Chapter 4. Vitamin E

4 Vitamin E Maret G Traber CONTENTS Introduction 154 History 154 Structures and Antioxidant Chemistry 154 Structure 154 Nomenclature 155 Chemical Properties 155 Antioxidant Network 156 Oxidized Vitamin E 156 Physiologic Relationships 156 Absorption 156 Lipoprotein Transport 157 Tissue Delivery 157 Storage Sites 158 Vitamin E-Specific Proteins 158 a-Tocopherol Transfer Protein 158 Other Tocopherol-Binding Proteins 159 Plasma Vitamin E Kinetics 160 Metabolism and Excretion 160 Cellular and Biochemical Functions 162 Nutritional Requirement 162 Adequacy of Vitamin E Intakes in Normal U.S Populations 163 Food Sources of Vitamin E 163 Deficiency Signs and Methods of Nutritional Assessment 164 Vitamin E Deficiency Caused by Genetic Defects in the a-Tocopherol Transfer Protein 164 Vitamin E Deficiency Caused by Genetic Defects in Lipoprotein Synthesis 165 Vitamin E Deficiency as a Result of Fat Malabsorption Syndromes 165 Pathology of Human Vitamin E Deficiency 166 Assessment of Vitamin E Status 166 Efficacy of Pharmacological Doses of Vitamin E 167 Conclusion 168 Acknowledgment 168 References 168 ß 2006 by Taylor & Francis Group, LLC INTRODUCTION Vitamin E is unique because it remains a vitamin without a specific function Other vitamins are cofactors, hormones, or have specific roles in metabolism Vitamin E deficiency symptoms are varied because the major function of vitamin E is that of a lipid-soluble antioxidant Therefore, vitamin E deficiency symptoms are dependent on a-tocopherol content, uptake and turnover, as well as susceptibility to and the degree of oxidative stress in a given tissue Furthermore, vitamin E concentrations depend on the presence of other antioxidants to maintain a-tocopherol in its unoxidized state (1) Importantly, vitamin E’s antioxidant function cannot be fulfilled by just any antioxidant Specifically, only a-tocopherol meets human vitamin E requirements (2) Plasma a-tocopherol is controlled by the hepatic a-tocopherol transfer protein (a-TTP) (3,4) and, in humans, a genetic defect in a-TTP results in severe vitamin E deficiency (5) a-TTP is necessary for the facilitated transfer of a-tocopherol from the liver to the plasma (6,7) This chapter describes vitamin E structures, antioxidant function, lipoprotein transport, and delivery to tissues Requirements, intake, human deficiency symptoms, and the role of vitamin E in the prevention of chronic disease are discussed HISTORY Vitamin E deficiency in rats fed rancid fat was first described in 1922 by Evans and Bishop (8) In 1936, Evans et al (9) isolated vitamin E from wheat germ and named this factor, ‘‘atocopherol;’’ a name derived from the Greek ‘‘tokos’’ (offspring) and ‘‘pherein’’ (to bear) with an ‘‘ol’’ to indicate that it was an alcohol Subsequently, b-tocopherol and g-tocopherol were isolated from vegetable oils (10), demonstrating that various forms of vitamin E exist, but that a-tocopherol is the most effective form in preventing vitamin E deficiency symptoms Today, it seems likely that the minor a-tocopherol contaminant in these vitamin E preparations provided their vitamin E biologic activity Specific vitamin E deficiency symptoms (e.g., fetal resorption, muscular dystrophy, and encephalomalacia) were observed in experimental animals fed vitamin E-deficient diets (11) The most popular, though most tedious and time-consuming, assay for the biologic activity of vitamin E is the fetal resorption assay (11) Here, vitamin E-depleted virgin female rats are mated with normal males After successful mating, various levels of single vitamin E forms are fed in several divided doses to the females, which are killed 20–21 days after mating The number of living, dead, and resorbed fetuses are counted and the percentage of live young determined Thus, the vitamin E biologic activity depends on the amount necessary to maintain the maximum number of live fetuses The results of this assay remain in use today to define the international units (IUs) of vitamin E activity Horwitt (12,13) attempted to induce vitamin E deficiency in men by feeding a diet low in vitamin E (2–4 mg a-tocopherol) for six years to volunteers at the Elgin State Hospital in Illinois After about two years, their serum vitamin E levels decreased into the deficient range Although their erythrocytes were more sensitive to peroxide-induced hemolysis, overt anemia did not develop The data from the Horwitt study was used in 2000 to set the recommended dietary allowance (RDA) for vitamin E (2) These latest RDAs are discussed later STRUCTURES AND ANTIOXIDANT CHEMISTRY STRUCTURE Vitamin E is the name for molecules with a-tocopherol antioxidant activity, including all tocol and tocotrienol derivatives (14) These antioxidants include four tocopherols and four ß 2006 by Taylor & Francis Group, LLC R1 Tocopherol HO CH3 R1 CH R2 CH3 R3 CH3 β γ CH3 H H CH3 δ H H CH3 CH3 CH3 α O R2 CH3 R3 R1 H CH3 H CH3 CH3 Tocotrienol HO CH3 O R2 R3 CH3 CH3 CH3 CH3 FIGURE 4.1 Vitamin E structures are shown The methyl groups