Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C(alpha) activation.

Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0-250 microm) of alpha-, gamma-, or delta-tocopherol or alpha-, gamma-, or delta-tocotrienol. Treatment with growth inhibitory doses of delta-tocopherol (100 microm), alpha-tocotrienol (50 microm), or gamma- or delta-tocotrienol (10 microm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC(alpha). However, these treatments were found to inhibit EGF-induced PKC(alpha) activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 microm alpha- or gamma-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC(alpha) activation.

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on preneoplastic (CL-S1), neoplastic (-SA), and highly malignant (+SA) mouse mammary epithelial cell growth and viability in vitro. Over a 5-day culture period, treatment with 0-120 microM alpha- and gamma-tocopherol had no effect on cell proliferation, whereas growth was inhibited 50% (IC50) as compared with controls by treatment with the following: 13, 7, and 6 microM tocotrienol-rich-fraction of palm oil (TRF); 55, 47, and 23 microM delta-tocopherol; 12, 7, and 5 microM alpha-tocotrienol; 8, 5, and 4 microM gamma-tocotrienol; or 7, 4, and 3 microM delta-tocotrienol in CL-S1, -SA and +SA cells, respectively. Acute 24-hr exposure to 0-250 microM alpha- or gamma-tocopherol (CL-S1, -SA, and +SA) or 0-250 microM delta-tocopherol (CL-S1) had no effect on cell viability, whereas cell viability was reduced 50% (LD50) as compared with controls by treatment with 166 or 125 microM delta-tocopherol in -SA and +SA cells, respectively. Additional LD50 doses were determined as the following: 50, 43, and 38 microM TRF; 27, 28, and 23 microM alpha-tocotrienol; 19, 17, and 14 microM gamma-tocotrienol; or 16, 15, or 12 microM delta-tocotrienol in CL-S1, -SA, and +SA cells, respectively. Treatment-induced cell death resulted from activation of apoptosis, as indicated by DNA fragmentation. Results also showed that CL-S1, -SA, and +SA cells preferentially accumulate tocotrienols as compared with tocopherols, and this may partially explain why tocotrienols display greater biopotency than tocopherols. These data also showed that highly malignant +SA cells were the most sensitive, whereas the preneoplastic CL-S1 cells were the least sensitive to the antiproliferative and apoptotic effects of tocotrienols, and suggest that tocotrienols may have potential health benefits in preventing and/or reducing the risk of breast cancer in women.

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0-120 microM alpha- and gamma-tocopherol had no effect, whereas 12.5-100m microM tocotrienol-rich fraction of palm oil (TRF), 100-120 microM delta-tocopherol, 50-60 microM alpha-tocotrienol, and 8-14 microM gamma- or delta-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0-250 microM alpha-, gamma-, and delta-tocopherol had no effect, whereas similar treatment with 100-250 microM TRF, 140-250 microM alpha-, 25-100 microM gamma- or delta-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, delta-tocopherol, and alpha-, gamma-, and delta-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent gamma- and delta-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

Tocotrienol: a review of its therapeutic potential.

OBJECTIVES: To summarize new knowledge surrounding the physiological activity of tocotrienol, a natural analogue of tocopherol. RESULTS: The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. In the vitamin E group, alpha-tocopherol is considered to be the most active form. However, recent research has suggested tocotrienol to be a better antioxidant. Moreover, tocotrienol has been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein B and lipoprotein(a) plasma levels. In addition, tocotrienol has been suggested to have an anti-thrombotic and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. CONCLUSION: The physiological activities of tocotrienol suggest it to be superior than alpha-tocopherol in many situations. Hence, the role of tocotrienol in the prevention of cardiovascular disease and cancer may have significant clinical implications. Additional studies on its mechanism of action, as well as, long-term intervention studies, are needed to clarify its function. From the pharmacological point-of-view, the current formulation of vitamin E supplements, which is comprised mainly of alpha-tocopherol, may be questionable.

alpha-Tocopherol increased nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats.

