Vitamin E Metabolism in Humans - UWA Research …...i Vitamin E Metabolism in Humans Michael William...

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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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Page 1: Vitamin E Metabolism in Humans - UWA Research …...i Vitamin E Metabolism in Humans Michael William Clarke Bachelor of Science (Medical Science) This thesis is presented for the degree

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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 …………………………………………………...29

1.10 Vitamin E and Drug Metabolism ………………………………………………..31

1.10.1 Cytochrome P450 Enzyme Activity …………………………….............31

1.10.2 CYP3A and Vitamin E …………………………………………………..31

1.10.3 PXR Activation and Hormone Synthesis ………………………………..33

1.11 Vitamin E Supplementation and All-cause Mortality …………………………...34

1.12 Conclusion …………………………………………………………………........35

1.13 References ……………………………………………………………………….37

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Page

CHAPTER 2: TOH METABOLISM AND OXIDATIVE STRESS IN FAMILIAL

HYPOBETALIPOPROTEINAEMIA

2.1 Introduction ……………………………………………………………………. 58

2.2 Subjects and Methods …………………………………………………………. 60

2.2.1 Subjects …………………………………………………………………60

2.2.2 Platelet Preparation ……………………………………………………. 60

2.2.3 Erythrocyte Preparation …………………………………………….. …61

2.2.4 HPLC Analysis of TOHs ……………………………………………… 61

2.2.5 GC-MS Analysis of TOH Metabolites …………………………………63

2.2.6 F2-isoprostanes ………………………………………………………… 64

2.2.7 Lipid Analysis …………………………………………………………. 64

2.2.8 FPLC Analysis of lipoproteins ………………………………………… 64

2.2.9 Statistical Analysis …………………………………………………….. 65

2.3 Results …………………………………………………………………………. 66

2.4 Discussion ………………………………………………………………………72

2.5 References ………………………………………………………………………76

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CHAPTER: 3 EFFECT OF MIXED TOHs ON SERUM γ-TOH, BLOOD CELL

γ-TOH AND BIOMARKERS OF PLATELET ACTIVATION IN SUBJECTS

WITH TYPE 2 DIABETES

3.1 Introduction …………………………………………………………………….. 80

3.2 Subjects and Methods …………………………………………………………...81

3.2.1 Study Protocol ………………………………………………………….. 81

3.2.2 Sample Preparation ……………………………………………………...83

3.2.3 HPLC analysis of TOHs with Electrochemical Detection ........................84

3.2.4 Urinary TOH Metabolites .........................................................................84

3.2.5 ELISA Assays ...........................................................................................84

3.2.6 Lipid Analysis ...........................................................................................84

3.2.7 Statistical Analysis ....................................................................................85

3.3 Results ...................................................................................................................85

3.3.1 Serum and Cellular TOH Analysis ...........................................................85

3.3.2 Urinary TOH Metabolite Excretion ..........................................................86

3.3.3 Markers of Platelet and Endothelial Function ..........................................91

3.4 Discussion .............................................................................................................91

3.5 References .............................................................................................................96

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CHAPTER 4: VITAMIN E SUPPLEMENTATION AND DRUG METABOLISM IN

HUMANS

4.1 Introduction .........................................................................................................103

4.1.1 Vitamin E, Drug Metabolism and PXR ..................................................103

4.1.2 Assessment of PXR mediated CYP3A4 activity ....................................105

4.2 Subjects and Methods .........................................................................................107

4.2.1 Subjects ...................................................................................................107

4.2.2 Study Protocol .........................................................................................107

4.2.3 Midazolam Area Under Curve (AUC) ....................................................108

4.2.4 Power Calculations to Determine Minimum Subject Numbers ..............109

4.2.5 Materials ..................................................................................................110

4.2.6 Midazolam Measurement using GCMS ..................................................110

4.2.7 Serum TOH and Urine TOH Metabolites ...............................................111

4.2.8 Statistics ..................................................................................................112

4.3 Results .................................................................................................................113

4.3.1 Serum α and γ-TOH Concentrations .......................................................113

4.3.2 Urine TOH Metabolites ..........................................................................113

4.3.3 Effect of TOH Supplementation on Midazolam AUC ...........................113

4.3.4 Hydroxymidazolam AUC .......................................................................113

Page

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4.4 Discussion ...........................................................................................................117

4.4.1 CYP3A4 and α-TOH Metabolism ..........................................................117

4.4.2 PXR activation by Vitamin E in Human cell Cultures ...........................118

4.4.3 Effect of Vitamin E on CYP450 mediated Drug Metabolism

in Animals ...............................................................................................118

4.4.4 Vitamin E and Drug Metabolism in Humans .........................................119

4.4.5 PXR and NF-κB ......................................................................................121

4.5 Conclusion ..........................................................................................................122

4.6 References ...........................................................................................................123

CHAPTER 5: THE EFFECTS OF SESAME INGESTION ON TOH METABOLISM AND

PLATELET FUNCTION IN HUMAN SUBJECTS WITH CHARACTERSTICS OF THE

METABOLIC SYNDROME

5.1 Introduction .........................................................................................................131

5.1.1 -TOH and its Metabolite γ-CEHC .........................................................132

5.1.2 Sesame Ingestion, TOH Metabolism and Risk Factors for

Cardiovascular Disease............................................................................133

5.2 Subjects and Study Design.......................................................................134

5.2.1 Subjects ...................................................................................................134

5.2.2 Study Design ...........................................................................................135

Page

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5.2.3 Power Calculation ...................................................................................136

5.2.4 Supplement Composition ........................................................................137

5.3 Methods ...............................................................................................................139

5.3.1 HPLC Analysis of TOHs with Electrochemical Detection .....................139

5.3.2 Plasma F2-isoprostanes ...........................................................................139

5.3.3 Urinary TOH Metabolites .......................................................................139

5.3.4 Elisa Assays ............................................................................................139

5.3.5 Platelet PFA 100 Analysis ......................................................................139

5.3.6 Routine Biochemistry .............................................................................140

5.3.7 Statistical Analysis ..................................................................................140

5.4 Results .................................................................................................................141

5.4.1 Serum TOH and Urine CEHC Concentrations .......................................141

5.4.2 Subject food intake during study period .................................................141

5.4.3 Effects of sesame on body weight, and biochemical parameters ............145

5.4.4 Effect of treatment on platelet and endothelial function .........................145

5.4.5 Effect of treatment on systemic inflammation and oxidative stress .......147

5.5 Discussion ...........................................................................................................148

5.5.1 Sesame and TOH metabolism .................................................................148

5.5.2 Sesame, lipid metabolism, inflammation and oxidative stress ...............149

Page

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5.5.3 Sesame, γ-TOH, platelet and endothelial function .................................150

5.6 Conclusion ..........................................................................................................152

5.7 References ...........................................................................................................153

CHAPTER 6: CONCLUSION AND FUTURE DIRECTIONS

6.1 Summary .............................................................................................................160

6.2 Conclusion ..........................................................................................................161

6.3 Future Directions ................................................................................................162

PERMISSIONS

CURRICULUM VITAE

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ACKNOWLEDGEMENTS

I would like to dedicate this thesis to my family. In particular, my wife Maria for her

support (both intellectual and emotional), and my two children Nadine and Annalise

(for making me smile even when things were difficult). I also wish to thank my

immediate family namely my mother May, father Laurence, brother Andrew and sister

Lorna. Their support of me has kept me positive over the years and I am extremely

grateful to them. I also wish to thank Lucy and Joe Ruscitto for their support.

I would also like to acknowledge the support of my supervisors, namely Professor

Kevin Croft and Clinical Professor John Burnett. These gentlemen have given me the

opportunity to push myself and complete this body of work. They also provided the

right balance with plenty of encouragement, sound scientific advice and without them

this work would not have been possible.

