vitamin e in human health and disease

Critical Reviews in Clinical Laboratory Sciences, 45(5):417–450 (2008)Copyright C© 2008 Informa Healthcare USA, Inc.ISSN: 1040-8363 print / 1549-781X onlineDOI: 10.1080/10408360802118625

VITAMIN E IN HUMAN HEALTH AND DISEASE

Michael W. Clarke and John R. Burnett � School of Medicine and Pharmacology,University of Western Australia, Crawley WA 6009, Australia and Department of CoreClinical Pathology and Biochemistry, PathWest Laboratory Medicine WA 6847, Royal PerthHospital, Wellington Street Campus, Perth, Australia

Kevin D. Croft � School of Medicine and Pharmacology, University of WesternAustralia, Crawley WA 6009, Australia

Referee Dr. Jean-Marc Zingg, Vascular Biology Laboratory, JM USDA Human NutritionResearch Center on Aging, Tufts University, Boston, MA, USA

� Vitamin E in nature is comprised of a family of tocopherols and tocotrienols. The most studied ofthese is α-tocopherol (α-TOH), because this form is retained within the body, and vitamin E deficiencyis corrected with this supplement. α-TOH is a lipid-soluble antioxidant required for the preservationof cell membranes, and it potentially acts as a defense against oxidative stress. Many studies haveinvestigated the metabolism, transport, and efficacy α-TOH in the prevention of sequelae associatedwith cardiovascular disease (CVD). Supplementation with vitamin E is considered to provide healthbenefits against CVD through its antioxidant activity, the prevention of lipoprotein oxidation, and theinhibition of platelet aggregation. However, the results from large prospective, randomized, placebo-controlled clinical trials with α-TOH have been largely negative. A recent meta-analysis suggeststhat α-TOH supplements may actually increase all-cause mortality; however, the mechanism for thisincreased risk is unknown. In vitro studies performed in human cell cultures and animal modelssuggest 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 efficacy of any drugmetabolized by CYP3A4 or MDR1. Other possibilities include an adverse effect of α-TOH on bloodpressure 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; α-CEHC, 2,5,7,8-tetramethyl-2 (2′-carboxy-ethy)-6-hydroxychroman, metabolite of α-TOH; α-TOH, alpha tocopherol; α-TQH2,

Address correspondence to Dr. John R. Burnett, Department of Core Clinical Pathology and Bio-chemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, Wellington Street, GPO Box X2213,Perth WA 6847, Australia. E-mail: [email protected]

417

418 M. W. Clarke et al.

α-TOH hydroquinone; α-TTP, α-TOH transfer protein; γ -CEHC, 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (metabolite of γ -TOH); γ -TOH, gamma tocopherol;γ YP, cylochrome P450, AAPH, 2,2′-azobis(2)-amidinopropane (a free radical generator);ABCA1, ATP-binding cassette transporter A1; apoE−/−mouse, apolipoprotein E doubleknock out mouse; ASAP, Antioxidant Supplementation in Atherosclerosis Prevention;ATBC, α-TOH, beta-Carotene Prevention Study; AVED, ataxia with vitamin E deficiency;CHAOS, Cambridge Heart Antioxidant Study; CHD, coronary heart disease; COX-2,cyclooxygenase-2; CoQ10, ubiquinone-10; CoQ10H2, ubiquinol-10, reduced form of coen-zyme Q; CVD, cardiovascular disease; CYP3a11, murine equivalent to human CYP3A4;CYP3A4, cytochrome P450 3A4; d6-RRR-α-TOH, deuterium labelled natural alpha toco-pherol; ECD, electrochemical detection; FHBL, familial hypobetalipoproteinemia; FIVE,familial isolated vitamin E deficiency; GC/MS, gas chromatography mass spectrometry;GISSI, Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico; HDL,high-density lipoprotein; HepG2, human hepatoma cell line; HPLC, high-performanceliquid 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 resis-tant protein 1; MTP, microsomal triglyceride transfer protein; NHBLI, National Heart,Blood and Lung Institute; NO2, nitrogen dioxide; PGE2, prostaglandin E2; PXR, preg-nane X receptor; RNOS, reactive nitrogen-oxide species; RRR-α-TOH, d-α-TOH, naturalalpha tocopherol; RXR, retinoic acid receptor; SPACE, Secondary Prevention with An-tioxidants of Cardiovascular Disease in Endstage Renal Disease; SR-BI, scavenger recep-tor class B type I; SRR-∝-TOH, dl-α-TOH, synthetic alpha tocopherol; TAP, tocopherol-associated protein; TBP, tocopherol-binding protein; TMP, tocopherol-mediated perox-idation; 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 veg-etable oil that cured sterility in rats maintained on a lard diet.1 Since then,research into the metabolism and nutritional requirements of vitamin E hasled to a substantial body of knowledge about its role in human health anddisease. 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 fourtocopherols (TOH), namely, (α, β, γ , δ), and four tocotrienols (α, β, γ , δ).The most studied is α-TOH. The name TOH comes from the Greek tocos(childbirth), phero (to bear), and ol (alcohol).3 These eight structurally sim-ilar compounds, together with their biological activity compared to α-TOHare shown in Table 1.4 The most recognized role for α-TOH is as a lipid-soluble antioxidant required for the preservation of cell membranes, whereit reacts quickly with peroxyl radicals to preserve polyunsaturated fatty acids.5

More recently, α-TOH has been implicated in the activation of a number ofgenes.6

Primary vitamin E deficiency is generally found only in prematureand low-birth-weight infants. Secondary causes include fat malabsorp-tion syndromes (e.g ., cystic fibrosis, chronic liver disease, abetalipopro-teinemia, and intestinal resection) and some hematological disorders

Vitamin E in Humans 419

TABLE 1 The Structures and Biological Activities of the Eight Naturally Occurring Forms ofVitamin E

Common Activity based Compared toName Structure on rat assay RRR-α-TOH

d-α-TOH 1.49 100%

d-β-TOH 0.75 50%

d-γ -TOH 0.15 10%

d-δ-TOH 0.05 3%

d-α-tocotrienol 0.75 50%

d-β-tocotrienol 0.08 5%

d-γ -tocotrienol Not known

d-δ-tocotrienol Not known

Adapted from Ref. 4.

(e.g ., β-thalassemia major, sickle-cell anemia, and glucose-6-phosphatedehydrogenase deficiency).3 Patients with vitamin E deficiency have abnor-mal erythrocyte membrane morphology due to oxidative stress, and the char-acteristic acanthocytosis is associated with a reduction in red cell half life.7

Long-term deficiency in vitamin E can lead to neurological abnormalities,including ataxia, hyporeflexia, blindness, and dementia.8


The effect of α-TOH on the oxidation of lipoproteins and its po-tential role as an antiatherogenic supplement have received consider-able attention in the last decade. Moreover, a number of studies havedescribed a role for α-TOH beyond its antioxidant function. α-TOHhas been shown to inhibit smooth-muscle cell proliferation,9 endothe-lial dysfunction10 and platelet aggregation11 by a protein kinase-C de-pendant mechanism; it has also been found to inhibit monocyte adhe-sion to endothelial cells12 and macrophage-mediated lipid peroxidation invitro.13 These diverse functions of α-TOH and the potential pro-oxidanteffects observed in some studies14 may account for the paradoxical re-sults observed with human clinical trials on the prevention of recurrentatherosclerosis.15

The dietary requirement for vitamin E is often ascribed to the intakeof polyunsaturated fatty acids within the diet.16 However, this generalizationmay not be appropriate in all situations as vitamin E may act as a pro-oxidantin smokers who consume a diet high in polyunsaturated fatty acids.17 Becauseof conflicting data about vitamin E supplementation, it is not surprising tosee disagreement about the recommended daily allowance for α-TOH inhumans and to observe that γ -TOH has not yet been included.18−20 A rec-ommended dietary intake of 15 mg α-TOH/day21 which would suggest thatall dietary needs can be met from α-TOH.19 However, because of recent ad-verse publicity associated with high-dose vitamin E supplementation,22−24 it isprudent to return to fundamental questions relating to the requirements forthese compounds and to re-evaluate the supposition that high-dose vitaminE supplements in the form of α-TOH are safe.