Antioxidant protection provided by different doses of alpha-tocopherol was compared by determining nitric oxide synthase (NOS) activity in blood vessels of spontaneously hypertensive rats (SHR) treated with alpha-tocopherol. SHR were divided into four groups namely hypertensive control (C), treatment with 17 mg of alpha-tocopherol/kg diet (alpha1), 34 mg of alpha-tocopherol/kg diet (alpha2), and 170 mg of alpha-tocopherol/kg diet (alpha3). Wister Kyoto (WKY) rats were used as normal control (N). Blood pressure were recorded from the tail by physiography every other night for the duration of the study period of 3 months. At the end of the trial, animals were sacrificed. The NOS activity in blood vessels was measured by [3H]arginine radioactive assay and the nitrite concentration in plasma by spectrophotometry at wavelength 554 nm using Greiss reagent. Analysis of data was done using Student's t test and Pearson's correlation. The computer program Statistica was used for all analysis. Results of our study showed that for all the three alpha-tocopherol-treated groups, blood pressure was significantly (P < .001) reduced compared to the hypertensive control and maximum reduction of blood pressure was shown by the dosage of 34 mg of alpha-tocopherol/kg diet (C: 209.56 +/- 8.47 mm Hg; alpha2: 128.83 +/- 17.13 mm Hg). Also, NOS activity in blood vessels of SHR was significantly lower than WKY rats (N: 1.54 +/- 0.26 pmol/mg protein, C: 0.87 +/- 0.23 pmol/mg protein; P < .001). Although alpha-tocopherol in doses of alpha1, alpha2, and alpha3 increased the NOS activity in blood vessels, after treatment only that of alpha2 showed a statistical significance (P < .01). Plasma nitrite concentration was significantly reduced in SHR compared to normal WKY rats (N: 54.62 +/- 2.96 mol/mL, C: 26.24 +/- 2.14 mol/mL; P < .001) and accordingly all three groups showed significant improvement in their respective nitrite level (P < .001). For all groups, NOS activity and nitrite level showed negative correlation with blood pressure. It was significant for NOS activity in hypertensive control (r = -0.735, P = .038), alpha1 (r = -0.833, P = .001), and alpha2 (r = -0.899, P = .000) groups. For plasma nitrite, significant correlation was observed only in group alpha1 (r = -0.673, P = .016) and alpha2 (r = -0.643, P = .024). Only the alpha2 group showed significant positive correlation (r = 0.777, P = .003) between NOS activity and nitrite level. In conclusion it was found that compared to WKY rats, SHR have lower NOS activity in blood vessels, which upon treatment with antioxidant alpha-tocopherol increased the NOS activity and concomitantly reduced the blood pressure. There was correlation of lipid peroxide in blood vessels with NOS and nitric oxide, which implies that free radicals may be involved in the pathogenesis of hypertension.

Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols.

The apoptosis-inducing properties of RRR-alpha-, beta-, gamma-, and delta-tocopherols, alpha-, gamma-, and delta-tocotrienols, RRR-alpha-tocopheryl acetate (vitamin E acetate), and RRR-alpha-tocopheryl succinate (vitamin E succinate) were investigated in estrogen-responsive MCF7 and estrogen-nonresponsive MDA-MB-435 human breast cancer cell lines in culture. Apoptosis was characterized by two criteria: 1) morphology of 4,6-diamidino-2-phenylindole-stained cells and oligonucleosomal DNA laddering. Vitamin E succinate, a known inducer of apoptosis in several cell lines, including human breast cancer cells, served as a positive control. The estrogen-responsive MCF7 cells were more susceptible than the estrogen-nonresponsive MDA-MB-435 cells, with concentrations for half-maximal response for tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol of 14, 15, 7, and 97 micrograms/ml, respectively. The tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol induced MDA-MB-435 cells to undergo apoptosis, with concentrations for half-maximal response of 176, 28, 13, and 145 micrograms/ml, respectively. With the exception of RRR-delta-tocopherol, the tocopherols (alpha, beta, and gamma) and the acetate derivative of RRR-alpha-tocopherol (RRR-alpha-tocopheryl acetate) were ineffective in induction of apoptosis in both cell lines when tested within the range of their solubility, i.e., 10-200 micrograms/ml. In summary, these studies demonstrate that naturally occurring tocotrienols and RRR-delta-tocopherol are effective apoptotic inducers for human breast cancer cells.

