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  • i

    Vitamin E Metabolism in Humans

    Michael William Clarke

    Bachelor of Science (Medical Science)

    This thesis is presented for the degree of

    Doctor of Philosophy of the University of

    Western Australia

    School of Medicine and Pharmacology

    2008

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    ABSTRACT

    Vitamin E is comprised of a family of tocopherols (TOH) and tocotrienols. The most

    studied of these is α-tocopherol (α-TOH), as this form is retained within the body and

    any deficiency of vitamin E is corrected with this supplement. α-TOH is a lipid-soluble

    antioxidant required for the preservation of cell membranes and potentially acts as a

    defense against oxidative stress. Individuals who have a primary vitamin E deficiency

    such as low birth weight infants, secondary vitamin E deficiency due to fat

    malabsorption such as in abetalipoproteinaemia, or a genetic defect in TOH transport

    require supplementation. There is debate as to whether vitamin E supplementation in

    other patient groups is required.

    Vitamin E supplementation has been recommended for persons with FHBL, a

    rare disorder of lipoprotein metabolism that leads to low serum α-TOH and decreased

    LDL cholesterol and apolipoprotein B concentrations. We examined the effect of

    truncated apoB variants on vitamin E metabolism and oxidative stress in persons with

    heterozygous FHBL. We used HPLC with electrochemical detection to measure α- and

    -TOH in serum, erythrocytes, and platelets, and GC-MS to measure urinary F2-

    isoprostanes and TOH metabolites as markers of oxidative stress and TOH intake,

    respectively. Erythrocyte α-TOH was decreased, but we observed no differences in

    lipid-adjusted serum TOHs, erythrocyte -TOH, platelet α- or -TOH, urinary F2-

    isoprostanes, or TOH metabolites. Taken together, our findings do not support the

    recommendation that persons with heterozygous FHBL should receive vitamin E

    supplementation.

    Supplementation with vitamin E is postulated to protect against cardiovascular

    disease (CVD) through its antioxidant activity, prevention of lipoprotein oxidation and

    inhibition of platelet aggregation. While some studies have demonstrated a potential

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    benefit of vitamin E on platelet function, -TOH, a major dietary form of vitamin E,

    may have protective properties that differ from those of α-TOH. Individuals with well

    controlled type 2 diabetes mellitus were given a supplement high in γ-TOH (60% -

    TOH) and compared with α-TOH supplementation and placebo. We measured serum

    and cellular TOH concentrations, markers of platelet function and soluble CD40 ligand

    (sCD40L) before and after the six week intervention. As expected, serum and cellular

    -TOH concentrations increased significantly with both TOH treatments. In contrast,

    supplementation with -TOH led to a decrease in both serum and cellular -TOH, while

    supplementation with mixed TOHs increased both  and -TOH. We did not observe

    an effect of either treatment on biomarkers of in-vivo platelet function. Taken together,

    our findings suggest TOH supplements do not inhibit platelet function or sCD40L

    release in type 2 diabetics. However, mixed TOH treatment results in high

    concentrations of both  and -TOH in serum and cells without the reduction in -TOH

    seen with high dose -TOH supplementation alone.

    About 50% of clinically relevant drug oxidations are mediated by the cytochrome

    (CYP) P4503A family, with the CYP3A4 accounting for most of this activity.

    Induction of CYP3A4 by vitamin E could lead to an increase in drug metabolism, by

    lowering the efficacy of any drug metabolised by this cytochrome. We hypothesised

    that up-regulation of CYP3A4 by α-TOH in the liver would decrease the concentration

    of midazolam in the plasma, a known CYP3A4 substrate. Healthy subjects were given

    an intravenous bolus (1 mg) of midazolam before and after treatment with α-TOH or

    placebo for a three (750 IU) and six week (1500 IU) period. Serum TOHs were

    measured by HPLC with electrochemical detection and plasma midazolam and urine

    TOH metabolites measured by GCMS. Serum α-TOH concentrations increased by

    100% and urinary α-TOH metabolite excretion increased 20-fold in the treatment

    groups compared with placebo. However, there was no effect on the midazolam area

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    under the curve in subjects taking α-TOH compared with placebo. Taken together,

    these findings refute the hypothesis that α-TOH supplementation (at the doses tested)

    interferes with hepatic CYP3A4 mediated drug metabolism in healthy subjects.