I wish to also thank a number of colleagues and friends who have supported my

studies and provided much needed scientific input into this work. Many thanks to

Sandy Musk, Mario Taranto, Richard Scarff and all of the staff in Special Chemistry at

Royal Perth Hospital (RPH) for their assistance and kind words. Finally, I would like to

thank a number of individuals from the University Department of Medicine at RPH for

their help namely; Noelene Atkins, Dr Jason Wu, Dr Natalie Ward, Professor Ian

Puddey and all of the staff on Level 3 and 4 for helping me at various stages. Special

thanks to Associate Professor Thomas Ledowski for his considerable input into the

Vitamin E and Drug Metabolism study.

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PERSONAL CONTRIBUTION OF THE AUTHOR

The author was involved in the design and implementation of all of these studies in

collaboration with the other investigators. These studies were supported by grants from

the Royal Perth Hospital (RPH) Medical Research Foundation, Raine Medical

Research Foundation, National Health & Medical Research Council, and National

Heart Foundation of Australia. We wish to thank Cognis for providing the TOHs and

Cardinal Health Australia for encapsulating the TOH supplements. The measurement

of urine TOH metabolites in all studies was performed by Dr Jason Wu and the

measurement of urine F2-isoprostanes was performed by Dr Henrietta Headlam. Dr J

Swanson provided the deuterium labeled TOH metabolite standards. The studies from

chapters 3 and 5 were coordinated by Dr Natalie Ward and Dr Jason Wu respectively.

Jim Thom and staff at the Hematology Department at RPH performed the PFA-100

assay of platelet activation. The author performed all of the tocopherol assays, FPLC

assays, midazolam and the majority of the ELISA assays described in these chapters

and designed and coordinated the study in chapter 4.

Signatures

Student: Michael William Clarke __________________________________

Supervisor: Professor Kevin D. Croft _______________________________

Co-supervisor: Clinical Professor John R. Burnett _____________________

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TABLES

Page

Chapter 1

Table 1. The structures and biological activities of the eight naturally

occurring forms of vitamin E ……………………………………………… 2

Table 2. Comparison of α and γ-TOH concentrations in different tissues

between humans and rodents ……………………………………………... 5

Table 3. Relative affinities of various TOH analogues for the α-TTP ……………... 11

Table 4. Correlation matrix obtained comparing α and γ-TOH from plasma

and platelets with plasma lipids …………………………………………… 24

Table 5. α- and γ-TOH contents (n = 20) of plasma, red blood cells,

platelets, and lymphocytes of human subjects supplemented with

0, 30 and 100 mg/d of dl-α-tocopheryl acetate ……………………………. 25

Chapter 2

Table 1. Characteristics for the normal and heterozygous FHBL subjects ………… 61

Table 2. TOH, TOH metabolite and F2-isoprostane concentrations in normal

and heterozygous FHBL subjects ………………………………………… 66

Table 3. Lipid, apoprotein, TOH, TOH metabolite, and F2-isoprostane

concentrations in homozygous FHBL and ABL subjects …………………71

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Page

Chapter 3

Table 1. Baseline characteristics for all subjects involved in the study

separated by treatment group ……………………………………………… 86

Table 2. Urinary excretion of TOH metabolites α, γ and δ – CEHC

before and after treatment ………………………………………………….. 87

Table 3. Effects of treatment on biomarkers of platelet function …………………… 92

Chapter 4

Table 1. Baseline characteristics for subjects for the TARDIS study ………………. 107

Chapter 5

Table 1. Demographic profile at baseline ……………………………………………137

Table 2. Composition of sesame and placebo bars …………………………………. 138

Table 3. Contents of nutrients in sesame and placebo bars ………………………… 138

Table 4. Macronutrient intake of volunteers per day during food bar

supplementation …………………………………………………………… 141

Table 5. Subjects body weight, plasma lipids, creatinine and -GT ………………... 146

Table 6. Platelet and endothelial function markers …………………………………. 146

Table 7. Serum inflammatory and oxidative stress biomarkers …………………….. 147

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FIGURES

Page

Chapter 1

Figure 1. TOH Transport in lipoproteins and cells ………………………………….. 7

Figure 2. Hepatocyte vitamin E transport and metabolism …………………………. 10

Figure 3. Pathways leading to the urinary excretion of α-TOH metabolites ……….. 19

Chapter 2

Figure 1a. FPLC chromatograms of a normal subject ………………………………. 67

Figure 1b. FPLC chromatograms from a heterozygous FHBL subject .…………….. 68

Figure 2. Blood film morphologies of human subjects with normal and abnormal

cell membranes, characterized by the presence of acanthocytes ………... 69

Chapter 3

Figure 1. Platelet-Derived Mediators of the Inflammatory Response ……………… 82

Figure 2. Post intervention serum concentrations of and -TOH,

corrected for baseline values ……………………………………………... 88

Figure 3. Post intervention red cell concentrations of and -TOH,

corrected for baseline values ……………………………………………... 89

Figure 4. Post intervention platelet concentrations (per 109 cells) of

and -TOH, corrected for baseline values ……………………………... 90

Chapter 4

Figure 1. Molecular mechanism of PXR action ……………………………………. 106

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Figure 2. Correlation between the 4 sample AUC and 2 sample AUC

used in this study …………………………………………………………. 110

Figure 3. A representative chromatogram from a human subject receiving

1mg I.V. midazolam ………………………………………………………112

Figure 4. Serum α and γ-TOH concentrations for visit 1 (baseline), visit 2

(following α-TOH, 750 IU for 3 weeks), visit 3 (following α-TOH,

1500 IU for 6 weeks) …………………………………………………….. 114

Figure 5. Urine α-CEHC concentrations for visit 1 (baseline), visit 2 (following

α-TOH, 750 IU for 3 weeks), visit 3 (following α-TOH, 1500 IU for

6 weeks) …………………………………………………………………. 115

Figure 6. Midazolam AUC separated by treatment group and visit 1

(baseline), visit 2 (following α-TOH, 750 IU for 3 weeks) and

visit 3 (following α-TOH, 1500 IU for 6 weeks) ……………………….. 116

Chapter 5

Figure 1. The chemical structures of 2 major lignans found in sesame seeds;

sesamin and sesamolin …………………………………………………. 134

Figure 2a. Serum α-TOH concentrations comparing treatment with sesame

versus placebo ………………………………………………………….. 142

Figure 2b. Serum γ-TOH concentrations comparing treatment with

sesame versus placebo …………………………………………………. 142

Figure 3a. Serum α-TOH concentrations corrected for plasma lipids

comparing treatment with sesame versus placebo ……………………... 143

Figure 3b. Serum γ-TOH concentrations corrected for plasma lipids

comparing treatment with sesame versus placebo ……………………... 143

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Figure 4a. α-CEHC urine metabolites comparing treatment with sesame

versus placebo …………………………………………………………. 144

Figure 4b. γ-CEHC urine metabolites comparing treatment with sesame

versus placebo …………………………………………………………. 144

Figure 4c. δ-CEHC urine metabolites comparing treatment with sesame versus

Placebo ………………………………………………………………… 145

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ABREVIATIONS

4-HNE 4-hydroxynonenal

AAPH 2,2'-azobis(2)-amidinopropane (a free radical generator)

ABCA1 ATP-binding cassette transporter A1

ABL Abetalipoproteinaemia

all-rac-α-TOH synthetic alpha tocopherol

AHA ATP-III American Heart Association Adult Treatment Panel III

apoB apolipoprotein B

apoE apolipoprotein E

apoE-/-

mouse apolipoprotein E double knock out mouse

ASAP Supplementation in Atherosclerosis Prevention (ASAP)

ATBC α-TOH, β-Carotene Prevention Study

AUC area under curve

AVED ataxia with vitamin E deficiency

BMI body mass index

BSTFA N, O-bis(trimethylsilyl)trifluoroacetamide

CHAOS Cambridge Heart Antioxidant Study (CHAOS) study

CHD coronary heart disease

CHOD-PAP (HiCo) total cholesterol assay (Roche)

