Isoprenoids: 1. Tocopherols and Tocotrienols (Vitamin E)

Tocopherols and tocotrienols constitute a series of related benzopyranols (or methyl tocols or tocochromanols) that are synthesised in plants and other photosynthetic organisms, where they have many important functions but especially as part of a complex web of antioxidants that protect plants from the activities of reactive oxygen species (ROS). First described in 1922 as a dietary factor essential to prevent foetal reabsorption in rats, it was soon understood that plants contained a fat-soluble vitamin (vitamin E) that is essential for innumerable aspects of animal development. Of the many related molecules, only one form, i.e., α‑tocopherol, is recognized as having vitamin E activity in humans in that it prevents a spectrum of human deficiency diseases termed 'ataxia', which is characterized by very low concentrations of α‑tocopherol in plasma. The progression of the disease can be prevented by the administration of α-tocopherol only, although the pathogenic mechanism appears to be uncertain. While all tocopherols are known to be powerful lipid-soluble antioxidants in vitro at least, α‑tocopherol has further indirect roles in signal transduction and gene expression in animal tissues. On the other hand, specific functions (non-vitamin E) for other tocopherol forms such as γ-tocopherol and the tocotrienols and their metabolites in animal tissues are now being revealed. Vegetable oils are a major dietary source of vitamin E for humans.

1.  Structure and Occurrence

Tocopherols are benzopyranols with a 20-carbon saturated phytyl tail (including the pyranol ring) and four chiral centres in total, with variable numbers of methyl groups attached to the benzene ring, and they have three chiral carbons, one at C2 in the chromanol ring and two in the side chain at C4′ and C8′ with R,R,R stereochemistry. The tocotrienols differ in having a 20-carbon geranylgeranyl tail with three trans double bonds at the 3', 7' and 11' positions relative to the ring system and only one chiral centre (C2). The four main constituents of the two classes are termed - alpha (5,7,8-trimethyl), beta (5,8-dimethyl), gamma (7,8-dimethyl) and delta (8-methyl). Plastochromanol-8 (γ-toco-octaenol) is an analogue of γ-tocotrienol with a much longer sidechain.

Formulae of tocopherols

The tocochromanols are only synthesised by plants and other oxygenic photosynthetic organisms, such as algae and some cyanobacteria, but they are essential components of the diet of animals. Of these, only natural R,R,R-α-tocopherol is now designated ‘vitamin E’, as explained below, although the other tocopherols are sometimes termed ‘vitamers’ (some claim incorrectly - nor should all forms be termed isomers strictly speaking). In the USA, the current recommended dietary allowance (RDA) is 15 mg α‑tocopherol daily for adults. In plants, there is a great range of tocochromanol contents and compositions, and photosynthetic plant tissues contain from 10 to 50 μg tocochromanols per g fresh weight. α‑Tocopherol only is present in photosynthetic membranes of plant leaves, while γ‑tocopherol and other forms are found principally in fruits, seeds and nuts. While tocopherols are present in all photosynthetic organisms, the tocotrienols are found only in certain plant families.

Seed oils are a major source for the human diet and the compositions of tocopherols in some unrefined oils are listed in Table 1. Sunflower and olive oils are good sources of α‑tocopherol and palm oil of the tocotrienols. In general, tocotrienols tend to be abundant only in monocots such as wheat, rice and barley (bran oils especially), though a major commercial source is palm oil (seeds and fruits). In leaf tissue, α-tocopherol is often the main form, while γ-tocopherol is the primary tocopherol of many seeds. Plastochromanol-8 was first found in leaves of the rubber tree (Hevea brasiliensis), but it has since been found in many other plants including rapeseed and maize, although usually at lower levels than of the tocopherols. In addition, tocopherol esters of fatty acids occur in plant tissues, where they may be an inert storage form, but esterified tocopherols remain unchanged during digestion in animals so they may not make a contribution to vitamin E activity.

