-tocopherol decreased platelet aggregation and delayed time to occlusive thrombus (all p < 0.05 vs. control). Both alpha- and gamma-tocopherol decreased arterial superoxide anion generation, lipid peroxidation and LDL oxidation (all p < 0.05 vs. control), and increased endogenous SOD activity (p < 0.05). The effects of gamma-tocopherol were more potent than those of alpha-tocopherol (p < 0.05). CONCLUSIONS: This study indicates that both alpha- and gamma-tocopherol decrease platelet aggregation and delay intraarterial thrombus formation, perhaps by an increase in endogenous antioxidant activity. Alpha-tocopherol is significantly more potent than alpha-tocopherol in these effects.

26. J Nutr Biochem 2001 Jun;12(6):318-329
Synergistic effect of tocotrienol-rich fraction (TRF(25)) of rice bran and lovastatin on lipid parameters in hypercholesterolemic humans.
Qureshi AA, Sami SA, Salser WA, Khan FA.
Advanced Medical Research, 8251 Raymond Road, 53719, Madison, WI, USA

Tocotrienols exert hypocholesterolemic action in humans and animals. Lovastatin is widely used for that purpose. Both agents work by suppressing the activity of beta-hydroxy-beta methylglutaryl coenzyme A reductase through different mechanisms, post-transcriptional vs competitive inhibition. A human study with 28 hypercholesterolemic subjects was carried out in 5 phases of 35 days each, to check the efficacy of tocotrienol-rich fraction (TRF(25)) of rice bran alone and in combination with lovastatin. After placing subjects on the American Heart Association (AHA) Step-1 diet (phase II), the subjects were divided into two groups, A and B. The AHA Step-1 diet was continued in combination with other treatments during phases III to V. Group A subjects were given 10 mg lovastatin, 10 mg lovastatin plus 50 mg TRF(25), 10 mg lovastatin plus 50 mg alpha-tocopherol per day, in the third, fourth, and fifth phases, respectively. Group B subjects were treated exactly to the same protocol except that in the third phase, they were given 50 mg TRF(25) instead of lovastatin.The TRF(25) or lovastatin plus AHA Step-1 diet effectively lower serum total cholesterol (14%, 13%) and LDL-cholesterol (18%, 15% P < 0.001), respectively, in hypercholesterolemic subjects. The combination of TRF(25) and lovastatin plus AHA Step-1 diet significantly reduces of these lipid parameters of 20% and 25% (P < 0.001) in these subjects. Substitution of TRF(25) with alpha-tocopherol produces insignificant changes when given with lovastatin. Especially significant is the increase in the HDL/LDL ratio to 46% in group (A) and 53% (P < 0.002) in group (B). These results are consistent with the synergistic effect of these two agents. None of the subjects reported any side-effects throughout the study of 25-weeks. In the present study, the increased effectiveness of low doses of tocotrienols (TRF(25)) as hypocholesterolemic agents might be due to a minimum conversion to alpha-tocopherol. The report also describes in vivo the conversion of gamma-[4-3H]-, and [14C]-desmethyl (d-P(21)-T3) tocotrienols to alpha-tocopherol.

27. Lipids 1993 Dec;28(12):1113-8
gamma-Tocotrienol as a hypocholesterolemic and antioxidant agent in rats fed atherogenic diets.
Watkins T, Lenz P, Gapor A, Struck M, Tomeo A, Bierenbaum M.
Kenneth L. Jordan Heart Fund, Montclair, New Jersey 07042.

