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Vitamin E: protective role of a Janus molecule

Institute of Biochemistry and Molecular Biology, University of Bern, 3012 Bern, Switzerland

¹Correspondence: Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland. E-mail: angelo.azzi{at}mci.unibe.ch

	ABSTRACT

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Since the discovery of vitamin E in 1922, its deficiency has been associated with various disorders, particularly atherosclerosis, ischemic heart disease, and the development of different types of cancer. A neurological syndrome associated with vitamin E deficiency resembling Friedreich ataxia has also been described. Whereas epidemiological studies have indicated the role of vitamin E in preventing the progression of atherosclerosis and cancer, intervention trials have produced contradictory results, indicating strong protection in some cases and no significant effect in others. Although it is commonly believed that phenolic compounds like vitamin E exert only a protective role against free radical damage, antioxidant molecules can exert other biological functions. For instance, the antioxidant activity of 17-ß-estradiol is not related to its role in determining secondary sexual characters, and the antioxidant capacity of all-trans-retinal is distinguished from its role in rhodopsin and vision. Thus, it is not unusual that -tocopherol (the most active form of vitamin E) has properties independent of its antioxidant/radical scavenging ability. The Roman god Janus, shown in ancient coins as having two faces in one body, inspired the designation of ‘Janus molecules’ for these substances. The new biochemical face of vitamin E was first described in 1991, with an inhibitory effect on cell proliferation and protein kinase C activity. After a decade, this nonantioxidant role of vitamin E is well established, as confirmed by authoritative studies of signal transduction and gene regulation. More recently, a tocopherol binding protein with possible receptor function has been discovered. Despite such important developments in understanding the molecular mechanism and the targets of vitamin E, its new Janus face is not fully elucidated. Greater knowledge of the molecular events related to vitamin E will help in selecting the parameters for clinical intervention studies such as population type, dose response effects, and possible synergism with other compounds.—Ricciarelli, R., Zingg, J.-M., Azzi, A. Vitamin E: protective role of a Janus molecule.

Key Words: tocopherol • antioxidant • atherosclerosis • intervention trials

	VITAMIN E

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
The term vitamin E was introduced by Evans and Bishop to describe a dietary factor important for reproduction in rats (1) . Natural vitamin E includes two groups of closely related fat-soluble compounds, the tocopherols and tocotrienols, each with the four , ß, , and analogs (Fig. 1 ). The eight analogous compounds are widely distributed in nature; the richest sources are latex lipids (8% w/v), followed by edible plant oils. Sunflower seeds contain almost exclusively -tocopherol (59.5 mg/g of oil), oil from soybeans contains the -, -, and -tocopherol (62.4, 20.4, and 11.0 mg/g oil), and palm oil contains high concentrations of tocotrienols (17.2 mg/g oil) and -tocopherol (18.3 mg/g oil) (2) . Although the antioxidant property of these molecules is similar, distinct biological effects can be distinguished at a molecular level. The specificity is the result of a selective retention of -tocopherol in the body and the preferential interactions of some of the compounds with molecular components of the cells.

View larger version (15K): [in this window] [in a new window]   Figure 1. Chemical structure of tocopherol and tocotrienol analogs.

	UPTAKE OF VITAMIN E INTO THE BODY IS A HIGHLY SELECTIVE PROCESS; LITTLE IS KNOWN ABOUT ITS CELLULAR AND INTRACELLULAR DISTRIBUTION

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Because of its hydrophobicity, dietary vitamin E requires special transport mechanisms in the aqueous environment of the plasma, body fluids, and cells. In humans, vitamin E is taken up together with dietary lipids and bile in the proximal part of the intestine. The tocopherols are assembled together with triglycerides, cholesterol, phospholipids, and apolipoproteins into chylomicrons. During chylomicron lipolysis, a part of vitamin E is distributed to tissues. Overexpression of lipoprotein lipase increases the transfer of tocopherol from chylomicrons into skeletal muscle cells (3) . The other part is captured by the liver with the chylomicron remnants. In the liver, -tocopherol is specifically recognized by the 32 kDa -tocopherol transfer protein (-TTP), incorporated into very low density lipoproteins (VLDL), then transported and delivered to peripheral cells. The LDL and high density lipoprotein (HDL) fractions combined contain 90% of the total serum vitamin E in humans (4) . The plasma phospholipid transfer protein facilitates the exchange of tocopherol between LDL and HDL (5) .