The purpose of this article is to (1) describe vitamin E transport in hu-mans and animal models, (2) examine the potential role of vitamin E inthe oxidation of lipoproteins and treatment of atherosclerosis, and (3) ex-plore the potential for vitamin E isoforms to alter the metabolism of clinicallyimportant 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 methyla-tion in the chromanol ring and the configuration of the methyl groups inthe phytyl-side chains. The degree of methylation in the chromanol ring(Table 1) determines the antioxidant activity of each form of vitamin E, withα-TOH having twice the antioxidant activity of γ -TOH.25 The biological ac-tivities of the different forms of vitamin E are expressed in internationalunits per milligram (IU/mg). The relative activities are based on an assayusing a biological system in which the amount of natural vitamin E requiredto prevent fetal resorption in rats deficient in vitamin E is compared to


TABLE 2 Comparison of α-and γ -TOH Concentrations in Different Tissues in Humans and Rodents

Humans Rats and Mice

γ -TOH α-TOH γ -TOH α-TOH

Plasma (µmol/l) 2–7 15–20 1.3–1.7 7.2–13.0Liver (nmol/g) – 20∗ 4.5–5.3 30.0–33.4Adipose (nmol/g) 176 ± 80 440 ± 279 29.5 ± 4.1 79.8 ± 6.9Muscle (nmol/g) 107 155 ± 163 3.6–5.6 15.1–22.7Skin (nmol/g) 180 ± 89 127 ± 74 3.0 ± 2.8 8.9 ± 3.0

Taken from Ref.32.∗Subject received 75 mg D3 RRR-α-TOH for seven days before sampling.168

RRR-α-TOH(d-α-tocopherol, natural alpha tocopherol).26 However, usingthis data to compare the biological activities of the different forms of naturalvitamin E can make interpretation difficult, because the human require-ments and tissue concentrations of the two most important natural forms ofvitamin E, namely α- and γ -TOH, are markedly different between humansand rats (Table 2).

α-TOH, the major form of vitamin E in humans, is the most lipid-solubleantioxidant and the most abundant TOH in human tissues.5,25 Syntheticvitamin E (SRR-α-TOH, dl-α-TOH) is an equal mixture of eight stereoisomersof α-TOH.4 All isomers have an identical chromanol group and hence equalantioxidant activity.27 Synthetic α-TOH contains only 12.5% pure RRR-α-TOH and equal amounts of the other forms. However, only the four 2R-α-TOH isoforms are efficiently retained in the body.27 The biological activitiesfor each isomer are known in the rat but not in humans.28 Further studiesin humans are required to establish the biological activity of the differentstereoisomers of synthetic vitamin E.

B. Intestinal Absorption of Vitamin E and Postprandial

Metabolism

The intestinal absorption of vitamin E requires the intake and digestionof dietary fat, which is enhanced by the production of bile acids from theliver.29 Dietary vitamin E bound in micelles formed within the intestine isabsorbed, along with triglycerides and cholesterol, by a passive process intoenterocytes. Chylomicrons containing vitamin E are assembled and secretedinto the lymph.25 Inhibition of the scavenger receptor class B type I (SR-BI)blocks up to 80% of α-TOH uptake, and suggests a role for SR-BI in intestinalTOH transport.30

In the circulation, chylomicrons interact with lipoprotein lipase (LPL) torelease non-esterified fatty acids and triglycerides (Figure 1). It is thought thatTOHs may be delivered to tissues such as muscle, adipocytes, and the brainduring this process as the transfer of TOH to fibroblasts was observed in vitro


FIGURE 1 TOH transport in lipoproteins and cells. Dietary vitamin E is absorbed through the intestineand then the TOHs are transported within the peripheral blood bound to lipoproteins and cells suchas platelets and red blood cells. The delivery of TOHs to tissues occurs via lipoprotein lipase-mediateddelipidation of chylomicrons and delivery from low-density lipoprotein (LDL) and HDL. There is pref-erential incorporation of RRR-α-TOH into very low-density lipoprotein (VLDL) mediated by α-TTP. TheHDL delivery of TOHs is probably important in individuals with low VLDL and LDL cholesterol. Thereis 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 lipidsduring LPL-mediated delipidation of lipoproteins. As chylomicron remnantsare formed, they can then exchange surface components, including TOHs,with high-density lipoprotein (HDL). HDL can then transfer TOHs to otherlipoproteins in the circulation.25 This pathway is particularly important forindividuals with abetalipoproteinemia and homozygous familial hypobeta-lipoproteinema because of absent or extremely low plasma levels ofapolipoprotein B-containing lipoproteins. Supplementation of these indi-viduals with high-dose vitamin E (100–150 mg/kg per day) can normalizeadipose 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 beengreatly facilitated by the use of deuterated TOHs to assess the distribution of


each form among lipoproteins.25,32 Studies in humans have demonstratedthat α-TOH is preferentially incorporated into very low-density lipoprotein(VLDL) particles, and γ -TOH is excreted in the bile33

Similarly, in rats, monkeys, and humans, the naturally occurring RRRstereoisomer of α-TOH is also preferentially incorporated into VLDL.34 Onereport studied patients with and without inherited disorders of lipoproteinmetabolism to elucidate the steps involved in the discrimination of the dif-ferent vitamin E isoforms.34 A subject with homozygous familial hypobe-talipoproteinemia (FHBL) and abnormal apolipoprotein B-100 productionshowed preferential enrichment of his “VLDL” fraction with d6-RRR-α-TOH(deuterium-labeled natural ∝-TOH), 24 hours after supplementation withd6-RRR-α-TOH acetate. Of interest, this patient had normal HDL d6-RRR-α-TOH concentrations, did not have symptoms of vitamin E deficiency, andhas not required vitamin E supplementation. Taken together, this complexstudy showed the importance of chylomicron and HDL metabolism for TOHdistribution to lipoproteins for patients with impaired transport of TOHsdue to rare inherited disorders of lipid and lipoprotein metabolism, anddemonstrated that VLDL particles, even abnormal ones, are preferentiallyenriched with RRR-α-TOH.34 Our own study examined oxidative stress andTOH metabolism in individuals who were heterozygous for FHBL and con-cluded that supplementation with vitamin E was not required in this group,because oxidative stress was not evident, nor did they exhibit any clinicalsigns of TOH deficiency.35

The importance of chylomicron delivery of TOHs to peripheral tissueshas been highlighted recently in a murine model.36 Genetically engineeredmice that specifically lack microsomal triglyceride transfer protein (MTP) inthe liver were fed deuterated α-TOH; the majority of TOH was replacedin peripheral tissues within one month, despite their inability to secreteVLDL.36 Recent in vitro studies using human fibroblasts and murine RAW264macrophages showed that the export of α-TOH to HDL was mediated, at leastin part, by the ATP-binding cassette transporter A1 (ABCA1).37 Moreover,ABCA1 was directly involved in the translocation of α-TOH to apoproteins .37

(Figure 2). Taken together, these studies show that vitamin E can be metabo-lized and delivered to tissues by a variety of routes and that low serum vitaminE concentrations do not necessarily equate to vitamin E deficiency. VitaminE deficiency may also be organ specific and depend on the environment inquestion, with serum tocopherol concentrations not necessarily indicative ofthose in peripheral tissues.

