Critical Reviews in Clinical Laboratory Sciences, 45(5):417450 (2008) Copyright C 2008 Informa Healthcare USA, Inc.

ISSN: 1040-8363 print / 1549-781X online DOI: 10.1080/10408360802118625

VITAMIN E IN HUMAN HEALTH AND DISEASE

Michael W. Clarke and John R. Burnett 2 School of Medicine and Pharmacology, University of Western Australia, Crawley WA 6009, Australia and Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA 6847, Royal Perth Hospital, Wellington Street Campus, Perth, Australia Kevin D. Croft 2 School of Medicine and Pharmacology, University of Western Australia, Crawley WA 6009, Australia
Referee Dr. Jean-Marc Zingg, Vascular Biology Laboratory, JM USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA, USA

Vitamin E in nature is comprised of a family of tocopherols and tocotrienols. The most studied of these is -tocopherol (-TOH), because this form is retained within the body, and vitamin E deciency is corrected with this supplement. -TOH is a lipid-soluble antioxidant required for the preservation of cell membranes, and it potentially acts as a defense against oxidative stress. Many studies have investigated the metabolism, transport, and efcacy -TOH in the prevention of sequelae associated with cardiovascular disease (CVD). Supplementation with vitamin E is considered to provide health benets against CVD through its antioxidant activity, the prevention of lipoprotein oxidation, and the inhibition of platelet aggregation. However, the results from large prospective, randomized, placebocontrolled clinical trials with -TOH have been largely negative. A recent meta-analysis suggests that -TOH supplements may actually increase all-cause mortality; however, the mechanism for this increased risk is unknown. In vitro studies performed in human cell cultures and animal models suggest that vitamin E might increase the hepatic production of cytochrome P450s and MDR1. Induction of CYP3A4 or MDR1 by vitamin E could potentially lower the efcacy of any drug metabolized by CYP3A4 or MDR1. Other possibilities include an adverse effect of -TOH on blood pressure in high-risk populations. Because of the wide popularity and use of vitamin E supplements, further research into potential adverse effects is clearly warranted. Keywords Antioxidant, atherosclerosis, CYP3A4, erythrocyte, metabolism, mortality, oxidative stress, platelet, tocopherol, vitamin E.

Abbreviations 4-HNE, 4-hydroxynonenal; -CEHC, 2,5,7,8-tetramethyl-2 (2 -carboxyethy)-6-hydroxychroman, metabolite of -TOH; -TOH, alpha tocopherol; -TQH2 ,
Address correspondence to Dr. John R. Burnett, Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, Wellington Street, GPO Box X2213, Perth WA 6847, Australia. E-mail: john.burnett@health.wa.gov.au

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-TOH hydroquinone; -TTP, -TOH transfer protein; -CEHC, 2,7,8-trimethyl-2-(carboxyethyl)-6-hydroxychroman (metabolite of -TOH); -TOH, gamma tocopherol; YP, cylochrome P450, AAPH, 2,2 -azobis(2)-amidinopropane (a free radical generator); ABCA1, ATP-binding cassette transporter A1; apoE/ mouse, apolipoprotein E double knock out mouse; ASAP, Antioxidant Supplementation in Atherosclerosis Prevention; ATBC, -TOH, beta-Carotene Prevention Study; AVED, ataxia with vitamin E deciency; CHAOS, Cambridge Heart Antioxidant Study; CHD, coronary heart disease; COX-2, cyclooxygenase-2; Co Q10 , ubiquinone-10; CoQ10 H2 , ubiquinol-10, reduced form of coenzyme Q; CVD, cardiovascular disease; CYP3a11, murine equivalent to human CYP3A4; CYP3A4, cytochrome P450 3A4; d6 -RRR--TOH, deuterium labelled natural alpha tocopherol; ECD, electrochemical detection; FHBL, familial hypobetalipoproteinemia; FIVE, familial isolated vitamin E deciency; GC/MS, gas chromatography mass spectrometry; GISSI, Gruppo Italiano per lo Studio della Sopravvivenza nellInfarto miocardico; HDL, high-density lipoprotein; HepG2, human hepatoma cell line; HPLC, high-performance liquid chromatography; HOPE, Heart Outcomes Prevention Evaluation Study; HTGL, hepatic triglyceride lipase; LDL, low-density lipoprotein; LOOH, lipid hydroperoxides; LPL, lipoprotein lipase; MDMs, monocyte-derived macrophages; MDR1, multidrug resistant protein 1; MTP, microsomal triglyceride transfer protein; NHBLI, National Heart, Blood and Lung Institute; NO2 , nitrogen dioxide; PGE2 , prostaglandin E2 ; PXR, pregnane X receptor; RNOS, reactive nitrogen-oxide species; RRR--TOH, d--TOH, natural alpha tocopherol; RXR, retinoic acid receptor; SPACE, Secondary Prevention with Antioxidants of Cardiovascular Disease in Endstage Renal Disease; SR-BI, scavenger receptor class B type I; SRR--TOH, dl--TOH, synthetic alpha tocopherol; TAP, tocopherolassociated protein; TBP, tocopherol-binding protein; TMP, tocopherol-mediated peroxidation; TO, tocopherol radical; TOH, tocopherol; VLDL, very low density lipoprotein; WHHL, Watanabe heritable hyperlipidemic.

I. INTRODUCTION In 1922, Evans and Bishop described factor X, a nutrient found in vegetable oil that cured sterility in rats maintained on a lard diet.1 Since then, research into the metabolism and nutritional requirements of vitamin E has led to a substantial body of knowledge about its role in human health and disease. High quantities of vitamin E in the US diet are found in cereals, oils (including soy), and salad dressings.2 Natural vitamin E is comprised of four tocopherols (TOH), namely, (, , , ), and four tocotrienols (, , , ). The most studied is -TOH. The name TOH comes from the Greek tocos (childbirth), phero (to bear), and ol (alcohol).3 These eight structurally similar compounds, together with their biological activity compared to -TOH are shown in Table 1.4 The most recognized role for -TOH is as a lipidsoluble antioxidant required for the preservation of cell membranes, where it reacts quickly with peroxyl radicals to preserve polyunsaturated fatty acids.5 More recently, -TOH has been implicated in the activation of a number of genes.6 Primary vitamin E deciency is generally found only in premature and low-birth-weight infants. Secondary causes include fat malabsorption syndromes (e.g., cystic brosis, chronic liver disease, abetalipoproteinemia, and intestinal resection) and some hematological disorders

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TABLE 1 The Structures and Biological Activities of the Eight Naturally Occurring Forms of Vitamin E Common Name d--TOH Activity based on rat assay 1.49 Compared to RRR--TOH 100%

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Structure

d--TOH

0.75

50%

d- -TOH

0.15

10%

d--TOH

0.05

3%

d--tocotrienol

0.75

50%

d--tocotrienol

0.08

5%

d- -tocotrienol

Not known

d--tocotrienol

Not known

Adapted from Ref. 4.