Table 1. Tocopherol and tocotrienol contents (mg/Kg) in some seed oils.
α-T* β-T γ-T δ-T α-TT* β-TT γ-TT δ-TT
palm 89 - 18 - 128 - 323 72
soybean 100 8 1021 421 - - - -
maize 282 54 1034 54 49 8 161 6
sunflower 670 27 11 1 - - - -
rapeseed 202 65 490 9 - - - -
* Abbreviations: T, tocopherol; TT, tocotrienol
Data from: Gunstone, F.D., Harwood, J.L. and Padley, F.B. The Lipid Handbook (Second Edition) (Chapman & Hall, London) (1994).

An unusual tocopherol that has been termed marine-derived α-tocomonoenol is found together with α-tocopherol in a wide range of marine fish species, where it appears to be a more efficient scavenger of free radicals at low temperatures. A related isomer with a Δ11 double bond has been found in palm oil and kiwi fruit. While pumpkin seeds contain both α- and γ-tocomonoenols, other plant species contain β-, γ- and δ-tocomonoenols with unsaturation in the terminal isoprene unit of the side chain. Tocochromenols or 3,4‑dehydrotocopherols, i.e., with a double bond in the pyranol ring, are known as well as more complex tocopherol-like molecules.

Formulae of some tocomonoenols

α-Tocopherol is a minor but ubiquitous component of the lipid constituents of animal cell membranes (non-raft domains), with estimates ranging from one molecule of tocopherol to 100 to 1000 molecules of phospholipid, depending on the membrane. The hydrophobic tail lies within the membrane, as might be expected, and the polar head group is orientated towards the surface but below the level of the phosphate moieties of the phospholipids. There may be some limited hydrogen bonding between the hydroxyl groups and phosphate depending on the degree of hydration of the membrane. On the other hand, there is a strong affinity of α-tocopherol for polyunsaturated fatty acids, where the chromanol unit may interact with the double bonds, suggesting that tocopherol is located deep within the membrane.

Formula of alpha-tocopheryl phosphateα-Tocopheryl phosphate has been detected at low levels in plasma, liver and adipose tissue (see below), and together with catabolic tocopherol metabolites, has important biological properties.

During the refining of vegetable oils, much of the natural tocopherols is lost or destroyed. Most commercial vitamin E is therefore prepared by chemical synthesis with trimethylhydroquinone and phytyl bromide as the precursors. The resulting product is a mixture of eight stereoisomers (from R,R,R- to S,S,S-methyl groups) of α‑tocopherol, with the various stereoisomers differing by a factor of two in biologic activity, as a consequence of the stereochemistry of position 2 in the chromanol ring (i.e., 2S-α- compared to 2R-α-tocopherol). It is usually administered as the acetate derivative in vivo. Tocopherols are not usually regarded as effective antioxidants in the polyunsaturated seed oils of commerce, and at higher concentrations they can even act as pro-oxidants, although the reasons for this are not understood.

2.  Biosynthesis and Functions of Tocochromanols in Plants

The mechanism of biosynthesis of tocopherols has been elucidated and involves coupling of phytyl diphosphate with homogentisic acid (2,5‑dihydroxyphenylacetic acid), followed by cyclization and methylation reactions. The plant chloroplast is the site of biosynthesis of all the intermediates, and most of the enzymes are located on the inner membrane of the chloroplast envelope, although there is increasing evidence that plastoglobules associated with the thylakoid membrane may be involved.

Biosynthesis of alpha-tocopherol

The aromatic amino acid tyrosine can be considered the basic precursor, and this is oxidized to p-hydroxypyruvic acid, which in the first committed step is converted to homogentisic acid by the enzyme p-hydroxyphenylpyruvate dioxygenase. Homogentisic acid is condensed with phytyl diphosphate, derived from phytol obtained from hydrolysis of chlorophyll, in a reaction catalysed by a prenyl transferase to yield 2-methyl-6-phytyl-plastoquinol, which is first methylated to form 2,3-dimethyl-5-phytyl-1,4-benzoquinol and then converted by the enzyme tocopherol cyclase to γ-tocopherol. A further methylation reaction produces α-tocopherol, while modifications to the pathway produce β- and δ‑tocopherols, together with plastoquinones and thence plastochromanol-8. Tocotrienols and tocomonoenols result from a similar series of reactions but with geranylgeranyl diphosphate and tetrahydro-geranylgeraniol diphosphate, respectively, as substrates in the condensation step. The isoprenoid precursors are synthesised by the non-mevalonate or 'MEP' pathway (see the web page on plant sterols).

