We evaluated the effects of vitamin E and beta-carotene on apolipoprotein (apo)E +/- female mice, which develop atherosclerosis only when fed diets high in triglyceride and cholesterol. Mice were fed a nonpurified control diet (5.3 g/100 g triglyceride, 0.2 g/100 g cholesterol), an atherogenic diet alone (15.8 g/100 g triglyceride, 1.25 g/100 g cholesterol, 0.5 g/100 g Na cholate) or the atherogenic diet supplemented with either 0.5 g/100 g (+)-alpha-tocopherol (mixed isomers); 0.5 g/100 g palm tocopherols (palm-E; 33% alpha-tocopherol, 16.1% alpha-tocotrienol, 2.3% beta-tocotrienol, 32.2% gamma-tocotrienol, 16.1% delta-tocotrienol); 1.5 g/100 g palm-E; or 0.01 g/100 g palm-carotenoids (58% beta-carotene, 33% alpha-carotene, 9% other carotenoids). Compared with mice fed the control diet, plasma cholesterol was fourfold greater in mice fed the atherogenic diet. Mice fed the 1.5 g/100 g palm-E supplement had 60% lower plasma cholesterol than groups fed the other atherogenic diets. Mice fed the atherogenic diet had markedly higher VLDL, intermediate density lipoprotein (IDL) and LDL cholesterol and markedly lower HDL cholesterol than the controls. Lipoprotein patterns in mice supplemented with alpha-tocopherol or palm carotenoids were similar to those of the mice fed the atherogenic diet alone, but the pattern in mice supplemented with 1. 5 g/100 g palm-E was similar to that of mice fed the control diet. In mice fed the atherogenic diet, the hepatic cholesterol plus cholesterol ester concentration was 4.4-fold greater than in mice fed the control diet. Supplementing with 1.5 g/100 g palm-E lowered hepatic cholesterol plus cholesterol ester concentration 66% compared with the atherogenic diet alone. Mice fed the atherogenic diet had large atherosclerotic lesions at the level of the aortic valve. With supplements of 0.5 g/100 g palm-E or 1.5 g/100 g palm-E, the size of the lesions was 92 or 98% smaller, respectively. The 0.5 g/100 g alpha-tocopherol and palm carotenoid supplements had no effect. Supplements did not alter mRNA abundance for apolipoproteins A1, E, and C3. The beneficial effect of tocotrienols on atherogenesis, the plasma lipoprotein profile and accumulation of hepatic cholesterol esters cannot be attributed to their antioxidant properties.

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

McIntyre BS, Briski KP, Gapor A, Sylvester PW.

Proc Soc Exp Biol Med. 2000 Sep;224(4):292-301.

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.

Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant, and antitumor properties

Qureshi AA, Mo H, Packer L, Peterson DM.

J Agric Food Chem. 2000 Aug;48(8):3130-40

Two novel tocotrienols were isolated from stabilized and heated rice bran, apart from the known alpha-, beta-, gamma-, and delta-tocopherols andtocotrienols. These new tocotrienols were separated by HPLC, using a normal phase silica column. Their structures were determined by ultraviolet, infrared, nuclear magnetic resonance, circular dichroism, and high-resolution mass spectroscopies and established as desmethyl tocotrienol [3, 4-dihydro-2-methyl-2-(4,8,12-trimethyltrideca-3′(E),7′(E), 11′-trienyl)-2H-1-benzopyran-6-ol] and didesmethy tocotrienol [3, 4-dihydro-2-(4,8,12-trimethyltrideca-3′(E),7′(E), 11′-trienyl)-2H-1-benzopyran-6-ol]. These tocotrienols significantly lowered serum total and LDL cholesterol levels and inhibited HMG-CoA reductase activity in chickens. They had much greater in vitro antioxidant activities and greater suppression of B16 melanoma cell proliferation than alpha-tocopherol and known tocotrienols. Results indicated that the number and position of methyl substituents in tocotrienols affect their hypocholesterolemic, antioxidant, and antitumor properties.

Vitamin E is a fat-soluble vitamin that consists of a group of tocols and tocotrienols with hydrophobic character, but possessing a hydroxyl substituent that confers an amphipathic character on them. The isomers of biological importance are the tocopherols, of which alpha-tocopherol is the most potent vitamin. Vitamin E partitions into lipoproteins and cell membranes, where it represents a minor constituent of most membranes. It has a major function in its action as a lipid antioxidant to protect the polyunsaturated membrane lipids against free radical attack. Other functions are believed to be to act as membrane stabilizers by forming complexes with the products of membrane lipid hydrolysis, such as lysophospholipids and free fatty acids. The main experimental approach to explain the functions of vitamin E in membranes has been to study its effects on the structure and stability of model phospholipid membranes. This review describes the function of vitamin E in membranes and reviews the current state of knowledge of the effect of vitamin E on the structure and phase behaviour of phospholipid model membranes.