In the lung, HDL is the primary source of vitamin E for type II pneumocytes, and its uptake is regulated by the expression of scavenger receptor SR-B1 (6) . In the brain, HDL-associated -tocopherol is transferred selectively into cells constituting the blood–brain barrier via scavenger receptor SR-BI (7) . Similarly, scavenger receptor SR-BI transports HDL-associated -tocopherol from the periphery into the liver, where again it is specifically recognized by -TTP, recycled, and secreted in VLDL (8) . When the -TTP gene is mutated, -tocopherol concentrations in serum and peripheral cells are very low, implying that uptake and absorption of -tocopherol by -TTP are essential to maintain an adequate amount of tocopherol in the organism. Thus, in addition to the dietary availability of -tocopherol, the expression level of liver -TTP protein may be critical in determining the -tocopherol level in plasma and peripheral cells. In peripheral cells, the highest content (150 µg/g tissue) is found in adipose tissue whereas erythrocytes have a relatively low content (2 µg/g tissue) of -tocopherol (9) .

Relative affinities of tocopherol analogs for -TTP calculated from the degree of competition for the alpha form are as follows: -tocopherol, 100%; ß-tocopherol, 38%; -tocopherol, 9%; -tocopherol, 2%; -tocopherol acetate, 2%; -tocopherol quinone, 2% (10) . The synthetic racemic tocopherol contains eight different side chain isomers, the RRR form (natural) and all the others containing S isomers. Some of the natural and the non-natural tocopherol isomers are excluded from the plasma and secreted with the bile (11 , 12) .

	WHAT IS THE ROLE OF THE TOCOPHEROL BINDING PROTEINS (TBPs) FOUND IN TISSUES?

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-TTP is expressed not only in the liver, but also in parts of the brain, where it may transport -tocopherol or perform other biological functions (13) in the retina (14) , lymphocytes, and fibroblasts (15) . Moreover, -TTP expression in the uterus plays an important role in supplying the vitamin to the labyrinthine trophoblast region of the placenta, explaining the fetal resorption occurring in rats fed a vitamin E-deficient diet (16) .

It is still unclear how many other -tocopherol binding proteins exist, and which mechanism regulates -tocopherol transfer and its concentration in peripheral cells. Recently, a novel tocopherol binding protein was identified, the 46 kDa tocopherol-associated protein (TAP) (17) . It appears to be ubiquitously expressed, although initial data indicated a preferential expression in brain, liver, and prostate. TAP may be specifically involved in the intracellular transport of tocopherol, for example, between membrane compartments and the plasma membrane. The similarity of TAP to the yeast secretory protein indicates that TAP may trigger similar functions, such as phospholipid/tocopherol signaling, phospholipid/tocopherol transport and secretion, or adjusting the tocopherol composition of membranes.

A 15 kDa TBP that preferentially binds -tocopherol may be responsible for intracellular distribution of -tocopherol (18) . The presence of a membrane TBP (TBPpm) in tissues may regulate their -tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of -tocopherol in these cells (19) .

The 75 kDa plasma phospholipid transfer protein, which is known to catalyze the exchange of phospholipids and other amphipathic compounds between lipid structures, also binds -tocopherol and facilitates the exchange of tocopherol between HDL and LDL (5) .

	VITAMIN E DEFICIENCY IS ASSOCIATED WITH A PRECISE AILMENT: CEREBELLAR ATAXIA

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Mutations of the -TTP gene lead to reduced -tocopherol concentrations in plasma and tissues, which lead ultimately to a severe syndrome named ataxia with vitamin E deficiency (AVED) (20) . These patients show loss of neurons, symptoms of retinal atrophy, massive accumulation of lipofuscin in neurons including dorsal root ganglions, and retinitis pigmentosa (21) .

As in hepatocellular carcinoma, reduced -TTP gene expression could also lead to reduced plasma level of -tocopherol (22) . Moreover, the uptake of dietary antioxidants (tocopherols, carotenoids, flavonoids) and transport by chylomicrons from intestine to the liver is impaired in abetalipoproteinemia and lipid malabsorption syndromes such as cholestatic liver disease, short-bowel syndrome, and cystic fibrosis, which often show symptoms similar to AVED (23) . It is unknown whether the degenerative neurological symptoms in patients with vitamin E deficiency syndromes are the result of insufficient protection by antioxidants or are due to a lack of specific and nonantioxidant effects mediated by -tocopherol. In these diseases, the transport of -tocopherol is impaired in either the liver or intestine by the complete absence of a transport pathway, leading to extreme low plasma -tocopherol levels (24) . It can be assumed that conditions may exist with partially impaired vitamin E uptake and transport, such as heterozygotic mutation of vitamin E binding proteins (25) or less penetrant mutations (26) , with consequent less severe symptoms or delayed outcome. In fact, the age of symptom onset in AVED patients depends on the type of mutation in the -TTP gene (26) .