(e.g., -thalassemia major, sickle-cell anemia, and glucose-6-phosphate dehydrogenase deciency).3 Patients with vitamin E deciency have abnormal erythrocyte membrane morphology due to oxidative stress, and the characteristic acanthocytosis is associated with a reduction in red cell half life.7 Long-term deciency in vitamin E can lead to neurological abnormalities, including ataxia, hyporeexia, blindness, and dementia.8

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The effect of -TOH on the oxidation of lipoproteins and its potential role as an antiatherogenic supplement have received considerable attention in the last decade. Moreover, a number of studies have described a role for -TOH beyond its antioxidant function. -TOH has been shown to inhibit smooth-muscle cell proliferation,9 endothelial dysfunction10 and platelet aggregation11 by a protein kinase-C dependant mechanism; it has also been found to inhibit monocyte adhesion to endothelial cells12 and macrophage-mediated lipid peroxidation in vitro.13 These diverse functions of -TOH and the potential pro-oxidant effects observed in some studies14 may account for the paradoxical results observed with human clinical trials on the prevention of recurrent atherosclerosis.15 The dietary requirement for vitamin E is often ascribed to the intake of polyunsaturated fatty acids within the diet.16 However, this generalization may not be appropriate in all situations as vitamin E may act as a pro-oxidant in smokers who consume a diet high in polyunsaturated fatty acids.17 Because of conicting data about vitamin E supplementation, it is not surprising to see disagreement about the recommended daily allowance for -TOH in humans and to observe that -TOH has not yet been included.1820 A recommended dietary intake of 15 mg -TOH/day21 which would suggest that all dietary needs can be met from -TOH.19 However, because of recent adverse publicity associated with high-dose vitamin E supplementation,2224 it is prudent to return to fundamental questions relating to the requirements for these compounds and to re-evaluate the supposition that high-dose vitamin E supplements in the form of -TOH are safe. The purpose of this article is to (1) describe vitamin E transport in humans and animal models, (2) examine the potential role of vitamin E in the oxidation of lipoproteins and treatment of atherosclerosis, and (3) explore the potential for vitamin E isoforms to alter the metabolism of clinically important drugs. II. VITAMIN E TRANSPORT IN HUMANS AND ANIMAL MODELS A. Structure and Properties of Vitamin E Isomers The isoforms of vitamin E differ in the degree and site of methylation in the chromanol ring and the conguration of the methyl groups in the phytyl-side chains. The degree of methylation in the chromanol ring (Table 1) determines the antioxidant activity of each form of vitamin E, with -TOH having twice the antioxidant activity of -TOH.25 The biological activities of the different forms of vitamin E are expressed in international units per milligram (IU/mg). The relative activities are based on an assay using a biological system in which the amount of natural vitamin E required to prevent fetal resorption in rats decient in vitamin E is compared to

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TABLE 2 Comparison of -and -TOH Concentrations in Different Tissues in Humans and Rodents Humans -TOH Plasma (mol/l) Liver (nmol/g) Adipose (nmol/g) Muscle (nmol/g) Skin (nmol/g) 27 176 80 107 180 89 -TOH 1520 20 440 279 155 163 127 74 Rats and Mice -TOH 1.31.7 4.55.3 29.5 4.1 3.65.6 3.0 2.8 -TOH 7.213.0 30.033.4 79.8 6.9 15.122.7 8.9 3.0

Taken from Ref.32. Subject received 75 mg D RRR--TOH for seven days before sampling.168 3

RRR--TOH(d--tocopherol, natural alpha tocopherol).26 However, using this data to compare the biological activities of the different forms of natural vitamin E can make interpretation difcult, because the human requirements and tissue concentrations of the two most important natural forms of vitamin E, namely - and -TOH, are markedly different between humans and rats (Table 2). -TOH, the major form of vitamin E in humans, is the most lipid-soluble antioxidant and the most abundant TOH in human tissues.5,25 Synthetic vitamin E (SRR--TOH, dl--TOH) is an equal mixture of eight stereoisomers of -TOH.4 All isomers have an identical chromanol group and hence equal antioxidant activity.27 Synthetic -TOH contains only 12.5% pure RRR-TOH and equal amounts of the other forms. However, only the four 2R-TOH isoforms are efciently retained in the body.27 The biological activities for each isomer are known in the rat but not in humans.28 Further studies in humans are required to establish the biological activity of the different stereoisomers of synthetic vitamin E. B. Intestinal Absorption of Vitamin E and Postprandial Metabolism The intestinal absorption of vitamin E requires the intake and digestion of dietary fat, which is enhanced by the production of bile acids from the liver.29 Dietary vitamin E bound in micelles formed within the intestine is absorbed, along with triglycerides and cholesterol, by a passive process into enterocytes. Chylomicrons containing vitamin E are assembled and secreted into the lymph.25 Inhibition of the scavenger receptor class B type I (SR-BI) blocks up to 80% of -TOH uptake, and suggests a role for SR-BI in intestinal TOH transport.30 In the circulation, chylomicrons interact with lipoprotein lipase (LPL) to release non-esteried fatty acids and triglycerides (Figure 1). It is thought that TOHs may be delivered to tissues such as muscle, adipocytes, and the brain during this process as the transfer of TOH to broblasts was observed in vitro

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FIGURE 1 TOH transport in lipoproteins and cells. Dietary vitamin E is absorbed through the intestine and then the TOHs are transported within the peripheral blood bound to lipoproteins and cells such as platelets and red blood cells. The delivery of TOHs to tissues occurs via lipoprotein lipase-mediated delipidation of chylomicrons and delivery from low-density lipoprotein (LDL) and HDL. There is preferential incorporation of RRR--TOH into very low-density lipoprotein (VLDL) mediated by -TTP. The HDL delivery of TOHs is probably important in individuals with low VLDL and LDL cholesterol. There is a ready exchange between erythrocytes and HDL, but it is unknown how platelets acquire their TOHs. Adapted From Ref. 25.

in the presence of bovine LPL.31 These tissues receive most of their lipids during LPL-mediated delipidation of lipoproteins. As chylomicron remnants are formed, they can then exchange surface components, including TOHs, with high-density lipoprotein (HDL). HDL can then transfer TOHs to other lipoproteins in the circulation.25 This pathway is particularly important for individuals with abetalipoproteinemia and homozygous familial hypobetalipoproteinema because of absent or extremely low plasma levels of apolipoprotein B-containing lipoproteins. Supplementation of these individuals with high-dose vitamin E (100150 mg/kg per day) can normalize adipose tissue TOH concentrations via HDL transport of TOHs to tissues.25 C. Hepatic Metabolism of Vitamin E The understanding of the absorption and transport of vitamin E has been greatly facilitated by the use of deuterated TOHs to assess the distribution of

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each form among lipoproteins.25,32 Studies in humans have demonstrated that -TOH is preferentially incorporated into very low-density lipoprotein (VLDL) particles, and -TOH is excreted in the bile33 Similarly, in rats, monkeys, and humans, the naturally occurring RRR stereoisomer of -TOH is also preferentially incorporated into VLDL.34 One report studied patients with and without inherited disorders of lipoprotein metabolism to elucidate the steps involved in the discrimination of the different vitamin E isoforms.34 A subject with homozygous familial hypobetalipoproteinemia (FHBL) and abnormal apolipoprotein B-100 production showed preferential enrichment of his VLDL fraction with d6 -RRR--TOH (deuterium-labeled natural -TOH), 24 hours after supplementation with d6 -RRR--TOH acetate. Of interest, this patient had normal HDL d6 -RRR-TOH concentrations, did not have symptoms of vitamin E deciency, and has not required vitamin E supplementation. Taken together, this complex study showed the importance of chylomicron and HDL metabolism for TOH distribution to lipoproteins for patients with impaired transport of TOHs due to rare inherited disorders of lipid and lipoprotein metabolism, and demonstrated that VLDL particles, even abnormal ones, are preferentially enriched with RRR--TOH.34 Our own study examined oxidative stress and TOH metabolism in individuals who were heterozygous for FHBL and concluded that supplementation with vitamin E was not required in this group, because oxidative stress was not evident, nor did they exhibit any clinical signs of TOH deciency.35 The importance of chylomicron delivery of TOHs to peripheral tissues has been highlighted recently in a murine model.36 Genetically engineered mice that specically lack microsomal triglyceride transfer protein (MTP) in the liver were fed deuterated -TOH; the majority of TOH was replaced in peripheral tissues within one month, despite their inability to secrete VLDL.36 Recent in vitro studies using human broblasts and murine RAW264 macrophages showed that the export of -TOH to HDL was mediated, at least in part, by the ATP-binding cassette transporter A1 (ABCA1).37 Moreover, ABCA1 was directly involved in the translocation of -TOH to apoproteins .37 (Figure 2). Taken together, these studies show that vitamin E can be metabolized and delivered to tissues by a variety of routes and that low serum vitamin E concentrations do not necessarily equate to vitamin E deciency. Vitamin E deciency may also be organ specic and depend on the environment in question, with serum tocopherol concentrations not necessarily indicative of those in peripheral tissues. D. TOH Transfer Proteins Vitamin E distribution and the role of TOH regulatory proteins has been the topic of a recent review.27 The -TOH transfer protein (-TTP) is a 32 kDa cytosolic lipid-binding protein that is found in a number of tissues but mainly