In plants, tocopherols are found mainly in the chloroplasts of green tissues, but they are also present in seeds, fruits, roots and tubers. They are vital molecules, limiting the damage from photosynthesis-derived reactive oxygen species during conditions of oxidative stress, including high-intensity light stress, and the mechanisms for this antioxidant activity are discussed below. However, recent studies seem to suggest that they are just one of many different components that are involved in photo-protection. Certainly, any tocochromanol peroxy radicals formed must be converted back to the original compounds by the concerted action of other plant antioxidants, such as ascorbate, glutathione, ubiquinol or lipoic acid, and antioxidant enzymes, including superoxide dismutase, catalase and peroxidases - the antioxidant network (see below). Tocopherols are essential for the control of non-enzymatic lipid peroxidation during seed dormancy and germination of seedlings. In their absence, elevated levels of malondialdehyde and phytoprostanes are formed, and there can be inappropriate activation of plant defence responses.

There is evidence that tocopherols play a part in intracellular signalling in plants in that they regulate the amounts of jasmonic acid in leaves (see our web page on plant lipoxins), via modulating the extent of lipid peroxidation and gene expression, and so they influence plant development and stress responses. By controlling the degree of lipid peroxidation in chloroplasts (redox regulation), they limit the accumulation of lipid hydroperoxides required for synthesis of jasmonic acid, which in turn regulates the expression of genes that affect abiotic stress conditions, including drought, salinity and extremes of temperature. The translocation of enzymes to the plasma membrane is regulated by tocopherols, possibly by modulating protein-membrane interactions, or by altering membrane microdomains (lipid rafts), or by competing for common binding sites within lipid transport proteins. In addition, tocopherols are required for the development of the cell walls in phloem transfer cells under cold conditions. It appears that α- and γ‑tocopherol and the tocotrienols may each have distinct functions, and γ-tocopherol is reportedly more potent than α‑tocopherol in protecting plants from the harmful effects of osmotic stresses and is important for the longevity of seeds. Efforts are underway to increase the tocopherol levels in plants by selective breeding and genetic manipulation with the aim of producing crops with greater potential health benefits to consumers and perhaps for the plants per se.

3.  Tocopherols Metabolism in Animals

In animals, the first step in the digestion of tocopherols in the diet is their dissolution with other lipids in mixed micelles in the gut. All tocopherol forms are absorbed to a similar extent by the intestines by means of transporters in the enterocyte apical membrane that have a broad specificity for hydrophobic molecules, such as cholesterol, vitamin D and carotenoids. These include scavenger receptor class B type I (SR-BI), the CD36 protein, and NPC1-like intracellular cholesterol transporter 1 (NPC1L1), although some passive diffusion cannot be ruled out. Transport across the enterocyte may involve cytoplasmic transporters or clathrin-coated vesicles before the tocopherols are incorporated into chylomicrons in free form in the Golgi apparatus for release into the lymph. At the liver, α-tocopherol is the only form taken up from the chylomicrons by a receptor-mediated mechanism with the aid of a specific tocopherol-binding protein (the α-tocopherol transfer protein (α-TTP)), i.e., a 30,500 Da cytosolic protein that has a marked affinity for α-tocopherol and can enhance its transfer between membranes. This recognizes α-tocopherol by the three methyl groups and hydroxyl on the chromanol ring and by the structure and orientation of the phytyl side chain. It is the chief regulator of whole body α‑tocopherol status and is expressed primarily in the cytosol of hepatocytes in the liver but has been reported (in much lower concentrations) in other tissues, such as the placenta.