Individuals with an inherent lower efficiency of tocopherol uptake may benefit most from supplemental intake of -tocopherol. In several epidemiological studies, low levels of -tocopherol have been associated with an increased risk for coronary artery disease and increased intake has been shown to be protective. If polymorphisms in transport and/or action of vitamin E exist, it could significantly affect the outcome of epidemiological studies, in which the initial plasma level and the individual efficiency of uptake and transport of -tocopherol often are not known.

	VITAMIN E PROTECTS HUMANS AGAINST VARIOUS DISORDERS

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Since its discovery in 1922, vitamin E supplementation has shown beneficial effects for numerous disorders, particularly atherosclerosis, ischemic heart disease, and development of different types of cancer (27 28 29) . The neurological symptoms of AVED are stabilized and sometimes reverted in patients after vitamin E treatment (30 , 31) .

It is evident that the biological role of vitamin E needs to be re-examined, since its simple antioxidant function is not sufficient to explain all the effects shown by the molecule.

	ANTIOXIDANT PROPERTIES OF -TOCOPHEROL

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Although it is commonly believed that phenolic compounds like vitamin E exert a protective role against free radical damage, antioxidant molecules can exert additional biological functions. The estrogen 17-ß-estradiol, for instance, has antioxidant capacity (32) , which has been proposed to protect women from coronary artery disease, but the determination of secondary sexual features is not mediated by its antioxidant activity (33) . All-trans-retinol is again a potent antioxidant (34) , but the main function of retinol in rhodopsin and vision is not related to this property.

Vitamin E is the major hydrophobic chain-breaking antioxidant that prevents the propagation of free radical reactions in the lipid components of membranes, vacuoles and plasma lipoproteins.

The antioxidant properties of vitamin E are well known and documented (35) . In particular, prevention by -tocopherol of LDL oxidation has been studied (36) . Although the correlation between the level of LDL oxidation and atherosclerosis is not always evident (37) , alternative studies have suggested that -tocopherol protection against LDL oxidation may be secondary to the inhibition of protein kinase C (PKC). This enzyme seems to be responsible for the release of reactive oxygen species and lipid oxidation (38 , 39) .

	PRO-OXIDANT PROPERTIES OF -TOCOPHEROL

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
In contrast to all the described antioxidant properties of vitamin E, it has been shown that lipid peroxidation of LDL is faster in the presence -tocopherol, and is substantially accelerated by enrichment of the vitamin in LDL, either in vitro or in vivo (40 , 41) . It was thus proposed that peroxidation is propagated within lipoprotein particles by the vitamin E radical (i.e., -tocopheroxyl radical) unless it became reduced by vitamin C or ubiquinol-10 (42) . However, the importance of pro-oxidation reactions of -tocopherol in vivo, under physiological conditions, appears to be questionable.

	ANTIALKYLATING PROPERTIES OF -TOCOPHEROL

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Nitric oxide released by macrophages during inflammation reacts with active oxygen to form peroxynitrite. Peroxynitrite nitrates protein and peroxidizes lipids. -Tocopherol (the principal form of vitamin E in the United States diet) and -tocopherol (the major form present in the European diet and in supplements), both protect against peroxynitrite-induced lipid oxidation (43) . Christen et al. reported that lipid hydroperoxide formation in liposomes is inhibited more effectively by -tocopherol than -tocopherol by a nonantioxidant mechanism (44) . However, Goss et al. (45) concluded that the presence of -tocopherol attenuates nitration of both -tocopherol and tyrosine, showing that nitration of -tocopherol becomes significant only after -tocopherol depletion. This would imply that -tocopherol alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species in vivo (45) .

	NONANTIOXIDANT EFFECTS OF -TOCOPHEROL

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
The nonantioxidant properties of tocopherol were discovered when, in several experimental models, the four tocopherol analogs had different effects, although they share a similar antioxidant capacity. It can be speculated that the selective uptake and transport of -tocopherol represents the evolutionary selection of a molecule with specific functions, different from its antioxidant properties.

In the sections below, we discuss the effect of -tocopherol at cellular level, focusing on the nonantioxidant properties shown by the molecule (Table 1 and Table 2).

View this table: [in this window] [in a new window]   Table 1. Inhibition of cell proliferation by -tocopherol in different cell lines

Effects of -tocopherol at cellular level
In 1991 inhibition of PKC activity was found to be at the basis of the vascular smooth muscle cell growth arrest induced by -tocopherol (46 , 47) . Many reports have subsequently confirmed the involvement of PKC in the effect of -tocopherol on different cell types, including monocytes, macrophages, neutrophils, fibroblasts. and mesangial cells (38 , 48 49 50) . -Tocopherol, but not ß-tocopherol, was found to inhibit thrombin-induced PKC activation and endothelin secretion in endothelial cells (51) . -Tocopherol, and not ß-tocopherol or trolox, inhibits the activity of PKC from monocytes, followed by inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) and by an impaired assembly of the NADPH-oxidase and of superoxide production (52) . -Tocopherol has the important biological effect of inhibiting the release of the proinflammatory cytokine, IL-1ß, via inhibition of the 5-lipoxygenase pathway (53) .