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FIGURE 2 Hepatocyte vitamin E transport and metabolism. Vitamin E forms enter hepatocytes from diet via chylomicrons or from endogenous lipoproteins LDL and HDL. When internalized, -TTP will selectively incorporate RRR--TOH into VLDL for export in preference to the other forms of vitamin E. The traditional role for vitamin E is depicted with the vitamin associated with the cell membrane protecting poly-unsaturated fatty acids from oxidation. The TOHs have been shown to bind to two intracellular proteins, namely, TBP and TAP, but the precise role for these has yet to be described. The proposed activation of PXR by vitamin E leads to the up-regulation of a number of genes including, CYP3A4 and MDR1, which, along with CYP4F2, metabolize the different forms of vitamin E and allow them to be excreted from the cell. Membrane transporters SR-BI and ABCA1 have also been implicated in vitamin E metabolism. Adapted from Refs.25,37,147,169 LDL, low density lipoprotein; HDL, high density lipoprotein; VLDL, very low density lipoprotein; VE, vitamin E forms; -TTP, -TOH transfer protein; TAP, tocopherol associated protein; TBP, tocopherol binding protein; PXR, pregnane X receptor; RXR, retinoic acid receptor; CYP3A4, cytochrome P450 3A4; MDR1, multidrug resistant protein 1 (or p-glycoprotein); CYP4F2, cytochrome P450 F2; SR-BI, scavenger receptor BI; ABCA1, ATP-binding cassette transporter A1; CEHCs, vitamin E metabolites (see text).

the liver,38 with some expression in rat brain, spleen, lung, and kidney, and in some regions in human brain.27 Previous studies in rats39 and humans33,34,40 showed that RRR--TOH was retained within lipoproteins. -TTP has a high afnity for RRR--TOH compared to other TOHs (Table 3), which may, in part, account for the biological potency of each form of vitamin E.41 It is thought that -TTP directly facilitates the incorporation of -TOH into VLDL.25 However, this theory remains controversial.27 In rat hepatoma cells expressing -TTP, more -TOH was secreted into the medium.42 When the cells were incubated with brefeldin A, an inhibitor of VLDL secretion, the export of -TOH was not affected, which suggested that the two processes are distinct.27 It has been postulated that -TTP is involved in retaining and recycling TOHs within hepatocytes by localizing within intracellular endosomes

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TABLE 3 Relative Afnities of Various TOH Analogs for -TTP Competitors -TOH -TOH -TOH -TOH -TOH acetate -TOH quinone SRR--TOH -Tocotrienol Trolox Taken from Ref. 41.

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Relative afnity (%) 100 38.1 9.3 8.9 0.6 1.6 0.3 1.7 0.1 1.5 0.1 10.5 0.4 12.4 2.3 9.1 1.2

and binding to TOHs. It has also been proposed that the efux of -TOH to the plasma membrane is facilitated by -TTP and that free -TOH may then be taken up by VLDL particles or other lipoproteins.27 A recent study has shown that the transporter ABCA1 may also be involved in facilitating -TTP-mediated secretion of TOHs from hepatocytes.43 However, further studies will be required to demonstrate whether this process occurs in human tissues. There are other proteins, called tocopherol associated proteins (TAP), that specically bind to TOHs. hTAP1, a 46 kDa protein, has recently been described in humans;44 it has sequence homology similar to -TTP and is found in the liver, prostate, and brain. However, a specic function for this protein has yet to be found.27 Two other TAP proteins, namely hTAP2 and hTAP3, are similar to hTAP1, and are involved in tocopherol-mediated cell signaling pathways.45 A recent report of a TAP (also known as supernatant protein factor) knockout mouse suggests a role for TAP in hepatic cholesterol synthesis.46 The relationship of this to tocopherol metabolism remains unclear. TOH-binding protein (TBP), a 14.2-kDa cystolic protein found in rat liver and heart, stimulates the transfer of -TOH from liposomes to mitochondria in vitro.47 Although this protein may be involved in intracellular trafcking of -TOH, a direct role for this protein has not been described.27 E. TOH Transfer Protein Deciency Patients with familial isolated vitamin E deciency (FIVE), also known as ataxia with vitamin E deciency (AVED), have been studied to examine the role of -TTP in TOH metabolism. First described in 1981,48 this rare autosomal recessive neurogenerative disease49 is often misdiagnosed as Friedreichs ataxia, but it can be distinguished by measuring -TOH concentrations (low in FIVE) or by molecular analysis of the frataxin gene on chromosome 9.50 Patients with severe vitamin E deciency develop hyporeexia, ataxia, limited upward gaze, muscle weakness, and constriction of their

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visual elds. Long-term vitamin E deciency can lead to blindness, dementia, and cardiac arrhythmias.51 Untreated patients with FIVE have plasma -TOH concentrations 1% of normal.27 These patients do not produce VLDL enriched with -TOH and therefore must rely on chylomicron metabolism to distribute dietary -TOH to tissues.27 However, they still require supplementation with 800 mg RRR -TOH twice daily to maintain plasma concentrations within the reference interval.49 Mice decient in -TTP have provided useful models to study disease processes where oxidative stress is thought to play a role.52,53 Tereswa et al. examined atherosclerotic lesion development in ApoE knockout mice (ApoE / ) that also had vitamin E deciency due to disruption of the -TTP gene.53 Vitamin E deciency associated with -TTP deciency promoted lesion formation in the proximal aorta in ApoE / mice. Moreover, -TOH concentrations were reduced by >85%, and the generation of F2 -isoprostanes was increased, indicating that lipid peroxidation was not suppressed in this region of the aorta. Whether this effect is evidence of an atheroprotective effect has been challenged because the effect of vitamin E supplementation was modest, and the -TOH concentrations were 100 times greater in the group with normal -TTP function.54 Taken together, these ndings53 are consistent with other work on ApoE / mice, which showed that dietary supplementation with vitamin E (2000 IU/kg chow) signicantly reduced F2 isoprostane concentrations in urine, plasma, and vascular tissue.55 However, another study of ApoE / mice showed a relatively small positive effect with vitamin E supplementation (0.2% wt/wt in diet).56 Furthermore, combining chow with vitamin E (0.05% wt/wt) with -carotene (0.05% wt/wt) showed no benet in this mouse model.57 Thus, the potential role of vitamin E in the prevention of atherosclerosis is controversial. III. VITAMIN E AND OXIDATIVE STRESS A. Vitamin E and Lipoprotein Oxidation Atherosclerosis has been described as a disease involving a number of different processes including an inammatory component58 and oxidation modication of lipoproteins.59 However, these two processes are not mutually exclusive. Vitamin E is thought to play a role both in regulating aspects of the immune system and in antioxidant defense of lipoproteins.60 No single oxidant responsible for LDL oxidation has been identied, and it is likely that many factors, including transition metals, 15-lipoxgenase, myeloperoxidasederived oxidants, and reactive nitrogen species, are involved.54 The antioxidant properties of vitamin E, which were studied in regard to their role in supplementation in humans, may provide an explanation for the paradoxical results obtained from clinical trials.61,62 Although -TOH is important, it is not the only determinant in the resistance of LDL to oxidation.63