Selective uptake of dietary tocotrienols into rat skin

Ikeda S, Niwa T, Yamashita K.

J Nutr Sci Vitaminol (Tokyo). 2000 Jun;46(3):141-3.

Using a vitamin E mixture extracted from palm oil, the tissue distribution of dietary tocotrienols and tocopherols was examined in rats and mice. Wistar rats (4-wk-old) were fed a diet containing 48.8 mg/kg alpha-tocopherol, 45.8 mg/kg alpha-tocotrienol and 71.4 mg/kg gamma-tocotrienol for 8 wk. Nude mice (BALB/c Slc-nu, 8-wk-old) and hairless mice (SKH1, 8-wk-old) were fed the same diet for 4 wk. alpha-Tocopherol was abundantly retained in the skin, liver, kidney and plasma of rats and mice. alpha-Tocotrienol and gamma-tocotrienol were detected slightly in the liver, kidney and plasma, while substantial amounts of these tocotrienols were detected in the skin of both rats and mice. The present study suggests that the skin is a unique tissue in respect to its ability to discriminate between various vitamin E analogs.

Natural vitamin E is composed of eight different vitamers (alpha-, beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols). As these eight vitamers have different antioxidant and biological activities, it is necessary to have quantitative data on each substance separately. The aim of this study was to find universal HPLC columns for the separation of all eight components and to test if a few columns of the same material (different batches) will give reproducible results. Normal-phase HPLC separations of vitamin E compounds in a prepared mixture (containing oat extracts, palm oil and tocopherol standards) were tried on six silica, three amino and one diol columns. As shown by calculations of retention factors (k), separation factors (alpha), numbers of theoretical plates (N) and resolutions (Rs), the best separations were obtained on three silica columns and two amino columns using 4 or 5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert.-butyl methyl ether in hexane as the mobile phase.

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.

Vitamin E: Non-antioxidant roles

Azzi A, Stocker A.

Prog Lipid Res. 2000 May;39(3):231-55.

Vitamin E was originally considered a dietary factor of animal nutrition especially important for normal reproduction. The significance of vitamin E has been subsequently proven as a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life processes. More recently alpha-tocopherol has been found to possess functions that are independent of its antioxidant/radical scavenging ability. Absorption in the body is alpha-tocopherol selective and other tocopherols are not absorbed or are absorbed to a lesser extent. Furthermore, pro-oxidant effects have been attributed to tocopherols as well as an anti-nitrating action. Non-antioxidant and non-pro-oxidant molecular mechanisms of tocopherols have been also described that are produced by alpha-tocopherol and not by beta-tocopherol. alpha-Tocopherol specific inhibitory effects have been seen on protein kinase C, on the growth of certain cells and on the transcription of some genes (CD36, and collagenase). Activation events have been seen on the protein phosphatase PP2A and on the expression of other genes (alpha-tropomyosin and Connective Tissue Growth Factor). Non-antioxidant molecular mechanisms have been also described for gamma-tocopherol, delta-tocopherol and tocotrienols.

A precise and selective liquid chromatographic procedure for determining tocopherol and tocotrienol isomers in vegetable oils, formulated preparations, and biscuits was developed and validated. The proposed method quantitates vitamin E in better conditions of recoverability and reproducibility than the standard saponification procedure. Tocopherols and tocotrienols were extracted in hexane from vegetable oils, passed through a silica Sep-pak, chromatographed on a mu-Bondapak C18 column with a mobile phase of methanol-water (95 + 5, v/v), identified at 292 nm, and detected with fluorescence procedure (excitation 296 nm, and emission 330 nm). The correlation coefficient on the calibration curve was 0.9995 over the range of 0.1 to 100 microg/mL. Overall recovery of vitamin E isomers was 93%; coefficients of variation for intra- and interday precision, < 2.25%. The results obtained from extraction methods 1 (with saponification) and 2 (without saponification) were compared by ANOVA test. Significant differences appeared between vitamin E isomers (p < or = 0.05).

Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60 (c-Src) kinase activation and death of HT4 neuronal cells

Sen CK, Khanna S, Roy S, Packer L.

J Biol Chem. 2000 Apr 28;275(17):13049-55.

HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death.Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.

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