Inhibition of PKC by -tocopherol in vascular smooth muscle cells is observed to occur at concentrations of -tocopherol close to those measured in healthy adults (54) . ß-Tocopherol per se is not very effective but prevents the inhibitory effect of -tocopherol. The mechanism involved is not related to the radical scavenging properties of these two molecules, which are essentially equal (55) . In vitro studies with recombinant PKC have shown that inhibition by -tocopherol is not caused by tocopherol-protein interaction. -Tocopherol does not inhibit PKC expression as well. Inhibition of PKC activity by -tocopherol occurs at a cellular level by producing dephosphorylation of the enzyme, whereby ß-tocopherol is much less potent (56) . Dephosphorylation of PKC occurs via protein phosphatase PP₂A, which is activated by the treatment with -tocopherol (56 57 58) .

One group (59) has reported that prevention of glomerular dysfunction in diabetic rats can be achieved by treatment with -tocopherol. Such a protection occurs through inhibition of PKC. In this case, however, -tocopherol would act on the diacylglycerol pathway by activating the enzyme diacylglycerol kinase with consequent diminution of diacylglycerol and PKC activation. In these studies, high glucose was responsible for increased diacylglycerol synthesis, which was counteracted, in the presence of -tocopherol, by the activation of diacylglycerol kinase.

Transcriptional regulation by -tocopherol
Recently, the possibility of gene regulation by -tocopherol has been analyzed (60) . Up-regulation of -tropomyosin expression by -tocopherol, and not by ß-tocopherol, once more suggests a nonantioxidant mechanism (61) . Long- and short-term -tocopherol supplementation inhibits liver collagen 1(I) gene expression (62) . Age-dependent increase of collagenase expression in human skin fibroblasts can be reduced by -tocopherol (63) .

In rats, the liver -TTP and its mRNA are modulated by dietary vitamin E deficiency (64) . Scavenger receptors, particularly important in the formation of atherosclerotic foam cells, are also modulated by -tocopherol. In smooth muscle cells and monocytes/macrophages, the oxidized LDL scavenger receptors SR-A and CD36 are down regulated at transcriptional level by -tocopherol but not by ß-tocopherol (65 66 67) . The relevance of CD36 expression in the onset of atherosclerosis has been clarified by Febbraio and co-workers, who have shown that disruption of the CD36 gene protects against atherosclerotic lesion development in mice (68) .

The following questions remain open. In some cases, differential effects of -tocopherol and ß-tocopherol have been found, pointing to a nonantioxidant mechanism at the basis of gene regulation (61 , 66) . In other cases, however, only -tocopherol has been tested, thus leaving the mechanism of -tocopherol action unclarified. Furthermore, the involvement of PKC has not always been assessed and it remains to be established whether the transcriptional regulation of certain genes is a consequence of PKC inhibition.

Inhibition of monocyte-endothelial adhesion
-Tocopherol enrichment of monocytes and polymorphonuclear leukocytes decreases agonist-induced and LDL-induced adhesion to human endothelial cells both in vivo and in vitro (69 70 71) . Monocytes as well as neutrophils diminution of adhesion induced by -tocopherol is dependent on the inhibition of adhesion molecule expression (72 73 74) . These events are relevant to the onset of inflammation as well as in the early stages of atherogenesis.

Inhibition of platelet adhesion and aggregation
-Tocopherol inhibits aggregation of human platelets by a PKC-dependent mechanism both in vitro and in vivo (49 , 75 76 77) . Another study has indicated that both - and -tocopherol decrease platelet aggregation and delay intra-arterial thrombus formation (76) . That -tocopherol was significantly more potent than -tocopherol suggests that a simple antioxidant mechanism is not applicable to these effects.

The studies reported above are consistent with the conclusions of Iuliano et al. (78) : that circulating LDL accumulates in human atherosclerotic plaques and that such accumulation by macrophages is prevented by -tocopherol in vivo. The protection by -tocopherol may not be due only to the prevention of LDL oxidation, but also to the down-regulation of the scavenger receptor CD36 and to the inhibition of PKC activity.