Scottish thistleα-TTP ultimately regulates the egress of α-tocopherol selectively from hepatocytes with the aid of the ATP-binding cassette proteins ABCA1 and ABCG for conveyance in the plasma lipoproteins, mainly in the very-low-density lipoproteins or VLDL (and thence to LDL) and HDL in humans, to the peripheral tissues (together with much smaller amounts of γ-tocopherol). Most of the other tocopherol forms are directed towards catabolism. Once in the circulation, tocopherol can exchange spontaneously between membranes and lipoproteins, and no specific transport protein for vitamin E in plasma has yet been described. Transfer of tocopherols from the VLDL to peripheral tissues occurs as triacylglycerols are hydrolysed by the enzyme lipoprotein lipase, while that in LDL is processed via the LDL receptor-mediated uptake pathway. In the brain, tocopherol is transported by apo-E enriched lipoproteins. Within cells of peripheral tissues, including the central nervous system, α-TTP functions in transporting α-tocopherol to wherever it is required in membranes, a process that appears to be aided by phosphatidylinositol metabolites. Concentrations of tocopherols can vary appreciably amongst tissues, with most in adipose tissue and adrenals, less in kidney, heart and liver, and least in the erythrocytes.

This "α-tocopherol salvage pathway" is due partly to this process and partly to selective oxidation (see below), and the result is a 20- to 30‑fold enrichment of α‑tocopherol in plasma (average concentration 22-28 μM) relative to the other tocopherols. The process of conservation of one specific tocopherol appears to determine the relative vitamin E activities of the tocopherols and tocotrienols in vivo, rather than their individual potencies as antioxidants as measured in model systems in vitro. Therefore, only α‑tocopherol (including synthetic material) or natural mixtures containing this can be sold under the label 'Vitamin E'. γ‑Tocopherol is the second most abundant form in plasma, and it is present in relatively greater proportions in skin, adipose tissue and skeletal muscle, where it has some specific biological properties that are distinct from those of α-tocopherol. Tocotrienols are not usually detected in tissues, although they are more potent antioxidants in vitro and they may have other functions.

Catabolism: The unwanted surplus of tocochromanols other than α-tocopherol may be excreted in the urine and faeces in the form of carboxy-chromanols, including the so-called 'Simon metabolites' - tocopheronic acids (carboxyethylhydroxychromans, CEHC) and tocopheronolactones, after oxidative cleavage of much of the phytyl tail, although these are normally detected in the form of conjugates as sulfate or glucuronidate esters. In liver cells, the first step in catabolism of γ‑tocopherol is ω‑hydroxylation by cytochrome P450 (CYP4F2) at the 13' carbon to form γ-13'‑hydroxychromanol in the endoplasmic reticulum, followed by ω‑oxidation in the peroxisomes to produce γ‑13'‑carboxychromanol and finally by stepwise β‑oxidation in the mitochondria to cut off two or three carbon moieties from the phytyl chain in each cycle.

Catabolism of gamma-tocopherol

In human cell cultures in vitro, carboxychromanol intermediates have been identified for all tocopherols together with forms in which the hydroxyl group is sulfated, and in the plasma of rodents, sulfated carboxychromanols are the main tocopherol metabolites. As the vitamin E ω‑hydroxylase has a high affinity for tocopherols other than the α-form and does not attack that bound to the α-tocopherol transfer protein, this provides a further specific enhancement of the α‑tocopherol concentration in plasma relative to the others. Some of these catabolic metabolites may have some biological activity of their own, and carboxyethylhydroxychromans derived from γ‑tocopherol were reported to induce apoptosis in cancer cells and to have anti-inflammatory effects by inhibition of cyclooxygenases and 5‑lipoxygenase (see below). Tocotrienols are catabolized in the same way but with additional steps in which the double bonds are reduced prior to oxidation; the final carboxyethylchromanols are the same as for tocopherols.

4.  Tocopherols as Antioxidants

Although the syndrome associated with a lack of vitamin E in the diet of animals has been well known for decades, the mode of action and specific locations of tocopherols in cell membranes are not clearly understood, and several theories have been proposed to explain their functions. From studies in vitro, it has long been believed that a major task is to act as an antioxidant by scavenging free radicals to inhibit, decrease, delay, or prevent oxidative damage to unsaturated lipids or other membrane constituents and thence to tissues, although there are other functions. Upon exposure to the free radical-generating agent carbon tetrachloride, vitamin E administration can prevent hepatotoxicity and lipid peroxidation, which is a cause of ferroptosis, an iron-dependent form of non-apoptotic cell death. In numerous non-biological systems such as foods, cosmetics and pharmaceutical preparations, tocopherols are invaluable additives as fat-soluble antioxidants. Although the discussion here is limited to the effects upon lipids, free radicals can cause damage to proteins, DNA, and indeed virtually any native substance in living organisms, and of course, tocopherols are only one class of many natural antioxidants.