D. TOH Transfer Proteins

Vitamin E distribution and the role of TOH regulatory proteins has beenthe topic of a recent review.27 The α-TOH transfer protein (α-TTP) is a 32 kDacytosolic lipid-binding protein that is found in a number of tissues but mainly


FIGURE 2 Hepatocyte vitamin E transport and metabolism. Vitamin E forms enter hepatocytes fromdiet via chylomicrons or from endogenous lipoproteins LDL and HDL. When internalized, α-TTP willselectively incorporate RRR-α-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 protect-ing poly-unsaturated fatty acids from oxidation. The TOHs have been shown to bind to two intracellularproteins, namely, TBP and TAP, but the precise role for these has yet to be described. The proposedactivation of PXR by vitamin E leads to the up-regulation of a number of genes including, CYP3A4 andMDR1, which, along with CYP4F2, metabolize the different forms of vitamin E and allow them to beexcreted from the cell. Membrane transporters SR-BI and ABCA1 have also been implicated in vitamin Emetabolism. Adapted from Refs.25,37,147,169 LDL, low density lipoprotein; HDL, high density lipoprotein;VLDL, very low density lipoprotein; VE, vitamin E forms; α-TTP, α-TOH transfer protein; TAP, toco-pherol associated protein; TBP, tocopherol binding protein; PXR, pregnane X receptor; RXR, retinoicacid 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 transporterA1; CEHCs, vitamin E metabolites (see text).

the liver,38 with some expression in rat brain, spleen, lung, and kidney, and insome regions in human brain.27 Previous studies in rats39 and humans33,34,40

showed that RRR-α-TOH was retained within lipoproteins. α-TTP has a highaffinity for RRR-α-TOH compared to other TOHs (Table 3), which may, inpart, account for the biological potency of each form of vitamin E.41

It is thought that α-TTP directly facilitates the incorporation of α-TOHinto VLDL.25 However, this theory remains controversial.27 In rat hepatomacells expressing α-TTP, more α-TOH was secreted into the medium.42 Whenthe cells were incubated with brefeldin A, an inhibitor of VLDL secretion, theexport of α-TOH was not affected, which suggested that the two processes aredistinct.27 It has been postulated that α-TTP is involved in retaining and recy-cling TOHs within hepatocytes by localizing within intracellular endosomes


TABLE 3 Relative Affinities of Various TOH Analogs for α-TTP

Competitors Relative affinity (%)

α-TOH 100β-TOH 38.1 ± 9.3γ -TOH 8.9 ± 0.6δ-TOH 1.6 ± 0.3α-TOH acetate 1.7 ± 0.1α-TOH quinone 1.5 ± 0.1SRR-α-TOH 10.5 ± 0.4α-Tocotrienol 12.4 ± 2.3Trolox 9.1 ±1.2

Taken from Ref. 41.

and binding to TOHs. It has also been proposed that the efflux of α-TOHto the plasma membrane is facilitated by α-TTP and that free α-TOH maythen be taken up by VLDL particles or other lipoproteins.27 A recent studyhas shown that the transporter ABCA1 may also be involved in facilitatingα-TTP-mediated secretion of TOHs from hepatocytes.43 However, furtherstudies will be required to demonstrate whether this process occurs in humantissues.

There are other proteins, called tocopherol associated proteins (TAP),that specifically bind to TOHs. hTAP1, a 46 kDa protein, has recently beendescribed in humans;44 it has sequence homology similar to α-TTP and isfound in the liver, prostate, and brain. However, a specific function for thisprotein has yet to be found.27 Two other TAP proteins, namely hTAP2 andhTAP3, are similar to hTAP1, and are involved in tocopherol-mediated cellsignaling pathways.45 A recent report of a TAP (also known as supernatantprotein factor) knockout mouse suggests a role for TAP in hepatic choles-terol synthesis.46 The relationship of this to tocopherol metabolism remainsunclear.

TOH-binding protein (TBP), a 14.2-kDa cystolic protein found in rat liverand heart, stimulates the transfer of α-TOH from liposomes to mitochondriain vitro.47 Although this protein may be involved in intracellular traffickingof α-TOH, a direct role for this protein has not been described.27

E. TOH Transfer Protein Deficiency

Patients with familial isolated vitamin E deficiency (FIVE), also knownas ataxia with vitamin E deficiency (AVED), have been studied to exam-ine the role of α-TTP in TOH metabolism. First described in 1981,48 thisrare autosomal recessive neurogenerative disease49 is often misdiagnosed asFriedreich’s ataxia, but it can be distinguished by measuring α-TOH con-centrations (low in FIVE) or by molecular analysis of the frataxin gene onchromosome 9.50 Patients with severe vitamin E deficiency develop hypore-flexia, ataxia, limited upward gaze, muscle weakness, and constriction of their


visual fields. Long-term vitamin E deficiency can lead to blindness, dementia,and cardiac arrhythmias.51 Untreated patients with FIVE have plasma α-TOHconcentrations ∼1% of normal.27 These patients do not produce VLDL en-riched with α-TOH and therefore must rely on chylomicron metabolism todistribute dietary α-TOH to tissues.27 However, they still require supplemen-tation with 800 mg RRR α-TOH twice daily to maintain plasma concentrationswithin the reference interval.49

Mice deficient in α-TTP have provided useful models to study disease pro-cesses where oxidative stress is thought to play a role.52,53 Tereswa et al. exam-ined atherosclerotic lesion development in ApoE knockout mice (ApoE −/−)that also had vitamin E deficiency due to disruption of the α-TTP gene.53

Vitamin E deficiency associated with α-TTP deficiency promoted lesion for-mation in the proximal aorta in ApoE −/− mice. Moreover, α-TOH concen-trations were reduced by >85%, and the generation of F2-isoprostanes wasincreased, indicating that lipid peroxidation was not suppressed in this re-gion of the aorta. Whether this effect is evidence of an atheroprotectiveeffect has been challenged because the effect of vitamin E supplementa-tion was modest, and the α-TOH concentrations were 100 times greater inthe group with normal α-TTP function.54 Taken together, these findings53

are consistent with other work on ApoE −/−mice, which showed that dietarysupplementation with vitamin E (2000 IU/kg chow) significantly reduced F2-isoprostane concentrations in urine, plasma, and vascular tissue.55 However,another study of ApoE −/−mice showed a relatively small positive effect withvitamin E supplementation (0.2% wt/wt in diet).56 Furthermore, combiningchow with vitamin E (0.05% wt/wt) with β-carotene (0.05% wt/wt) showedno benefit in this mouse model.57 Thus, the potential role of vitamin E inthe 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 ofdifferent processes including an inflammatory component58 and oxidationmodification of lipoproteins.59 However, these two processes are not mutuallyexclusive. Vitamin E is thought to play a role both in regulating aspects ofthe immune system and in antioxidant defense of lipoproteins.60 No singleoxidant responsible for LDL oxidation has been identified, and it is likely thatmany factors, including transition metals, 15-lipoxgenase, myeloperoxidase-derived oxidants, and reactive nitrogen species, are involved.54

The antioxidant properties of vitamin E, which were studied in regard totheir role in supplementation in humans, may provide an explanation for theparadoxical results obtained from clinical trials.61,62 Although α-TOH is im-portant, it is not the only determinant in the resistance of LDL to oxidation.63


Ubiquinol-10 (CoQ10H2), the reduced form of coenzyme Q, is a well-studiedco-antioxidant for α-TOH60 and is an effective lipid-soluble antioxidant atphysiological concentrations.64 Ubiquinone-10 (CoQ10) is reduced duringintestinal absorption to CoQ10H2.56

The disappearance of antioxidants from LDL isolated from healthy sub-jects has been examined under different oxidizing conditions.65 An initiallag period (because of contaminating ascorbic acid) was followed by detec-tion of lipid-peroxidation products, which coincided with the consumptionof CoQ10H2. This occurred even though 80% of the endogenous carotenoidsand 95% of the endogenous α-TOH were still present.65

The relative roles of L-ascorbic acid (vitamin C), α-TOH, and CoQ10H2

in protecting LDL from oxidation in vitro have been described.66 When con-centrations of co-antioxidants ascorbate and CoQ10H2 are low, the α-TOHradical (TO·) can act to transfer, rather than trap, electrons. Also, CoQ10H2

can act as a better antioxidant than α-TOH in LDL because the semiquinoneradical formed can leave the lipoprotein particle rather than promote fur-ther peroxidation.66 The rate of radical stress is important in determiningthe amounts co-antioxidants available to interact with α-TOH and this inturn influences the role of α-TOH within the LDL particle.