COX-2 cyclooxygenase-2

CV coefficient of variation

CVD cardiovascular disease

CYP cytochrome P450 enzyme

Cyp3a11 murine equivalent to human CYP3A4

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d-α-tocopherol natural alpha tocopherol

d6-RRR-α-TOH deuterium labelled natural alpha tocopherol

DHA docosahexanoic acid

dl-α-TOH synthetic alpha tocopherol

DPB diastolic blood pressure

ECD electrochemical detection

EDTA ethylenediaminetetraacetic acid (chelating agent)

EPA eicosapentanoic acid

FHBL familial hypobetalipoproteinaemia

FIVE familial isolated vitamin E deficiency

FPLC fast performance liquid chromatography

FXN frataxin

GCMS gas chromatography mass spectrometry

GISSI Gruppo Italiano per lo Studio della Sopravvivenza

nell'Infarto miocardico (GISSI-Prevenzione trial)

GPO-PAP triglyceride assay (Roche)

HDL high density lipoprotein

HepG2 human hepatoma cell line

HOPE Heart Outcomes Prevention Evaluation Study (HOPE) trial

HPLC high performance liquid chromatography

hTAP1 human TOH-associated protein

HTGL hepatic triglyceride lipase

ICAM intracellular adhesion molecule

IL interleukin

LDL low density lipoprotein

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LOOH lipid hydroperoxides

LPL lipoprotein lipase

LS180 intestinal human adenocarcinoma cell line

MDM monocyte derived macrophage

MDR1 multidrug resistant protein 1

McARH777 rat hepatoma cell line

MCP-1 monocyte chemoattractant protein 1

MMP matrix metalloproteinase

mRNA messenger RNA

MRP multi-drug resistance associated protein

MTBSTFA N-Methyl-N- (Tert-Butyldimethylsilyl)trifluoroacetamide

(derivatization reagent)

MTP microsomal triglyceride transfer protein

NHANES National Health and Nutrition Examination Survey

NHBLI National Heart, Blood and Lung Institute

NF-B nuclear transcription factor-B

NO nitric oxide

NO2 nitrogen dioxide

OH- hydroxyl radical

PGE2 prostaglandin E2

PFA platelet function assay

PPAR peroxisome proliferator-activated receptor

PRP platelet-rich plasma

PSGL-1 P-selectin glycoprotein ligand 1 receptor

PXR pregnane X receptor

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RAW264 murine macrophage cell line

RNOS reactive nitrogen oxide species

RRR-α-TOH natural alpha tocopherol

RXR retinoic acid receptor

SD standard deviation

SEM standard error of mean

SIM selected ion monitoring

SPACE Prevention with Antioxidants of Cardiovascular Disease in

Endstage Renal Disease (SPACE) trial

sCD40L soluble CD40 ligand

SNP single nucleotide polymorphism

sP-selectin soluble P-selectin

SR-BI scavenger receptor class B type I

SREPB sterol regulatory element binding protein

SBP systolic blood pressure

TAP tocopherol associated protein

TBP tocopherol binding protein

TMCS trimethylchlorosilane

TMP tocopherol-mediated peroxidation

TO· tocopherol radical

TOH tocopherols

TNF tumour necrosis factor

TX thromboxane

Ubiquinol-10 (CoQ10H2) reduced form of coenzyme Q

Ubiquinone (CoQ10) coenzyme Q

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VCAM vascular-cell adhesion molecule

VE vitamin E forms

VLDL very low density lipoprotein

vWf von Willebrand factor

WHHL Watanabe heritable hyperlipidaemic

α-CEHC 2,5,7,8-tetramethyl-2(2'-carboxyethy)-6-hydroxychroman

(metabolite of α-tocopherol)

α-TQH2 α-tocopherol hydroquinone

α-TTP α-tocopherol transfer protein

δ-CEHC 2,8,-dimethyl-2-(2-carboxyethyl)-6-hydroxychroman

(metabolite of δ-tocopherol)

δ-TOH delta tocopherol

γ-CEHC 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hdroxychroman

(metabolite of gamma tocopherol)

-GT gamma-glutamyl transferase

γ-TOH gamma tocopherol

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* A version of this chapter has been accepted for publication in Critical Reviews in

Clinical Laboratory Sciences 2008.

1

CHAPTER 1

INTRODUCTION

1.1 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 (α, β, γ, δ) and four

tocotrienols (α, β, γ, δ). Of the eight natural forms of vitamin E, the most studied is α-

TOH. The name TOH comes from the Greek tocos (childbirth), phero (to bear) and ol

(alcohol).3 The term “vitamin E” refers to a group of eight structurally similar

compounds that demonstrate the biological activity of α-TOH to varying degrees (Table

1).4 The most recognised role for α-TOH is as a lipid-soluble antioxidant required for

the preservation of cell membranes, by reacting quickly with peroxyl radicals to

preserve polyunsaturated fatty acids.5 More recently, α-TOH has been implicated in the

activation of a number of genes.6

Primary vitamin E deficiency is generally only found in premature and low birth

weight infants. Secondary causes include fat malabsorption syndromes (e.g. cystic

fibrosis, chronic liver disease, abetalipoproteinaemia (ABL) and intestinal resection)

and some haematological disorders (e.g. β-thalassaemia major, sickle-cell anaemia and

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2

glucose-6-phosphate dehydrogenase deficiency).3 Patients with vitamin E deficiency

have abnormal erythrocyte membrane morphology due to oxidative stress and

Common Name Structure Activity based Compared to

on rat assay RRR- -TOH

d--TOH 1.49 100%

d--TOH 0.75 50%

d--TOH 0.15 10%

d--TOH0.05 3%

d--tocotrienol 0.75 50%

d--tocotrienol 0.08 5%

d--tocotrienol

d--tocotrienol

Not Known

Not Known

O

HO

O

O

HO

O

HO

HO

O

HO

O

HO

O

HO

O

HO

Table 1. The structures and biological activities of the eight naturally occurring

forms of vitamin E

Adapted from ref 4

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3

the characteristic 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 potential role as an

antiatherogenic supplement has received considerable attention in the last decade.

Moreover, a number of studies have also described a role for α-TOH beyond its

antioxidant function. α-TOH has been shown to inhibit smooth muscle cell

proliferation,9 endothelial dysfunction

10 and platelet aggregation

11 by a protein kinase-

C dependant mechanism. α-TOH has also been found to inhibit monocyte adhesion to

endothelial cells 12

and macrophage-mediated lipid peroxidation in-vitro.13

These

diverse functions of α-TOH and the potential pro-oxidant effects observed in some

studies 14

may account for the paradoxical results observed with human clinical trials in

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 generalisation 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

Given the conflicting data about

vitamin E supplementation, it is not surprising to see disagreement about the

recommended daily allowance set for α-TOH in humans and that -TOH has not yet

been included.18

19

20

A dietary intake of 15 mg α-TOH/day has been recommended,21

suggesting that all dietary needs can be met from α-TOH.19

However, given the recent

adverse publicity associated with high dose vitamin E supplementation,22

23

24

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 α-TOH are safe.

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4

The purpose of this chapter 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.

1.2 Vitamin E Transport in Humans and Animal Models

1.2.1 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 configuration of the methyl groups of 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 α-TOH having twice the antioxidant activity of γ-TOH, as

assessed by chemical reactivity in assay systems.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 where the amount

of natural vitamin E required to prevent foetal resorption in rats deficient in vitamin E is

compared to d-α-TOH.26

However, using these data for comparing the biological

activities of the different forms of natural vitamin E can make interpretation difficult.

This is mainly because the human requirements and tissue concentrations of the two

most important natural forms of vitamin E, namely α- and γ-TOH, are markedly

different between humans and rats (Table 2).

α-TOH, the major form of vitamin E in humans, is the most lipid-soluble

antioxidant and the most abundant TOH in human tissues.25

5 Synthetic vitamin E (dl-

α-TOH or all-rac-α-TOH), is an equal mixture of eight stereoisomers of α-TOH.4 All

isomers have an identical chromanol group and hence equal antioxidant 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

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5

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 stereoisomer‟s of synthetic vitamin E.