Because of their lipophilic character, tocopherols are located in the membranes or with storage lipids where they may be available immediately to interact with lipid hydroperoxides, such as those described in more detail in our web pages on isoprostanes, reactive aldehydes and oxidized phospholipids. In brief, Reactive Oxygen Species (ROS), of which innumerable forms exist, can be derived by enzymatic or non-enzymatic means and produce superoxide anions and other peroxyl radicals. Superoxide radicals (O2•-) ultimately generate highly toxic hydroxyl (OH) or alkoxyl radicals, which can abstract a hydrogen atom from bis-allylic methylene groups of polyunsaturated fatty acids under aerobic conditions in vivo in animals and plants to generate lipid peroxyl radicals (LOO) and hydroperoxy-fatty acids. Singlet oxygen (1O2 or O=O) is the most active ROS (non-radical) in photosynthetic tissues of plants. As radical generation is not enzymatic, all methylene groups between two cis double bonds can potentially be involved in the reaction, although not necessarily to the same degree. Tocopherols react rapidly in a non-enzymic manner unlike many other cellular antioxidants, which are dependent on enzymes, to scavenge lipid peroxyl radicals, i.e., the chain-carrying species that propagate lipid peroxidation. In model systems in vitro, all the tocopherols (α > γ > β > δ) and tocotrienols are good antioxidants, with the tocotrienols being the most potent.

Lipid oxidation proceeds by a chain process mediated by free radicals, in which the lipid peroxyl radical serves as a chain carrier. In the initial step of chain propagation, a hydrogen atom is abstracted from the target lipid by the peroxyl radical as shown -

Oxidation mechanism - 1/2

Tocopherol terminates or inhibits autoxidation cycles by first transferring a hydrogen atom to an oxidized lipid within a cell membrane (LOO) to produce a molecule of lipid hydroperoxide (LOOH) and the tocopherol radical (TO). Next, the TO radical further reacts with another LOO radical in a coupling reaction with the result that one tocopherol molecule prevents lipid peroxidation at two sites.

Oxidation mechanism - 3

The potency of an antioxidant is determined by the relative rates of reactions (1) and (2). When a tocopherol radical is formed, it is stabilized by delocalization of the unpaired electron about the fully substituted chromanol ring system, thus rendering it relatively unreactive and preventing propagation of the chain reaction. This explains the high first-order rate constant for hydrogen transfer from α‑tocopherol to peroxyl radicals, as studies of the relative rates of chain propagation to chain inhibition by α-tocopherol in model systems have demonstrated that α-tocopherol is able to scavenge peroxyl radicals much more rapidly than the peroxyl radical can react with a lipid substrate. α-Tocopherol is most efficient at providing protection against peroxyl radicals in a membrane environment.

Reaction of the tocopherol radical with a lipid peroxyl radical, as illustrated, yields 8α-substituted tocopherones, which are readily hydrolysed to 8α‑hydroxy tocopherones that rearrange spontaneously to form α-tocopherol quinones. In an alternative pathway, the tocopheroxyl radical reacts with the lipid peroxyl radical to form epoxy-8α-hydroperoxytocopherones, which hydrolyse and rearrange to epoxyquinones. Tocopherol dimers and trimers may be formed as minor products.

Tocopherol as an antioxidant

Reactive nitrogen species (RNS): Free radical-mediated lipid peroxidation is the major pathway of lipid oxidation taking place in humans, and while α‑tocopherol is a major antioxidant, it does not scavenge nitrogen dioxide radicals, carbonate anion radicals and hypochlorite efficiently. Vitamin E forms with an unsubstituted 5‑position, such as γ-tocopherol, are an exception to the rule that the various tocopherols have comparable antioxidant properties in that they can trap electrophiles, including reactive nitrogen species, which are enhanced during inflammation.