LDL from healthy subjects has been examined before and after sup-plementation with RRR-α-TOH (1 g/day) and/or CoQ10 (100 mg/day)for a total of five days.67 Native LDL contained 8.5 ± 2 molecules of α-TOH and 0.5 to 0.8 CoQ10H2 molecules per particle. Incubation of LDLin Ham’s F10 medium containing transition metal ions depleted LDL ofα-TOH, and this depletion increased in the presence of monocyte-derivedmacrophages (MDMs). When LDL was incubated in vitro with α-TOH, theconcentrations of α-TOH increased 6- to 7-fold in the LDL particles. Fur-thermore, these particles were more easily oxidized than native LDL both inthe presence and absence of monocyte MDMs, which suggests a pro-oxidantrole for α-TOH.67 In supplemented subjects, LDL α-TOH concentrationsincreased 2- to 3-fold and CoQ10H2 concentrations increased 3- to 4-fold.LDL was more susceptible to oxidation in subjects receiving only α-TOHand more resistant in those receiving CoQ10. Of interest, those receivingco-supplementation had LDL that was more resistant to oxidation thannative LDL or LDL incubated with α-TOH. Taken together, these resultswere explained using the model of TOH-mediated peroxidation (TMP),with CoQ10H2 inhibiting TMP and protecting LDL from oxidation.67 A de-tailed review of TMP has implications for using α-TOH for the prevention ofatherosclerosis.68

α-TOH hydroquinone (α-TQH2), derived from TOH quinone, has beenshown to effectively inhibit oxidation of α-TOH, and CoQ10H2, along withsurface and core lipids, by a number of oxidants in vitro.69 It has been de-scribed as the most efficient lipophilic antioxidant, because it effectivelyregenerates TO· to α-TOH and decreases consumption of CoQ10H2. These


investigators suggest that α-TQH2 may be a potential therapeutic agent,69

but this awaits confirmation 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-α-TOH.56 Twenty-four weeks of supplementation with vitaminE 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 increasedsignificantly in all tissues measured. Supplementation with vitamin E andCoQ10 decreased lesion size at the sites examined (aortic root ∼30%, aorticarch ∼50%, and descending thoracic aorta ∼80%). Vitamin E alone de-creased lesion size only in the aortic root. The inhibition of lesion size aftercombined supplementation was associated with a decrease in aortic concen-trations of lipid hydroperoxides (LOOH). However, supplementation withvitamin E alone did not lower the aortic concentrations of LOOH. Takentogether, these results were consistent with the theory of TMP and that com-bined supplementation with both vitamin E and CoQ10 may be more anti-atherogenic than vitamin E alone.56

Long-term antioxidant supplementation (12 months) with α-TOH,probucol, and CoQ10 in Watanabe heritable hyperlipidemic (WHHL) rabbitswith doses approximating therapeutic doses in humans (300 mg SRR-α-TOHand CoQ10, 1 g probucol) had no effect on aortic lesion size but decreasedcopper-induced ex vivo lipid peroxidation.70 Probucol also decreased lipidperoxidation by copper, but CoQ10 had no effect. However, vitamin E failedto decrease the amount of lipid-standardized LOOH and correspondinghydroxides.70 This finding is also consistent with the model of TMP in theabsence of sufficient co-antioxidants.68

The concentrations of oxidized lipids, α-TOH, and its oxidation prod-ucts in human lesions were determined during different stages in atheroscle-rotic lesion development.71 Oxidation of α-TOH occurred early in the dis-ease and exceeded lipid peroxidation, and lesions were not depleted ofα-TOH. The products of α-TOH oxidation, tocopherylquinone, and toco-pheryl epoxides, were <20% of the total TOHs, with tocopherylquinone themajor product formed. Using an in vitro assay, Terentis et al. determinedthat tocopherylquinone is generated in the presence of two electron (non-radical) oxidants such as hypochlorite and peroxynitrite. They also foundthat the oxidation products formed were similar to those formed when LDLis oxidized in the presence of nitrite.71 Because oxidized lipids co-exist withα-TOH in atherosclerotic lesions,72 this study calls into question the rationalefor using antioxidants such as α-TOH to prevent atherosclerosis.71

More recently a study of the effects of vitamin E supplementation onatherosclerosis in mice with vitamin E deficiency showed that any benefitwas small and dependent upon the degree of pre-existing deficiency.73 Thelake of effect on aortic F2-isoprostane and oxidized lipid formation suggested


that vitamin E supplementation may not reduce oxidative stress within thevessel wall.73 Moreover, supplementing healthy individuals with α-TOH hadno effect on lipid peroxidation as measured by urinary concentrations of4-hydroxynonenal (4-HNE) or F2-isoprostanes; this challenges the rationalefor supplementing healthy subjects with α-TOH to inhibit oxidative stress.74

Recent human intervention studies examining the antioxidant potentialof RRR-α-TOH administration with 1,200 IU/d for two years75 and 1,600IU/d for 16 weeks76 showed a significant reduction in F2-isoprostane pro-duction; this is consistent with the concept that high doses of RRR-α-TOHare required to reduce oxidative stress in humans. However, another recentstudy in subjects with essential hypertension demonstrated a reduction inisoprostane formation and blood pressure with a lower dose of α-TOH (400IU/d) but in combination with vitamin C (100 mg/d).77 Taken together,these studies suggest that RRR-α-TOH supplements may reduce oxidativestress in human populations with high oxidative stress, but the effect mayoccur only with higher doses or in combination with co-antioxidants.