Although not all scientific groups agree on the molecular details, PKC inhibition is accepted as a common denominator of cellular events regulated by -tocopherol: cell proliferation, cell adhesion, enhancement of immune response, free radical production and gene expression. However, the molecular mechanisms at the basis of these events are not yet fully elucidated. A few observations, such as PP2A (56) and diacylglycerol kinase (79) activation, 5-lipoxygenase (80) and cyclooxygenase (81) inhibition, still miss a mechanistic explanation. On the other hand, the expression of several genes such as CD36 (66) , SR class A (65) , collagenase (63) , and ICAM-1 (72) appears to be regulated by -tocopherol in a PKC independent way. A further understanding of the molecular events at the basis of -tocopherol gene regulation is part of ongoing studies.

In conclusion, numerous events are related to nonantioxidant properties of -tocopherol (Table 2) , both at transcriptional and posttranscriptional level. However, whether -tocopherol acts by a pleiotropic mechanism, or it binds to a receptor capable of regulating different reactions, still remains unknown.

View this table: [in this window] [in a new window]   Table 2. Effects of -tocopherol and their supposed molecular mechanisms

	VITAMIN E PROTECTS AGAINST ATHEROSCLEROSIS IN SEVERAL ANIMAL STUDIES, BUT NOT IN ALL

TOP ABSTRACT VITAMIN E UPTAKE OF VITAMIN E... WHAT IS THE ROLE... VITAMIN E DEFICIENCY IS... VITAMIN E PROTECTS HUMANS... ANTIOXIDANT PROPERTIES OF... PRO-OXIDANT PROPERTIES OF... ANTIALKYLATING PROPERTIES OF... NONANTIOXIDANT EFFECTS OF... VITAMIN E PROTECTS AGAINST... PROTECTION AGAINST HUMAN... ARTERIAL IMAGING STUDIES CONTROLLED INTERVENTION TRIALS CONCLUSIONS REFERENCES

 
Hypercholesterolemia can lead to enhanced plasma LDL concentration, their increased oxidative modification, and impaired endothelial function. Experimental results have shown that vitamin E can prevent some of these events, improving the endothelial function. In cholesterol-fed rabbits, for example, 50 IU/day synthetic vitamin E reduced plasma LDL and vessel wall oxidation within 6 days (82) . In a different study (83) , vitamin E (50 mg/kg) fully prevented cholesterol-induced atherosclerotic lesions in rabbits. Note that in this experimental model, control animals were given a vitamin E-poor diet (5–10 ppm), which probably increased the efficiency of treatments. In Watanabe hyperlipidemic rabbits, 0.5% w/w synthetic vitamin E added to the food inhibited LDL oxidation and caused a 32% reduction of the atherosclerotic area in the aortic arch region (84) . In contrast, New Zealand White rabbits, fed a 1% cholesterol diet and 10,000 IU/kg -tocopheryl acetate, showed significantly more intima atherosclerotic proliferation than the controls (85) .

In atherosclerosis-susceptible apolipoprotein E (Apo E) knockout mice, vitamin E deficiency, caused by disruption of the -tocopherol transfer protein gene, increased the severity of atherosclerotic lesions in the proximal aorta (86) .

In a different study (87) , carried out with male monkeys, vitamin E was found to significantly inhibit the progression of the disease, as measured by the thickness of carotid artery. Their plasma concentration of vitamin E was proportional to the resistance of the carotid artery to stenosis. In this study, individual variations in blood levels were achieved by the same dose of vitamin E, underlining the need of results that include a dose-response study (88) .

That the effect of -tocopherol in animal models may not be due to its antioxidant properties is also suggested by results obtained using probucol, a powerful inhibitor of atherosclerosis in several animal models. However, probucol not only failed to decrease, but actively increased atherogenesis in LDLR^-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL (89) . This suggests that the reduction of atherosclerosis observed in some animal models is due to intracellular effects which are absent in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the HDL decrease in murine models (90) . A dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits has also been shown. It is suggested that, in this animal model, aortic accumulation of oxidized lipid is not required for the initiation and progression of atherogenesis. Moreover, during the development of the disease, -tocopherol is not depleted in the lesions (91) .

The above-cited examples, which are not intended to cover the literature, show that an antioxidant treatment may have both proatherogenic and antiatherogenic effects in different experimental animal models. Moreover, it appears that LDL oxidation does not constantly correlate with atherogenic events.

The reason for the lack of responsiveness to atherogenic conditions in different animals is far from being understood. Of the two most commonly used experimental animals, rabbits and rats, the former are prone to atherosclerosis induced by hypercholesterolemic diets, whereas the latter are resistant. However, the knockout mouse for Apo E becomes prone to atherogenesis (68) . The subsequent knockout of the scavenger receptor gene CD36 protects the mouse, indicating that a particular genetic combination of Apo and scavenger receptor type may be at the basis of the animal susceptibility to atherosclerosis. One can also speculate that high expression of certain genes (for instance CD36) in certain animals or certain parts of the aorta may favor plaque deposition. On this genetic basis, nutritional intake of compounds enhancing or diminishing the expression of CD36 may favor or retard atherosclerosis.