Table 2. Comparison of α and γ-TOH concentrations in different tissues between

humans and rodents

Humans Rats and Mice

γ-TOH α-TOH γ-TOH α-TOH

Plasma (mol/L) 2-7 15-20 1.3-1.7 7.2-13.0

Liver (nmol/g) − 20* 4.5-5.3 30.0-33.4

Adipose (nmol/g) 176 ± 80 440 ± 279 29.5 ± 4.1 79.8 ± 6.9

Muscle (nmol/g) 107 155 ± 163 3.6-5.6 15.1-22.7

Skin (nmol/g) 180 ± 89 127 ± 74 3.0 ± 2.8 8.9 ± 3.0

Taken from ref. 29

* Subject received 75 mg D3 RRR-α-TOH for 7 days before sampling 30

1.2.2 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 and bile pigments from the liver.31

Dietary vitamin E bound in micelles formed within the intestine is absorbed by a

passive process into enterocytes along with triglycerides and cholesterol. 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 α-TOH uptake,

implicating SR-BI in intestinal TOH transport.32

In the circulation, chylomicrons interact with lipoprotein lipase (LPL) to release

non-esterified 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

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6

transfer of TOH to fibroblasts was observed in-vitro in the presence of bovine LPL.33

These tissues receive most of their lipids during LPL-mediated delipidation of

lipoproteins. As chylomicron remnants are formed, they can then exchange surface

components with high-density lipoprotein (HDL), including TOHs. HDL can then

transfer TOHs to other lipoproteins in the circulation.25

This pathway is particularly

important for individuals with abetalipoproteinaemia and homozygous familial

hypobetalipoproteinaemia (FHBL), due to absent or extremely low plasma levels of

apoB-containing lipoproteins. Supplementation of these individuals with high dose

vitamin E (100-150 mg/kg/day) can normalise adipose tissue TOH concentrations via

HDL transport of TOHs to tissues.25

1.2.3 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 each form

between lipoproteins.25

29

Studies in humans have demonstrated that α-TOH is

preferentially incorporated into very low density lipoprotein (VLDL) particles and γ-

TOH excreted in the bile.34

Similarly, in rats, monkeys and humans the naturally

occurring RRR stereoisomer of α-TOH is also preferentially incorporated into VLDL.35

One study used patients with and without inherited disorders of lipoprotein metabolism

to elucidate the steps involved in the discrimination of the different vitamin E

isoforms.35

A subject with homozygous FHBL and abnormal apo B-100 production

showed preferential enrichment of his „VLDL‟ fraction with d6-RRR-α-TOH, 24 hours

after supplementation with d6-RRR-α-TOH acetate. Of interest, this patient had normal

HDL d6-RRR-α-TOH concentrations, did not have symptoms of vitamin E deficiency

and has not required vitamin E supplementation.

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7

α-TOH,

SRR-α-TOH,

-TOH

α-TOH,

SRR-α-TOH,

-TOH

CHYLOMICRONSCHYLOMICRONS

LPL

α-TOH, SRR-α-TOH,

- TOH, Fatty acids

α-TOH,

SRR- α-TOH,

-TOH

REMNANTSREMNANTS

α-TOH,

SRR-α-TOH,

-TOH

LDLLDL

HDLHDL

α-TOH,

SRR-α-TOH,

-TOH

LPL, HTGL

LiverLiver

VLDLVLDL

BILE

SRR-α-TOH,

-TOH

ErythrocytesErythrocytes

-CEHCURINE

Platelets

α-TOH, SRR-α-TOH,

- TOH

Tissue

Tocopherols

Intestine

Vitamin E

from diet or

supplements

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 the LPL-mediated delipidation

of chylomicrons and delivery from LDL and HDL. There is a preferential incorporation

of the RRR-α-TOH into VLDL mediated by the α-TTP (see text). 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

LDL indicates low density lipoprotein; HDL, high density lipoprotein; VLDL, very low

density lipoprotein; LPL, lipoprotein lipase; HTGL, hepatic triglyceride lipase; α-TOH,

α-tocopherol; γ-TOH, γ-tocopherol; and SRR-α-TOH, synthetic α-tocopherol.

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8

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-α-TOH.35

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, given that oxidative stress was not evident nor

did they exhibit any clinical signs of TOH deficiency.36

The importance of chylomicron delivery of TOHs to peripheral tissues has been

highlighted recently in a murine model.37

The genetically engineered mice that

specifically lack microsomal triglyceride transfer protein (MTP) in the liver were fed

deuterated α-TOH, with the majority of TOH replaced in peripheral tissues within one

month, despite the inability to secrete VLDL.37

Recent in-vitro studies using human

fibroblasts and murine RAW264 macrophages showed that the export of α-TOH to

HDL was mediated, at least in part, by the ATP-binding cassette transporter A1

(ABCA1).38

Moreover, ABCA1 was directly involved in the translocation of α-TOH to

apoproteins 38

(Figure 2). Taken together, these studies show that vitamin E can be

metabolised and delivered to tissues by a variety of routes and that low serum vitamin E

concentrations does not always equate to vitamin E deficiency. Vitamin E deficiency

may also be organ-specific and depend on the environment in question, with serum

TOH concentrations not necessarily indicative of those in peripheral tissues.

1.2.4 TOH Transfer Proteins

Vitamin E distribution and the role of TOH regulatory proteins has been the topic of a

recent review.27

The α-TOH transfer protein (α-TTP) is a 32 kDa cytosolic lipid-

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9

binding protein that is found mainly in the liver,39

with some expression in rat brain,

spleen, lung, kidney and in some regions in the human brain.27

Previous studies in rats

40 and humans

34 41

35

showed that RRR-α-TOH was retained within lipoproteins. The

α-TTP has a high affinity for RRR-α-TOH compared to other TOHs (Table 3) which

may, in part, account for the biological potency of each form of vitamin E.42

It is thought that the α-TTP directly facilitates the incorporation of α-TOH into

VLDL.25

However, this remains controversial.27

In the rat hepatoma cell line

McARH7777 expressing the α-TTP, more α-TOH was secreted into the medium.43

When the cells were incubated with Brefeldin A, an inhibitor of VLDL secretion, the

export of α-TOH was not affected, suggesting that the two processes are distinct.27

It

has been postulated that the role of α-TTP is to effectively remove vitamin E from

sorting endosomes to prevent its elimination.27

Furthermore, it has also been proposed

that the efflux of α-TOH to the plasma membrane is facilitated by the α-TTP and that

the free α-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

the α-TTP mediated secretion of TOHs from hepatocytes.44

However, further studies

will be required to demonstrate whether this process occurs in human tissues.

There are other binding proteins that specifically bind to TOHs. TOH-associated

protein (hTAP1), a 46 kDa protein, has recently been described in humans.45

hTAP1

has sequence homology similar to the α-TTP and is found in the liver, prostate and

brain. However, a specific function for this protein has yet to be found.27

There are two

other TAP proteins, namely hTAP2 and hTAP3, and these are similar to hTAP1 with

both these proteins involved in TOH mediated cell signaling pathways.46

Recently a

TAP (also known as supernatant protein factor) knockout mouse has been reported

suggesting a role for the TAP in hepatic cholesterol synthesis.47

However, the

relationship of this finding to TOH metabolism remains unclear.

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10

Chylomicron

Remnants

LDL

SR-BI

HDL

VLDL TTP

TAP

TBP

VE

PXR

VLDL

LDL R

Nucleus

VE

VE

PXR

Cytoplasm

DNA

CEHCs

(SRR, γ-TOH)

CYP 3A4

MDR1

MDR1VE

Urine

Bile

ABC-A1

RRR

RRR

VEAPOE R

VE

VE

HDLVE

CYP 4F2

Cell Membrane

VE

SR-BI

RXR

RXR

VE

VE

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 internalised the α-TTP will selectively incorporate

the 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 protecting poly-unsaturated fatty acids from oxidation. The TOHs have been

shown to bind to intracellular proteins namely the 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 metabolise 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

48

38

49

LDL indicates low density lipoprotein; HDL, high density lipoprotein; VLDL, very low

density lipoprotein; VE, vitamin E forms; α-TTP, α-tocopherol transfer protein; TAP,

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11

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; and CEHCs,

vitamin E metabolites (see text).

