Vitamin E: action, metabolism and perspectives

Natural vitamin E includes four tocopherols and four tocotrienols. RRR-alpha-tocopherol is the most abundant form in nature and has the highest biological activity. Although vitamin E is the main lipid-soluble antioxidant in the body, not all its properties can be assigned to this action. As antioxidant, vitamin E acts in cell membranes where prevents the propagation of free radical reactions, although it has been also shown to have pro-oxidant activity. Non-radical oxidation products are formed by the reaction between alpha-tocopheryl radical and other free radicals, which are conjugated to glucuronic acid and excreted through the bile or urine. Vitamin E is transported in plasma lipoproteins. After its intestinal absorption vitamin E is packaged into chylomicrons, which along the lymphatic pathway are secreted into the systemic circulation. By the action of lipoprotein lipase (LPL), part of the tocopherols transported in chylomicrons are taken up by extrahepatic tissues, and the remnant chylomicrons transport the remaining tocopherols to the liver. Here, by the action of the "alpha-tocopherol transfer protein", a major proportion of alpha-tocopherol is incorporated into nascent very low density lipoproteins (VLDL), whereas the excess of alpha-tocopherol plus the other forms of vitamin E are excreted in bile. Once secreted into the circulation, VLDL are converted into IDL and LDL by the action of LPL, and the excess of surface components, including alpha-tocopherol, are transferred to HDL. Besides the LPL action, the delivery of alpha-tocopherol to tissues takes place by the uptake of lipoproteins by different tissues throughout their corresponding receptors. Although we have already a substantial information on the action, effects and metabolism of vitamin E, there are still several questions open. The most intriguing is its interaction with other antioxidants that may explain how foods containing small amounts of vitamin E provide greater benefits than larger doses of vitamin E alone.     

Absorption,Transport and Metabolism of Vitamin E

Vitamin E includes eight naturally occurring fat-soluble nutrients called tocopherols and dietary intake of vitamin E activity is essential in many species. α-Tocopherol has the highest biological activity and the highest molar concentration of lipid soluble antioxidant in man. Deficiency of vitamin E may cause neurological dysfunction, myopathies and diminished erythrocyte life span. α-Tocopherol is absorbed via the lymphatic pathway and transported in association with chylomicrons. In plasma α-tocopherol is found in all lipoprotein fractions, but mostly associated with apo B-containing lipoproteins in man. In rats approximately 50% of α-tocopherol is bound to high density lipoproteins (HDL). After intestinal absorption and transport with chylomicrons α-tocopherol is mostly transferred to parenchymal cells of the liver were most of the fat-soluble vitamin is stored. Little vitamin E is stored in the non-parenchymal cells (endothelial, stellate and Kupffer cells). α-Tocopherol is secreted in association with very low density lipoprotein (VLDL) from the liver. In the rat about 90% of total body mass of α-tocopherol is recovered in the liver, skeletal muscle and adipose tissue. Most α-tocopherol is located in the mitochondrial fractions and in the endoplasmic reticulum, whereas little is found in cytosol and peroxisomes. Clinical evidence from heavy drinkers and from experimental work in rats suggests that alcohol may increase oxidation of α-tocopherol, causing reduced tissue concentrations of α-tocopherol. Increased demand for vitamin E has also been observed in premature babies and patients with malabsorption, but there is little evidence that the well balanced diet of the healthy population would be improved by supplementation with vitamin E.     

Unleashing the untold and misunderstood observations on vitamin E

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

Increased α-tocopherol content in soybean seed overexpressing the <Emphasis Type="Italic">Perilla frutescens γ-tocopherol methyltransferase</Emphasis> gene

Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, γ-tocopherol, the biosynthetic precursor to α-tocopherol, is the predominant form found in the seed, whereas α-tocopherol is the most bioactive component. This suggests that the final step of the α-tocopherol biosynthetic pathway catalyzed by γ-tocopherol methyltransferase (γ-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active α-tocopherol form could have significant health benefits. In order to increase the soybean seed α-tocopherol content, the γ-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens γ-TMT gene resulted in a 10.4-fold increase in the α-tocopherol content and a 14.9-fold increase in the β-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that α-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the α-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.     