IV. TOH METABOLITES

A. α-TOH

Because strong epidemiological evidence supports a role for vitamin Ein heart disease prevention,78,79 the measurement and study of productsderived from α-TOH oxidation have received considerable attention. Uri-nary metabolites of vitamin E have been studied since the 1950s.80 Thefirst published data describing urinary metabolites of α-TOH from rabbitsand humans appeared in 1956.81 Known as the “Simon metabolites,” bothtocopheronic acid and the subsequent tocopheronolactone were detectedin urine after high-dose supplementation with α-TOH.81 These metaboliteswere generally thought to arise following the antioxidant action of vitaminE in vivo with the chroman ring being opened after oxidation.80 This ex-planation has been challenged with the suggestion that tocopheronolactoneis produced from 2,5,7,8-tetramethyl-2 (2′-carboxyethy)-6-hydroxychroman(α-CEHC, a metabolite of ∝-TOH) in the presence of oxygen from sam-ple handling.82 In this study, subjects receiving greater than 50 mg perday of α-TOH reached a plasma threshold of 7–9 µmol α-TOH/g totallipid and had detectable α-CEHC concentrations, determined using a high-performance liquid chromatography (HPLC) method with electrochemi-cal detection (ECD). The concentrations of α-CEHC (µmol/24 h) corre-lated well with α-TOH concentrations (µmol/g total plasma lipid) whenthe threshold was reached. It was concluded that α-TOH can undergo ω-oxidation without prior oxidation and that α-CEHC is therefore the majorurinary metabolite of α-TOH produced by healthy humans (Figure 3). Fur-thermore, as plasma concentrations of α-TOH can be raised only 3-fold,83


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


regardless of how much α-TOH is given, the investigators proposed that uri-nary concentrations of α-CEHC are a marker of adequate vitamin E intake.They raised the possibility that high concentrations of α-TOH may indeed beharmful and that high urinary concentrations of α-CEHC may reflect this.82

They also acknowledged that the formation of Simon metabolites may occurin vivo, but probably only during episodes of oxidative stress.82

In contrast, other investigators have shown that both tocopheronolac-tone and α-CEHC were present in the urine of subjects not taking α-TOHsupplements.80,84,85 These later studies employed more sensitive gas chro-matography/mass spectrometry (GC/MS) methods, which could explainthe different findings.80 The use of sensitive methods to measure both serumand urine metabolites of vitamin E, with emphasis on correct sample han-dling and processing, should lead to a greater understanding of the sig-nificance of these metabolites in both supplemented and unsupplementedindividuals.

B. γ -TOH and Its Major Metabolite γ -CEHC

α-TTP is the main regulator of α-TOH incorporation into lipoproteins,whereas it plays only a small part in γ -TOH metabolism. Instead γ -TOHis metabolized to 2,7,8-trimethyl-2- (β-carboxyethyl)-6-hydroxychroman (γ -CEHC), via a cytochrome P450 enzyme in the liver,86 which is then excretedin the urine .87 (Figure 2).

There has been considerable recent interest in the role of γ -TOH inhuman health, and it has been the topic of a recent review.32γ -TOH com-prises around 70% of the total TOH dietary intake in the US population. It isfound in soybeans, corn, and walnuts and other nuts, and oils derived fromsoy, corn, and sesame are rich in γ -TOH.32

There has been greater interest in the role of α-TOH versus γ -TOH inhuman nutrition. The concentrations of α-TOH are greater than γ -TOH intissues, and the biological activity of γ -TOH is ∼10% of α-TOH, as deter-mined by the rat fetal resorption assay.88 The difference in activity relatesin part to different plasma and tissue concentrations of the two TOHs inhumans and rodents (Table 2). Tissue concentrations of γ -TOH are higherin humans than in rodents, particularly in skin and muscle, and these levelsprobably reflect the different ways in which each species metabolizes the twoforms of TOH.32

In a case-control study, γ -TOH concentrations in patients with coro-nary heart disease (CHD) were lower than in healthy, age-matched controls,whereas the concentrations of α-TOH were not different between the twogroups.89 Similarly, γ -TOH concentrations were significantly lower in CHDpatients than in controls, with no corresponding decrease in plasma α-TOHor CoQ10H2 concentrations in the patient group.90

There is some evidence that γ -TOH is superior to α-TOH at detoxi-fying nitrogen dioxide (NO2) and that it is more effective at inhibiting


peroxynitrite-induced lipid peroxidation of phosphatidylcholine lipo-sosmes.91 This inhibition occurs because reactive nitrogen-oxide species(RNOS) can be trapped by γ -TOH, which leads to the formation of 5-nitro-γ -TOH.92 γ -TOH is highly reactive toward RNOS because it hasone less methyl group and thus a reactive position available to trap elec-trophiles, whereas α-TOH is fully substituted in the chromanol ring.32 RNOS,like peroxynitrite, can rapidly cross phospholipid membranes and oxida-tize proteins, DNA, lipids, redox metal centers and methionine.93 A re-cent study also found evidence for increased nitration of γ -TOH in sub-jects with CHD but concluded that a larger trial must be conducted toclearly demonstrate the efficacy of this marker.94 It has been suggestedthat the supplementation of patients in clinical trials with only α-TOHmay be inappropriate, as α-TOH displaces γ -TOH in the plasma.92 How-ever, based on the recent work highlighting the potential importance ofγ -TOH in human health,27 displacing it from the circulation may not bebeneficial.

The major metabolite of γ -TOH is γ -CEHC. γ -CEHC, first describedin 1996 after a 30-year search for a natriuretic factor that may control theconcentrations of extracellular fluid within the body, also has properties thatmay be important for human health.95 0.6 mg of γ -CEHC was subsequentlypurified from 800 l of human urine and was used to demonstrate that thecompound could reversibly inhibit the 70pS potassium (K+) channel whilenot inhibiting the sodium (Na+) pump. The natriuretic properties of γ -CEHC are not shared by α-CEHC.95

γ -TOH and γ -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 thesecells.96 Taken together, these findings suggest possible anti-inflammatoryroles for γ -TOH and γ -CEHC, which may be important in preventing CHDand cancer.96 This awaits confirmation in an appropriately powered humanclinical trial.

V. VITAMIN E DISTRIBUTION IN CELLS

A. Erythrocyte Vitamin E

The erythrocyte membrane has an organized structure that gives the cellits characteristic doughnut-shaped appearance. Hydrogen peroxide has beenused to induce hemolysis of erythrocytes in vitro in subjects who were depletedof vitamin E,97 and the amount of inducible hemolysis is related to the relativeamounts of oxidizable polyunsaturated fatty acids and protective vitamin E98

The percent hemolysis of erythrocytes was shown to be inversely proportionalto the amount of TOH (at physiological levels in blood) added to the reactionmixture. However, in vivo, the plasma concentration of vitamin E did notdirectly correlate with the percent hemolysis of erythrocytes by this assay.98


Taken together, these results suggest that plasma TOH concentrations maynot provide an accurate reflection of the vitamin E status of an individualand that measurement of vitamin E concentrations in other cells or tissuesmay provide better information regarding long-term intake of vitamin E.

In recent years, in a series of experiments, Simon et al. have examinederythrocyte vitamin E, specifically α-TOH, content in asymptomatic menwho are at risk for developing atherosclerosis.99 This study, which exam-ined erythrocyte α-TOH concentrations and erythrocyte hemolysis with 2,2′-azobis(2)-amidinopropane (AAPH), found that α-TOH concentrations werelower with increased erythrocyte hemolysis in hypercholesterolemic mencompared to normocholesterolemic men, even though plasma concentra-tions were normal.99 The same group examined the transfer of vitamin Ebetween erythrocytes and HDL and concluded that the uptake of α-TOH byerythrocytes is not impaired in hypercholesterolemic subjects and that thelower concentrations of erythrocyte vitamin E seen in this group could be dueto impaired delivery to tissues. They also cited a previous study100 in whichα-TOH moved to LDL when the LDL: HDL ratio was high in vitro, which mayaccount for the low concentrations of erythrocyte vitamin E seen in these hy-percholesterolemic patients.101 This group recently compared erythrocyteand plasma vitamin E concentrations to carotid intima-media thickness in261 men at risk for cardiovascular disease(CVD).102 They found a negativecorrelation between carotid intima-media thickness and erythrocyte vitaminE concentrations (P < 0.01), but not plasma or HDL α-TOH concentra-tions. They suggested that this inverse relationship may indicate that cellularα-TOH may be effective at inhibiting the early stages of disease.102 Furtherstudies may clarify the efficacy of erythrocyte vitamin E as a marker of diseaserisk 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. sup-plemented rats with varying amounts of d-α-tocopheryl acetate over a 10-weekperiod.103 They found that within groups of rats fed the same diet, the repro-ducibility of the dose response was the most consistent for platelets. They alsofound that platelets provided a more sensitive indicator of vitamin E intakethan erythrocytes or plasma.103 Vatassery examined healthy male subjectsand observed that α-TOH and γ -TOH concentrations in plasma correlatedwell with total lipid, cholesterol, and triglyceride concentrations but plateletconcentrations of α-TOH and γ -TOH did not104 (Table 4). This finding con-firmed a previous observation showing that platelet TOHs correlated poorlywith red cell and plasma TOH concentrations.105 However, platelet TOHconcentrations compared well to plasma concentrations when expressed permg lipid.104 Because platelet TOH concentrations do not directly depend on