Table 3. Relative affinities of various TOH analogues for the α-TTP

Competitors Relative affinity (%)

α-TOH 100

-TOH 38.1 9.3

-TOH 8.9 0.6

-TOH 1.6 0.3

α–TOH acetate 1.7 0.1

α–TOH quinone 1.5 0.1

SRR-α-TOH 10.5 0.4

α-Tocotrienol 12.4 2.3

Trolox 9.1 1.2

Taken from ref. 42

The TOH-binding protein (TBP), a 14.2-kDa cystolic protein, found in rat liver

and heart, stimulates the transfer of α-TOH from liposomes to mitochondria in-vitro.50

Although this protein may be involved in intracellular trafficking of α-TOH, a direct

role for this protein has not been described.27

1.2.5 TOH Transfer Protein Deficiency

Patients with familial isolated vitamin E deficiency (FIVE), also known as ataxia with

vitamin E deficiency (AVED), have been studied to examine the role of the α-TTP in

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12

TOH metabolism. First described in 1981, 51

this rare autosomal recessive

neurogenerative disease 52

is often misdiagnosed as Friedreich‟s ataxia, but can be

distinguished by measuring α-TOH concentrations (low in FIVE) or by molecular

analysis of the frataxin gene (FXN) on chromosome 9.53

Patients with severe vitamin E

deficiency develop hyporeflexia, 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.54

Untreated patients with FIVE have plasma α-TOH

concentrations ~1% of normal.27

These patients do not produce VLDL enriched with α-

TOH and therefore must rely on chylomicron metabolism to distribute dietary α-TOH to

tissues.27

However, they still require supplementation with 800 mg RRR α-TOH twice

daily to maintain plasma concentrations within the reference interval.52

Studies using mice deficient in the α-TTP have provided useful models of disease

processes where oxidative stress is thought to play a role.55

56

Tereswa et al. examined

atherosclerotic lesion development in ApoE knockout mice (ApoE -/-

) that also had

vitamin E deficiency due to disruption of the α-TTP gene.56

Vitamin E deficiency

associated with α-TTP deficiency promoted atherosclerotic lesion formation in the

proximal aorta in ApoE -/-

mice. Moreover, a >85% reduction in α-TOH concentrations

was found and the generation of F2-isoprostanes was increased indicating lipid

peroxidation was not suppressed in this region of the aorta. Whether this effect is

evidence for an atheroprotective effect has been challenged, as the effect of vitamin E

supplementation was modest, given that the α-TOH concentrations were 100 times

greater in the group with normal α-TTP function.57

Taken together, these findings are

consistent with other work in the ApoE -/-

mice which showed that dietary

supplementation with vitamin E (2000 IU/kg chow) significantly reduced F2-isoprostane

concentrations in urine, plasma and vascular tissue.58

However, another study in the

same animal model observed a relatively small positive effect with vitamin E

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13

supplementation (0.2% wt/wt).59

Furthermore, combining chow with vitamin E

(0.05% wt/wt) with β-carotene (0.05% wt/wt) showed no benefit in this murine model.60

Thus, the potential role of vitamin E in the prevention of atherosclerosis remains

controversial.

1.3 Vitamin E and Oxidative Stress

Atherosclerosis has been described as a disease involving a number of different

processes including an inflammatory component 61

and the oxidation modification of

lipoproteins.62

However, these two processes are not mutually exclusive. Vitamin E is

thought to play a role in both regulating aspects of the immune system and forming an

important part of the antioxidant defense of lipoproteins.63

No single oxidant

responsible for LDL oxidation has been identified, and it is likely that many factors

could be involved, including transition metals, 15-lipoxgenase, myeloperoxidase

derived oxidants and reactive nitrogen species.57

The antioxidant properties of vitamin E have been examined to support a role for

supplementation in humans and may provide an explanation for the paradoxical results

obtained from clinical trials.64

65

Although α-TOH is important, it is not the only

determinant in the resistance of LDL to oxidation.66

Ubiquinol-10 (CoQ10H2), the

reduced form of coenzyme Q, is a well-studied co-antioxidant for α-TOH 63

and is an

effective lipid-soluble antioxidant at physiological concentrations.67

Ubiquinone-10

(CoQ10) is reduced during intestinal absorption to the antioxidant CoQ10H2.59

The disappearance of antioxidants from LDL isolated from healthy subjects has

been examined after exposure to different oxidising conditions.68

An initial lag period

was observed, followed by the detection of lipid peroxidation products, coinciding with

the consumption of CoQ10H2. It was determined that contaminating ascorbate in the

medium was responsible for the initial lag phase of lipid peroxidation, which was then

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followed by the complete consumption of CoQ10H2. This occurred even though 80%

of the endogenous carotenoids and 95% of the endogenous α-TOH were still present.68

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

protecting LDL from oxidation in-vitro have been described.69

When concentrations of

co-antioxidants ascorbate and CoQ10H2 are low, then the α-TOH radical can act to

transfer electrons rather than trapping them. In addition CoQ10H2 is a better antioxidant

than α-TOH in LDL because the semiquinone radical formed can leave the lipoprotein

particle rather than promote further peroxidation.69

The rate of radical stress is

important in determining the amounts of co-antioxidants available to interact with α-

TOH and this in turn influences the role of α-TOH within the LDL particle.

LDL from healthy subjects has been examined before and after supplementation

with d-α-TOH (1 g/day) and /or CoQ10 (100 mg/day) for a total of 5 days.70

Native

LDL contained 8.5 2 molecules of α-TOH and 0.5 to 0.8 CoQ10H2 molecules per

particle. This LDL was depleted of α-TOH after incubation in the Ham‟s F10 medium

containing transition metal ions and this depletion was increased in the presence of

monocyte derived macrophages (MDMs). When the LDL was incubated in-vitro with

α-TOH, the concentrations of α-TOH increased 6- to 7-fold in the LDL particle.

Furthermore, these LDL particles were more easily oxidised than native LDL both in

the presence and absence of MDMs, suggesting a pro-oxidant role for α-TOH.70

In

supplemented subjects, LDL α-TOH concentrations increased 2- to 3-fold and CoQ10H2

concentrations increased 3- to 4-fold. LDL was more susceptible to oxidation in

subjects receiving only α-TOH, whereas those receiving CoQ10 had LDL more resistant

to oxidation. Of interest, those receiving co-supplementation had LDL more resistant to

oxidation than native or LDL incubated with α-TOH. Taken together, these results were

consistent with the model of TOH-mediated peroxidation (TMP) with CoQ10H2

inhibiting TMP and protecting LDL from oxidation.70

A detailed review of TMP has

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been published, with implications for using α-TOH for the prevention of

atherosclerosis.71

α-TOH hydroquinone (α-TQH2) is derived from TOH quinone and has been

shown to effectively inhibit oxidation of α-TOH, CoQ10H2 along with surface and core

lipids by a number of oxidants in-vitro.72

It has been described as the most efficient

lipophilic antioxidant and effectively regenerates the TO· to α-TOH and decreases

consumption of CoQ10H2. α-TQH2 may have potential as a therapeutic agent,72

but this

awaits confirmation in clinical trials.