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Ubiquinol-10 (CoQ10 H2 ), the reduced form of coenzyme Q, is a well-studied co-antioxidant for -TOH60 and is an effective lipid-soluble antioxidant at physiological concentrations.64 Ubiquinone-10 (CoQ10 ) is reduced during intestinal absorption to CoQ10 H2 .56 The disappearance of antioxidants from LDL isolated from healthy subjects has been examined under different oxidizing conditions.65 An initial lag period (because of contaminating ascorbic acid) was followed by detection of lipid-peroxidation products, which coincided with the consumption of CoQ10 H2 . This occurred even though 80% of the endogenous carotenoids and 95% of the endogenous -TOH were still present.65 The relative roles of L-ascorbic acid (vitamin C), -TOH, and CoQ10 H2 in protecting LDL from oxidation in vitro have been described.66 When concentrations of co-antioxidants ascorbate and CoQ10 H2 are low, the -TOH radical (TO) can act to transfer, rather than trap, electrons. Also, CoQ10 H2 can act as a better antioxidant than -TOH in LDL because the semiquinone radical formed can leave the lipoprotein particle rather than promote further peroxidation.66 The rate of radical stress is important in determining the amounts co-antioxidants available to interact with -TOH and this in turn inuences the role of -TOH within the LDL particle. LDL from healthy subjects has been examined before and after supplementation with RRR--TOH (1 g/day) and/or CoQ10 (100 mg/day) for a total of ve days.67 Native LDL contained 8.5 2 molecules of TOH and 0.5 to 0.8 CoQ10 H2 molecules per particle. Incubation of LDL in Hams F10 medium containing transition metal ions depleted LDL of -TOH, and this depletion increased in the presence of monocyte-derived macrophages (MDMs). When LDL was incubated in vitro with -TOH, the concentrations of -TOH increased 6- to 7-fold in the LDL particles. Furthermore, these particles were more easily oxidized than native LDL both in the presence and absence of monocyte MDMs, which suggests a pro-oxidant role for -TOH.67 In supplemented subjects, LDL -TOH concentrations increased 2- to 3-fold and CoQ10 H2 concentrations increased 3- to 4-fold. LDL was more susceptible to oxidation in subjects receiving only -TOH and more resistant in those receiving CoQ10 . Of interest, those receiving co-supplementation had LDL that was more resistant to oxidation than native LDL or LDL incubated with -TOH. Taken together, these results were explained using the model of TOH-mediated peroxidation (TMP), with CoQ10 H2 inhibiting TMP and protecting LDL from oxidation.67 A detailed review of TMP has implications for using -TOH for the prevention of atherosclerosis.68 -TOH hydroquinone (-TQH2 ), derived from TOH quinone, has been shown to effectively inhibit oxidation of -TOH, and CoQ10 H2 , along with surface and core lipids, by a number of oxidants in vitro.69 It has been described as the most efcient lipophilic antioxidant, because it effectively regenerates TO to -TOH and decreases consumption of CoQ10 H2 . These

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investigators suggest that -TQH2 may be a potential therapeutic agent,69 but this awaits conrmation in clinical trials. Atherosclerosis development and lipid peroxidation products have been examined in ApoE / mice on a high-fat diet supplemented with CoQ10 and/or RRR--TOH.56 Twenty-four weeks of supplementation with vitamin E and CoQ10 increased plasma concentrations of vitamin E 3-fold and CoQ10 7-fold; the majority were located in VLDL. Aortic concentrations of CoQ10 concentrations increased >10-fold, and vitamin E concentrations increased signicantly in all tissues measured. Supplementation with vitamin E and CoQ10 decreased lesion size at the sites examined (aortic root 30%, aortic arch 50%, and descending thoracic aorta 80%). Vitamin E alone decreased lesion size only in the aortic root. The inhibition of lesion size after combined supplementation was associated with a decrease in aortic concentrations of lipid hydroperoxides (LOOH). However, supplementation with vitamin E alone did not lower the aortic concentrations of LOOH. Taken together, these results were consistent with the theory of TMP and that combined supplementation with both vitamin E and CoQ10 may be more antiatherogenic than vitamin E alone.56 Long-term antioxidant supplementation (12 months) with -TOH, probucol, and CoQ10 in Watanabe heritable hyperlipidemic (WHHL) rabbits with doses approximating therapeutic doses in humans (300 mg SRR--TOH and CoQ10 , 1 g probucol) had no effect on aortic lesion size but decreased copper-induced ex vivo lipid peroxidation.70 Probucol also decreased lipid peroxidation by copper, but CoQ10 had no effect. However, vitamin E failed to decrease the amount of lipid-standardized LOOH and corresponding hydroxides.70 This nding is also consistent with the model of TMP in the absence of sufcient co-antioxidants.68 The concentrations of oxidized lipids, -TOH, and its oxidation products in human lesions were determined during different stages in atherosclerotic lesion development.71 Oxidation of -TOH occurred early in the disease and exceeded lipid peroxidation, and lesions were not depleted of -TOH. The products of -TOH oxidation, tocopherylquinone, and tocopheryl epoxides, were <20% of the total TOHs, with tocopherylquinone the major product formed. Using an in vitro assay, Terentis et al. determined that tocopherylquinone is generated in the presence of two electron (nonradical) oxidants such as hypochlorite and peroxynitrite. They also found that the oxidation products formed were similar to those formed when LDL is oxidized in the presence of nitrite.71 Because oxidized lipids co-exist with -TOH in atherosclerotic lesions,72 this study calls into question the rationale for using antioxidants such as -TOH to prevent atherosclerosis.71 More recently a study of the effects of vitamin E supplementation on atherosclerosis in mice with vitamin E deciency showed that any benet was small and dependent upon the degree of pre-existing deciency.73 The lake of effect on aortic F2 -isoprostane and oxidized lipid formation suggested

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that vitamin E supplementation may not reduce oxidative stress within the vessel wall.73 Moreover, supplementing healthy individuals with -TOH had no effect on lipid peroxidation as measured by urinary concentrations of 4-hydroxynonenal (4-HNE) or F2 -isoprostanes; this challenges the rationale for supplementing healthy subjects with -TOH to inhibit oxidative stress.74 Recent human intervention studies examining the antioxidant potential of RRR--TOH administration with 1,200 IU/d for two years75 and 1,600 IU/d for 16 weeks76 showed a signicant reduction in F2 -isoprostane production; this is consistent with the concept that high doses of RRR--TOH are required to reduce oxidative stress in humans. However, another recent study in subjects with essential hypertension demonstrated a reduction in isoprostane formation and blood pressure with a lower dose of -TOH (400 IU/d) but in combination with vitamin C (100 mg/d).77 Taken together, these studies suggest that RRR--TOH supplements may reduce oxidative stress in human populations with high oxidative stress, but the effect may occur only with higher doses or in combination with co-antioxidants. IV. TOH METABOLITES A. -TOH Because strong epidemiological evidence supports a role for vitamin E in heart disease prevention,78,79 the measurement and study of products derived from -TOH oxidation have received considerable attention. Urinary metabolites of vitamin E have been studied since the 1950s.80 The rst published data describing urinary metabolites of -TOH from rabbits and humans appeared in 1956.81 Known as the Simon metabolites, both tocopheronic acid and the subsequent tocopheronolactone were detected in urine after high-dose supplementation with -TOH.81 These metabolites were generally thought to arise following the antioxidant action of vitamin E in vivo with the chroman ring being opened after oxidation.80 This explanation has been challenged with the suggestion that tocopheronolactone is produced from 2,5,7,8-tetramethyl-2 (2 -carboxyethy)-6-hydroxychroman (-CEHC, a metabolite of -TOH) in the presence of oxygen from sample handling.82 In this study, subjects receiving greater than 50 mg per day of -TOH reached a plasma threshold of 79 mol -TOH/g total lipid and had detectable -CEHC concentrations, determined using a highperformance liquid chromatography (HPLC) method with electrochemical detection (ECD). The concentrations of -CEHC (mol/24 h) correlated well with -TOH concentrations (mol/g total plasma lipid) when the threshold was reached. It was concluded that -TOH can undergo oxidation without prior oxidation and that -CEHC is therefore the major urinary metabolite of -TOH produced by healthy humans (Figure 3). Furthermore, as plasma concentrations of -TOH can be raised only 3-fold,83