TABLE 4 Correlation of α-and γ -TOH Concentrations in Plasma and Platelets with Plasma TotalLipid, Cholesterol, and Triglyceride

Plasma Platelets

α-TOH γ -TOH α-TOH γ -TOH

Total lipid 0.79∗ 0.60∗ 0.22 0.15Cholesterol 0.58∗ 0.46∗ 0.27 0.17Triglyceride 0.78∗ 0.61∗ 0.08 0.09

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

lipid concentrations, it follows that they should not be influenced by any freeexchange between the two compartments. These investigators recommendassaying baseline platelet vitamin E concentrations before supplementationto assess nutritional adequacy.104 This is supported by human data showingthat platelet vitamin E determination provided the most sensitive indicatorfor dietary intake of vitamin E.106 (Table 5). Of interest, when comparingthe concentrations of TOH found in both erythrocytes and platelets, therelationship was stronger when the values were corrected for plasma lipidconcentrations.106

VI. VITAMIN E AND PLATELET FUNCTION

An increase in platelet aggregation and adherence to endothelium isan important factor in lesion progression and plaque stability in vivo,58,107

and oxidative stress within platelets may potentially contribute to throm-bus formation.108 The effects of vitamin E isomers on platelet function arelargely determined by the type of vitamin E used. Early work by Nordoy

TABLE 5 α- and γ -TOH Concentrations (n = 20) of Plasma, Red Blood Cells, Platelets, andLymphocytes of Human Subjects Supplemented with 0, 30, and 100 mg/d of dl-α-Tocopheryl Acetate

Plasma Plasma RBC Platelets µmol/µmol/l µmol/g lipid µmol/l 10 g protein Lymphocytes

α-TOH (mg/d)0

30100

23.9 ± 1.2a

29.0 ± 1.2b

36.0 ± 1.9c

4.2 ± 0.1a

4.9 ± 0.1b

6.3 ± 0.2c

5.1 ± 0.1a

6.0 ± 0.1b

7.9 ± 0.2c

4.3 ± 0.1a

5.5 ± 0.2b

6.9 ± 0.2c

2.1 ± 0.1a

2.5 ± 0.1b

2.7 ± 0.1c

γ -TOH (mg/d)0

30100

5.8 ± 0.5a

3.6 ± 0.5b

2.4 ± 0.3c

1.0 ± 0.1a

0.7 ± 0.0b

0.5 ± 0.0c

1.4 ± 0.1a

1.0 ± 0.1b

0.7 ± 0.0c

1.1 ± 0.1a

0.8 ± 0.1b

0.5 ± 0.0c

0.7 ± 0.1a

0.4 ± 0.0b

0.2 ± 0.0c

∗Numbers in the same column with different letter superscripts are significantly different by thepaired t test (P < 0.05).

Taken from Ref. 106.


and Strom105 showed that incubation of platelets with synthetic SRR-α-TOHdid not result in any significant uptake of α-TOH by the platelets. Plateletshave been shown to readily take up natural RRR-α-TOH and demonstrate acorresponding decrease in platelet aggregation, whereas the synthetic formwas poorly incorporated and did not affect the aggregation of platelets.109

More recent studies have also examined the efficacy of synthetic SRR-α-TOHon platelet function in healthy individuals with neither study showing anybeneficial effect.110,111

The effect of RRR-α-TOH on platelet aggregation appears to involveinhibition of protein kinase C within platelets and an increase of platelet-derived nitric oxide.11 Human studies have shown a reduction in ex vivoplatelet aggregation in subjects following supplementation with α-TOH112,113

and supplements containing high amounts of γ -TOH.114,115

The efficacy of vitamin E supplements in preventing platelet activationmay depend on many factors, including baseline TOH levels, the presenceof co-antioxidants within platelets or the plasma, and whether natural or syn-thetic forms are used. The prevention of arachidonic acid-mediated α-TOHoxidation within platelets can be reversed by co-incubation with ascorbic acidor glutathione.116 This may have implications because RRR-α-TOH in com-bination with ascorbic acid has been shown to inhibit atherosclerotic diseaseprogression in subjects with hypercholesterolemia,117 and platelet activationmay be important in this patient population.118

Any potential benefit can also be complicated by patient adherence toconventional drug treatments, such as aspirin and statins, known to effect af-fect platelet function. We have examined the effects of 500 mg of RRR-α-TOHand 500 mg of a mixed TOH supplement on markers of platelet activationin well-controlled diabetic subjects, many of whom were taking conventionaltreatments. We observed no benefit in relation to platelet function in spiteof the significant increase in platelet TOH concentrations.119 Thus, it is un-likely that TOH treatment alone will gain acceptance as a viable strategy toinhibit platelet function. Further studies are required to determine if naturalTOHs supplements, in combination with other agents such as ascorbic acid,can significantly inhibit platelet aggregation in vivo.

VII. REPORTING VITAMIN E CONCENTRATIONS

As plasma lipid concentrations influence plasma TOH levels, it has beenrecommended that the concentration of α-TOH be expressed per mg oflipid.120 In a study of 85 alcoholic patients, the ratio of TOH to choles-terol plus triglycerides was the best tool for identifying deficiency (sensitiv-ity 95%, specificity 99%).121 Patients with liver disease, for example, mayhave elevated lipid concentrations, but a deficiency in vitamin E may bemissed if the lipid concentrations are not taken into account. Conversely, pa-tients with low lipid concentrations, for example, with heterozygous familial


hypobetalipoproteinemia, may be classified as vitamin E deficient when theyare not.121

VIII. VITAMIN E AND ATHEROSCLEROSIS

In 1991 the National Heart, Blood and Lung Institute (NHBLI) con-vened a workshop to review current knowledge about the oxidative modi-fication hypothesis of lipoproteins and their implication in the pathogen-esis of atherosclerosis.122 It was thought that the use of naturally occur-ring antioxidants in clinical trials for the prevention of atherosclerosis wassafe. Since then a number of human trials have been conducted. A meta-analysis of the effects of high-dose versus low-dose vitamin E supplemen-tation on cardiovascular mortality found that, for observational studies,the test for overall effect favored high vitamin E intake [odds ratio 0.67(0.54–0.83)]. In contrast, the analysis from six major intervention trials wereequivocal.123

The results from human trials of vitamin E supplementation have beencontroversial. It has been suggested that such studies would have benefitedfrom the measurement of markers of in vivo lipid peroxidation, like F2-isoprostanes, to establish any effect on oxidative stress.62 The different dosesand forms of TOH given to different populations might, in part, explain theparadoxical results obtained from these trials. In countries where a “Mediter-ranean diet” is consumed, a protective effect against atherosclerosis has beenshown.124 The population in the Gruppo Italiano per lo Studio della Soprav-vivenza nell’Infarto miocardico (GISSI-Prevenzione trial),125 which typicallyconsumed a Mediterranean-style diet rich in antioxidants, still developedCVD and may not have benefited from the 300 IU of synthetic vitamin Egiven.61 A subsequent follow-up of the participants in the GISSI-Prevenzionetrial revealed that treatment with vitamin E led to a 50% increase in conges-tive heart failure in subjects with left ventricular dysfunction.24