This study was designed to determine whether incorporation of gamma-tocotrienol or alpha-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 gamma-tocotrienol (50 mg/kg) and alpha-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 gamma-tocotrienol resulted in similar, though quantitatively smaller, decrements in these plasma values. Plasma alpha-tocopherol concentrations were lowest in rats fed menhaden oil without either chromanol. Though plasma alpha-tocopherol did not rise with gamma-tocotrienol supplementation at 50 mg/kg, gamma-tocotrienol at 100 mg/kg of ration spared plasma alpha-tocopherol, which rose from 0.60 0.2 to 1.34 0.4 mg/dL (P < 0.05). The highest concentration of alpha-tocopherol was measured in plasma of animals fed a ration supplemented with alpha-tocopherol at 500 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

28. J Nutr 2001 Jan;131(1):161S-163S
Vitamin E: mechanisms of action as tumor cell growth inhibitors.
Kline K, Yu W, Sanders BG.
Division of Nutrition and. School of Biological Sciences, The University of Texas at Austin, Austin, TX 78712, USA. k.kline@mail.utexas.edu
http://www.nutrition.org/cgi/content/full/131/1/161S?view=full&pmid=11208955

29. J Nutr 1994 May;124(5):607-14
The chemoprevention of cancer by mevalonate-derived constituents of fruits and vegetables.
Elson CE, Yu SG.
Department of Nutritional Sciences, University of Wisconsin, Madison 53706-1571.

Anutritive isoprenoid constituents of fruits, vegetables, cereal grains and essential oils exhibit a spectrum of anticarcinogenic activities. The induction of hepatic Phase II detoxifying activities by dietary isoprenoids appears to underlie their blocking action. The second anticarcinogenic action of the dietary isoprenoids, suppression of the growth of chemically initiated and transplanted tumors is, we suggest, secondary to the inhibition of mevalonate pathway activities. Mevinolin, a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity, depletes cells of the intermediate products of the pathway that are required for the posttranslational modification of proteins, a process giving the proteins lipophilic anchors that bind to membranes. As a consequence, nuclear lamins and ras oncoproteins remain in nascent states, and cells do not proliferate. gamma-Tocotrienol, perillyl alcohol, geraniol and d-limonene suppress hepatic HMG-CoA reductase activity, a rate-limiting step in cholesterol synthesis, and modestly lower serum-cholesterol levels of animals. These isoprenoids also suppress tumor growth. The HMG-CoA reductase of neoplastic tissues differs from that of sterologenic tissues in being markedly resistant to sterol feedback inhibition. Our review suggests that the mevalonate pathway of tumor tissues is uniquely sensitive to the inhibitory actions of the dietary isoprenoids.

30. J Nutr 1999 Apr;129(4):804-13
Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.
Mo H, Elson CE.
Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.

Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable and grain consumption on cancer risk.

31. J Nutr 1997 May;127(5):668-74
Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo.
He L, Mo H, Hadisusilo S, Qureshi AA, Elson CE.
Department of Nutritional Sciences, University of Wisconsin, Madison 53706, USA.

Sundry mevalonate-derived constituents (isoprenoids) of fruits, vegetables and cereal grains suppress the growth of tumors. This study estimated the concentrations of structurally diverse isoprenoids required to inhibit the increase in a population of murine B16(F10) melanoma cells during a 48-h incubation by 50% (IC50 value). The IC50 values for d-limonene and perillyl alcohol, the monoterpenes in Phase I trials, were 450 and 250 micromol/L, respectively; related cyclic monoterpenes (perillaldehyde, carvacrol and thymol), an acyclic monoterpene (geraniol) and the end ring analog of beta-carotene (beta-ionone) had IC50 values in the range of 120-150 micromol/L. The IC50 value estimated for farnesol, the side-chain analog of the tocotrienols
(50 micromol/L) fell midway between that of alpha-tocotrienol (110 micromol/L) and those estimated for gamma- (20 micromol/L) and delta- (10 micromol/L) tocotrienol. A novel tocotrienol lacking methyl groups on the tocol ring proved to be extremely potent (IC50, 0.9 micromol/L). In the first of two diet studies, experimental diets were fed to weanling C57BL female mice for 10 d prior to and 28 d following the implantation of the aggressively growing and highly metastatic B16(F10) melanoma. The isomolar (116 micromol/kg diet) and the
Vitamin E-equivalent (928 micromol/kg diet) substitution of d-gamma-tocotrienol for dl-alpha-tocopherol in the AIN-76A diet produced 36 and 50% retardations, respectively, in tumor growth (P < 0.05). In the second study, melanomas were established before mice were fed experimental diets formulated with 2 mmol/kg d-gamma-tocotrienol, beta-ionone individually and in combination. Each treatment increased (P < 0.03) the duration of host survival. Our finding that the effects of individual isoprenoids were additive suggests the possibility that one component of the anticarcinogenic action of plant-based diets is the tumor growth-suppressive action of the diverse isoprenoid constituents of fruits, vegetables and cereal grains.