Atherosclerosis development and lipid peroxidation products has been examined

in ApoE -/-

mice on a high fat diet, supplemented with CoQ10 and/or RRR-α-TOH.59

24

weeks of supplementation with vitamin E and CoQ10 increased plasma concentrations of

vitamin E 3-fold and CoQ10 7-fold with the majority located in VLDL. Aortic

concentrations of CoQ10 increased >10-fold and vitamin E increased significantly in all

tissues measured. Supplementation with vitamin E and CoQ10 decreased lesion size at

the three 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 anti-atherogenic than

vitamin E given alone.59

Long-term antioxidant supplementation (12 months) with α-TOH, probucol and

CoQ10 in Watanabe heritable hyperlipidaemic (WHHL) rabbits with doses

approximating therapeutic doses given to humans (300 mg dl-α-TOH and CoQ10, 1g

probucol) had no effect on aortic lesion size, but did decrease copper-induced ex-vivo

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lipid peroxidation.73

Probucol also decreased lipid peroxidation by copper, whereas

CoQ10 had no effect. However, vitamin E failed to reduce the amount of lipid-

standardised LOOH and corresponding hydroxides.73

This finding is also consistent

with the model of TMP in the absence of sufficient co-antioxidants.71

The concentrations of oxidised lipids, α-TOH and its oxidation products in human

lesions were determined during different stages in atherosclerotic lesion development.74

Oxidation of α-TOH occurred early in the disease, exceeded lipid peroxidation and

lesions were not depleted of α-TOH. The products of α-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 (non-

radical) oxidants such as hypochlorite and peroxynitrite. They also found that the

oxidation products formed were similar to those formed when LDL is oxidised by

monocytes in the presence of nitrite.74

Given that oxidised lipids co-exist with α-TOH

in atherosclerotic lesions, 75

this study calls into question the rational for using

antioxidants such as α-TOH to prevent atherosclerosis.74

More recently, the effects of vitamin E supplementation on atherosclerosis in mice

with vitamin E deficiency have shown that any benefit was small and dependant upon

the degree of pre-existing deficiency.76

There was no effect on aortic F2-isoprostane

formation or oxidised lipid formation suggesting that vitamin E supplementation may

not reduce oxidative stress within the vessel wall.76

Moreover, supplementing healthy

individuals with α-TOH had no effect on lipid peroxidation as measured by urinary

concentrations of 4-hydroxynonenal (4-HNE) or F2-isoprostanes challenging the

rationale for supplementing healthy subjects with α-TOH to inhibit oxidative stress.77

Recent human intervention studies have examined the antioxidant potential of

RRR-α-TOH showing a significant reduction in F2-isoprostane production with 1200

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IU/day for 2 years 78

and 1600 IU/day for 16 weeks,79

consistent with the concept that

high doses of RRR-α-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 α-TOH (400 IU/day) but

in combination with vitamin C (100 mg/day).80

Taken together, these studies suggest

that RRR-α-TOH supplements may reduce oxidative stress in human populations with

high oxidative stress, but the effect might only occur with higher doses or in

combination with co-antioxidants.

1.4 TOH Metabolites

1.4.1 α-TOH

Given the strong epidemiological evidence supporting a role for diets rich in vitamin E

in CHD prevention,81

82

the measurement and study of the products derived from α-

TOH oxidation have received considerable attention. The urinary metabolites of

vitamin E have been studied since the 1950‟s.83

The first published data describing the

urinary metabolites of α-TOH from rabbits and humans appeared in 1956.84

Known as

the „Simon metabolites‟, both tocopheronic acid and the subsequent tocopheronolactone

were detected in the urine, after high dose supplementation with α-TOH.84

These

metabolites have generally been accepted to arise following the antioxidant action of

vitamin E in-vivo with the chroman ring being opened after oxidation.83

This

conclusion has been challenged with the suggestion that tocopheronolactone is produced

from 2,5,7,8-tetramethyl-2 (2'-carboxyethy)-6-hydroxychroman (α-CEHC) in the

presence of oxygen from sample handling.85

In this study, subjects receiving greater

than 50 mg of α-TOH per day obtained a plasma threshold of 7-9 μmol α-TOH/g total

lipid and had detectable α-CEHC concentrations, determined using a high performance

liquid chromatography (HPLC) method with electrochemical detection (ECD). The

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concentrations of α-CEHC (μmol/24 h) correlated well with α-TOH concentrations

(μmol/g total plasma lipid) when the threshold was reached. It was concluded that α-

TOH can undergo ω-oxidation, without prior oxidation, and that α-CEHC is therefore

the major urinary metabolite of α-TOH produced by healthy humans (Figure 3).

Furthermore, as plasma concentrations of α-TOH can only be raised 3-fold 86

regardless

of how much α-TOH is given, the investigators proposed that urinary concentrations of

α-CEHC are a marker of adequate vitamin E intake. They also raised the possibility

that high concentrations of α-TOH may indeed be harmful and that high urinary

concentrations of α-CEHC could reflect harmful vitamin E concentrations.85

The

investigators also acknowledged that the formation of Simon metabolites might occur

in-vivo, but probably only during episodes of oxidative stress.85

In contrast, other investigators have shown that both tocopheronolactone and α-

CEHC were present in the urine of subjects not taking α-TOH supplements.83

87

88

These later studies employed more sensitive gas chromatography/mass spectrometry

(GC/MS) methods and this could explain the different findings from earlier work using

HPLC.83

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 significance of these metabolites in both supplemented and

unsupplemented individuals.

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RRR -TOH

A B

tocopheroxyl radical

oxidation

side chain oxidation

tocopheronolactone

+ROO -+ e

tocopherolquinone

tocopherol hydroquinone

-2 e +2 e

tocopheronic acid

-CEHC

==

OH

HO

OH

HO

OH

OHC

OH

O= O

= O

OH

O

O

=C

OH

O

side chain oxidation

OH

HO

O

HO HOO2

O

Figure 3 Pathways leading to the urinary excretion of α-TOH metabolites

The commonly accepted pathway A vs. the newly proposed pathway B.

Taken from ref 85

RRR-α-TOH indicates natural α-tocopherol;

α-CEHC, 2,5,7,8-tetramethyl-2 (2‟-carboxyethy)-6-hydroxychroman (metabolite of α-

tocopherol)

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1.4.2 -TOH and its major metabolite -CEHC

The α-TTP is the main regulator of α-TOH incorporation into lipoproteins, whereas it

only plays a small part in γ-TOH metabolism. Instead γ-TOH is metabolised to 2,7,8-

trimethyl-2- (β-carboxyethyl)-6-hydroxychroman (γ-CEHC) via a cytochrome P450

enzyme in the liver,89

which is then excreted in the urine 90

(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.29

-TOH comprises around 70% of the total

TOH dietary intake in the United States population and is found within soybeans, corn,

walnuts and other nuts. Oils derived from soy, corn and sesame are therefore rich in -

TOH.29

There has been a greater interest in the role of α-TOH versus -TOH in human

nutrition. The concentrations of α-TOH are greater than -TOH in tissues and the

biological activity of -TOH is ~10% of α-TOH, as determined by the rat foetal

resorption assay.91

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 human than in rodents, particularly in skin and

muscle and these levels probably reflect the different way each species metabolise each

form of TOH.29

In a case-control study, γ-TOH concentrations in patients with CHD were lower

than healthy, age-matched controls whereas the concentrations of α-TOH were not

different between the two groups.92

Similarly, γ-TOH concentrations were significantly

lower in CHD patients than in controls with no corresponding decrease in plasma α-

TOH or CoQ10H2 concentrations in the patient group.93

There is some evidence that -TOH is superior to α-TOH at detoxifying nitrogen

dioxide (NO2) and that it is more effective at inhibiting peroxynitrite-induced lipid

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peroxidation of phosphatidylcholine liposomes.94

This inhibition occurs because

reactive nitrogen oxide species (RNOS) can be trapped by γ-TOH, which leads to the

formation of 5-nitro--TOH.95

The high reactivity of γ-TOH towards RNOS is because

it has one less methyl group leaving a reactive position available to trap electrophiles,

whereas α-TOH is fully substituted in the chromanol ring.29

RNOS like peroxynitrite

can rapidly cross phospholid membranes and oxidatively modify proteins, DNA, lipids,

redox metal centres as well as methionine.96

A recent study has also found evidence for

the increased nitration of -TOH in subjects with CHD, but concluded that a larger trial

must be conducted to clearly demonstrate the efficacy of this marker.97

It has been

suggested that the supplementation of patients in clinical trials with only α-TOH may be

inappropriate as α-TOH displaces -TOH in the plasma.95

However, given the recent

work highlighting the potential importance of γ-TOH in human health,27

displacing it

from the circulation may not be beneficial.