	PROTECTION AGAINST HUMAN ATHEROSCLEROSIS HAS BEEN OBSERVED IN SUBJECTS TAKING HIGH VITAMIN E QUANTITIES WITH THE DIET

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Fred Gey, a pioneer in the study of micronutrients protection against cardiovascular disease (CVD) and cancer, has made the observation that "... CVD mortality in the currently available European study populations is by far more strongly correlated to vitamin E than to the classical risk factors total plasma, cholesterol and blood pressure..." (92) .

A study of 16 European populations showed a strong inverse correlation between blood levels of vitamin E and the risk of CVD death (92) . However, in a cohort of the MONICA study, a significant correlation was not found between serum vitamin E concentration and the risk of myocardial infarction (MI) (93) .

In a large European case control study, the EURAMIC study, adipose tissue levels of -tocopherol and ß-carotene were measured in subjects with acute MI (94) . No protective effect attributable to -tocopherol was found, and the authors suggested that -tocopherol levels obtained from the diet, without supplementation, may not have been high enough (94) .

Prospective cohort studies use dietary questionnaires and self-recalled intake levels of vitamin E. The largest study of this type reported to date is the Nurses‘ Health Study, which involved more than 120,000 female nurses, followed for 8 years (95) . The results revealed that those women who obtained vitamin E exclusively from diet had a small and nonsignificant reduction in the relative risk for developing CVD, whereas women in the highest quintile of vitamin E intake from supplement use had a relative risk of nonfatal MI or death from coronary disease of 0.54 (96) . The second of these related studies, the Health Professional Follow-Up Study, started in 1986 and involved more than 40,000 male professionals who were free of heart disease and diabetes (97) . Subjects were followed for 4 years and, again, dietary intakes of vitamin E were found to be strongly, but not significantly, correlated with reduced risk for coronary heart disease or death (96) .

	ARTERIAL IMAGING STUDIES

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High-resolution ultrasound measurements of the arterial wall thickness (IMT) can measure the extent of atherosclerosis at early, subclinical stages (98 , 99) . The EVA trial involved a total of 1389 French subjects for a 4 year period. In the subjects‘ red blood cells the levels of vitamin E was found to be significantly associated with less thickening of the arterial wall (100) .

In the Kupio Ischemic Heart Disease Study, the relation between vitamin E and ß-carotene plasma levels and the status of carotid IMT was examined over 12 months in 216 men with high LDL cholesterol levels (101) . There was a very significant inverse correlation between the progression of carotid artery narrowing and the vitamin E plasma levels as well as those of ß-carotene.

More recently, the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study, has analyzed the effect of vitamin E and C on 3-year progression of carotid atherosclerosis (102) . A randomized sample of 520 smoking and nonsmoking men and postmenopausal women with serum cholesterol >/= 5.0 mmol/L were studied. Twice a day, subjects were given a formulation of 91 mg vitamin E, 250 mg of slow release vitamin C, a combination of these, or a placebo. Atherosclerotic progression, measured by IMT, and calculated over semiannual assessments, was reduced by 74% in the male population receiving the formulation with both vitamins. No effect on the arterial wall thickness has been found in the female group.

Using data from the Cholesterol Lowering Atherosclerosis Study (CLAS) (103) , Azen and co-workers have studied the association of self-selected supplementary antioxidant vitamin intake on the rate of progression of early preintrusive atherosclerosis (104) . Less carotid IMT progression was found for high supplementary vitamin E users (>/=100 IU per day) when compared with low vitamin E users (0.008 vs. 0.023 mm/y). However, no effect of vitamin E within the group receiving lipid-lowering drugs was found.

The Study to Evaluate Carotid Ultrasound changes in patients treated with vitamin E (SECURE) (105) , a substudy of the HOPE trial, was a prospective, double-blind trial that evaluated the effects of long-term treatment with the angiotensin-converting enzyme inhibitor ramipril and vitamin E on atherosclerosis progression in high-risk patients. A total of 732 patients >/= 55 years of age who had vascular disease or diabetes and at least one other risk factor and who did not have heart failure or a low left ventricular ejection fraction were randomly assigned to receive ramipril 2.5 mg/day or 10 mg/day and vitamin E 400 IU/day, or matching placebos. Average follow-up was 4.5 years. Atherosclerosis progression was evaluated by IMT. There were no differences in atherosclerosis progression rates between patients on vitamin E and those on placebo.

The majority of these studies indicate that vitamin E protects against carotid thickening, but less supportive results are provided by the SECURE trial.