Vitamin E requirements, transport, and metabolism: Role of α-tocopherol-binding proteins

Vitamin E (RRR-α-tocopherol) is a lipid-soluble antioxidant that is present in the membranes of intracellular organelles. There it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E that differ in their structure and in biological activity in vivo and in vitro. Although γ-tocopherol is a more effective free radical scavenger than α-tocopherol in vitro, the reverse is true in vivo, suggesting that the tocopherol distribution systems favor the localization of α-tocopherol at the sites where it is required. Vitamin E is transported in plasma primarily by lipoproteins, but little is known of how it is transported intracellularly. A 30 kDa α-tocopherol-binding protein in the liver cytoplasm may regulate plasma vitamin E concentrations by preferentially incorporating the vitamin E homologue, RRR-α-tocopherol (α-tocopherol), into nascent very low density lipoproteins. However, this α-tocopherol-binding protein is unique to the hepatocyte, whereas α-tocopherol is present in the cells of all major tissues. Moreover α-tocopherol accumulates at those sites within the cell where oxygen radical production is greatest and thus where it is most required; in the membranes of heavy mitochondria, light mitochondria, and endoplasmic reticulum. This raises the question of how the lipid-soluble α-tocopherol is transported intracellularly in different tissues. We have identified a new α-tocopherol-binding protein of molecular mass 14.2 kDa in the cytosol of heart and liver. This protein specifically binds α-tocopherol in preference to the δ- and γ-homologues but does not bind oleate. Studies on immunoreactivity and ligand specificity of the protein suggest that it is not a fatty acid-binding protein. The 14.2 kDa α-tocopherol-binding protein stimulates the transfer of α-tocopherol from liposomes to mitochondria in vitro by 8 to 10 fold. We suggest that this low molecular mass TBP may be responsible for the intracellular transport and distribution of α-tocopherol in the tissues.     

Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid. We postulate that similar mechanisms may be important in the regulation of LPL activity at the vascular endothelium.     

Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV

Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent chylomicrons.     

The two faces of α- and γ-tocopherols: an in vitro and ex vivo investigation into VLDL, LDL and HDL oxidation

BackgroundVitamin E and its derivatives, namely, the tocopherols, are known antioxidants, and numerous clinical trials have investigated their role in preventing cardiovascular disease; however, evidence to date remains inconclusive. Much of the in vitro research has focused on tocopherol's effects during low-density lipoprotein (LDL) oxidation, with little attention being paid to very LDL (VLDL) and high-density lipoprotein (HDL). Also, it is now becoming apparent that γ-tocopherol may potentially be more beneficial in relation to cardiovascular health.ObjectivesDo α- and γ-tocopherols become incorporated into VLDL, LDL and HDL and influence their oxidation potential in an in vitro and ex vivo situation?DesignFollowing (i) an in vitro investigation, where plasma was preincubated with increasing concentrations of either α- or γ-tocopherol and (ii) an in vivo 4-week placebo-controlled intervention with α- or γ-tocopherol. Tocopherol incorporation into VLDL, LDL and HDL was measured via high-pressure liquid chromatography, followed by an assessment of their oxidation potential by monitoring conjugated diene formation.ResultsIn vitro: Both tocopherols became incorporated into VLDL, LDL and HDL, which protected VLDL and LDL against oxidation. However and surprisingly, the incorporation into HDL demonstrated pro-oxidant properties. Ex vivo: Both tocopherols were incorporated into all three lipoproteins, protecting VLDL and LDL against oxidation; however, they enhanced the oxidation of HDL.ConclusionsThese results suggest that α- and γ-tocopherols display conflicting oxidant activities dependent on the lipoprotein being oxidized. Their pro-oxidant activity toward HDL may go some way to explain why supplementation studies with vitamin E have not been able to display cardioprotective effects.     

Microbiological production of tocopherols: current state and prospects

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.     

Biological functions and metabolic fate of vitamin E revisited

Information accumulated lately has confirmed the essentiality of vitamin E for humans and provided a better understanding of its biological function and metabolic fate. The discovery of -tocopherol transfer protein, which preferentially binds to RRR--tocopherol, not only provides conclusive evidence of the essentiality of vitamin E for humans, but also sheds light on the superiority of RRR--tocopherol biologically over other isomers. The presence of tocopherol regeneration systems and multiple interdependent antioxidant systems is largely responsible for the lack of a widespread deficiency in humans and the difficulty to deplete vitamin E in the adult. The bulk of excess tocopherols consumed is excreted to feces unchanged or to urine with the side chain shortened but the chroman ring intact. The ability of dietary vitamin E to mediate mitochondrial superoxide generation affords a possible mode of action of vitamin E at the tissue levels. By decreasing the generation and/or the levels of reactive oxygen/nitrogen species, dietary vitamin E not only protects against oxidative damage, but also modulates the expression and/or activation of redox-sensitive biological response modifiers that regulate important cellular events.     