Effects of gamma-tocotrienol on ApoB synthesis, degradation, and secretion in HepG2 cells.

gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen.

Effects of tocotrienol on the intracellular translocation and degradation of apolipoprotein B: possible involvement of a proteasome independent pathway.

gamma-Tocotrienol (gamma-T3), a HMG CoA reductase inhibitor, was previously shown to stimulate the intracellular degradation of apolipoprotein B (apoB) in HepG2 cells. The aim of this study was to explore the effects of gamma-T3 on the proteasome dependent co-translational degradation and the proteasome independent post-translational degradation of apoB. Previous studies have shown that apoB translocation across the endoplasmic reticulum (ER) membrane governs the co-translational degradative pathway of apoB. Therefore, we first examined the effects of gamma-T3 on this pathway using a specific translocation assay derived from HepG2 cells. Our results indicated that gamma-T3 reduced the efficiency of apoB translocation across the ER membrane, suggesting that co-translational degradation may be partially involved. Evidence of an ER associated post-translational degradation was also provided upon pre-treating digitonin-permeabilized HepG2 cells with a proteasome inhibitor, lactacystin. When chased for 2h, ER degradation of apoB was observed and was further enhanced in the presence of gamma-T3 versus untreated control, in spite of proteasome inhibition. Combined with the ability of ALLN, a proteasome and cysteine protease inhibitor, to block the post-translational degradation of apoB, the data suggest that gamma-T3 diverted more apoB to a cytosolic proteasomal dependent and possibly an ER-associated proteasomal independent degradation pathways.

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.

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 combinations with tamoxifen should be considered as a possible improvement in breast cancer therapy.

Palm oil tocotrienols and plant flavonoids act synergistically with each other and with Tamoxifen in inhibiting proliferation and growth of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells in culture.

Palm oil, unlike many other dietary oils, does not increase the yield of chemically-induced mammary tumors in rats when fed at high levels in the diet. This difference appears to be due to the vitamin E fraction of palm oil, which is rich in tocotrienols, since palm oil stripped of this fraction does increase tumor yields. Experiments in our laboratory have shown that tocotrienols inhibit proliferation and growth of both MDA-MB-435 and MCF-7 cells in culture much more effectively than a-tocopherol. In addition, it was found that combinations of tocotrienols with Tamoxifen, a drug widely used for treatment of breast cancer, inhibit these cells more effectively than either tocotrienols or Tamoxifen alone. The present studies have now shown synergistic effects between tocotrienols and a number of other flavonoids from various plant sources, including citrus fruits, in the inhibition of both MDA-MB-435 and MCF-7 cells (IC50s 0.05-25 and 0.02-5 µg/mL respectively). In the MCF-7 cells, 1:1:1 combinations of tocotrienols, flavonoids and Tamoxifen were even more effective, with the best combination being d-tocotrienol, hesperetin and Tamoxifen (IC50 0.0005 µg/mL). These results suggest that diets containing palm oil may reduce the risk of breast cancer, particularly when eaten with other plant foods containing flavonoids, and may also enhance the effectiveness of Tamoxifen for treatment of breast cancer.

Modulation of vascular endothelial cell function by palm oil antioxidants.