	CONTROLLED INTERVENTION TRIALS

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The CHAOS trial is a secondary prevention trial that enrolled subjects with established heart disease (106) . 2002 subjects were randomized to receive vitamin E or placebo. 546 subjects in the tocopherol group were given 800 IU/day and the remainder were given 400 IU/day, but the two groups were combined for statistical analysis. After 510 days, those on vitamin E experienced a significant 77% reduction in the risk for nonfatal MI.

The GISSI trial is a secondary prevention trial that enrolled 11,324 Italian patients who had survived a MI (107) . There were four treatments groups, one of them involving 300 mg/day of synthetic vitamin E. The results of this trial are so complex that they still remain unclear. In an initial analysis, the results did not reach statistical significance for the vitamin E group (107 , 108) . Later on, the GISSI trial has been reviewed and further analyzed (109 110 111) ; Salen et al. (110) comment: "Cardiovascular mortality was significantly reduced by vitamin E in GISSI and the effect on overall survival showed a very favorable trend..."

The Linxian China Study is a primary prevention trial testing four combinations of micronutrients on overall mortality and cancer mortality (112) . The subjects were randomized to receive placebo, or synthetic vitamin E (30 IU) together with ß-carotene, and selenium. After supplements were given for 5.25 years, small but significant reductions in total (relative risk of 0.91) and cancer (relative risk of 0.87) mortality were observed in subjects receiving ß-carotene, vitamin E, and selenium but not the other tested nutrients.

The ATBC trial tested the effects of vitamin E, ß-carotene, and both micronutrients together in heavy smokers (113) . A group of 29,133 Finnish male were given synthetic vitamin E (50 mg), ß-carotene, both, or a placebo. Surprisingly, the ATBC trial found an increased risk for lung cancer in the group receiving ß-carotene rather than a placebo (114) . The subjects on vitamin E experienced 32% lower risk of prostate cancer and a 41% lower mortality from prostate cancer (115) . No statistically significant benefit for heart disease was found for either micronutrient (116) . Like the Linxian study, the ATBC trial used a much smaller dose of synthetic vitamin E than the CHAOS and the GISSI studies.

In the Heart Outcomes Prevention Evaluation (HOPE) study (117) , 9541 patients defined as being at high risk for cardiovascular disease, were given ramipril, vitamin E (400 IU/day), both of them, or a placebo. Patients were followed for a mean of 4.6 years. Primary outcomes, defined as myocardial infarction, stroke or cardiovascular disease death, did not differ by vitamin E treatment whether given alone or in combination with ramipril. The authors believed that perhaps longer follow-up was needed, although their data do not suggest a trend in that direction.

In a smaller trial, the Secondary Prevention with Antioxidants of Cardiovascular Disease in Endstage Renal Disease (SPACE) (118) , 196 hemodialysis patients with preexisting cardiovascular disease received 800 IU/day vitamin E or matching placebo. The primary end point was a composite variable consisting of myocardial infarction (fatal and non fatal), ischemic stroke, peripheral vascular disease, and unstable angina. Secondary outcomes included each of the component outcomes, total mortality and cardiovascular-disease mortality. After a median of 519 days, a 46% reduction was attained in the primary end point, contributed largely to the reduction in total myocardial infarction (70%). No significant differences in total mortality were detected. The authors comment (118) : "These findings are consistent with those of the CHAOS..."

The protection against atherosclerosis observed in subjects taking high vitamin E quantities can be criticized because of possible vitamin E-associated confounding factors that may be actually responsible for the effects attributed to vitamin E. However, the rather general positiveness shown by the epidemiological studies, carried out with different populations and in different locations, speaks for a significant benefit of vitamin E.

In the most important intervention studies, CHAOS and SPACE are consistent with each other. Although the population was different in size and type, the protective effect of vitamin E was remarkably similar (around 50% against myocardial infarction). A disturbing result in the CHAOS study is that no decrease in the total mortality was observed. CHAOS found a decrease in nonfatal myocardial infarction but an increase in fatal myocardial infarction after treatment with RRR -tocopherol. Pro-oxidant effects of -tocopherol may have caused early, fatal myocardial infarction, associated with rupture of unstable plaques. The protective effects in CHAOS may be the result of a longer adaptation process associated with regulation of gene expression and inhibition of smooth muscle cells proliferation. The same consideration may apply to the HOPE study. Similar critical observations have been recently made by Pryor in a very thorough paper (88) .

As pointed out by Jialal and co-workers (109) , a careful analysis of the GISSI study reveals that -tocopherol supplementation resulted in the following significant effects when the more appropriate four-way analysis was undertaken: 20% reduction in cardiovascular deaths, 23% reduction in cardiac death, 25% reduction in coronary death, 35% reduction in sudden death, despite the primary end point not being statistically significant. Moreover, patients were on a Mediterranean diet that was rich in antioxidants, which could also have confounded the benefits of -tocopherol.