32. Nutr Cancer 1992;18(1):1-29
Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence.
Block G, Patterson B, Subar A.
Dept. of Social and Administrative Health Sciences, School of Public Health,
University of California, Berkeley 94720.

Approximately 200 studies that examined the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary are reviewed. A statistically significant protective effect of fruit and vegetable consumption was found in 128 of 156 dietary studies in which results were expressed in terms of relative risk. For most cancer sites, persons with low fruit and vegetable intake (at least the lower one-fourth of the population) experience about twice the risk of cancer compared with those with high intake, even after control for potentially confounding factors. For lung cancer, significant protection was found in 24 of 25 studies after control for smoking in most instances. Fruits, in particular, were significantly protective in cancers of the esophagus, oral cavity, and larynx, for which 28 of 29 studies were significant. Strong evidence of a protective effect of fruit and vegetable consumption was seen in cancers of the pancreas and stomach (26 of 30 studies), as well as in colorectal and bladder cancers (23 of 38 studies). For cancers of the cervix, ovary, and endometrium, a significant protective effect was shown in 11 of 13 studies, and for breast cancer a protective effect was found to be strong and consistent in a meta analysis. It would appear that major public health benefits could be achieved by substantially increasing consumption of these foods.

33. Proc Soc Exp Biol Med 1999 Sep;221(4):294-311
Isoprenoid-mediated inhibition of mevalonate synthesis: potential application to cancer.
Elson CE, Peffley DM, Hentosh P, Mo H.
Department of Nutritional Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. elson@nutrisci.wisc.edu

Pure and mixed isoprenoid end products of plant mevalonate metabolism trigger actions that suppress 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity. These actions modulate HMG CoA reductase mRNA translation and the proteolytic degradation of HMG CoA reductase. Such post-transcriptional events, we propose, are activated directly by acyclic isoprenoids and indirectly by cyclic isoprenoids. Isoprenoids, acting secondarily to the dominant
transcriptional effector of sterologenesis, modestly lower cholesterol levels, if and only if, sterologenesis is not repressed by a saturating imput of dietary cholesterol. An anomaly associated with tumor growth-a sterol feedback-resistant HMG CoA reductase activity-ensures a pool of sterologenic pathway intermediates. Such intermediates provide lipophilic anchors essential for membrane attachment and biological activity of growth hormone receptors, nuclear lamins A and B, and oncogenic ras. Tumor HMG CoA reductase retains high sensitivity to the
isoprenoid-mediated secondary regulation. Repression of mevalonate synthesis by plant-derived isoprenoids reduces ras and lamin B processing, arrests cells in G1, and initiates cellular apoptosis. This unique tumor cell-specific sensitivity allows isoprenoids to be used for tumor therapy, an application emulating that of the statins, but one free of adverse effects. When evaluated at levels provided by a typical diet, isoprenoids individually have no impact on cholesterol synthesis and tumor growth. Nonetheless, isoprenoid-mediated activities are additive, and, sometimes synergistic. Therefore, the combined actions of the estimated 23,000 isoprenoid constituents of plant materials, acting in concert with other chemopreventive phytochemicals, may explain the lowered cancer risk associated with a diet rich in plant products. In contrast, that lowering of cancer risk does not correspond to supplemental intake of other dietary factors associated with fruits, vegetables, and cereal grains, namely fiber, beta-carotene, vitamin C, and vitamin E, and only weakly to supplemental folate.