on the chromanol head determine whether the molecule is a-, b- or g-, or d-, while the tail determines whether the molecule is a tocopherol or a tocotrienol tocotrienols, which have similar chromanol structures: trimethyl (a-), dimethyl (b- or g-), and monomethyl (d-) Tocotrienols differ from tocopherols in that they have an unsaturated tail However, in 2000, the Food and Nutrition Board (FNB) (2) defined a-tocopherol as the only form that meets human vitamin E requirements, because only a-tocopherol has been shown to reverse human vitamin E deficiency symptoms Unlike most other vitamins, chemically synthesized a-tocopherol is not identical to the naturally occurring form a-Tocopherol synthesized by condensation of trimethyl hydroquinone with racemic isophytol (15) contains eight stereoisomers, arising from the three chiral centers (2,40 , and 80 , Figure 4.1), and is designated all-rac-a-tocopherol (incorrectly called D,L-atocopherol) The FNB (2) defined that only 2R-a-tocopherol forms meet human vitamin E requirements Thus, only half of the stereoisomers in all-rac-a-tocopherol meet the vitamin E requirement Vitamin E supplements often contain a-tocopherol esters, including a-tocopheryl acetate, succinate, or nicotinate The ester form is not an antioxidant and thus has a long shelf life Vitamin E esters are readily hydrolyzed in the gut and are absorbed as a-tocopherol (16) NOMENCLATURE The FNB (2) definition of vitamin E has led to confusion about vitamin E units The vitamin E unit currently used on supplement labels was defined by the U.S Pharmacopoeia (17) The IU of vitamin E equals mg all-rac-a-tocopheryl acetate, 0.67 mg RRR-a-tocopherol, or 0.74 mg RRR-a-tocopheryl acetate However, the FNB (Table 6.1 in (2)) defined the vitamin E requirement in milligrams of 2R-a-tocopherol and provided conversion factors, such that all-rac is equal to 1=2 RRR-a-tocopherol To estimate the number of milligrams of 2R-a-tocopherol, IU all-rac-a-tocopherol (or its esters) must be multiplied by 0.45; whereas IU RRR-a-tocopherol (or its esters) is multiplied by 0.67 CHEMICAL PROPERTIES All vitamin E forms act as lipid-soluble chain-breaking antioxidants (18) Vitamin E is a potent peroxyl radical scavenger and especially protects PUFA within phospholipids of biological membranes and in plasma lipoproteins When lipid hydroperoxides are oxidized to peroxyl radicals (ROO.), these react 1,000 times faster with vitamin E (Vit E-OH) than with PUFA (RH) (19) The chromanol hydroxyl group reacts with a peroxyl radical to form a hydroperoxide and the chromanoxyl radical (Vit E-O.): ß 2006 by Taylor & Francis Group, LLC In the presence of vitamin E, ROO þ Vit E-OH ! ROOH þ Vit E-O In the absence of vitamin E, ROO þ RH ! ROOH þ R R þ O2 ! ROO In this way, vitamin E acts as a chain-breaking antioxidant, preventing further autooxidation of lipids ANTIOXIDANT NETWORK Vitamin E interacts with other antioxidants to remain in the unoxidized form The chromanoxyl radical Vit E-O reacts with vitamin C (or other reductants serving as hydrogen donors, AH), oxidizing the other antioxidant and reducing vitamin E Vit E-O þ AH ! Vit E-OH þ A Biologically important antioxidants that regenerate chromanols from chromanoxyl radicals include ascorbate (vitamin C) and thiols, especially glutathione Various metabolic processes can then reduce these other antioxidants This phenomenon has led to the idea of vitamin E recycling, where vitamin E is restored by other antioxidants Since the a-tocopheroxyl radical can readily be reduced to a-tocopherol, the amount of vitamin E that is recycled is likely much larger than the amount that is further oxidized OXIDIZED VITAMIN E The primary oxidation product of a-tocopherol is a-tocopheryl quinone, which can be conjugated to yield the glucuronide after reduction to the hydroquinone The glucuronide can be excreted into bile or further degraded in the kidneys to a-tocopheronic acid, which is excreted in the urine (20) Other oxidation products, including dimers and trimers, as well as other adducts have also been described (18) Specific vitamin E oxidation products have been generated in vitro (21,22) These include 4a,5-epoxy- and 7,8-epoxy-8a(hydroperoxy)tocopherones and their respective hydrolysis products, 2,3-epoxy-tocopherol quinone and 5,6-epoxy-a-tocopherol quinone However, these products are formed during in vitro oxidation; their importance in vivo is unknown PHYSIOLOGIC RELATIONSHIPS ABSORPTION The absorption of vitamin E from the intestinal lumen is dependent on processes necessary for fat digestion and uptake into enterocytes (23) Pancreatic esterases are required for release of free fatty acids from dietary triglycerides Esterases are also required for the hydrolytic cleavage of tocopheryl esters, present in dietary supplements Bile acids, monoglycerides, and free fatty acids are important components of mixed micelles Bile acids are required for the formation of mixed micelles and are essential for vitamin E absorption In the absence of both pancreatic