In general, we recognize the following factors to be at the basis of the irregular results from observational and clinical studies. First, the observational studies are in general showing a positive outcome of high vitamin E intake. This may be caused by different reasons: The observational studies initiate the protection earlier, and other substances associated with vitamin E may be actually responsible, or coresponsible, for the alleged effect of vitamin E. In particular, vitamin C may play a central role in the effect of vitamin E; however, this association has been used only in one study (ASAP), which had a positive outcome. Another important factor, that may have determined the observed uncertainty in the clinical trials, is the selection of the stage of the disease. Observational studies include longtime prevention in individuals that may have been at a very early stage of disease. In the clinical studies, the selection of the population in terms of relatively old age and previous coronary heart disease may not have been ideal since irreversible arterial damage may have been already created. But if the same population had, until the time of admission into the trial, a diet rich in vitamins E and C, their lesions may have been less serious and still in a treatable phase. However, if the population had already very high plasma levels of vitamins C and E, the supplementation would be less effective. Source and dosages of vitamin E have been also different in different studies and no clear instructions have been given regarding the time of vitamin E uptake. Furthermore, no one has considered that certain individuals absorb vitamin E very poorly.

In the SPACE study, Boaz and co-workers (118) postulate that the positive results were due to the effect against the strong oxidative stress present in hemodialysis patients, but the effect of vitamin E supplementation on oxidative status was not measured. It has been seen that supplementation with antioxidants can indeed be protective or worsen damage. After oxidative damage has begun, in fact, transition metal ions liberated after administration of a powerful antioxidant could promote damage (119) . Another important variable not sufficiently considered in clinical intervention studies regards the end point(s) of the trial. There is almost unanimous agreement in the literature that vitamin E prevents age-dependent carotid artery thickening. It is clear, however, that ischemic heart disease is a complex condition, where only one component is narrowing of the coronary arteries. Although in the early stages of the disease vitamin E may prevent plaque formation (inhibiting radical production and associated LDL oxidation, inhibiting adhesion of monocytes, inhibiting CD36 expression, etc.), the only effect of the vitamin in later stages can be of diminishing platelet aggregation, improving endothelial function, and protecting from reperfusion injury.

	CONCLUSIONS

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Three relevant questions can be posed from this study. 1) Is it possible that not all the effects of -tocopherol relevant to the protection against atherosclerosis in animals have a counterpart in the prevention of the human pathology? 2) What can be the basis for the contradictory results obtained by different clinical trials? 3) Should we abandon the idea that -tocopherol may help protect against atherosclerosis, or should we improve the trial conditions?

We will not answer these questions, but try instead to discuss them.

Although biochemical, cellular, and molecular biology ideas about -tocopherol have increased dramatically, many molecular phenomena are still far from been fully elucidated. Model studies (68) have shown that CD36 knockout protects against atherosclerotic lesion development in Apo E^-/- mice. In human, four different Apo E alleles can be present. It has been shown that the Apo E genotype may influence carotid atherosclerosis in its early stages and that, in particular, the E4 allele favors the disease (120 , 121) . However, the clinical intervention studies discussed above have not considered Apo E and CD36 polymorphisms, possibly influencing the effect of -tocopherol treatment. They also neglected to measure the basal level of -tocopherol in plasma before and after the supplementation. Very few studies (for instance, the ASAP study) have considered the possibility that pro-oxidant effects of -tocopherol may be protected by ascorbic acid. Finally, the existence of -tocopherol binding proteins should be taken into consideration: the complexities of -tocopherol absorption and metabolism are another variable in the puzzle of in vivo -tocopherol function.

Clinical aspects are also worth some comments. It is possible that the wrong stage of disease (irreversible) was chosen in some studies; age is not the sole element in determining the phase of the disease. In the HOPE study, too few clinical events unexpectedly took place in the placebo group, making the statistical analysis less sensitive. Furthermore, women show a much slower progression of atherosclerosis, another factor to be considered.

It appears that vitamin E treatments should be started much earlier, continue for a longer period, and be consumed with vitamin C for its effect to become measurable. The population of choice should be selected according to age, Apo E genotype, gender, and vitamin E status.

-Tocopherol, a Janus molecule, is beginning to reveal its second, nonantioxidant function. It is possible that novel reactions and novel genes, found to be under -tocopherol control, may help clarify the relationships between molecular and clinical events.

	ACKNOWLEDGMENTS

 
This study was supported by the Swiss National Science Foundation, the Foundation for the Research on Nutrition in Switzerland, and Bayer Vital (Germany). R.R. is recipient of a Telethon-Italia fellowship.

	REFERENCES

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