The “unexpected role” of vitamin E in free radical-induced hemolysis of human erythrocytes

Vitamin E involves a group of tocopherols and tocotrienols, in which α-tocopherol with the highest biological activity plays a more efficient role in advanced lesions with aged oxidized tissues. However, the results of the present study reveal that a large amount of endogenous α-tocopherol in human low-density lipoprotein (LDL) in the absence of any other antioxidants may initiate additional free radical propagation under low concentration of free radical initiator (i.e., 2,2′-azobis(2-amidinopropane hydrochloride) [AAPH], a water-soluble free radical source) to peroxide polyunsaturated fatty acids in LDL in the manner of α-tocopherol-mediated peroxidation (TMP). Whether the addition of high concentration of exogenous α-tocopherol to human erythrocytes under low concentration of AAPH can also drive TMP is the concern in this research work. Moreover, the hemolysis extent of human erythrocytes peroxidized by AAPH is followed easily by the determination of the hemoglobin outside the erythrocytes. A series of observations on various concentrations of AAPH-induced hemolysis in the presence of various concentrations of exogenous α-tocopherol demonstrates that the high concentration of exogenous α-tocopherol, coupled with low concentration of AAPH, can initiate TMP in the free-radical-induced peroxidation of human erythrocytes system as well. This result provides direct evidence to support TMP theory and expands its application into in vitro erythrocytes system.     

RRR- and SRR-alpha-tocopherols are secreted without discrimination in human chylomicrons, but RRR-alpha-tocopherol is preferentially secreted in very low density lipoproteins

Five subjects ingested in a single oral dose containing 50 mg each of 2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate) with natural stereochemistry, and of 2S,4'R,8'R-alpha-(5-C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate). These are two of eight stereoisomers in synthetic vitamin E. By day 1 the plasma and red blood cells were enriched fourfold with d6-RRR-alpha-tocopherol (P less than 0.004). The ratio of d6-RRR-/d2-SRR- further increased over the succeeding 4 days, because the d3-SRR- decreased at a faster rate than did the d6-RRR-stereoisomer. Plasma and lipoproteins were isolated at intervals during the first day, and daily for 3 days, from four additional subjects fed a mixture of equal amounts of the deuterated tocopherols. The plasma contained similar concentrations of the two forms until 11 h, when the d6-RRR-alpha-tocopherol concentration became significantly greater (P less than 0.05). The chylomicrons contained similar concentrations of the two deuterated tocopherols, but the VLDL (very low density lipoproteins) became preferentially enriched in d6-RRR-alpha-tocopherol by 11 h. The pattern of the deuterated tocopherols shows that during chylomicron catabolism all of the plasma lipoproteins were labeled equally with both tocopherols, but that during the subsequent VLDL catabolism the low and high density lipoproteins became enriched in d6-RRR-alpha-tocopherol. These results suggest the existence of a mechanism in the liver for assembling VLDL preferentially enriched in RRR- relative to SRR-alpha-tocopherol.     

Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism.

To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations.     

Lipoprotein lipase release from BFC-1 beta adipocytes. Effects of triglyceride-rich lipoproteins and lipolysis products.

Lipoprotein lipase (LPL), synthesized by adipocytes and myocytes, must be transported to the luminal endothelial cell surface where it then interacts with circulating lipoproteins. The first step in this extracellular LPL transport pathway is LPL release from the surface of LPL-synthesizing cells. Because hydrolysis of triglyceride (TG)-rich lipoproteins releases LPL from the apical surface of endothelial cells, we hypothesized that the same substances dissociate LPL from adipocytes. 125I-LPL was bound to the surface of brown adipocytes (BFC-1 beta). LPL binding to the adipocyte surface was greater than to endothelial cell surfaces. Using low concentrations of heparin, more LPL was released from endothelial cells than BFC-1 beta, suggesting that the affinity of LPL binding to the adipocytes was greater than LPL affinity for endothelial cells. Greater than 3-fold more LPL was released from the cell surface when very low density lipoproteins (VLDL) were added to culture medium containing 3% bovine serum albumin. LPL remaining on the cell surface decreased with VLDL addition. Endogenously produced LPL activity was also released from the cells by VLDL. Low and high density lipoproteins did not release 125I-LPL or LPL activity from the adipocytes. To assess whether lipolysis was necessary for LPL release, BFC-1 beta were incubated with TG-rich lipoproteins from a patient with apoCII deficiency. The apoCII-deficient lipoproteins did not release LPL unless an exogenous source of apoCII was added. Apolipoproteins E and Cs and high molar ratios of oleic acid:bovine serum albumin did not release surface-associated LPL. Lysolecithin (25 and 100 microM), but not lecithin, monoglycerides, or diglycerides, released adipocyte surface LPL. Because lysolecithin also released LPL during a 4 degrees C incubation, cellular metabolic functions are not required for LPL dissociation from the cells. Lysolecithin also inhibited LPL binding to endothelial cells; however, this effect was abrogated by addition of bovine serum albumin. We hypothesize that lipolysis products from TG-rich lipoproteins release adipocyte surface LPL, which can then be transported to the luminal endothelial cell surface.     

