In 1922, Herbert Evans and Katherine Bishop discovered what they called ‘substance X’, an essential factor for successful reproduction in rats . Three years later, this ‘substance X’ was assigned its vitamin status and the letter E, being the next serial alphabetical designation after the preceding discovery of the vitamins A-D . In 1936, Evans and co-workers isolated an alcohol with the biological activity of vitamin E from wheat germ oil and proposed the name α‑tocopherol (Greek: tokos = child birth; phero = to bear; and -ol, indicating an alcohol) .
Structures and stereochemistry
Vitamin E is a generic name for all substances exerting the biological activity (see below) of α‑tocopherol (αT). The eight recognized natural vitamin E compounds (subsequently referred to as ‘vitamers’) consist of a chroman head substituted with a 16-carbon side-chain and are classified into tocopherols, with a saturated side-chain, and tocotrienols, with an unsaturated side-chain with three isolated double bonds. The Greek letters α, β, γ, and δ are added as prefixes to denote the number and positions of methyl groups linked to the chroman head (Figure 1). The phytyl side chain of the tocopherols has three chiral centres at positions 2, 4', and 8', which can be either in the R- or S-conformation, giving rise to eight different stereoisomers (RRR, RSR, RRS, RSS, SRR, SSR, SRS, and SSS) for each tocopherol.The naturally occurring tocopherols exist solely as RRR-stereoisomers; synthetic tocopherols are composed of an equimolar mixture of all eight stereoisomers, a so-called all racemic (all rac) mixture .
Occurrence and dietary intake
Vitamin E is exclusively synthesised by photosynthetic organisms. Plants accumulate αT in their green tissues, while γT and δT are mainly present in seeds, and tocotrienols are predominant in cereal grains and palm oil [5, 6]. The richest sources of vitamin E are vegetable oils, with wheat germ, safflower, and sunflower oils being particularly rich in αT, and soybean, corn, and sesame oils in γT. Other good sources of vitamin E include lipid-rich plant parts such as nuts, seeds and grains. The dietary intake of vitamin E in Western diets is mainly from fats and oils used in margarine, mayonnaise, salad dressings, and also from fortified foods such as breakfast cereals and fruit juices. In contrary to other Western populations where αT is the predominant form in the diet, γT is the major dietary form of vitamin E in the USA due to the widespread use of soybean and corn oils . The naturally occurring tocopherols exist solely as RRR-stereoisomers. Synthetic tocopherols, on the other hand, are composed of an equimolar mixture of all eight stereoisomers, a so-called all racemic (all rac) mixture. The vitamer most frequently used in supplements and fortified foods is αT (mostly all rac-αT, but also RRR-αT); often in the form of esters with acetate, succinate or nicotinate to improve its storage stability .
Absorption, transport, and metabolism
The intestinal absorption of vitamin E generally parallels the absorption of dietary fat. Vitamin E is taken up into enterocytes and secreted with chylomicrons into the lymphatic system. The chylomicrons pass through the thoracic duct into the systemic circulation where a fraction of the transported vitamin E is transferred to high-density lipoproteins (HDL), from where it can easily be distributed to all circulating lipoproteins, or tissues. Chylomicron degradation ultimately results in the formation of chylomicron remnants, which are taken up into the liver. Right to this point, the extent of vitamin E absorption and transport to the liver appears to be similar for all vitamers . From the liver, (natural) RRR-αT is preferentially secreted into the blood stream with lipoproteins, involving a cytosolic α‑tocopherol transfer protein (TTP) with pronounced selectivity towards the 2R‑isomers (R-configuration at carbon 2) . Hosomi and co-workers  determined the affinities of TTP for some E-vitamers relative to that for RRR‑αT and observed the following lower values: RRR-βT, 38%; RRR-γT, 9%; RRR‑δT, 2%; SRR-αT, 11%; and α-tocotrienol, 12%. Interestingly, the affinity values for the tocopherols are comparable to their biological activities and, in part, reflect their plasma concentrations. Therefore, TTP was proposed as the primary determinant of the discrimination and the biological activity of the vitamers in the body . See “Own related research” (below) for a discussion of why this notion might be incorrect.
The lipid-soluble vitamin E is degraded to water-soluble carboxyethyl hydroxychroman (CEHC) metabolites by side-chain degradation without modification of the chromanol head and excreted in urine . The first step in the metabolism of tocopherols and tocotrienols consists of a terminal ω‑hydroxylation of the side-chain by cytochrome P450 (CYP) isozymes followed by a stepwise shortening of the tail by β-oxidation [16-18]. In vitro, tocopherol-ω-hydroxylase, the enzyme(s) initiating vitamin E metabolism, exhibited similar binding affinities for αT and γT, but exhibited much higher catalytic activity towards γT, suggesting a central role for this enzyme in the selective retention of αT in the body and the regulation of γT plasma concentrations (Sontag & Parker, 2002). Our own findings in human subjects, showing that urinary excretion as well as plasma concentrations of γCEHC exceed those of αCEHC by a factor of ~4-5 and ~14-16, respectively, further support this notion [15, Frank et al. unpublished observations]. In agreement, up to ~50% of ingested γT was excreted in urine as the corresponding γCEHC metabolite , while only 1-3% of the consumed αT dose was converted to urinary αCEHC in humans . Furthermore, when Traber and co-workers compared the urinary excretion of deuterated αCEHC derived from RRR-αT and all rac-αT, they found 2-4 times more “all rac”-metabolites . In the fruit fly Drosophila melanogaster, the selective accumulation of αT has been associated with CYP- but not TTP-activity .