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FIGURE 3 Pathways leading to the urinary excretion of -TOH metabolites. The commonly accepted pathway A vs. the proposed pathway B. Taken from Ref. 82. RRR--TOH, natural -TOH; -CEHC, 2,5,7,8tetramethyl-2 (2 -carboxyethy)-6-hydroxychroman (metabolite of -TOH).

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regardless of how much -TOH is given, the investigators proposed that urinary concentrations of -CEHC are a marker of adequate vitamin E intake. They raised the possibility that high concentrations of -TOH may indeed be harmful and that high urinary concentrations of -CEHC may reect this.82 They also acknowledged that the formation of Simon metabolites may occur in vivo, but probably only during episodes of oxidative stress.82 In contrast, other investigators have shown that both tocopheronolactone and -CEHC were present in the urine of subjects not taking -TOH supplements.80,84,85 These later studies employed more sensitive gas chromatography/mass spectrometry (GC/MS) methods, which could explain the different ndings.80 The use of sensitive methods to measure both serum and urine metabolites of vitamin E, with emphasis on correct sample handling and processing, should lead to a greater understanding of the signicance of these metabolites in both supplemented and unsupplemented individuals. B. -TOH and Its Major Metabolite -CEHC -TTP is the main regulator of -TOH incorporation into lipoproteins, whereas it plays only a small part in -TOH metabolism. Instead -TOH is metabolized to 2,7,8-trimethyl-2- (-carboxyethyl)-6-hydroxychroman ( CEHC), via a cytochrome P450 enzyme in the liver,86 which is then excreted in the urine .87 (Figure 2). There has been considerable recent interest in the role of -TOH in human health, and it has been the topic of a recent review.32 -TOH comprises around 70% of the total TOH dietary intake in the US population. It is found in soybeans, corn, and walnuts and other nuts, and oils derived from soy, corn, and sesame are rich in -TOH.32 There has been greater interest in the role of -TOH versus -TOH in human nutrition. The concentrations of -TOH are greater than -TOH in tissues, and the biological activity of -TOH is 10% of -TOH, as determined by the rat fetal resorption assay.88 The difference in activity relates in part to different plasma and tissue concentrations of the two TOHs in humans and rodents (Table 2). Tissue concentrations of -TOH are higher in humans than in rodents, particularly in skin and muscle, and these levels probably reect the different ways in which each species metabolizes the two forms of TOH.32 In a case-control study, -TOH concentrations in patients with coronary heart disease (CHD) were lower than in healthy, age-matched controls, whereas the concentrations of -TOH were not different between the two groups.89 Similarly, -TOH concentrations were signicantly lower in CHD patients than in controls, with no corresponding decrease in plasma -TOH or CoQ10 H2 concentrations in the patient group.90 There is some evidence that -TOH is superior to -TOH at detoxifying nitrogen dioxide (NO2 ) and that it is more effective at inhibiting

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peroxynitrite-induced lipid peroxidation of phosphatidylcholine lipososmes.91 This inhibition occurs because reactive nitrogen-oxide species (RNOS) can be trapped by -TOH, which leads to the formation of 5nitro- -TOH.92 -TOH is highly reactive toward RNOS because it has one less methyl group and thus a reactive position available to trap electrophiles, whereas -TOH is fully substituted in the chromanol ring.32 RNOS, like peroxynitrite, can rapidly cross phospholipid membranes and oxidatize proteins, DNA, lipids, redox metal centers and methionine.93 A recent study also found evidence for increased nitration of -TOH in subjects with CHD but concluded that a larger trial must be conducted to clearly demonstrate the efcacy of this marker.94 It has been suggested that the supplementation of patients in clinical trials with only -TOH may be inappropriate, as -TOH displaces -TOH in the plasma.92 However, based on the recent work highlighting the potential importance of -TOH in human health,27 displacing it from the circulation may not be benecial. The major metabolite of -TOH is -CEHC. -CEHC, rst described in 1996 after a 30-year search for a natriuretic factor that may control the concentrations of extracellular uid within the body, also has properties that may be important for human health.95 0.6 mg of -CEHC was subsequently puried from 800 l of human urine and was used to demonstrate that the compound could reversibly inhibit the 70pS potassium (K+ ) channel while not inhibiting the sodium (Na+ ) pump. The natriuretic properties of CEHC are not shared by -CEHC.95 -TOH and -CEHC have also been shown to inhibit cyclooxygenase-2 (COX-2) activity in human macrophages and epithelial cells. This, in turn, reduced the COX-2 catalyzed synthesis of prostaglandin E2 (PGE2 ) by these cells.96 Taken together, these ndings suggest possible anti-inammatory roles for -TOH and -CEHC, which may be important in preventing CHD and cancer.96 This awaits conrmation in an appropriately powered human clinical trial. V. VITAMIN E DISTRIBUTION IN CELLS A. Erythrocyte Vitamin E The erythrocyte membrane has an organized structure that gives the cell its characteristic doughnut-shaped appearance. Hydrogen peroxide has been used to induce hemolysis of erythrocytes in vitro in subjects who were depleted of vitamin E,97 and the amount of inducible hemolysis is related to the relative amounts of oxidizable polyunsaturated fatty acids and protective vitamin E98 The percent hemolysis of erythrocytes was shown to be inversely proportional to the amount of TOH (at physiological levels in blood) added to the reaction mixture. However, in vivo, the plasma concentration of vitamin E did not directly correlate with the percent hemolysis of erythrocytes by this assay.98

