Vitamin E: non-antioxidant roles

Abstract

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 α-tocopherol has been found to possess functions that are independent of its antioxidant/radical scavenging ability. Absorption in the body is α-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 α-tocopherol and not by β-tocopherol. α-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 (α-tropomyosin and Connective Tissue Growth Factor). Non-antioxidant molecular mechanisms have been also described for γ-tocopherol, δ-tocopherol and tocotrienols.

Introduction

The term “Vitamin E”, introduced in 1922 by Evans and Bishop [1] described a dietary factor in animal nutrition considered at the time to be especially important for normal reproduction. After feeding female rats accidentally with rancid fat they observed a deficiency syndrome, in which foetal resorption was the most characteristic symptom. From the fact that adding fresh salad to the diet reversed the symptoms they concluded that plants contained a specific factor being responsible for the observations. The multiple nature of the vitamin began to appear in 1936, when two compounds with vitamin E activity were isolated and characterized from wheat germ oil [2]. These compounds were designated as α- and β-tocopherol, deduced from the Greek expressions “tokos” (childbirth) and “phorein” (to bring forth). In the following years two additional tocopherols, γ- and δ-tocopherol [3], [4] as well as the tocotrienols [5] were isolated from edible plant oils, so that today a total of four tocopherols and four tocotrienols are known to occur in nature. The American Food and Nutrition Board in 1968 officially recognized the essential nature of vitamin E. Researchers have since confirmed the significance of vitamin E as a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life processes.

Unlike other vitamins that represent one well-defined chemical structure, natural vitamin E includes two groups of closely related fat-soluble compounds, tocopherols and tocotrienols. The compounds of both groups are all derivatives of 6-chromanol. The first group derives from tocol, which carries a saturated isoprenoid C-16 side chain and three chiral centres with configuration R at position 2, 4′ and 8′ (Fig. 1).

The members of the second group have a triply unsaturated side chain at the positions 3′, 7′, and 11′. Within one group the members are designated α, β, γ and δ depending on the number and the position of the methyl groups attached to the aromatic ring [6].

The tocopherols are naturally occurring phenolic benzopyrans, which display antioxidant activities in vivo and in vitro. Since their initial discovery, they have been investigated to elucidate their mechanism of action and to identify potential metabolites. Much interest has been focused on their reactivity towards peroxy radicals as well as on their remarkable regiospecificity towards oxidation and electrophilic substitution [7], [8]. Burton and Ingold [9] have studied extensively the effects of chemical structure of phenolic compounds on the reactivity towards peroxyl radicals. By measuring the rate constant for hydrogen abstraction from the tocopherols and related phenols they found that α-tocopherol had the highest value (k₁=2.35×10⁶ M⁻¹ s⁻¹ at 30°C) of all the compounds examined. It was concluded that the rate constant k₁ is determined primarily by the bond dissociation energy of the phenolic O–H bond, which is influenced by stereoelectronic effects as well as by constituent effects. They could demonstrate that the heterocyclic chromanol ring has an optimised structure for resonance stabilization of the unpaired electron of the α-tocopheroxyl radical and that electron-donating substituents e.g. methyl groups, increase this effect [9] (Fig. 2).

Besides its radical trapping properties, α-tocopherol can act as a strong reductant and as electrophilic agent in chemical reactions, depending on its environment. It has recently been shown that the chemical oxidation of α-tocopherol proceeds in a two-electron process, which does not involve the formation of any radical intermediate [10]. This mechanistic approach has led to a general understanding of the oxidation mechanism of α-tocopherol. The results indicate that the oxidation of α-tocopherol by non-radical processes is always accompanied by the formation of a transition state, or perhaps intermediate, which is characterized by a para-quinoid structure and its mesomeric ortho-quinone methide (Fig. 3). Investigations on the chemistry of the methide indicate that this important intermediate is reactive towards nucleophilic agents and also towards protic solvents. Reactions of the ortho-quinone methide that is formed by the phenolic OH-group and a ring methyl group always occur in the 5-position, never by the 7-methyl group. The preference of the 5-position, the so-called “Mills–Nixon-Effect”, has been calculated and ascribed to simple changes in conjugative effects, as already discussed by Behan et al. [11]. Oxidative coupling in the absence of methyl groups, (e.g. in γ-tocopherol) still occurs in the 5-position for the same reasons. In the light of these results, previously reported investigations on the reactivity of α- and γ-tocopherol towards reactive oxygen and nitrogen species have to be re-evaluated [12], [13].