34. J Nutr 1995 Jun;125(6 Suppl):1666S-1672S
Suppression of mevalonate pathway activities by dietary isoprenoids: protective roles in cancer and cardiovascular disease.
Elson CE.
Department of Nutritional Sciences, University of Wisconsin-Madison 53706, USA.

Diet-cancer and diet-cardiovascular disease interrelationships may be explained by the mevalonate-suppressive action of isoprenoid end products of plant secondary metabolism. Assorted monoterpenes, sesquiterpenes, carotenoids and tocotrienols posttranscriptionally down regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, a key activity in the sterologenic pathway. The modest decrease in cholesterol synthesis is associated with a concomitant lowering of low-density lipoprotein cholesterol. The reductase activity in tumor tissues differs from that of liver in being resistant to sterol feedback regulation. Tumor reductase activity retains sensitivity to posttranscriptional regulation. As a consequence, the isoprenoid-mediated suppression of mevalonate synthesis depletes tumor tissues of two intermediate products, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are incorporated
posttranslationally into growth control-associated proteins. At 10-fold higher concentrations, monoterpenes inhibit the protein isoprenyl transferases that catalyze this incorporation. At levels of intake likely provided by a diet based on Food Pyramid guidelines, assorted isoprenoids decrease cardiovascular disease risk and suppress the growth of initiated cells. At pharmacological levels of intake, isoprenoids block the initiation phase of chemical carcinogenesis. Isoprenoids targeted to the inhibition of the isoprenylation of oncogenic forms of ras proteins may offer a novel approach to chemotherapy. Adjunctive isoprenoids might decrease the level of competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase required to manage hypercholesterolemia.

35. Nutrition 1993 May-Jun;9(3):229-32
Long-term administration of tocotrienols and tumor-marker enzyme activities during hepatocarcinogenesis in rats.
Rahmat A, Ngah WZ, Shamaan NA, Gapor A, Abdul Kadir K.
Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia,
Jalan Raja Muda Abdul Aziz, Kuala Lumpur.

The effects of long-term administration of tocotrienol on hepatocarcinogenesis in rats induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated by determining the activities of gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), glutathione S-transferases (GSTs), and glutathione (GSH) levels in blood and liver. Twenty-eight male 7- to 8-wk-old Rattus norwegicus rats, weighing 120-160 g, were used in this study. The rats were divided into four treatment groups: a control group on a basal diet, a group fed a basal diet supplemented with tocotrienol (30 mg/kg food), a group treated with DEN/AAF, and a group treated with DEN/AAF and fed a diet supplemented with tocotrienol (30 mg/kg food). Blood was collected monthly, and GGT, ALP, and GSH levels were determined. The rats were killed after 9 mo, and the livers were examined morphologically. Grayish white nodules (2/liver) were found in all the DEN/AAF-treated rats (n = 10), but only one of the rats treated with DEN/AAF and supplemented with tocotrienol (n = 6) had liver nodules. A significant increase in the level of blood and liver GSH, ALP, and GGT activities was observed in the DEN/AAF-treated rats. Liver GSTs were similarly increased with DEN/AAF treatment. Tocotrienol supplementation attenuated the impact of the carcinogens in the rats.

36. Comp Biochem Physiol C 1993 Sep;106(1):237-40
Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol.
Ong FB, Wan Ngah WZ, Shamaan NA, Md Top AG, Marzuki A, Khalid AK.
Jabatan Biokimia, Fakulti Perubatan, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala, Lumpur.

The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated. 2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1-2 days. 3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1-3 days. 4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 microM tocotrienol at 1-3 days in culture compared to the control.

37. J Nutr 1997 May;127(5):668-74
Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo.
He L, Mo H, Hadisusilo S, Qureshi AA, Elson CE.
Department of Nutritional Sciences, University of Wisconsin, Madison 53706, USA.