The major metabolite of γ-TOH is γ-CEHC. γ-CEHC, first described in 1996

after a 30 year search for a natriuretic factor that may control the concentrations of

extracellular fluid within the body, also has properties that may be important for human

health.98

0.6 mg of -CEHC was subsequently purified from 800 L of human urine and

this was then used to demonstrate that the compound could reversibly inhibit the 70pS

potassium (K+) channel whilst not inhibiting the sodium (Na

+) pump. The natriuretic

properties of γ-CEHC are not shared by α-CEHC.98

-TOH and its major metabolite -CEHC have also been shown to inhibit

cyclooxygenase-2 (COX-2) activity in human macrophages and epithelial cells. This in

turn reduced the synthesis of prostaglandin E2 (PGE2) by these cells. PGE2 is generated

from the oxidative metabolism of arachidonic acid during inflammation and this is

catalyzed by COX-2.99

Taken together, these findings suggest possible anti-

inflammatory roles for -TOH and -CEHC, which in turn may be important in

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preventing CHD and cancer.99

This awaits confirmation in an appropriately powered

human clinical trial.

1.5 Vitamin E Distribution in Cells

1.5.1 Erythrocyte Vitamin E

The erythrocyte membrane has an organised structure, which gives the red cell its

characteristic doughnut shaped appearance. Hydrogen peroxide has been used to induce

haemolysis of erythrocytes in-vitro in subjects who were depleted of vitamin E,100

and

the amount of inducible haemolysis is related to the relative amounts of oxidisable

polyunsaturated fatty acids and of protective vitamin E.101

The percent haemolysis of

erythrocytes was shown to be inversely proportional to the amount of TOH added to the

reaction mixture and that the amount of TOH added was consistent with the

concentrations found within the blood. However, in-vivo the plasma concentration of

vitamin E did not directly correlate with the percent haemolysis of erythrocytes by this

assay.101

Taken together, these results suggest that plasma TOH concentrations may be

misleading as to 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, specifically α-TOH content in asymptomatic men who are at risk for

developing atherosclerosis.102

This study examined erythrocyte α-TOH concentrations

and erythrocyte haemolysis with 2,2'-azobis(2)-amidinopropane (AAPH) and found α-

TOH concentrations were lower with increased erythrocyte haemolysis in

hypercholesterolaemic men compared to normocholesterolaemic men, even though

plasma concentrations of α-TOH were normal.102

The same group examined the

transfer of vitamin E between erythrocytes and HDL and concluded that the uptake of

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α-TOH by erythrocytes is not impaired in hypercholesterolaemic subjects and that the

lower concentrations of erythrocyte vitamin E seen in this group could be due to

reduced delivery to tissues. They also cite a previous study 103

in which α-TOH moved

to LDL, when the LDL: HDL ratio is high in-vitro, which may account for the low

concentrations of erythrocyte vitamin E seen in these patients.104

This group recently

compared erythrocyte vitamin E concentrations and plasma vitamin E concentrations to

carotid-intima-media thickness in 261 men at risk for cardiovascular disease (CVD).105

They found a negative correlation between carotid-intima-media thickness and

erythrocyte vitamin E concentrations (P<0.01), but not with plasma or HDL α-TOH

concentrations. It was suggested that this inverse relationship may indicate that cellular

α-TOH is possibly effective at inhibiting the early stages of disease.105

Further studies

may clarify the efficacy of erythrocyte vitamin E as a marker of disease risk and any

role in homeostasis.

1.5.2 Platelet Vitamin E

In an effort to establish the best marker of adequate vitamin E nutrition, platelet vitamin

E concentrations have been examined. In rats supplemented with varying amounts of d-

α-tocopheryl acetate over a 10-week period the response to dose was the most consistent

for platelets and that platelets provided a more sensitive indicator of vitamin E intake

over erythrocytes or plasma.106

In healthy male subjects α-TOH and -TOH

concentrations in plasma have been shown to correlate well with total lipid, cholesterol

and triglyceride concentrations, but platelet concentrations of α-TOH and -TOH did

not 107

(Table 4). This finding confirms a previous observation showing that platelet

TOHs correlated poorly with erythrocyte and plasma TOH concentrations.108

However,

platelet TOH concentrations compared well to plasma concentrations when expressed

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per mg lipid.107

Given that platelet TOH concentrations do not directly depend on lipid

concentrations then it follows that they should not be influenced by any free exchange

between the two compartments. These investigators recommend assaying baseline

platelet vitamin E concentrations before supplementation to assess nutritional

adequacy,107

a concept supported by human data showing platelet vitamin E

determination provided the most sensitive indicator for dietary intake of vitamin E 109

(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.109

Table 4. Correlation matrix obtained comparing α and γ-TOH from plasma and

platelets with plasma lipids.

Plasma Platelets

α-TOH -TOH α-TOH -TOH

Total Lipid 0.79 * 0.60 * 0.22 0.15

Cholesterol 0.58 * 0.46 * 0.27 0.17

Triglyceride 0.78 * 0.61 * 0.08 0.09

* Correlations are statistically significant at a P value of <0.001

Taken from ref 107

1.6 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 61

110

with oxidative stress within

platelets potentially contributing to thrombus formation.111

The effects of vitamin E

isomers on platelet function are largely determined by the type of vitamin E used. Early

work by Norday and Strom 108

showed that incubation of platelets with synthetic dl-α-

TOH did not result in any significant uptake of α-TOH by the platelets. Platelets have

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been shown to readily take up the natural RRR-α-TOH with a corresponding decrease

in platelet aggregation, whereas the synthetic form was poorly incorporated and did not

affect the aggregation of platelets.112

More recent studies have shown a lack of benefit

of synthetic dl-α-TOH on platelet function in healthy individuals.113

114

Table 5. α- and γ-TOH contents (n = 20) of plasma, red blood cells, platelets, and

lymphocytes of human subjects supplemented with 0, 30 and 100 mg/d of dl-α-

tocopheryl acetate.

Plasma Plasma RBC Platelets Lymphocytes

μmol/L μmol/g lipid μmol/L μmol/10 g protein

α–TOH (mg/day)

0

30

100

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/day)

0

30

100

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 the paired t test (P<0.05)

Taken from ref 109

The effect of RRR-α-TOH on platelet aggregation appears to involve inhibition of

protein kinase C within platelets and an increase of platelet-derived nitric oxide (NO).11

Human studies have shown a reduction in ex-vivo platelet aggregation in subjects

following supplementation with α-TOH 115

116

and high amounts of γ-TOH.117

118

The efficacy of vitamin E supplements in preventing platelet activation may

depend on many factors including baseline TOH levels, the presence of co-antioxidants

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present within platelets or the plasma and whether natural or synthetic forms are used.

The prevention of arachidonic acid mediated α-TOH oxidation within platelets can be

reversed by co-incubation with ascorbic acid or glutathione.119

This may have

implications given that RRR-α-TOH in combination with ascorbic acid has been shown

to inhibit atherosclerotic disease progression in subjects with hypercholesterolaemia 120

and platelet activation may be important in this patient population.121

Any potential benefit can also be complicated by patient adherence to

conventional drug treatments known to affect platelet function, such as aspirin and

statins.122

We have examined the effects of 500 mg of RRR-α-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 benefit

in relation to platelet function, in spite of the significant increase in platelet TOH

concentrations.123

Given these findings, 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 significantly inhibit platelet aggregation in-vivo.