Probably the best known “‘negative” trial for vitamin E supplementationwas the Heart Outcomes Prevention Evaluation Study (HOPE).126 In thistrial, 772 of the 4,761 patients who were at high risk for CVD received 400IU of natural source vitamin E for a mean follow-up of 4.5 years. Therewas no significant difference in the incidence of secondary cardiovascularoutcomes or in death from any cause and no significant adverse effects ofvitamin E supplementation. In a similar fashion to the GISSI-Prevenzionetrial, a subsequent follow-up study revealed an increase in the risk for heartfailure with vitamin E treatment.23

The Secondary Prevention with Antioxidants of Cardiovascular Diseasein Endstage Renal Disease (SPACE) trial127 in hemodialysis patients showedpositive effects with vitamin E supplementation (800 IU of natural RRR-α-TOH) in a patient group that was probably under considerable oxidativestress.128,129 These patients were also given a number of other antioxidants


as supplements, with 40% of all participants taking vitamin C.127 It has beensuggested that vitamin C in combination with vitamin E may in fact explainthe positive results.130,131

The Cambridge Heart Antioxidant Study (CHAOS)132 examined 2,002patients with angiographically proven coronary atherosclerosis. Subjects whowere given either 800 IU (546 subjects) or 400 IU (489 subjects) of nat-ural α-TOH had a 77% decrease in non-fatal myocardial infarction; how-ever, there was a non-significant increase in cardiovascular deaths in thesubjects receiving α-TOH. It was suggested that the apparent rise in fa-tal myocardial infarction could be related to transition-ion release fromunstable plaques, with α-TOH potentially acting as a pro-oxidant in thissetting.61

The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP)trial looked at the progression of carotid atherosclerosis in smoking and non-smoking men and post-menopausal women over a 3-year period.133 A total of520 patients were given, twice daily, 91 mg (136 IU) RRR-α-TOH, 250 mg slow-release vitamin C, a combination of the two, or placebo. The most significantfinding was that, in men taking the combination of the two, the proportionwho experienced progression was reduced by 74% (95% confidence interval36–89%) compared to placebo.133

The largest trial conducted to examine the effects of statin and antiox-idant therapy involved over 20,500 subjects with a variety of clinical condi-tions and examined prolonged use (>5 years) of simvastatin 40 mg with a“cocktail” of antioxidant vitamins (650 mg synthetic vitamin E, 250 mg vi-tamin C and 20 mg β-carotene).134 The results provide positive results forstatin therapy but there was no effect for the antioxidants used; however,they concluded that this ‘cocktail’ does not cause harm.134 This is in con-trast to a previous trial in which simvastatin, niacin and antioxidant vitaminswere given in combination to determine any clinical benefit.135 Antioxidantuse significantly impaired the benefits obtained from niacin and simvastatinwhen used concurrently; the protective increase in HDL2 with simvastatinplus niacin was attenuated by simultaneous therapy with antioxidants. Theuse of antioxidant vitamins for the treatment of CVD has been questionedfollowing these results and the predominantly negative results from largeclinical trials.135,136

IX. VITAMIN E AND PRE-ECLAMPSIA

Pre-eclampsia is a disorder that affects between 2 to 3% of pregnanciesand is estimated to cause ∼ 60,000 deaths worldwide. It is characterizedby high blood pressure and the presence of proteinuria and generally oc-curs in the second half of pregnancy. This condition involves a maternalinflammatory response, activation of maternal vascular endothelial cells, en-dothelial dysfunction, and leucocyte activation. Because oxidative stress has


been implicated in the pathogenesis of pre-eclampsia, there is considerableinterest in the potential for antioxidants to prevent this condition.137

A recent placebo-controlled randomized trial examined the potentialfor vitamin C (1,000 mg/day) and vitamin E (RRR-α-TOH 400 IU/day)to prevent pre-eclampsia in women with a variety of risk factors.138 Thestudy involved 2,395 subjects from 25 hospitals (1,196 in the vitamin groupand 1,199 in the placebo group) and each were treated daily from thesecond trimester until birth. The primary end point was pre-eclampsiaand the main secondary endpoints were low birth weight (<2.5 kg) andsmall size for gestational age. There was no difference for the primary out-come of pre-eclampsia; however, there was a reduction in the birth weightof babies whose mothers took the antioxidant treatment versus those onplacebo.138

A subsequent study conducted in Australia examined the potential ben-efit with the same doses of vitamin E and C to prevent pre-eclampsia innulliparous women.139 This study found no effect on the occurrence of pre-eclampsia or low birth weight, but other adverse outcomes were common inthe treatment group, including increased gestational hypertension, severegestational hypertension, antenatal hospitalization for hypertension, the useof antihypertensive agents, and the induction of labor for hypertension.139

Taken together, these findings suggest that the use of antioxidants for theprevention of pre-eclampsia is not warranted and may in fact be harmful.

A recent study has reported benefit with vitamin E and C used incombination to reduce blood pressure and oxidative stress in untreatedhypertensives.77 However, because of recent analyses suggesting potentialadverse consequences with the use of α-TOH in disease prevention in high-risk populations22,23 and our own data showing an increase in hypertensionfollowing supplementation with vitamin E in diabetic subjects taking otherdrugs,140 further studies must be performed to ascertain the safety of theagents, 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 themetabolism of drugs has been emphasized recently.141 The estimated num-ber of deaths annually in the USA due to adverse drug reactions is thought tonumber at least 100,000.142 The CYP3A isoforms are probably the most im-portant of all the drug-metabolizing enzymes in humans as they metabolize∼ 50% of all drug oxidations,141 and high quantities of this enzyme are foundin both the liver (29% of total) and the intestine (70% of total)143 CYP3A4 isthe most studied form of CYP3A in humans and is the most abundant isoformfound in both the liver and the intestine.144


The induction of CYP3A4 in humans through activation of the pregnaneX receptor (PXR) has been well described.141 Examples of drugs that induceCYP3A4 expression through this pathway include rifampicin and St. John’swort (hyperforin); such induction can lead to increased metabolism of otherdrugs, including calcium-channel blockers and HIV-protease inhibitors.141

The induction of CYP3A4 in primary human hepatocytes through activationof PXR has been observed following incubation with St. John’s wort extractfor 30 hours.145 A 14-day course of St. John’s wort extract induced the activityof CYP3A4 measured through the pharmacokinetics of alprazolam in humanvolunteers.146 It has been hypothesized that α-TOH can interfere with drugmetabolism by increasing the expression of CYP3A4 within the liver andthereby increasing the metabolism of certain drugs.147

B. CYP3A and Vitamin E

Studies have been performed to examine the capacity for α-TOH toincrease CYP3A expression in vivo in a mouse model. A 2- to 3-fold in-crease in CYP3a11 mRNA (murine equivalent to human CYP3A4) followingα-TOH (20 mg/kg) for three months has been reported.148 Importantly,γ -tocotrienol did not induce CYP3a11 mRNA. In mice given high doseγ -TOH supplementation for five weeks, the increase in CYP3a protein con-centrations correlated with hepatic α-TOH, but not with hepatic γ -TOHconcentrations.149

A number of different forms of vitamin E have been shown to activategene expression through activation of human PXR in HepG2 (a human hep-atoma cell line) cells in culture; RRR-α-TOH was able to increase PXR activity2- to 3-fold following incubation for 48 hours, but activation was higher withrifampicin (a known inducer) and also with α- and γ -tocotrienol.150