    Sesame lignans are natural components of sesame seed oil and there is evidence

    that these lignans can inhibit CYP450 enzymes, in particular, those responsible for

    vitamin E metabolism. We hypothesised that sesame seed ingestion would increase

    serum γ-TOH, lower plasma lipids and inhibit platelet function in human subjects with

    at least one cardiovascular risk factor. We used HPLC with electrochemical detection

    to measure α- and -TOH in serum and GC-MS to measure F2-isoprostanes and TOH

    metabolites as markers of oxidative stress and TOH intake, respectively. We used high-

    sensitive C-reactive protein as a measure of systemic inflammation. Platelet function

    was assessed using the PFA-100 platelet aggregation assay. Although serum -TOH

    increased by 17%, we observed no effect on lipid metabolism, markers of inflammation,

    oxidative stress or platelet function following treatment with ~25 g/day sesame seeds

    for five weeks. Our findings challenge the hypothesis that sesame seed ingestion

    provides beneficial cardiovascular effects.

    In summary, we have studied the metabolism and transport of both α- and γ-TOH in

    humans to evaluate the requirements for supplementation and the effects of vitamin E

    on platelet function and CYP3A4 activity. Specialised techniques using HPLC were

    developed to measure serum and cellular TOH concentrations both in supplemented and

    un-supplemented individuals. We also used GCMS to provide a sensitive, accurate

    assessment of TOH metabolites and midazolam pharmacokinetics in humans after

    vitamin E supplementation. We have examined the role vitamin E has on important

    biochemical endpoints, with emphasis on the implications for TOH supplementation in

    subjects at risk of CVD.

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    CONTENTS

    Page

    TITLE PAGE …………………………………………………………………………….. i

    ABSTRACT ……………………………………………………………………................ii

    TABLE OF CONTENTS ………………………………………………………………....v

    ACKNOWLEDGEMENTS ………………………………………………………….....xiii

    PERSONAL CONTRIBUTION OF THE AUTHOR …………………………………..xiv

    LIST OF TABLES ………………………………………………………………………xv

    LIST OF FIGURES ………………………………………………………………….....xvii

    ABBREVIATIONS ………………………………………………………………...........xx

    CHAPTER 1: INTRODUCTION

    1.1 Introduction ……………………………………………………………….............1

    1.2 Vitamin E Transport in Humans and Animal Models .........……………………...4

    1.2.1 Structure and Properties of Vitamin E Isomers ……………………..........4

    1.2.2 Intestinal Absorption of Vitamin E and Postprandial Metabolism ….........5

    1.2.3 Hepatic Metabolism of Vitamin E …………………………………..........6

    1.2.4 TOH Transfer Proteins ...............................................................................8

    1.2.5 TOH Transfer Protein Deficiency …………………………………….....11

    1.3 Vitamin E and Oxidative Stress ………………………………………………....13

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    Page

    1.4 TOH Metabolites …………………………………………………………..........17

    1.4.1 α-TOH ……………………………………………………………….…..17

    1.4.2 -TOH and its major metabolite -CEHC …………………………….....20

    1.5 Vitamin E Distribution in Cells ………………………………………………....22

    1.5.1 Erythrocyte Vitamin E ..............................................................................22

    1.5.2 Platelet Vitamin E ……………………………………………………….23

    1.6 Vitamin E and Platelet Function ………………………………………………...24

    1.7 Reporting Vitamin E Concentrations ……………………………………...........26

    1.8 Vitamin E and Atherosclerosis ………………………………………………….27

    1.9 Vitamin E and Pre-Eclampsia …………………………………………