The enzyme nitric oxide synthase can continuously produce nitric oxide (NO), which has many vital functions in human physiology, but in excess, this can react with superoxide to produce more reactive species such as nitrogen dioxide (NO2), peroxynitrite (ONOO-) and peroxynitrite radicals, which are potent and versatile oxidants that can attack a wide range of biological targets. These can induce lipid peroxidation and nitrate aromatic compounds and unsaturated fatty acids, while isomerizing cis-double bonds in fatty acids to the trans-configuration. γ-Tocopherol is much superior to α‑tocopherol in detoxifying the NO2 radical and peroxynitrite with formation of 5‑nitro-γ-tocopherol. This occurs in vivo, and the concentrations of 5-nitro-γ-tocopherol have been shown to be elevated in the plasma of subjects with coronary heart disease and in carotid-artery atherosclerotic plaque. For the same reason, γ‑tocopherol may have a functional role in the prevention of DNA damage over time.

Reaction of gamma-tocopherol with NO2

As part of this process, the less reactive nitric oxide can then be regenerated at beneficial physiological concentrations. In contrast, α-tocopherol can react with NO2 to form a reactive nitrosating agent, and it does not effectively reduce NO2 to NO in the absence of light.

The antioxidant network: In plant and animal tissues, tocopherols can be regenerated from the tocopheroxyl radicals in a redox cycle mediated by other endogenous antioxidants, including vitamins A and C and coenzyme Q, and this must greatly extend their biological potency. Vitamin C (ascorbic acid) may be required for regeneration of α‑tocopherol in aqueous systems, although it can act at the surface of membranes, while in turn it is oxidized to dehydroascorbic acid. This can be regenerated to the reduced form by glutathione (GSH) with production of glutathione disulfide (GSSG), which can subsequently be reduced enzymatically by glutathione reductase with NAD(P)H as a cofactor. In plants, an NAD(P)H-dependent quinone oxidoreductase is involved at an early stage of the regeneration process, while tocopherol cyclase, an enzyme involved in the biosynthesis of tocopherols, re-introduces the chromanol ring.

The antioxidant network

Thus, tocopherols are only one component of a complex web of metabolites and enzymes in tissues that have antioxidant activities and act by various mechanisms, including the stimulation of genes involved in signalling responses to environmental stresses. One antioxidant mechanism involves removal of free radicals and reactive species by enzymes such as superoxide-dismutase, catalase and glutathione peroxidase, while electron donors, such as glutathione, tocopherols, ascorbic acid, vitamin K, coenzyme Q and thioredoxin, scavenge free radicals also. Metal-binding proteins such as transferrin, metallothionein, haptoglobin and ceruloplasmin have antioxidant activity by sequestering pro-oxidant metal ions, such as iron and copper, although some metals such as selenium and zinc are in fact antioxidants. Other antioxidants, including flavonoids, carotenoids and phenolic acids as well as tocopherols, enter animal tissues via the food chain.

5.  Biological Functions of Tocochromanols in Animals

Vitamin E deficiency has been detected in patients with fat malabsorption, cystic fibrosis, Crohn's disease, liver disease and pancreatic insufficiency, and in premature infants. Impairment of the normal functions of the immune system has been demonstrated in animals and humans in vitamin E deficiency, and this can be corrected by vitamin E repletion. Although there are various proposals for the pathogenic mechanism, none as yet appears to be generally accepted. After the discovery of the effects of vitamin E on fertility in studies with laboratory animals, it was found to be required for the development of tissues and organs such as brain and nerves, muscle and bones, skin, bone marrow and blood, most of which are specific to α-tocopherol, although there is no evidence for an effect of vitamin E on fertility in humans, as was originally found in the rat. The rare genetic disorder "Ataxia with Isolated Vitamin E Deficiency” or "AVED” is the result of mutations in the gene coding for α-TTP. It is caused by the death of cerebellar Purkinje cells, but administration of α‑tocopherol prevents this and the subsequent development of clinical symptoms of the disease. Vitamin E deficiency can be a factor in non-alcoholic hepatosteatosis.