and biliary secretions, only negligible amounts of vitamin E are absorbed Thus, human vitamin E deficiency occurs as a result of fat malabsorption (24) The bioavailability of vitamin E appears also to be dependent on the fat content of the meal Hayes et al (25) reported that plasma a-tocopherol concentrations doubled when a-tocopheryl acetate (100–200 mg=day) was provided as a microdispersion in milk, compared with providing the same dose in orange juice Vitamin E absorption is relatively poor when it is consumed without fat, as was observed when vitamin E pills were consumed without food (26) ß 2006 by Taylor & Francis Group, LLC It is well known that increasing dietary fat increases absorption of vitamin E supplements (26,27) Roodenberg et al (28) suggested that a 3% fat intake was sufficient for optimal vitamin E bioavailability However, they measured bioavailability as increased plasma a-tocopherol concentrations following one week of supplementation with 50 mg a-tocopherol in either 50 g of a low- or high-fat spread, such that hot meals contained either less than 6.5 g fat or less than 45 g fat Thus, dissolving the vitamin E in the spread may have allowed normal vitamin E absorption During fat absorption, enterocytes synthesize chylomicrons that contain triglycerides, free and esterified cholesterol, phospholipids, and apolipoproteins (especially apolipoprotein [apo] B48) In addition, fat-soluble vitamins, carotenoids, and other fat-soluble dietary components are incorporated into chylomicrons Chylomicrons are then secreted into the lymph The movement of vitamin E through the absorptive cells is not well understood; no intestinal TTPs have been described Even in healthy individuals, the efficiency of vitamin E absorption is low (0.8 mg=g) PATHOLOGY OF HUMAN VITAMIN E DEFICIENCY The primary manifestations of human vitamin E deficiency include spinocerebellar ataxia, skeletal myopathy, and pigmented retinopathy (24) Hypo- or a-reflexia is the earliest symptom observed By the end of the first decade of life, untreated patients with chronic cholestatic hepatobiliary disease have a combination of spinocerebellar ataxia, neuropathy, and opthalmoplegia The progression of neurologic symptoms appears to be dependent on the level of oxidative stress accompanying the vitamin E deficiency The large-caliber, myelinated axons in peripheral sensory nerves are the predominant target in vitamin E deficiency in humans (120) In deficient humans, diminished amplitudes of sensory nerve action potential are common, whereas delayed conduction velocity, an indicator of demyelination, is unusual Thus, axonal degeneration rather than demyelination is the primary sensory nerve abnormality However, motor nerve demyelination has also been reported (121) Axonal dystrophy has been observed in the posterior columns of the spinal cord and the dorsal and ventral spinocerebellar tracts (120) Specifically, swollen, dystrophic axons (spheroids) have been observed in the gracille and cuneate nuclei of the brain stem Lipofuscin accumulation has been observed in dorsal sensory neurons and peripheral Schwann cell cytoplasm Electromyographic studies show denervation injury of muscles in patients with advanced vitamin E deficiency Somatosensory-evoked potential testing has shown a central delay in sensory conduction, correlating with degeneration of the posterior columns of the spinal cord ASSESSMENT OF VITAMIN E STATUS The FNB determined that a plasma concentration of less than 12 mmol a-tocopherol=L was associated with an increased tendency for hemolysis (2) The usual plasma a-tocopherol concentration in normal subjects is approximately 20 mmol=L Although low serum or plasma a-tocopherol concentrations are indicative of vitamin E deficiency, measurement of plasma levels are insufficient for patients with various forms of lipid malabsorption Calculation of effective plasma a-tocopherol concentrations needs to take into account plasma lipid levels when lipids are high or low These are calculated by dividing the plasma a-tocopherol by the sum of plasma cholesterol and triglycerides (122) For example, Sokol et al (123) showed that plasma vitamin E concentrations were in the normal range in patients with vitamin E deficiency resulting from cholestatic liver disease because they had extraordinarily high circulating lipid levels Adipose tissue concentrations can also be used as an indicator of long-term vitamin E status (48,49,124) Any patient presenting with peripheral neuropathies or retinitis pigmentosa with unknown causes should be evaluated to assess if they are vitamin E-deficient The ataxia of Friedreich’s ataxia is so remarkably similar to that of AVED patients that plasma concentrations ß 2006 by Taylor & Francis Group, LLC of vitamin E in all patients with ataxia should definitely be measured or their genotypes defined (125) EFFICACY OF PHARMACOLOGICAL DOSES OF VITAMIN E Vitamin E has antioxidant benefits and is generally considered to be nontoxic even in relatively high doses (>1000 mg) (2) Moreover, several studies have reported that vitamin E is associated with decreased chronic disease risk The Women’s Health Study, a 10 year prevention trial in normal, healthy women, found that 600 IU vitamin E decreased cardiovascular mortality by 24% and in women over 65 by 49% (107) Antioxidant treatment with the combination of vitamins E and C slowed atherosclerotic progression in intimal thickness of coronary and carotid arteries in hypercholesterolemic (126) and in heart-transplant patients (127) Epidemiologic studies indicate a beneficial role of vitamin E supplements in decreasing risk of degenerative diseases, such as cardiovascular disease and atherosclerosis (128,129), cancer (130), and cataract formation (131) The Cache County Study reported that antioxidant use (vitamin E > 400 IU and vitamin C > 500 mg) was associated with reduced Alzheimer’s disease prevalence and incidence in the elderly (132) Regular vitamin E supplement use for ten years or more was associated with a lower risk of dying of amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease) (133) It is, therefore, not surprising that vitamin E supplements are taken daily by more than 35 million people in the United States (112) Another important area of investigation is the maintenance of immune function Meydani et al (134–136) have demonstrated in a series of trials that immune function is compromised in the elderly, and that vitamin E supplements can improve immune responses No clinical trial in healthy people has shown that any supplemental dose of vitamin E causes adverse effects, with the exception of an increased tendency to bleed (137) However, an antioxidant intervention trial suggested adverse vitamin E effects in patients taking antihyperlipidemic drug therapy The intervention study was a three year, double-blind trial of antioxidants (vitamins E and C, b-carotene, and selenium) or placebos in 160 subjects taking both simvastatin and niacin (138,139) Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that is widely used in the treatment of hypercholesterolemia The protective increase in HDL2 with simvastatin plus niacin was attenuated by concurrent therapy with antioxidants The average stenosis progressed by 3.9% with placebos, 1.8% with antioxidants ( p ¼ 0.16 for the comparison with the placebo group), and 0.7% with simvastatin–niacin plus antioxidants ( p ¼ 0.004) and regressed by 0.4% with simvastatin–niacin alone ( p < 0.001) (139) The other study reporting adverse vitamin E effects was the Women’s Angiographic Vitamin and Estrogen (WAVE) Trial, a randomized, double-blind trial of 423 postmenopausal women with at least one coronary stenosis at baseline coronary angiography In postmenopausal women on hormone replacement therapy, all-cause mortality was increased in women assigned to antioxidant vitamins compared with placebo (HR, 2.8; 95% CI, 1.1–7.2; p ¼ 0.047) (140) Finally, the HopeToo trial suggested that patients at high risk for coronary heart disease taking vitamin E were at increased risk of leftventricular dysfunction, but no specific mechanism was proposed (141) One possibility for adverse vitamin E effects is vitamin E-dependent alterations in xenobiotic metabolism (142) Studies in mice showed that hepatic cytochrome P450 3a (analogous to CYP3A in humans) protein concentrations were correlated with hepatic a-tocopherol concentrations (89) CYP3A4 is responsible for the metabolism of more than 50% of prescription drugs, including statins (143) Both simvastatin (143) and estrogen (144) are metabolized by CYP3A4, lending support to the hypothesis that a-tocopherol in pharmacologic doses stimulates drug metabolism, potentially decreasing beneficial drug concentrations (145) These data suggest that a-tocopherol could stimulate xenobiotic metabolism, but clearly further studies are needed ß 2006 by Taylor & Francis Group, LLC CONCLUSION Vitamin E is clearly a lipid-soluble antioxidant There remains huge enthusiasm for the concept that a-tocopherol has some specific molecular role of regulation of cellular functions, but efforts to document such a role along with its physiological significance have been limited Numerous investigators have used gene chip technology to demonstrate specific vitamin E or a-tocopherol effects (146–152), but no consistent findings have emerged Clinical trials to demonstrate that vitamin E supplements reverse chronic diseases have largely been disappointing However, disease prevention shows more promise, but it is much more difficult to carry out the very long trials necessary to show benefit (107,153) Overall, it is important to recognize that vitamin E is a required nutrient, that its concentrations are regulated in vivo, that it has antioxidant benefits, and its role may be to prevent oxidative damage caused during lipid peroxidation Thus, those people consuming diets that are low in a-tocopherol may be at risk for increased oxidative damage because they 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