Natural-source and synthetic vitamin E are not identical. Unlike naturally occurring α-tocopherol, the synthetic form of vitamin E, all-rac-α-tocopherol, contains eight different stereoisomers arising from the three stereocenters of the molecule. The bioavailability and the biopotency of the various tocopherol derivatives and stereoisomers have been extensively studied in mammals by several authors [14], [15], [16], [17], [18]. Most studies assessing the biological activities of tocopherols are based on the classical rat foetal gestation–resorption assay [1], [19] (Table 1).

The data of the rat gestation–resorption assay show a preference for the most abundant natural derivative of vitamin E, RRR-α-tocopherol. Despite the fact that the synthetic stereoisomers of α-tocopherol must possess, for theoretical reasons, equal antioxidant properties, they have impaired relative biological activities. This indicates that structural features of α-tocopherol are of vital importance and that the antioxidant properties of tocopherols are not necessarily reflecting their biological activities. Consequently, alterations in biological activity of tocopherols are structure specific, including the presence or absence of ring methyl groups, stereochemistry of the chiral carbon centres, branching or desaturation of the side chain, respectively.

Numerous supplementation studies have been carried out in animals in order to assess the relative bioavailability of RRR-α-tocopherol compared to its stereoisomers in plasma and tissues [14], [16], [17], [20]. In these studies a ratio of 1.36 for natural RRR-α-tocopherol relative to all-rac-α-tocopherol was established. In a previous study, the biodiscrimination of the eight α-tocopherol stereoisomers was followed over a period of 90 days. The stereoisomer profiles showed a remarkable preference in the accumulation of 2R forms in brain (74%), adipose tissue (74%), liver (70%) and plasma (86%) [21]. The somewhat lesser accumulation effect in liver was explained by its involvement in primary biodiscrimination. Time dependent changes in serum concentrations of all eight α-tocopherol stereoisomers were also assessed in humans under comparable conditions [22]. The authors of the human studies consistently conclude that the bioavailability of RRR-α-tocopherol is up to three times higher than that of all-rac-α-tocopherol. In phase studies it was also clearly shown that all 2R epimers compared to the 2S epimers are preferentially retained in man, which implies the existence of tocopherol binding factors with stereospecificity towards the natural RRR-stereoisomer of α-tocopherol [15].

The space filling models of the R- and S-epimers of α-tocopherol indicate that the configuration at C-2 has a major impact on the three dimensional structure of the molecule (Fig. 4). Changing the configuration from R to S at C-2 leads to inversion of the angle between the phytyl tail and the chromanol ring. The influence of the chiral centres at C-4′ and C-8′ on structural changes in the phytyl tail are less pronounced but have an impact on the biological activity of the corresponding isomers (see Table 1).

Vitamin E is an essential nutrient in the human body and thus it must be provided by foods and supplements [23]. The eight isomers of vitamin E are widely distributed in nature. Vitamin E has been detected in varying compositions (4–160 μg/g fresh weight) in all plants having been examined so far [24]. The richest sources of natural vitamin E are latex lipids with an exceptional high tocopherol content (8% w/v) followed by edible oils originating from plants (cf. Table 2) [25]. Sunflower seeds contain almost exclusively α-tocopherol as single-isomer product [26]. The strong correlation between the content of tocopherols and the amount of unsaturated fatty acids in plant oils suggests that vitamin E represents the most important antioxidant in plant tissues [27]. In contrast to plants mammalian tissues contain almost exclusively α-tocopherol. The highest content of α-tocopherol, (150 μg/g) is found in adipose tissue whereas erythrocytes have a relatively low content (2 μg/g) [28]. Investigations on the vitamin E content of microorganisms have revealed a non-uniform picture. The groups of Skinner, Powls and Woggon have investigated the α-tocopherol content of phototrophic algae [29], [30], [31]. Significant amounts of α-tocopherol were detected in the green algae Chlorella (7.6 μg/g dry weight), Stichococcus bacillaris (134.2 μg/g dry weight), Dunaliella salina (63.8 μg/g dry weight) and Cladophora stichotoma (0.7 μg/g dry weight) as well as in the blue green alga Anabaena variabilis (213.5 μg/g dry weight) and also in the brown algae Macrocystis integrifolia (12.2 μg/g dry weight) and Fucus distichus (11.1 μg/g dry weight). No tocopherol has been found in the red algae Gigartina corymbifera, Drionitis lanceolata and in five yeast strains including Torula 1N.