While there are many fat-soluble antioxidants in the diet, only α-tocopherol is a vitamin. There appears to be little doubt that tocopherols inhibit many of the enzymes associated with inflammation in vitro in animals and may contribute to the amelioration and treatment of some chronic diseases. Paradoxically, it has been argued that data on the effects of vitamin E on biomarkers of oxidative stress in vivo are inconsistent because oxidized metabolites of vitamin E, i.e., that have reacted as antioxidants, are barely detectable in tissues, and vitamin E maintenance in vivo does not appear to have been clearly associated with its regeneration.

There appear to be significant differences between results obtained in studies with laboratory animals in comparison to those in humans. Thus, suggestions that dietary supplements of vitamin E may reduce the rate of oxidation of lipids in low-density lipoproteins in humans and thence the incidence or severity of atherosclerosis have not been confirmed by clinical intervention studies, although benefits in some conditions have been claimed. Indeed, there are suggestions that excessive vitamin E supplementation may even be harmful. One study has suggested that relatively high doses of natural α-tocopherol over a long period are required to demonstrate a significant reduction in the levels in urine of F2 isoprostanes, which are considered to be the most reliable marker for oxidative stress in vivo. It has even been suggested that tocopherol may be protected from functioning as an antioxidant in some tissues in vivo through the network of cellular antioxidant defences, such that tocopherols are utilized only when other antioxidants are exhausted, although there is no experimental proof of this hypothesis. While many studies indicate that γ-tocopherol appears to have significant beneficial effects in protecting cells from inflammatory damage, epidemiologic studies are not easy to interpret.

Scottish thistleAt the cellular level, RRR-α-tocopherol ameliorates diabetic nephropathy by activating diacylglycerol kinase alpha (DGKα) and not by an antioxidant effect. The membrane-bound 67 kDa laminin receptor (67LR) has a hydrophobic pocket that is a novel binding site for tocopherol, and this association mediates the activation of DGKα. To date, this appears to be the only known receptor for α-tocopherol.

Tocopherol has been shown to inhibit protein kinase C, and in the process, it inhibits the assembly and radical producing activity of NADPH oxidase in monocytes. As vitamin E suppresses the expression of xanthine oxidase, a source of reactive oxygen species, in the liver, it is possible that α-tocopherol can diminish the levels of free radicals by preventing their production and not by scavenging them. Its physical presence in membranes adjacent to polyunsaturated fatty acids may limit autoxidation.

With the discovery that the antioxidant effects of various tocopherols and tocotrienols have little relation to their vitamin E activities in vivo has come the realization that they have other functions in tissues, most of which are again specific to α‑tocopherol. Most current research is concerned with how vitamin E and its metabolites act in signalling and controversially in the regulation of gene activity. By preventing the increase of peroxidized lipids that alter both metabolic pathways and gene expression profiles within tissues and cells, it may act indirectly as a regulator of genes connected with tocopherol catabolism, lipid uptake, collagen synthesis, cellular adhesion, inflammation, the immune response and cell signalling. Vitamin E affects several transcription factors in this manner, including peroxisome proliferator-activated receptor gamma (PPARγ), nuclear factor erythroid-derived 2 (NRF2), nuclear factor kappa B (NFκB), RAR-related orphan receptor alpha (RORα), estrogen receptor beta (ERβ) and the pregnane X receptor (PXR).

α-Tocopherol and its metabolites are believed to modulate the activity of several enzymes involved in signal transduction, including protein kinases and phosphatases, lipid kinases and phosphatases, and other enzymes involved in lipid metabolism, but especially those with inflammatory properties such as lipoxygenases, cyclooxygenase-2 and phospholipase A2. While the credentials of tocopherols as antioxidants in vivo have sometimes been doubted, this does not preclude a role in the inhibition of oxidative enzymes in relation to the function of the immune system. Vitamin E regulates T cell function directly by its effects upon T cell membrane integrity, signal transduction and cell division, and it functions indirectly by affecting eicosanoids and related inflammatory mediators generated from other immune cells. Various tocopherols and tocotrienols have been shown to suppress COX-2 involvement in prostaglandin (PGD2 and PGE2) synthesis in lipopolysaccharide-activated macrophages.

