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Isoprenoids: 1. Tocopherols and Tocotrienols (Vitamin E)

Tocopherols constitute a series of related benzopyranols (or methyl tocols) that are synthesised in plant tissues and are especially abundant in vegetable oils. First described in 1922 as a dietary factor essential to prevent fetal reabsorption in rats, they were soon recognized as a vitamin that was essential for innumerable aspects of animal development. In addition, they were shown to be powerful lipid-soluble antioxidants and subsequently to have regulatory roles in signal transduction and gene expression.

1.   Structure and Occurrence

In the tocopherols, the C16 side chain is saturated, and in the tocotrienols it contains three trans double bonds. Together, these two groups are termed the tocochromanols. In essence, the tocopherols have a 20-carbon phytyl tail (including the pyranol ring), with variable numbers of methyl groups attached to the benzene ring, while the tocotrienols have a 20-carbon geranylgeranyl tail with double bonds at the 3', 7' and 11' positions attached to the ring system. The side-chain methyl groups of natural tocopherols have R,R,R stereochemistry. 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). In contrast, the tocotrienols have a single chiral centre. Plastochromanol-8 is an analogue of γ-tocotrienol with a much longer side-chain.

Formulae of tocopherols

These compounds 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, and collectively they are termed ‘vitamin E’ (the individual tocopherols are ‘vitamers’). The recommended daily intake for vitamin E varies between 3 and 15 mg/day according to the country and age of the person. 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. Tocopherols are present in all photosynthetic organisms, but the tocotrienols are found only in certain plant families, and α-tocopherol is often the main tocochromanol in leaves. 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 seeds and fruits, especially of monocots such as wheat, rice and barley, though a major commercial source is palm oil. In leaf tissue, α-tocopherol is often the main isomer, while γ-tocopherol is the primary tocopherol of many seeds. Plastochromanol-8 was first found in leaves of the rubber tree (Hevea brasiliensis), but has since been found in many other plants including rapeseed and maize, but usually at lower levels than of the tocopherols.

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.

Formulae of some less common tocols

α-Tocopheryl phosphate has recently been detected at low levels in liver and adipose tissue, and it is possible that it may be a common constituent of animal and plant tissues. Although the α-tocopherol kinase required for its biosynthesis has yet to be identified, early studies suggest that it has distinctive biological properties in tissues.

α-Tocopherol is a minor but ubiquitous component of the lipid constituents of animal cell membranes, with estimates ranging from one molecule of tocopherol to from 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.

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 the 2 position (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 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 is well understood, 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, 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 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 in the plastid also by the non-mevalonate or 'MEP' pathway (see the web page on plant sterols)

In plants, tocochromanols are found almost exclusively in the chloroplasts, where they were long believed to be the most important of the antioxidant molecules, limiting the damage from photosynthesis-derived reactive oxygen species during conditions of oxidative stress, including high-intensity light stress. The mechanisms for this antioxidant activity are discussed below. However, recent studies seem to suggest that they are just one of a number of 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, for example by ascorbate and glutathione. On the other hand, there is no doubt that 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 also play a part in intracellular signalling in plants in that they regulate the amounts of jasmonic acid (see our web page on plant lipoxins) in leaves, via modulating the extent of lipid peroxidation and gene expression, and so influence plant development and stress responses. Thus, 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 a number of stress conditions. The translocation of enzymes to the plasma membrane is regulated by tocopherols, possibly by modulating protein-membrane, 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.

3.   Tocopherols Metabolism in Animals

In animals, all tocopherols are absorbed to a similar extent in the intestines by mechanisms that are still obscure, and they are transported to the liver in chylomicrons mainly, where most of the tocochromanols are taken up by a receptor-mediated mechanism. This process is facilitated by a specific tocopherol-binding protein (the α-tocopherol transfer protein), i.e. a 30,500D cytosolic protein that has a marked affinity for α-tocopherol and can enhance its transfer between membranes. α-Tocopherol is then selectively conveyed to the plasma lipoproteins, mainly the very-low-density lipoproteins or VLDL (and thence to LDL) and HDL in humans, for transport to other tissues (together with much smaller amounts of γ-tocopherol).

Transfer of tocopherols from the VLDL to peripheral tissues is promoted by the enzyme lipoprotein lipase, while that in LDL is processed via the LDL receptor-mediated uptake pathway. Within cells of peripheral tissues, the α-tocopherol transfer protein 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.

The "α-tocopherol salvage pathway" is partly due 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. Thus, 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. Only α-tocopherol (including synthetic material) or natural mixtures containing this can be sold under the label 'Vitamin E'. γ-Tocopherol is the second abundant form in plasma, and it is relatively abundant in skin, adipose tissue and skeletal muscle, where it has some specific biological properties that are distinct from those of α-tocopherol. Although tocotrienols are more potent antioxidants in vitro, they are not usually detected in tissues.

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

Catabolism of alpha-tocopherol

Various carboxychromanol intermediates have been identified for all of the tocopherols together with sulfated forms of these in human cell cultures in vitro. As the vitamin E ω-hydroxylase has a high affinity for the 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..