Vitamin E requires, because of its hydrophobicity, special transport mechanisms in the aqueous environment of plasma, body fluids and cells. In humans, vitamin E is taken up in the proximal part of the intestine depending on the amount of food lipids, bile and pancreatic esterases. It is emulsified together with the fat-soluble components of the food. Lipolysis and emulsification of the formed lipid droplets then lead to the spontaneous formation of mixed micelles, which are absorbed at the brush border membrane of the mucosa by passive diffusion. Together with triglycerides, phospholipids, cholesterol and apolipoproteins, the tocopherols are re-assembled to chylomicrons by the Golgi of the mucosa cells [32]. The chylomicrons are stored as secretory granula and eventually excreted by exocytosis to the lymphatic compartment from where they reach the blood stream via the ductus thoracicus. The rather high clearance rate (24–48 h) of a bolus of vitamin E from the plasma and the concomitant rapid uptake by the liver parenchyma indicates that the intravascular degradation of the chylomicrons to remnants by the endothelial lipoprotein lipase (LPL) is a prerequisite for the hepatic uptake of tocopherols [33]. Most probably the exchange between apolipoproteins of the chylomicrons (type AI, AII and B₄₈) and HDL (type C and E) triggers the formation of the remnants and in this way favours the rapid uptake of the tocopherols via the hepatic receptors for apo-E and apo-B [34], [35], [36]. This hypothesis is supported by the fact that, comparing subcellular compartments the highest concentrations of α-tocopherol are found in lysosomes (14.6 mmol/mol lipid) [37] (Fig. 5).

In contrast to the unspecific uptake of vitamin E from food by the liver cells, the specific α-tocopherol transfer protein (α-TTP) mediates the transfer of α-tocopherol from the hepatic lysosomes into lipoproteins [38]. This protein specifically separates α-tocopherol from all incoming tocopherols and promotes its net mass transfer into VLDL [39]. α-TTP has been shown to possess both stereospecificity as well as regiospecificity towards the most abundant isomer of vitamin E, RRR-α-tocopherol. As a consequence of the selective transfer mechanism, major parts of the natural homologues and non-natural isomers of α-tocopherol are excluded from the plasma and secreted with the bile [40]. Several studies have shown that α-TTP is expressed only in liver in significant amounts. Thus, the incorporation of extracellular α-tocopherol into extrahepatic tissues would relate to a series of unknown processes. Because of its hydrophobicity, α-tocopherol is mainly transported in association with lipoproteins in the plasma compartment. All plasma lipoproteins can constitute α-tocopherol vehicles, and the contribution of distinct lipoprotein fractions to α-tocopherol transport actually depends on their relative proportions in one given plasma sample [41]. The plasma phospholipid transfer protein (PLTP), which is known to catalyse the exchange of phospholipids and other amphipatic compounds between lipid structures has been shown to facilitate the exchange of α-tocopherol between HDL and LDL [42].

Recently a cytosolic tocopherol binding protein with broad tissue distribution has been discovered in our group [43]. The function of this protein is still unknown and therefore it was given the name of tocopherol-associated protein (TAP). So far TAP has been shown to be ubiquitous, but more highly expressed in adult liver, prostate and brain tissue. Sequence homology of TAP ascribes it to a family of hydrophobic ligand binding proteins including α-TTP [44]. Another member of this family is the phosphatidylinositol-transfer protein (SEC14). This protein catalyses the transfer of phospholipids between membrane bilayers and plays an essential role in yeast Golgi function [45]. X-ray data of SEC14 provide structural insights in its function and new information concerning the architecture of the entire family of evolutionary conserved proteins [46] (Fig. 6).

The structural homology of TAP with phosphatidylinositol-transfer protein (SEC14) and its broad tissue distribution make TAP a possible candidate responsible for the regulation of tissue α-tocopherol levels. Nevertheless, the mechanisms by which vitamin E is transported, regulated within cells and how it is involved in cellular signalling still remains obscure. Furthermore, the possibility that α-tocopherol acts similar to the retinol derivatives, is being considered. The major urinary metabolite (α-CEHC) of α-tocopherol has been discovered recently [47]. It appears in human urine after vitamin E supplementation and is formed directly from α-tocopherol without previous oxidative splitting of the chromane ring. The correlation of tocopherol intake and urinary excretion of α-CEHC was examined in human volunteers supplemented with RRR-α-tocopherol in the range from 0 to 800 mg/d. The analysis revealed that α-CEHC was only excreted above a daily intake of 150 mg α-tocopherol. This amount was interpreted as an indicator of plasma saturation by vitamin E (ca. 80 μM) and may be considered as marker of maximum vitamin E intake [48]. If prevention of oxidative damage and promotion of an optimal health status are the objectives, current estimates implicate that roughly an amount ten times higher than that recommended to prevent symptoms of deficiency is needed [49]. In the light of our current knowledge it might be reasonable to conclude that human supplementation with RRR-α-tocopherol is preferable in disease prevention.