It has been established that the 13'-carboxy metabolite of α-tocopherol (α-T-13'-COOH) and other tocopherol ω-carboxylates are potent allosteric inhibitors of 5‑lipoxygenase, a key enzyme in the biosynthesis of the inflammatory leukotrienes. α-T-13'-COOH accumulates in immune cells and inflamed exudates both in vitro and in vivo in mice, and it has even been suggested that the immune regulatory and anti-inflammatory functions of α‑tocopherol depend on this endogenous metabolite.

Formula of alpha-T-13'-COOH

α-Tocopherol has a stimulatory effect on the dephosphorylation enzyme, protein phosphatase 2A, which cleaves phosphate groups from protein kinase C, leading to its deactivation. The mechanism may involve the binding of vitamin E directly to enzymes in order to compete with their substrates, or it may change their activities by redox regulation. It may compete for common binding sites within lipid transport proteins and so could alter the traffic of lipid mediators indirectly with effects upon their signalling functions and enzymatic metabolism. For example, it binds to albumin as well as to a specific α-tocopherol-associated protein (TAP), and in the latter form, it inhibits the phosphoinositide 3-kinase. It has been suggested that vitamin E may have a secondary role in stabilizing the structure of membranes, or it may interact with enzymes in membranes to interfere with binding to specific membrane lipids, or it may affect membrane microdomains such as lipid rafts.

Evidence suggests that the biological activities of β-, γ- and δ-tocopherols do not reflect their behaviour as chemical antioxidants, but anti-inflammatory, antineoplastic and natriuretic actions have been reported. Some non-antioxidant effects of γ-tocopherol in tissues in relation to reactive nitrogen oxide species have been observed, but the specificity of these in vivo is not yet certain, while anti-inflammatory properties have been described that have been attributed to a chain-shortened metabolite. Beneficial effects against cancer cells in vitro have been observed that have been ascribed to scavenging of reactive nitrogen species, since such effects are not seen with α-tocopherol. While α-tocopherol has no effect on cancer, there is some evidence that its isomers may influence tumour cells by activating the mitogen-activated protein kinase (MAPK) signalling pathway.

Tocotrienols have been shown to have neuroprotective effects and to inhibit cholesterol synthesis. They reduce the growth of breast cancer cells in vitro, possibly by influencing gene expression by interaction with the oestrogen receptor-β. When administered in combination with either standard antitumour agents as in chemotherapy or with natural compounds with anticancer activity, they are reported to exert a synergistic antitumour effect on cancer cells. γ-Tocotrienol is reported to be an inducer of apoptosis via endoplasmic reticulum stress, while α-tocotrienol may be neuroprotective by inhibition of lipoxygenase activity. Although anti-obesity and anti-diabetic effects have been observed in mice, clinical trials with humans appear to have given inconclusive results. These properties are largely distinct from those of the tocopherols, and the pharmaceutical potential of tocotrienols against cancer, bone resorption, diabetes, and skin, cardiovascular and neurological diseases is currently being studied.

The biological functions of α-tocopheryl phosphate are slowly being revealed. As well as being a possible storage or a transport (water-soluble) form of tocopherol, it is involved in cellular signalling and regulates several genes, including those involved in angiogenesis and vasculogenesis, in a different manner from α‑tocopherol per se. As it lacks the free hydroxyl group, it cannot act directly as an antioxidant, and some consider it to be the biologically active form of the vitamin, although the phosphate bond is rapidly hydrolysed in aqueous systems. It is certainly more active in a number of biological systems in vitro than α-tocopherol, so these effects cannot be ascribed to the hydrolysed molecule, and in some instances, it is antagonistic to α-tocopherol, as in its activity towards phosphatidylinositol 3-kinase. On the other hand, activation requires a kinase, while a phosphatase is needed to make the system reversible, but neither has yet been identified. Synthetic phosphate derivatives of γ-tocopherol and α-tocopheryl succinate are known to have potent anti-cancer properties.

6.  Analysis

Tocopherols can be analysed by gas chromatography, both with flame-ionization and mass spectrometric detection, but the methods that are usually recommended involve high-performance liquid chromatography with fluorescence detection, or more definitively with tandem mass spectrometry. Related methods are used for ubiquinones and the isoprenoid alcohols. Countercurrent chromatography has been employed as a mild method for preperative isolation of isomers.

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