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 location of tocopherols in cell membranes are not clearly understood. Several theories have been proposed to explain their functions. It is evident that a major task is to act as antioxidants to prevent free radical damage to unsaturated lipids or other membrane constituents and thence to tissues. For example, vitamin E administration can prevent lipid peroxidation and hepatotoxicity upon exposure to the free radical-generating agent carbon tetrachloride. Although there are suggestions that this activity may be secondary to more important biological functions (see below), there is no doubt that tocopherols are powerful antioxidants in vitro and in vivo. In non-biological systems such as foods, cosmetics, pharmaceutical preparations and so forth, they are certainly invaluable antioxidant additives.

Because of their lipophilic character, tocopherols are located in the membranes or with storage lipids where they are immediately available to interact with lipid hydroperoxides. They 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.

In general, the oxidation of lipids is known to proceed by a chain process mediated by a free radical, 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

The main function of α-tocopherol is to scavenge the lipid peroxyl radical before it is able to react with the lipid substrate as -

Oxidation mechanism - 2

The potency of an antioxidant is determined by the relative rates of reactions (1) and (2). When a tocopheroxyl radical is formed, it is stabilized by delocalisation of the unpaired electron about the fully substituted chromanol ring system rendering it relatively unreactive, thus preventing propagation of the chain reaction. This also 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.

In biological systems, oxidant radicals can spring from a number of sources, including singlet oxygen, alkoxyl radicals, superoxide, peroxynitrite, nitrogen dioxide and ozone. α-Tocopherol is most efficient at providing protection against peroxyl radicals in a membrane environment.

Reaction of the tocopheroxyl 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 also be formed as minor products.

Tocopherol as an antioxidant

Vitamin E forms with an unsubstituted 5-position, such as γ-tocopherol, are an exception to the rule that the various tocopherols have similar antioxidant properties in that they are able to trap electrophiles, including reactive nitrogen species, which are enhanced during inflammation. Thus, γ-tocopherol is superior to α-tocopherol in detoxifying NO2 and peroxynitrite with formation of 5-nitro-γ-tocopherol.

Reaction of gamma-tocopherol with NO2

In plant and animal tissues, tocopherols can be regenerated from the tocopheroxyl radicals in a redox cycle mediated by a number of endogenous antioxidants, including vitamins A and C and coenzyme Q, and this must greatly extend their biological potency. Vitamin C (ascorbate) may be especially important in aqueous systems, although it may also act at the surface of membranes. Many aspects of the enzymology of this process have still to be determined, but in plants an NAD(P)H-dependent quinone oxidoreductase is involved at an early stage, while tocopherol cyclase, an enzyme involved in the biosynthesis of tocopherols, re-introduces the chromanol ring.

While there appears little doubt that tocopherols inhibit many of the enzymes associated with inflammation in vitro, it has been argued that data on the effects of vitamin E on biomarkers of oxidative stress in vivo are inconsistent. Oxidized metabolites of vitamin E, i.e. that have reacted as antioxidants, are barely detectable in tissues. Thus, suggestions that dietary supplements of vitamin E may reduce the rate of oxidation of lipids in low-density lipoproteins and thence the incidence or severity of atherosclerosis now appear to be unfounded, although benefits in some conditions have been claimed. Indeed, there are suggestions that excessive vitamin E supplementation may even be harmful. A recent 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 of F2 isoprostanes in the urine, which are considered to be the most reliable marker for oxidative stress in vivo. This subject is highly contentious and I prefer to leave further discussion to clinical experts.

5.   Other Biological Functions of Tocochromanols in Animals

After the discovery of the effects of vitamin E on fertility, animal studies then documented the importance of the vitamin 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. With the discovery that the antioxidant effects of various tocopherols and tocotrienols have little relation to their vitamin E activities in vivo has come a belief that they have may have other functions in tissues, most of which are specific to α-tocopherol. There are many fat-soluble antioxidants in the diet but only α-tocopherol is a vitamin. It has even been suggested that tocopherol may be protected from functioning as an antioxidant in some tissues in vivo through a network of cellular antioxidant defences, such that only when other antioxidants are exhausted are the tocopherols utilized. However, there is no experimental proof of this hypothesis. Most current research is concerned with how the different forms of vitamin E act in signalling and the regulation of genes. While it is certainly true that most other vitamins are essential cofactors for specific enzymes or transcription factors, no receptor that binds specifically to vitamin E has yet been discovered.

Scottish thistleOther proposed functions, some of which are not generally accepted, include a role as a regulator of genes connected with tocopherol catabolism, lipid uptake, collagen synthesis, cellular adhesion, inflammation, the immune response and cell signalling. It is also 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, such as lipoxygenases, cyclooxygenase-2 and phospholipase A2. For example, 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 also compete for common binding sites within lipid transport proteins, and so may alter the traffic of lipid mediators indirectly with affects upon their signalling functions and enzymatic metabolism. 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. Some non-antioxidant effects of γ-tocopherol in tissues in relation to reactive nitrogen oxide species have been observed, but the specificity of these is not yet certain.

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. These properties are largely distinct from those of the tocopherols, and the pharmaceutical potential of tocotrienols against cancer, bone resorption, diabetes, and cardiovascular and neurological diseases are currently being studied.

The biological functions of α-tocopheryl phosphate are slowly being revealed. In addition to being a possible storage or a transport (water-soluble) form of tocopherol, it is involved in cellular signalling and regulates a number of genes, including those involved in angiogenesis and vasculogenesis. 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. Related methods are used for ubiquinones and the isoprenoid alcohols.

Recommended Reading

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