Sundry mevalonate-derived constituents (isoprenoids) of fruits, vegetables and cereal grains suppress the growth of tumors. This study estimated the concentrations of structurally diverse isoprenoids required to inhibit the increase in a population of murine B16(F10) melanoma cells during a 48-h incubation by 50% (IC50 value). The IC50 values for d-limonene and perillyl
alcohol, the monoterpenes in Phase I trials, were 450 and 250 micromol/L, respectively; related cyclic monoterpenes (perillaldehyde, carvacrol and thymol), an acyclic monoterpene (geraniol) and the end ring analog of beta-carotene (beta-ionone) had IC50 values in the range of 120-150 micromol/L. The IC50 value estimated for farnesol, the side-chain analog of the tocotrienols
(50 micromol/L) fell midway between that of alpha-tocotrienol (110 micromol/L) and those estimated for gamma- (20 micromol/L) and delta- (10 micromol/L) tocotrienol. A novel tocotrienol lacking methyl groups on the tocol ring proved to be extremely potent (IC50, 0.9 micromol/L). In the first of two diet studies, experimental diets were fed to weanling C57BL female mice for 10 d prior to and 28 d following the implantation of the aggressively growing and highly metastatic B16(F10) melanoma. The isomolar (116 micromol/kg diet) and the
Vitamin E-equivalent (928 micromol/kg diet) substitution of d-gamma-tocotrienol for dl-alpha-tocopherol in the AIN-76A diet produced 36 and 50% retardations, respectively, in tumor growth (P < 0.05). In the second study, melanomas were established before mice were fed experimental diets formulated with 2 mmol/kg d-gamma-tocotrienol, beta-ionone individually and in combination. Each treatment increased (P < 0.03) the duration of host survival. Our finding that the effects of individual isoprenoids were additive suggests the possibility that one component of the anticarcinogenic action of plant-based diets is the tumor growth-suppressive action of the diverse isoprenoid constituents of fruits, vegetables and cereal grains.

38. Lipids 1995 Dec;30(12):1139-43
Effect of tocotrienols on the growth of a human breast cancer cell line in culture.
Nesaretnam K, Guthrie N, Chambers AF, Carroll KK.
Palm Oil Research Institute of Malaysia, Kuala Lumpur, Malaysia.

The tocotrienol-rich fraction (TRF) of palm oil consists of tocotrienols and some alpha-tocopherol (alpha-T). Tocotrienols are a form of vitamin E having an unsaturated side-chain, rather than the saturated side-chain of the more common tocopherols. Because palm oil has been shown not to promote chemically-induced mammary carcinogenesis, we tested effects of TRF and alpha-T on the proliferation, growth, and plating efficiency (PE) of the MDA-MB-435 estrogen-receptor-negative human breast cancer cells. TRF inhibited the proliferation of these cells with a concentration required to inhibit cell proliferation by 50% of 180 microgram/mL whereas alpha-T had no effect at concentrations up to 1000 microgram/mL as measured by incorporation of [3H]thymidine. The effects of TRF and alpha-T also were tested in longer-term growth experiments, using concentrations of 180 and 500 microgram/mL. We found that TRF inhibited the growth of these cells by 50%, whereas alpha-T did not. Their effect on the ability of these cells to form colonies also was studied, and it was found that TRF inhibited PE, whereas alpha T had no effect. These results suggest that the inhibition is due to the presence of tocotrienols in TRF rather than alpha T.

39. Lipids 1998 May;33(5):461-9
Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status.
Nesaretnam K, Stephen R, Dils R, Darbre P.
Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, England. sarnesar@porim.gov.my

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-MB-231 human breast cancer cells, and effects were compared with those of alpha-tocopherol (alphaT). 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 microg/mL. MDA-MB-231 cells were also inhibited by TRF but with a linear dose-response such that 20 microg/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 gamma- and delta-fractions were the most inhibitory. Complete inhibition of MCF7 cell growth was achieved at 6 microg/mL of gamma-tocotrienol/delta-tocotrienol (gammaT3/deltaT3) in the absence of estradiol and 10 microg/mL of deltaT3 in the presence of estradiol, whereas complete suppression of MDA-MB-231 cell growth was not achieved even at concentrations of 10 microg/mL of deltaT3. By contrast to these inhibitory effects of tocotrienols, alphaT 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.