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Taken together, these results suggest that plasma TOH concentrations may not provide an accurate reection of the vitamin E status of an individual and that measurement of vitamin E concentrations in other cells or tissues may provide better information regarding long-term intake of vitamin E. In recent years, in a series of experiments, Simon et al. have examined erythrocyte vitamin E, specically -TOH, content in asymptomatic men who are at risk for developing atherosclerosis.99 This study, which examined erythrocyte -TOH concentrations and erythrocyte hemolysis with 2,2 azobis(2)-amidinopropane (AAPH), found that -TOH concentrations were lower with increased erythrocyte hemolysis in hypercholesterolemic men compared to normocholesterolemic men, even though plasma concentrations were normal.99 The same group examined the transfer of vitamin E between erythrocytes and HDL and concluded that the uptake of -TOH by erythrocytes is not impaired in hypercholesterolemic subjects and that the lower concentrations of erythrocyte vitamin E seen in this group could be due to impaired delivery to tissues. They also cited a previous study100 in which -TOH moved to LDL when the LDL: HDL ratio was high in vitro, which may account for the low concentrations of erythrocyte vitamin E seen in these hypercholesterolemic patients.101 This group recently compared erythrocyte and plasma vitamin E concentrations to carotid intima-media thickness in 261 men at risk for cardiovascular disease(CVD).102 They found a negative correlation between carotid intima-media thickness and erythrocyte vitamin E concentrations (P < 0.01), but not plasma or HDL -TOH concentrations. They suggested that this inverse relationship may indicate that cellular -TOH may be effective at inhibiting the early stages of disease.102 Further studies may clarify the efcacy of erythrocyte vitamin E as a marker of disease risk and any role in homeostasis. B. Platelet Vitamin E In an effort to establish the best marker of adequate vitamin E nutrition, platelet vitamin E concentrations have been examined. Lehmann et al. supplemented rats with varying amounts of d--tocopheryl acetate over a 10-week period.103 They found that within groups of rats fed the same diet, the reproducibility of the dose response was the most consistent for platelets. They also found that platelets provided a more sensitive indicator of vitamin E intake than erythrocytes or plasma.103 Vatassery examined healthy male subjects and observed that -TOH and -TOH concentrations in plasma correlated well with total lipid, cholesterol, and triglyceride concentrations but platelet concentrations of -TOH and -TOH did not104 (Table 4). This nding conrmed a previous observation showing that platelet TOHs correlated poorly with red cell and plasma TOH concentrations.105 However, platelet TOH concentrations compared well to plasma concentrations when expressed per mg lipid.104 Because platelet TOH concentrations do not directly depend on

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TABLE 4 Correlation of -and -TOH Concentrations in Plasma and Platelets with Plasma Total Lipid, Cholesterol, and Triglyceride Plasma -TOH Total lipid Cholesterol Triglyceride 0.79 0.58 0.78 -TOH 0.60 0.46 0.61 -TOH 0.22 0.27 0.08 Platelets -TOH 0.15 0.17 0.09

Correlations are statistically signicant at a P value of <0.001. Taken from Ref. 104.

lipid concentrations, it follows that they should not be inuenced by any free exchange between the two compartments. These investigators recommend assaying baseline platelet vitamin E concentrations before supplementation to assess nutritional adequacy.104 This is supported by human data showing that platelet vitamin E determination provided the most sensitive indicator for dietary intake of vitamin E.106 (Table 5). Of interest, when comparing the concentrations of TOH found in both erythrocytes and platelets, the relationship was stronger when the values were corrected for plasma lipid concentrations.106 VI. VITAMIN E AND PLATELET FUNCTION An increase in platelet aggregation and adherence to endothelium is an important factor in lesion progression and plaque stability in vivo,58,107 and oxidative stress within platelets may potentially contribute to thrombus formation.108 The effects of vitamin E isomers on platelet function are largely determined by the type of vitamin E used. Early work by Nordoy
TABLE 5 - and -TOH Concentrations (n = 20) of Plasma, Red Blood Cells, Platelets, and Lymphocytes of Human Subjects Supplemented with 0, 30, and 100 mg/d of dl--Tocopheryl Acetate Plasma mol/l -TOH (mg/d) 0 30 100 -TOH (mg/d) 0 30 100 23.9 1.2a 29.0 1.2b 36.0 1.9c 5.8 0.5a 3.6 0.5b 2.4 0.3c Plasma mol/g lipid 4.2 0.1a 4.9 0.1b 6.3 0.2c 1.0 0.1a 0.7 0.0b 0.5 0.0c RBC mol/l 5.1 0.1a 6.0 0.1b 7.9 0.2c 1.4 0.1a 1.0 0.1b 0.7 0.0c Platelets mol/ 10 g protein 4.3 0.1a 5.5 0.2b 6.9 0.2c 1.1 0.1a 0.8 0.1b 0.5 0.0c

Lymphocytes 2.1 0.1a 2.5 0.1b 2.7 0.1c 0.7 0.1a 0.4 0.0b 0.2 0.0c

Numbers in the same column with different letter superscripts are signicantly different by the paired t test (P < 0.05). Taken from Ref. 106.

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and Strom105 showed that incubation of platelets with synthetic SRR--TOH did not result in any signicant uptake of -TOH by the platelets. Platelets have been shown to readily take up natural RRR--TOH and demonstrate a corresponding decrease in platelet aggregation, whereas the synthetic form was poorly incorporated and did not affect the aggregation of platelets.109 More recent studies have also examined the efcacy of synthetic SRR--TOH on platelet function in healthy individuals with neither study showing any benecial effect.110,111 The effect of RRR--TOH on platelet aggregation appears to involve inhibition of protein kinase C within platelets and an increase of plateletderived nitric oxide.11 Human studies have shown a reduction in ex vivo platelet aggregation in subjects following supplementation with -TOH112,113 and supplements containing high amounts of -TOH.114,115 The efcacy of vitamin E supplements in preventing platelet activation may depend on many factors, including baseline TOH levels, the presence of co-antioxidants within platelets or the plasma, and whether natural or synthetic forms are used. The prevention of arachidonic acid-mediated -TOH oxidation within platelets can be reversed by co-incubation with ascorbic acid or glutathione.116 This may have implications because RRR--TOH in combination with ascorbic acid has been shown to inhibit atherosclerotic disease progression in subjects with hypercholesterolemia,117 and platelet activation may be important in this patient population.118 Any potential benet can also be complicated by patient adherence to conventional drug treatments, such as aspirin and statins, known to effect affect platelet function. We have examined the effects of 500 mg of RRR--TOH and 500 mg of a mixed TOH supplement on markers of platelet activation in well-controlled diabetic subjects, many of whom were taking conventional treatments. We observed no benet in relation to platelet function in spite of the signicant increase in platelet TOH concentrations.119 Thus, it is unlikely that TOH treatment alone will gain acceptance as a viable strategy to inhibit platelet function. Further studies are required to determine if natural TOHs supplements, in combination with other agents such as ascorbic acid, can signicantly inhibit platelet aggregation in vivo. VII. REPORTING VITAMIN E CONCENTRATIONS As plasma lipid concentrations inuence plasma TOH levels, it has been recommended that the concentration of -TOH be expressed per mg of lipid.120 In a study of 85 alcoholic patients, the ratio of TOH to cholesterol plus triglycerides was the best tool for identifying deciency (sensitivity 95%, specicity 99%).121 Patients with liver disease, for example, may have elevated lipid concentrations, but a deciency in vitamin E may be missed if the lipid concentrations are not taken into account. Conversely, patients with low lipid concentrations, for example, with heterozygous familial

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hypobetalipoproteinemia, may be classied as vitamin E decient when they are not.121 VIII. VITAMIN E AND ATHEROSCLEROSIS In 1991 the National Heart, Blood and Lung Institute (NHBLI) convened a workshop to review current knowledge about the oxidative modication hypothesis of lipoproteins and their implication in the pathogenesis of atherosclerosis.122 It was thought that the use of naturally occurring antioxidants in clinical trials for the prevention of atherosclerosis was safe. Since then a number of human trials have been conducted. A metaanalysis of the effects of high-dose versus low-dose vitamin E supplementation on cardiovascular mortality found that, for observational studies, the test for overall effect favored high vitamin E intake [odds ratio 0.67 (0.540.83)]. In contrast, the analysis from six major intervention trials were equivocal.123 The results from human trials of vitamin E supplementation have been controversial. It has been suggested that such studies would have beneted from the measurement of markers of in vivo lipid peroxidation, like F2 isoprostanes, to establish any effect on oxidative stress.62 The different doses and forms of TOH given to different populations might, in part, explain the paradoxical results obtained from these trials. In countries where a Mediterranean diet is consumed, a protective effect against atherosclerosis has been shown.124 The population in the Gruppo Italiano per lo Studio della Sopravvivenza nellInfarto miocardico (GISSI-Prevenzione trial),125 which typically consumed a Mediterranean-style diet rich in antioxidants, still developed CVD and may not have beneted from the 300 IU of synthetic vitamin E given.61 A subsequent follow-up of the participants in the GISSI-Prevenzione trial revealed that treatment with vitamin E led to a 50% increase in congestive heart failure in subjects with left ventricular dysfunction.24 Probably the best known negative trial for vitamin E supplementation was the Heart Outcomes Prevention Evaluation Study (HOPE).126 In this trial, 772 of the 4,761 patients who were at high risk for CVD received 400 IU of natural source vitamin E for a mean follow-up of 4.5 years. There was no signicant difference in the incidence of secondary cardiovascular outcomes or in death from any cause and no signicant adverse effects of vitamin E supplementation. In a similar fashion to the GISSI-Prevenzione trial, a subsequent follow-up study revealed an increase in the risk for heart failure with vitamin E treatment.23 The Secondary Prevention with Antioxidants of Cardiovascular Disease in Endstage Renal Disease (SPACE) trial127 in hemodialysis patients showed positive effects with vitamin E supplementation (800 IU of natural RRR-TOH) in a patient group that was probably under considerable oxidative stress.128,129 These patients were also given a number of other antioxidants