Biological functions of vitamin E
When Evans and Bishop studied the duration of the oestrous cycle in response to dietary changes in laboratory rats, they discovered that the absence of a substance “X” (later identified as αT) resulted in foetal death and resorption . During the following years, a multitude of vitamin E deficiency syndromes, such as muscular dystrophy and neuronal dysfunction, were described in various species , but no specific function could be ascribed to the vitamin. Decades later, the antioxidant activity of αT was discovered and assumed to be its major function in vivo . Recently, other biological activities of vitamin E, supposedly unrelated to its antioxidant properties, have been reported. These include roles in cellular signalling, gene expression, immune response, and apoptosis . However, the notion that αT might exert biological functions independent of its antioxidant activity is currently a matter of debate. Some researchers, spearheaded by Prof. Azzi, put forward the idea that αT or its derivative α-tocopheryl phosphate may act as cellular signalling molecules or redox sensors. Azzi’s rationale builds on the observation that other biomolecules, such as 17β‑estradiol, have distinct non-antioxidant physiological functions while also exerting antioxidant activity in a range of in vitro systems. He proposes that αT (or α‑tocopheryl phosphate) acts as a ligand for as yet unknown transcription factors or receptors thus affecting signal transduction and gene expression and that this signalling function is not compatible with that of an antioxidant molecule .
An opposing school of thought, led by Prof. Traber, promotes the idea that all biological activities exerted by αT, including the regulation of gene expression and cellular signalling cascades, can be explained on the basis of its antioxidant protection of polyunsaturated fatty acids and the resulting effects on membrane qualities (integrity, fluidity, phase separation, etc.) .
To date, the most convincing evidence that the vitamin function of αT may be that of an antioxidant protecting membrane lipids comes from studies in TTP knockout (TTP‑/‑) mice that lack the ability to retain αT in the body and consequently exhibit extremely low plasma and tissue αT concentrations, develop neurological dysfunctions, and are infertile . Jishage and co-workers created such an TTP-/- mouse model to study the prevention of the best established and most widely accepted vitamin E-deficiency symptom, namely foetal resorption, in response to high-dosage αT or BO-653 (a synthetic antioxidant) supplementation and observed the restoration of fertility in female mice supplemented with either antioxidant as compared to control mice fed a normal rodent chow. In this study, unspecific antioxidant activity was the mode of action restoring fertility .
Own related research
Based on the binding affinities of TTP for the different vitamin E congeners , published literature frequently states that TTP is the means by which the body achieves approximately 10-times higher plasma and tissue concentrations of αT as compared to γT. Our own research, on the other hand, showed that feeding of sesamin or alkylresorcinols for 4 weeks resulted in significantly increased plasma and tissue γT concentrations (up to ~1500%) in wild-type rats (known to express TTP), while αT concentrations remained unaffected (Figure 2) [11, 12].
Figure 2. Mean plasma concentrations of α- and γ-tocopherols (mg/ml) in rats fed sesamin-fortified (2 g/kg diet) or control diets for 4 weeks; error bars indicate SEM. No significant differences were observed between treatments for α-tocopherol concentrations; γ‑tocopherol concentrations were significantly different (P<0.0001, unpaired Student’s t-test).In order to study whether the observed increase in γT levels was due to an inhibition of γT metabolism, we incubated HepG2 cells together with sesamin or alkylresoricinols in the presence of tocopherols and determined the formation of the respective metabolites. Sesamin almost completely inhibited tocopherol side-chain degradation and cereal alkylresorcinols inhibited it, dose-dependently, by 20-80% (Figure 3) .
Figure 3. Inhibition of γ-tocopherol metabolism by alkylresorcinols in HepG2 cells. Rye alkylresorcinols = purified (99%) rye AR (C15:0 – C25:0); C15:0 = synthetic pentadecylresorcinol. Sesamin is a positive control; n=3. Bars not sharing a common subscript letter are statistically different (P<0.05); error bars indicate SEM.
To verify the inhibition of γT metabolism by sesame lignans in humans, sesame oil (treatment) or corn oil (control) muffins together with deuterium-labelled d6-αT and d2-γT were given to volunteers. Blood and urine samples were collected for 72 hours and analysed for deuterated and non-deuterated tocopherols and their metabolites. Consumption of sesame oil muffins significantly inhibited vitamin E metabolism and resulted in a reduced urinary excretion of d2-γCEHC (Figure 4) [13-15].
Figure 4. Urinary excretion of d2-γ-CEHC (nmol/g creatinine) over 24 h in women and men consuming d6-α- and d2-γ-tocopheryl acetate together with sesame oil or corn oil muffins, respectively. Repeated measures MANOVA revealed significant effects of oily type (P<0.030) and sex (P<0.055); no significant oil type-by-sex interactions were observed.
In conclusion, our own research supports the notion that vitamin E metabolism, rather than TTP, is responsible for the significantly higher concentrations of αT in the body as compared to all other vitamin E forms, although conclusive and direct proof is still lacking.
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