Several cardiovascular risk factors including, hypercholesterolaemia and hypertension, lead to diseased blood vessels due to endothelial cell dysfunction. Recent studies also indicate that such alterations in blood vessel function may involve free radical related mechanism(s). Therefore, in the present study, two different preparations of palm oils with variable antioxidant profiles, as well as a purified antioxidant fraction extracted from unprocessed palm oil (tocotrienol-rich-factor; TRF), were tested for their ability to influence blood vessel dysfunction in the spontaneously hypertensive rat (SHR). Adult SHRs were fed a synthetic diet supplemented (5% w/w) with either physically refined palm oil (PO), golden palm cooking oil (Nutrolein; GPO) or olive oil (OO; control diet). Antioxidant rich diet (TRF diet) was prepared by supplementing the OO diet with 0.2% (w/w) TRF. After 12 weeks of pre-feeding, segments of thoracic aorta were used to evaluate vascular function. Compared to the normotensive Wistar-Kyoto (WKY) control rats, aortic rings from the SHR showed impaired endothelium dependent relaxation to acetylcholine (ACh) which was restored by dietary TRF (p<0.05, ANOVA and Tukey's test). In addition, the paradoxical increase in tension in control hypertensive vessels observed at higher doses of ACh was prevented by TRF and also by the PO and GPO diets. Although the development of thromboxane-like constrictor response, after the inhibition of nitric oxide in hypertensive vessels, was unaffected by test diets, both TRF and GPO feeding prevented the amplification of this unwanted constriction by a threshold dose (7.2x10-10 M) of noradrenaline. Results suggest a modulatory role for minor constituents of edible oils and are in agreement with the recently reported benefits of natural antioxidants against cardiovascular diseases.

Palm oil antioxidant effects in patients with hyperlipidaemia and carotid stenosis-2 year experience.

Antioxidants appear to play a role in the prevention of atherosclerosis. Here, we investigated the antioxidant properties of a γ-tocotrienol and α-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 a 24-month period in 50 patients with cerebrovascular disease. Change in stenosis was measured with bilateral duplex ultrasonography. These studies revealed apparent carotid atherosclerotic regression in eight and progression in two of the 25 antioxidant patients, while none of the control group exhibited regression and ten of 25 showed progression (p<0.01). Serum α-tocopherol doubled while tocotrienols were undetectable throughout the study. Serum thiobarbituric acid reactive substances, decreased in the treatment group from 1.08 ± 0.14 to 0.80 ± 0.14 µM (p<0.05) after 24 months, and in the placebo group, they increased nonsignificantly from 0.99 ± 0.16 to 1.06 ± 0.17 µM. Both antioxidant and placebo groups displayed significantly increased 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. Palm oil tocols appear to benefit the course of carotid atherosclerosis.

Dietary alpha-tocopherol attenuates the impact of gamma-tocotrienol on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in chickens.

The concentration-dependent impact of gamma-tocotrienol on serum cholesterol can be traced to the posttranscriptional down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. gamma-Tocotrienol also suppresses tumor growth. Palmvitee, the tocopherol and tocotrienol-rich fraction of palm oil, is the sole commercial source of gamma-tocotrienol. Contrary to the universal findings of the efficacy of gamma-tocotrienol there are conflicting reports of the impact of Palmvitee on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, serum cholesterol concentrations and tumor development. These conflicting reports led us to examine the impact of alpha-tocopherol on the cholesterol-suppressive action of gamma-tocotrienol. Control and experimental diets were fed to groups of White Leghorn chickens (n = 10) for 26 d. The control diet was supplemented with 21 nmol alpha-tocopherol/g. All experimental diets provided 141 nmol of blended tocols/g diet. The alpha-tocopherol and gamma-tocotrienol concentrations of the experimental diets ranged from 21 to 141 and 0 to 120 nmol/g, respectively. We now report that including alpha-tocopherol in tocol blends containing adequate gamma-tocotrienol to suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase activity results in an attenuation of the tocotrienol action (P < 0.001). A summary of results from studies utilizing different Palmvitee preparations shows that effective preparations consist of 15-20% alpha-tocopherol and approximately 60% gamma- (and delta-) tocotrienol, whereas less effective preparations consist of > or = 30% alpha-tocopherol and 45% gamma- (and delta-) tocotrienol.

Response of hypercholesterolemic subjects to administration of tocotrienols.