40. Int J Food Sci Nutr 2000;51 Suppl:S95-103
Tocotrienols inhibit growth of ZR-75-1 breast cancer cells.
Nesaretnam K, Dorasamy S, Darbre PD.
Palm Oil Research Institute of Malaysia, PO Box 10620, Kuala Lumpur 50720, Malaysia.

The vitamin E component of palm oil provides a rich source of tocotrienols which have been shown previously to be growth inhibitory to two human breast cancer cell lines: responsive MCF7 cells and unresponsive MDA-MB-231 cells. Data presented here shows that the tocotrienol-rich fraction (TRF) of palm oil and individual fractions (alpha, gamma and delta) can also inhibit the growth of another responsive human breast cancer cell line, ZR-75-1. At low concentrations in the absence of oestrogen tocotrienols stimulated growth of the ZR-75-1 cells, but at higher concentrations in the presence as well as in the absence of oestradiol, tocotrienols inhibited cell growth strongly. As for MCF7 cells, alpha-tocopherol had no effect on growth of the ZR-75-1 cells in either the absence or presence of oestradiol. In studying the effects of tocotrienols in combination with antioestrogens, it was found that TRF could further inhibit growth of ZR-75-1 cells in the presence of tamoxifen (10(-7) M and 10(-8) M). Individual tocotrienol fractions (alpha, gamma, delta) could inhibit growth of ZR-75-1 cells in the presence of 10(-8) M oestradiol and 10(-8) M pure antioestrogen ICI 164,384. The immature mouse uterine weight bioassay confirmed that TRF could not exert oestrogen antagonist action in vivo. These results provide evidence of wider growth-inhibitory effects of tocotrienols beyond MCF7 and MDA-MB-231 cells, and with an oestrogen-independent mechanism of action, suggest a possible clinical advantage in combining administration of tocotrienols with antioestrogen therapy.

41. J Nutr 1997 Mar;127(3):544S-548S
Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination.
Guthrie N, Gapor A, Chambers AF, Carroll KK.
Department of Biochemistry, The University of Western Ontario, London, Canada.

Tocotrienols are a form of vitamin E, having an unsaturated isoprenoid side-chain rather than the saturated side-chain of tocopherols. The tocotrienol-rich fraction (TRF) from palm oil contains alpha-tocopherol and a mixture of alpha-, gamma- and delta-tocotrienols. Earlier studies have shown that tocotrienols display anticancer activity. We previously reported that TRF, alpha-, gamma- and delta-tocotrienols inhibited proliferation of estrogen receptor-negative MDA-MB-435 human breast cancer cells with 50% inhibitory concentrations (IC50) of 180, 90, 30 and 90 microg/mL, respectively, whereas alpha-tocopherol had no effect at concentrations up to 500 microg/mL. Further experiments with estrogen receptor-positive MCF-7 cells showed that tocotrienols also inhibited their proliferation, as measured by [3H] thymidine incorporation. The IC50s for TRF, alpha-tocopherol, alpha-, gamma- and delta-tocotrienols were 4, 125, 6, 2 and 2 microg/mL, respectively. Tamoxifen, a widely used synthetic antiestrogen inhibits the growth of MCF-7 cells with an IC50 of 0.04 microg/mL. We tested 1:1 combinations of TRF, alpha-tocopherol and the individual tocotrienols with tamoxifen in both cell lines. In the MDA-MB-435 cells, all of the combinations were found to be synergistic. In the MCF-7 cells, only 1:1 combinations of gamma- or delta-tocotrienol with tamoxifen showed a synergistic inhibitory effect on the proliferative rate and growth of the cells. The inhibition by tocotrienols was not overcome by addition of excess estradiol to the medium. These results suggest that tocotrienols are effective inhibitors of both estrogen receptor-negative and -positive cells and that combin