Alpha-tocopherol is secreted from rat liver in very low density lipoproteins

Three separate studies were carried out to test the hypothesis that rat liver secretes vitamin E (alpha-tocopherol) within very low density lipoproteins (VLDL). i) When the clearance of plasma chylomicrons (CM) and VLDL was blocked by the administration of Triton WR-1339, alpha-tocopherol concentrations increased linearly with time in both classes of triacylglycerol-rich lipoproteins, although accumulation rates within VLDL exceeded those within CM. For fasted rats, appearance of alpha-tocopherol in VLDL persisted at slightly reduced rates. alpha-Tocopherol and triglycerides in the VLDL fraction responded to Triton WR-1339 administration by coordinate increases. In contrast to the situation in serum, alpha-tocopherol concentrations decreased in the liver following injection of Triton. ii) In order to inhibit the secretion of hepatic lipoproteins containing apolipoprotein B (apoB), rats were fed a diet containing orotic acid. This resulted in a reduction of apoB and alpha-tocopherol concentrations in serum and VLDL, whereas the vitamin E content of liver was increased. iii) In primary cultures of hepatocytes, alpha-tocopherol was secreted into the culture media predominantly within VLDL. We, therefore, conclude that the liver secretes alpha-tocopherol within VLDL and in this way contributes to the maintenance of serum vitamin E concentrations.     

Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry

A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.     

Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Increasing vitamin E activity in economically important oil crops such as perilla will enhance the nutritional value of these crops. Perilla (Perilla frutescens Britt) represents an important oil crop in Asian countries, including Korea. Using Agrobacterium-mediated transformation, we have engineered perilla with the γ-tocopherol methyltransferase (γ-TMT) gene under the control of seed-specific vicillin promoter. Molecular characterization including PCR, Southern and Northern blots confirmed that the γ-TMT transgene was successfully inherited to and expressed in the progeny plants. The γ -TMT transgene was specifically expressed in immature seeds of transgenic plants, leading to efficient conversion of γ-tocopherol to α-tocopherol and dramatic increase in seed α-tocopherol content, as detected by high performance liquid chromatography analysis. We also showed that such a high α-tocopherol content phenotype was transmitted to the progeny plants. In addition, there was no significant change in fatty acid composition in transgenic seeds as compared with untransformed control Yeupsil cultivar, suggesting the lack of interplay between the fatty acid and tocopherol biosynthesis pathways. This was the first report on over expression of the γ-TMT gene in transgenic perilla displaying desirable high α-tocopherol content phenotype. Since α-tocopherol has the highest vitamin E activity, the transgenic perilla with high α-tocopherol content in seeds developed in this study will benefit both human and animal health.     

High dietary level of synthetic vitamin E on lipid peroxidation,membrane fatty acid composition and cytotoxicity in breast cancer xenograft and in mouse host tissue

Background  

d-α-tocopherol is a naturally occurring form of vitamin E not previously known to have antitumor activity. Synthetic vitamin E (sE) is a commonly used dietary supplement consisting of a mixture of d-α-tocopherol and 7 equimolar stereoisomers. To test for antilipid peroxidation and for antitumor activity of sE supplementation, two groups of nude mice bearing a MDA-MB 231 human breast cancer tumor were fed an AIN-76 diet, one with and one without an additional 2000 IU/kg dry food (equivalent to 900 mg of all-rac-α-tocopherol or sE). This provided an intake of about 200 mg/kg body weight per day. The mice were killed at either 2 or 6 weeks after the start of dietary intervention. During necropsy, tumor and host tissues were excised for histology and for biochemical analyses.