A recent review of these and other studies suggested that high-dose α-TOH supplementation may interfere with drug metabolism through activa-tion of CYP3A4, whereas γ -TOH and the tocotrienols may not, because ofincreased metabolism and excretion of these compounds in the liver.147

Subcutaneous α-TOH injections in rats caused a significant increase inliver α-TOH concentrations at day 18 with a concomitant increase in livermicrosomal CYP3A protein and P-glycoprotein (MDR1, multidrug resistantprotein 1).151 While this study did not relate this effect to drug metabolismdirectly, it suggested that α-TOH may increase CYP3A in humans and maypotentially affect the metabolism of certain drugs.151 Another considerationis the differential effects of vitamin E forms on PXR-mediated drug trans-porters in different tissues. For example, rats given subcutaneous α-TOHinjections showed significant increases in MDR1, but there was no effect onCYP3A protein expression in lung tissue.152

The use of combined antioxidants, RRR-α-TOH and vitamin C, in hu-mans showed a significant reduction in cyclosporin trough levels.153−155


However it was unclear which antioxidant had the effect or which drug me-tabolizing pathway was affected, because cyclosporine metabolized by bothCYP3A and MDR1.156

Clearly any effect of vitamin E on these enzymes in human tissues in vivoneeds to be determined. Studies in animals often demonstrate a differentresponse with known inducers of PXR when compared to humans, becauseof differences in the amino acid sequences in the ligand binding domain forthis receptor.157

Importantly, it takes two to three weeks for steady-state levels of CYP3Ato 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 animportant consideration in the design of studies examining potential inter-actions following induction of CYP3A4 or using vitamin E in the preventionof disease. Whether any forms of vitamin E can induce CYP3A4 or MDR1 inhumans in vivo has not yet been determined.

C. PXR Activation and Hormone Synthesis

Activation of PXR in humans may have undesirable effects in some pop-ulations. A recent study examined the effects of altered xenobiotic receptoractivity on adrenal steroid homeostasis in transgenic mice that had liver-specific expression of activated human PXR.158 The observed increase incorticosterone and aldosterone output caused disrupted circadian rhythmand increased expression of steroidogenic enzymes involved in the produc-tion of these steroids.158 Any effect of vitamin E on PXR activation mayaffect hormone synthesis pathways in humans and needs to be consideredwhen designing clinical trials. The α-TOH, beta-Carotene Prevention Study(ATBC) study examined the effect of long-term α-TOH supplementation(50 mg SRR-α-TOH acetate/day for five to eight years) on prostate cancerincidence and demonstrated a 32% reduction compared to placebo.159 Afollow-up study of 200 men participating in the ATBC trial showed a signifi-cant reduction in both androstenedione and testosterone in the TOH groupcompared to placebo.160 There was a significant negative correlation betweenserum α-TOH and androgens in this group of men.160 The investigators sug-gested that the reduction in hormone production is one mechanism by whichTOHs may reduce the incidence and mortality from prostate cancer. A recentpublication from the ATBC study found that high serum α-TOH concentra-tions were associated with a reduced risk of prostate cancer.161 Studies of theassociation between serum α-TOH concentrations and all-cause mortalitydemonstrated a significant reduction in risk, which was greater with increas-ing concentrations of α-TOH; however no additional benefit was gainedbeyond 13–14 mg/l (30 µmol/l).161 Whether vitamin E supplementationcan alter the production of hormones in vivo across different populations


remains an important question for further investigation in relation to bothpotential benefits and possible adverse effects.

XI. VITAMIN E SUPPLEMENTATION AND ALL-CAUSE

MORTALITY

Studies have investigated the metabolism and efficacy of α-TOH in theprevention of sequelae associated with CVD. Some have shown promisingresults in secondary prevention in conditions associated with oxidative stress.However, the results from large primary prevention clinical trials with α-TOHhave been largely negative.162

A meta-analysis of >130,000 participants from 19 clinical trials across awide range of vitamin E intake (16.5 IU to 2,000 IU) concluded that high-dose vitamin E supplements (≥400 IU/day as α-TOH) increase all-causemortality.22 This conclusion was challenged in a subsequent analysis163 thatsuggested that the adverse effect on mortality was only significant at dosesabove 2,000 IU/day, a dose much higher than the recommended upperlimit of 1,600 IU/day.21 This conclusion is in contrast to recent data on thelong-term effects of lower-dose α-TOH supplementation in subjects fromthe HOPE23 and GISSI-Prevenzione trials24 in which subjects received 400IU/day with a seven-year follow-up and 300 mg/day with a 3.5 year follow-up, respectively. These studies reported an increase in heart failure in theHOPE trial and heart failure in subjects with left ventricular dysfunction inthe GISSI-prevenzione trial. The relationship between antioxidant vitaminsand blood pressure was examined as a part of the Third National Healthand Nutrition Examination Survey, which showed a higher odds ratio forhypertension in subjects with higher serum vitamin E concentrations afteradjustment for a number of variables, including age, sex, race, diabetes,BMI, and dietary sodium intake.164 The relationship between α-TOH sup-plementation and blood pressure is supported by our own findings that showthat RRR-α-TOH (750 IU/d) supplementation for six weeks increased sys-tolic and diastolic blood pressure in well-treated diabetic subjects.140 Themechanism(s) for any adverse effect of a high dose of α-TOH is unknown.One explanation is that α-TOH may affect drug metabolism and therebythe efficacy of drugs used to treat subjects at risk of sequelae associated withCVD.

The most comprehensive analysis to date of studies of antioxidant sup-plementation for primary and secondary prevention of disease included68 randomized trials with 232,606 participants.136 This study on 26 tri-als on vitamin E, in which vitamin E was given either alone or in com-bination (following exclusion of trials with a high bias risk or use ofselenium), included 105,065 participants and demonstrated an increasein relative risk of all-cause mortality compared to placebo.136 Whetherthis increased risk is also present in healthy populations has not been


determined, and it is clear that this remains an important question for futureresearch.

XII. CONCLUSION

A normal diet contains both TOHs and tocotrienols along with a numberof other co-antioxidants and polyphenols. These may, in turn, confound anystudies that look at individual supplements to prevent disease. Because of thedisappointing results from clinical trials, and if one accepts that the primaryrole of α-TOH in preventing atherosclerosis is via the inhibition of in vivolipid peroxidation, then supplementation of α-TOH in healthy individualsto prevent atherosclerosis may not be warranted. However, α-TOH is clearlyrequired by individuals with clinical deficiency in the vitamin, such as thosewith abetaliproteinemia, who require supplementation to maintain normalneurological function. However, the supposition that antioxidants are safein all populations has not been substantiated.

As the roles of the different isomers of vitamin E in health and disease arefurther investigated, it will be important to continue to examine its effectson different cells within the body. Until the full importance of γ -TOH is es-tablished and the precise role of α-TOH has been elucidated, confusion willremain as to the correct amount of each form of vitamin E needed to meetthe nutritional requirements of humans. Further studies are needed to deter-mine if significant clinical drug interactions result from co-supplementationwith vitamin E.

The observation that individuals with a higher baseline serum vitamin Econcentration have a reduced risk of all-cause mortality165 probably reflectsthe importance of adequate dietary TOH intake. Data from the US popula-tion suggest that only 8.0% of men and 2.4% of women meet the estimated av-erage requirement (12 mg) for α-TOH intake,2 and it has been suggested thatthis 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 in-dividuals may have either too much, or too little, vitamin E intake, and bothcases may be deleterious to health.

The question of whether an appropriate dose of one or more formsof vitamin E, given singularly or with other compounds, has a role in theprevention and treatment of disease remains to be answered in future clinicaltrials examining supplementation of TOHs in humans.

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