The cholesterol-suppressive actions of Palmvitee and gamma-tocotrienol were assessed in hypercholesterolemic subjects after acclimation to the American Heart Association Step 1 dietary regimen for four and eight weeks, respectively. The four-week dietary regimen alone elicited a 5% decrease (P < 0.05) in the cholesterol level of the 36 subjects. Subjects continuing on the dietary regimen for a second four-week period experienced an additional 2% decrease in their cholesterol levels. Dietary assessments based on unanticipated recalls of 24-h food intake records suggest that significant reductions in energy and fat, predominantly in saturated fat, intakes are responsible. The subjects experienced significant Palmvitee- and gamma-tocotrienol-mediated decreases in cholesterol. The group of subjects acclimated to the dietary regimen for four weeks responded to Palmvitee (a blend of tocols providing 40 mg alpha-tocopherol, 48 mg alpha-tocotrienol, 112 mg gamma-tocotrienol, and 60 mg delta-to-cotrienol/day for four weeks) with a 10% decrease in cholesterol (P < 0.05). Dietary assessments showed no further change in energy and fat intakes. alpha-Tocopherol attenuated the cholesterol-suppressive action of the tocotrienols. The second group of subjects, acclimated to the dietary regimen for eight weeks, received 200 mg gamma-tocotrienol/d for four weeks. The cholesterol-suppressive potency of this alpha-tocopherol-free preparation was calculated to be equivalent to that of the mixture of tocotrienols (220 mg) used in the prior study. Cholesterol levels of the 16 subjects in the second group decreased 13% (P < 0.05) during the four-week trial. Plasma apolipoprotein B and ex vivo generation of thromboxane B2 were similarly responsive to the tocotrienol preparations, whereas neither preparation had an impact on high density lipoprotein cholesterol and apolipoprotein A-1 levels.

Antioxidant effects of tocotrienols in patients with hyperlipidemia and carotid stenosis.

Antioxidants may have a role in the prevention of atherosclerosis. In the present 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.

gamma-Tocotrienol as a hypocholesterolemic and antioxidant agent in rats fed atherogenic diets.

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)

Long-term administration of tocotrienols and tumor-marker enzyme activities during hepatocarcinogenesis in rats.

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.

Long-term tocotrienol supplementation and glutathione-dependent enzymes during hepatocarcinogenesis in the rat.

The effects of long-term administration of tocotrienol on hepatocarcinogenesis in rats induced by diethyl nitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated by the determination of plasma and liver gamma-glutamyl transpeptidase (GGT), cytosolic glutathione reductase (GSSG-Rx), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST). Twenty-eight male Rattus norwegicus rats (120-160g) were divided according to treatments into four groups: control group, tocotrienol - supplemented diet group (30mg/kg food), DEN/AAF-treated group and DEN/AAF treated plus tocotrienol-supplemented-diet group (30mg/kg food). The rats were sacrificed after nine months. The results obtained indicated no difference in the morphology and histology of the livers of control and tocotrienol-treated rats. Greyish-white neoplastic nodules (two per liver) were found in all the DEN/ AAF treated rats (n-10) whereas only one nodule was found in one of the carcinogen treated rats receiving tocotrienol supplementation (n-6). Histological examination showed obvious cellular damage for both the DEN/AAF-treated rats and the tocotrienol-supplemented rats but were less severe in the latter. Treatment with DEN/AAF caused increases in GGT, GSH-Px, GST and GSSG-Rx activities when compared to controls. These increases were also observed when tocotrienol was supplemented with DEN/AAF but the increases were less when compared to the rats receiving DEN/AAF only.

Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (palmvitee).

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.

Effect of a palm-oil-vitamin E concentrate on the serum and lipoprotein lipids in humans.

The effect of a capsulated palm-oil-vitamin E concentrate (palmvitee) on human serum and lipoprotein lipids was assessed. Each palmvitee capsule contains approximately 18, approximately 42, and approximately 240 mg of tocopherols, tocotrienols, and palm olein, respectively. All volunteers took one palmvitee capsule per day for 30 consecutive days. Overnight fasting blood was taken from each volunteer before and after the experiment. Serum lipids and lipoproteins were analyzed by using the enzymatic CHOD-PAP method. Our results showed that palmvitee lowered both serum total cholesterol (TC) and low-density-lipoprotein cholesterol (LDL-C) concentrations in all the volunteers. The magnitude of reduction of serum TC ranged from 5.0% to 35.9% whereas the reduction of LDL-C values ranged from 0.9% to 37.0% when compared with their respective starting values. The effect of palmvitee on triglycerides (TGs) and HDL-C was not consistent. Our results show that the palmvitee has a hypocholesterolemic effect.