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as supplements, with 40% of all participants taking vitamin C.127 It has been suggested that vitamin C in combination with vitamin E may in fact explain the positive results.130,131 The Cambridge Heart Antioxidant Study (CHAOS)132 examined 2,002 patients with angiographically proven coronary atherosclerosis. Subjects who were given either 800 IU (546 subjects) or 400 IU (489 subjects) of natural -TOH had a 77% decrease in non-fatal myocardial infarction; however, there was a non-signicant increase in cardiovascular deaths in the subjects receiving -TOH. It was suggested that the apparent rise in fatal myocardial infarction could be related to transition-ion release from unstable plaques, with -TOH potentially acting as a pro-oxidant in this setting.61 The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) trial looked at the progression of carotid atherosclerosis in smoking and nonsmoking men and post-menopausal women over a 3-year period.133 A total of 520 patients were given, twice daily, 91 mg (136 IU) RRR--TOH, 250 mg slowrelease vitamin C, a combination of the two, or placebo. The most signicant nding was that, in men taking the combination of the two, the proportion who experienced progression was reduced by 74% (95% condence interval 3689%) compared to placebo.133 The largest trial conducted to examine the effects of statin and antioxidant therapy involved over 20,500 subjects with a variety of clinical conditions and examined prolonged use (>5 years) of simvastatin 40 mg with a cocktail of antioxidant vitamins (650 mg synthetic vitamin E, 250 mg vitamin C and 20 mg -carotene).134 The results provide positive results for statin therapy but there was no effect for the antioxidants used; however, they concluded that this cocktail does not cause harm.134 This is in contrast to a previous trial in which simvastatin, niacin and antioxidant vitamins were given in combination to determine any clinical benet.135 Antioxidant use signicantly impaired the benets obtained from niacin and simvastatin when used concurrently; the protective increase in HDL2 with simvastatin plus niacin was attenuated by simultaneous therapy with antioxidants. The use of antioxidant vitamins for the treatment of CVD has been questioned following these results and the predominantly negative results from large clinical trials.135,136 IX. VITAMIN E AND PRE-ECLAMPSIA Pre-eclampsia is a disorder that affects between 2 to 3% of pregnancies and is estimated to cause 60,000 deaths worldwide. It is characterized by high blood pressure and the presence of proteinuria and generally occurs in the second half of pregnancy. This condition involves a maternal inammatory response, activation of maternal vascular endothelial cells, endothelial dysfunction, and leucocyte activation. Because oxidative stress has

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been implicated in the pathogenesis of pre-eclampsia, there is considerable interest in the potential for antioxidants to prevent this condition.137 A recent placebo-controlled randomized trial examined the potential for vitamin C (1,000 mg/day) and vitamin E (RRR--TOH 400 IU/day) to prevent pre-eclampsia in women with a variety of risk factors.138 The study involved 2,395 subjects from 25 hospitals (1,196 in the vitamin group and 1,199 in the placebo group) and each were treated daily from the second trimester until birth. The primary end point was pre-eclampsia and the main secondary endpoints were low birth weight (<2.5 kg) and small size for gestational age. There was no difference for the primary outcome of pre-eclampsia; however, there was a reduction in the birth weight of babies whose mothers took the antioxidant treatment versus those on placebo.138 A subsequent study conducted in Australia examined the potential benet with the same doses of vitamin E and C to prevent pre-eclampsia in nulliparous women.139 This study found no effect on the occurrence of preeclampsia or low birth weight, but other adverse outcomes were common in the treatment group, including increased gestational hypertension, severe gestational hypertension, antenatal hospitalization for hypertension, the use of antihypertensive agents, and the induction of labor for hypertension.139 Taken together, these ndings suggest that the use of antioxidants for the prevention of pre-eclampsia is not warranted and may in fact be harmful. A recent study has reported benet with vitamin E and C used in combination to reduce blood pressure and oxidative stress in untreated hypertensives.77 However, because of recent analyses suggesting potential adverse consequences with the use of -TOH in disease prevention in highrisk populations22,23 and our own data showing an increase in hypertension following supplementation with vitamin E in diabetic subjects taking other drugs,140 further studies must be performed to ascertain the safety of the agents, particularly in relation to hypertension. X. VITAMIN E AND DRUG METABOLISM A. Cytochrome P450 Enzyme Activity The importance of the cytochrome P450 (CYP) enzyme system for the metabolism of drugs has been emphasized recently.141 The estimated number of deaths annually in the USA due to adverse drug reactions is thought to number at least 100,000.142 The CYP3A isoforms are probably the most important of all the drug-metabolizing enzymes in humans as they metabolize 50% of all drug oxidations,141 and high quantities of this enzyme are found in both the liver (29% of total) and the intestine (70% of total)143 CYP3A4 is the most studied form of CYP3A in humans and is the most abundant isoform found in both the liver and the intestine.144

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The induction of CYP3A4 in humans through activation of the pregnane X receptor (PXR) has been well described.141 Examples of drugs that induce CYP3A4 expression through this pathway include rifampicin and St. Johns wort (hyperforin); such induction can lead to increased metabolism of other drugs, including calcium-channel blockers and HIV-protease inhibitors.141 The induction of CYP3A4 in primary human hepatocytes through activation of PXR has been observed following incubation with St. Johns wort extract for 30 hours.145 A 14-day course of St. Johns wort extract induced the activity of CYP3A4 measured through the pharmacokinetics of alprazolam in human volunteers.146 It has been hypothesized that -TOH can interfere with drug metabolism by increasing the expression of CYP3A4 within the liver and thereby increasing the metabolism of certain drugs.147 B. CYP3A and Vitamin E Studies have been performed to examine the capacity for -TOH to increase CYP3A expression in vivo in a mouse model. A 2- to 3-fold increase in CYP3a11 mRNA (murine equivalent to human CYP3A4) following -TOH (20 mg/kg) for three months has been reported.148 Importantly, -tocotrienol did not induce CYP3a11 mRNA. In mice given high dose -TOH supplementation for ve weeks, the increase in CYP3a protein concentrations correlated with hepatic -TOH, but not with hepatic -TOH concentrations.149 A number of different forms of vitamin E have been shown to activate gene expression through activation of human PXR in HepG2 (a human hepatoma cell line) cells in culture; RRR--TOH was able to increase PXR activity 2- to 3-fold following incubation for 48 hours, but activation was higher with rifampicin (a known inducer) and also with - and -tocotrienol.150 A recent review of these and other studies suggested that high-dose TOH supplementation may interfere with drug metabolism through activation of CYP3A4, whereas -TOH and the tocotrienols may not, because of increased metabolism and excretion of these compounds in the liver.147 Subcutaneous -TOH injections in rats caused a signicant increase in liver -TOH concentrations at day 18 with a concomitant increase in liver microsomal CYP3A protein and P-glycoprotein (MDR1, multidrug resistant protein 1).151 While this study did not relate this effect to drug metabolism directly, it suggested that -TOH may increase CYP3A in humans and may potentially affect the metabolism of certain drugs.151 Another consideration is the differential effects of vitamin E forms on PXR-mediated drug transporters in different tissues. For example, rats given subcutaneous -TOH injections showed signicant increases in MDR1, but there was no effect on CYP3A protein expression in lung tissue.152 The use of combined antioxidants, RRR--TOH and vitamin C, in humans showed a signicant reduction in cyclosporin trough levels.153155

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However it was unclear which antioxidant had the effect or which drug metabolizing pathway was affected, because cyclosporine metabolized by both CYP3A and MDR1.156 Clearly any effect of vitamin E on these enzymes in human tissues in vivo needs to be determined. Studies in animals often demonstrate a different response with known inducers of PXR when compared to humans, because of differences in the amino acid sequences in the ligand binding domain for this receptor.157 Importantly, it takes two to three weeks for steady-state levels of CYP3A to increase in humans following typical induction by a number of agents, and the reversal of this effect also takes several weeks to occur.141 This is an important consideration in the design of studies examining potential interactions following induction of CYP3A4 or using vitamin E in the prevention of disease. Whether any forms of vitamin E can induce CYP3A4 or MDR1 in humans in vivo has not yet been determined.

C. PXR Activation and Hormone Synthesis Activation of PXR in humans may have undesirable effects in some populations. A recent study examined the effects of altered xenobiotic receptor activity on adrenal steroid homeostasis in transgenic mice that had liverspecic expression of activated human PXR.158 The observed increase in corticosterone and aldosterone output caused disrupted circadian rhythm and increased expression of steroidogenic enzymes involved in the production of these steroids.158 Any effect of vitamin E on PXR activation may affect hormone synthesis pathways in humans and needs to be considered when designing clinical trials. The -TOH, beta-Carotene Prevention Study (ATBC) study examined the effect of long-term -TOH supplementation (50 mg SRR--TOH acetate/day for ve to eight years) on prostate cancer incidence and demonstrated a 32% reduction compared to placebo.159 A follow-up study of 200 men participating in the ATBC trial showed a signicant reduction in both androstenedione and testosterone in the TOH group compared to placebo.160 There was a signicant negative correlation between serum -TOH and androgens in this group of men.160 The investigators suggested that the reduction in hormone production is one mechanism by which TOHs may reduce the incidence and mortality from prostate cancer. A recent publication from the ATBC study found that high serum -TOH concentrations were associated with a reduced risk of prostate cancer.161 Studies of the association between serum -TOH concentrations and all-cause mortality demonstrated a signicant reduction in risk, which was greater with increasing concentrations of -TOH; however no additional benet was gained beyond 1314 mg/l (30 mol/l).161 Whether vitamin E supplementation can alter the production of hormones in vivo across different populations

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remains an important question for further investigation in relation to both potential benets and possible adverse effects. XI. VITAMIN E SUPPLEMENTATION AND ALL-CAUSE MORTALITY Studies have investigated the metabolism and efcacy of -TOH in the prevention of sequelae associated with CVD. Some have shown promising results in secondary prevention in conditions associated with oxidative stress. However, the results from large primary prevention clinical trials with -TOH have been largely negative.162 A meta-analysis of >130,000 participants from 19 clinical trials across a wide range of vitamin E intake (16.5 IU to 2,000 IU) concluded that highdose vitamin E supplements (400 IU/day as -TOH) increase all-cause mortality.22 This conclusion was challenged in a subsequent analysis163 that suggested that the adverse effect on mortality was only signicant at doses above 2,000 IU/day, a dose much higher than the recommended upper limit of 1,600 IU/day.21 This conclusion is in contrast to recent data on the long-term effects of lower-dose -TOH supplementation in subjects from the HOPE23 and GISSI-Prevenzione trials24 in which subjects received 400 IU/day with a seven-year follow-up and 300 mg/day with a 3.5 year followup, respectively. These studies reported an increase in heart failure in the HOPE trial and heart failure in subjects with left ventricular dysfunction in the GISSI-prevenzione trial. The relationship between antioxidant vitamins and blood pressure was examined as a part of the Third National Health and Nutrition Examination Survey, which showed a higher odds ratio for hypertension in subjects with higher serum vitamin E concentrations after adjustment for a number of variables, including age, sex, race, diabetes, BMI, and dietary sodium intake.164 The relationship between -TOH supplementation and blood pressure is supported by our own ndings that show that RRR--TOH (750 IU/d) supplementation for six weeks increased systolic and diastolic blood pressure in well-treated diabetic subjects.140 The mechanism(s) for any adverse effect of a high dose of -TOH is unknown. One explanation is that -TOH may affect drug metabolism and thereby the efcacy of drugs used to treat subjects at risk of sequelae associated with CVD. The most comprehensive analysis to date of studies of antioxidant supplementation for primary and secondary prevention of disease included 68 randomized trials with 232,606 participants.136 This study on 26 trials on vitamin E, in which vitamin E was given either alone or in combination (following exclusion of trials with a high bias risk or use of selenium), included 105,065 participants and demonstrated an increase in relative risk of all-cause mortality compared to placebo.136 Whether this increased risk is also present in healthy populations has not been

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determined, and it is clear that this remains an important question for future research. XII. CONCLUSION A normal diet contains both TOHs and tocotrienols along with a number of other co-antioxidants and polyphenols. These may, in turn, confound any studies that look at individual supplements to prevent disease. Because of the disappointing results from clinical trials, and if one accepts that the primary role of -TOH in preventing atherosclerosis is via the inhibition of in vivo lipid peroxidation, then supplementation of -TOH in healthy individuals to prevent atherosclerosis may not be warranted. However, -TOH is clearly required by individuals with clinical deciency in the vitamin, such as those with abetaliproteinemia, who require supplementation to maintain normal neurological function. However, the supposition that antioxidants are safe in all populations has not been substantiated. As the roles of the different isomers of vitamin E in health and disease are further investigated, it will be important to continue to examine its effects on different cells within the body. Until the full importance of -TOH is established and the precise role of -TOH has been elucidated, confusion will remain as to the correct amount of each form of vitamin E needed to meet the nutritional requirements of humans. Further studies are needed to determine if signicant clinical drug interactions result from co-supplementation with vitamin E. The observation that individuals with a higher baseline serum vitamin E concentration have a reduced risk of all-cause mortality165 probably reects the importance of adequate dietary TOH intake. Data from the US population suggest that only 8.0% of men and 2.4% of women meet the estimated average requirement (12 mg) for -TOH intake,2 and it has been suggested that this might be attained through the diet as opposed to supplementation.166 Because the use of vitamin E supplements is high in US adults,167 many individuals may have either too much, or too little, vitamin E intake, and both cases may be deleterious to health. The question of whether an appropriate dose of one or more forms of vitamin E, given singularly or with other compounds, has a role in the prevention and treatment of disease remains to be answered in future clinical trials examining supplementation of TOHs in humans. REFERENCES
[1] Evans HM, Bishop KS. On the existence of a hitherto unrecognised dietary factor essential for reproduction. Science 1922; 56: 650651. [2] Maras JE, Bermudez OI, Qiao N, Bakun PJ, Boody-Alter EL, Tucker KL. Intake of alpha-tocopherol is limited among US adults. J Am Diet Assoc 2004; 104: 567575.

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