1.7 Reporting Vitamin E Concentrations

As plasma lipid concentrations influence plasma TOH levels, it has been recommended

that the concentration of α-TOH be expressed per mg of lipid.124

In a study of 85

alcoholic patients, the ratio of TOH: cholesterol + triglycerides were the best tool for

identifying deficiency (sensitivity 95%, specificity 99%).125

Patients with liver disease,

for example, may have elevated lipid concentrations, but a deficiency in vitamin E may

be missed if the lipid concentrations are not taken into account. Conversely, patients

with low lipid concentrations, for example heterozygous FHBL, may be classed as

vitamin E deficient when they are not.125

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1.8 Vitamin E and Atherosclerosis

In 1991, the National Heart, Blood and Lung Institute (NHBLI) convened a workshop

to review current knowledge about the oxidative modification hypothesis of lipoproteins

and their implication in the pathogenesis of atherosclerosis.126

It was felt 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 meta-analysis

of the effects of high versus low vitamin E supplementation on cardiovascular mortality

found that for observational studies the test for overall effect favoured high vitamin E

intake, odds ratio 0.67 (0.54-0.83). In contrast, the analysis from six major intervention

trials were equivocal.127

The results from human trials of vitamin E supplementation have been

controversial and have generated considerable discussion. It has been suggested that

such studies would have benefited from the measurement of markers of in-vivo lipid

peroxidation, like F2-isoprostanes, to establish any effect on oxidative stress.64

The

different doses and forms of TOH given to different populations might, in part, explain

the paradoxical results obtained from clinical trials. In countries where a

„Mediterranean diet‟ is consumed, a protective effect of this diet against atherosclerosis

has been shown.128

The population in the Gruppo Italiano per lo Studio della

Sopravvivenza nell'Infarto miocardico (GISSI-Prevenzione trial), 129

who typically

consumed a Mediterranean style diet rich in antioxidants, still developed CVD and may

not have benefited from the 300 IU/day of synthetic vitamin E given.65

A subsequent

follow-up of the participants in the GISSI-Prevenzione trial has 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 has been

the Heart Outcomes Prevention Evaluation Study (HOPE) trial.130

In this trial, 772 of

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the 4761 patients who were at high risk for CVD, received 400 IU/day of RRR α-TOH

for a mean follow-up of 4.5 years. There was no significant difference in the incidence

of secondary cardiovascular outcomes or in death from any cause and no significant

adverse effects of vitamin E supplementation. In a similar fashion to the GISSI trial, a

subsequent follow-up study has 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) trial 131

in hemodialysis patients showed positive

effects with vitamin E supplementation (800 IU/day of natural RRR α-TOH) in a patient

group who were probably under considerable oxidative stress.132

133

These patients

were also given a number of other antioxidants as supplements, with 40% of all

participants taking vitamin C.131

It has been suggested that vitamin C in combination

with vitamin E may in fact explain the positive results.134

135

The Cambridge Heart Antioxidant Study (CHAOS) study 136

examined 2002

patients with angiographically proven coronary atherosclerosis. Subjects were given

either 800 IU/day (546 subjects) or 400 IU/day (489 subjects) of natural α-TOH and

there was a 77 % decrease of non-fatal myocardial infarction, however, there was a non-

significant increase in cardiovascular deaths in the subjects receiving α-TOH. It has

been suggested that the apparent rise in fatal myocardial infarction could be related to

transition-ion release from unstable plaques, with α-TOH potentially acting as a pro-

oxidant in this setting.65

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.137

A total of 520 patients were given

twice daily 91 mg (136 IU) d-α-TOH, 250 mg slow-release vitamin C, a combination of

the two or placebo. The most significant finding was that in men taking the

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combination of the two, the proportion who experienced progression was reduced by

74% (95 CI 36-89%) compared to placebo.137

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 has 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 β-carotene).138

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.138

This is in

contrast to a previous trial in which simvastatin, niacin and antioxidant vitamins were

given in combination to determine any clinical benefit.139

Antioxidant use significantly

impaired the benefits obtained from niacin and simvastatin when used concurrently,

with the protective increase in HDL2 with simvastatin plus niacin 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.139

140

1.9 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 characterised by hypertension, the presence of

proteinuria and generally occurs in the second half of pregnancy. This condition

involves a maternal inflammatory response, activation of maternal vascular endothelial

cells, endothelial dysfunction and leucocyte activation. Oxidative stress has been

implicated in the pathogenesis of pre-eclampsia and there is considerable interest in the

potential for antioxidants to prevent this condition.141

A recent placebo-controlled randomised trial examined the potential for vitamin C

(1000 mg/day) and vitamin E (RRR α-TOH 400 IU/day) to prevent pre-eclampsia in

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women with a variety of risk factors.142

The study involved 2395 subjects from 25

hospitals (1196 in vitamin group and 1199 in 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 birth weight of babies whose mothers took the

antioxidant treatment versus those on placebo.142

A subsequent study conducted in Australia examined the potential benefit with the

same doses of vitamin E and C to prevent pre-eclampsia in nulliparous women.143

This

study found no effect in regards to the occurrence of pre-eclampsia or low birth weight,

but other adverse outcomes were common in the treatment group including; increased

gestational hypertension, severe gestational hypertension, antenatal hospitalisation for

hypertension, the use of antihypertensive agents and the induction of labour for

hypertension.143

Taken together, these findings suggest the use of antioxidants for the

prevention of pre-eclampsia is not warranted and may in fact be harmful.

A recent study has reported benefit with vitamin E and C used in combination to

reduce blood pressure and oxidative stress in untreated hypertensives.80

However,

given recent analysis suggesting potential adverse consequences with the use of α-TOH

in disease prevention in high risk populations 23

22

and our own data showing an

increase in hypertension following supplementation with vitamin E in diabetic subjects

taking other drugs, 144

further studies must be performed to elicit any adverse effects of

the agents, particularly in relation to hypertension.

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1.10 Vitamin E and Drug Metabolism

1.10.1 Cytochrome P450 Enzyme Activity

The importance of the cytochrome P450 (CYP) enzyme system for the metabolism of

drugs has been emphasised recently 145

with the estimated number of deaths annually in

the United States due to adverse drug reactions thought to number at least 100,000.146

The CYP3A isoforms are probably the most significant class in humans as they perform

~ 50% of all drug oxidations 145

and high quantities of this enzyme are found in both the

liver (29% of total) and the intestine (70% of total).147

CYP3A4 is the most studied

form of CYP3A in humans and is the most abundant isoform found within both the liver

and the intestine.148

The induction of CYP3A4 in humans through activation of the pregnane X

receptor (PXR) has been well described.145

Examples of drugs that induce CYP3A4

expression through this pathway include rifampicin and St. John‟s wort (hyperforin) and

this can lead to increased metabolism of other drugs including calcium-channel blockers

and HIV-protease inhibitors.145

The induction of CYP3A4 in primary human

hepatocytes through activation of PXR has been observed following incubation with St

John‟s wort extract for 30 h.149

A 14 day course of St John‟s wort extract induced the

activity of CYP3A4 measured through the pharmacokinetics of alprazolam in human

volunteers.150

It has been hypothesised that α-TOH can interfere with drug metabolism

through increasing the expression of CYP3A4 within the liver and thereby increasing

the metabolism of certain drugs.49

1.10.2 CYP 3A and Vitamin E

Studies have been performed to examine the capacity for α-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 α-TOH (20 mg/kg/day) for 3 months

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has been reported.151

Importantly, γ-tocotrienol did not induce Cyp3a11 mRNA. In

mice high dose γ-TOH supplementation for 5 weeks increased CYP3a protein

concentrations which correlated with hepatic α-TOH, but not with hepatic γ-TOH

concentrations.152

A number of different forms of vitamin E have been shown to activate gene

expression through activation of human PXR in HepG2 cells in culture, with RRR-α-

TOH able to increase PXR activity 2-to 3-fold following incubation for 48 h, but

activation was higher with rifampicin (known inducer) and also with α and γ-

tocotrienol.153

These and other studies have been recently reviewed and it has been suggested

that high dose α-TOH supplementation might interfere with drug metabolism through

activation of CYP3A4, whereas γ-TOH and the tocotrienols might not, due to increased

metabolism and excretion in the liver of these compounds.49

Subcutaneous α-TOH injections given to rats caused a significant increase in liver

α-TOH concentrations at day 18 with a concomitant increase in liver microsomal

CYP3A protein and P-glycoprotein (MDR1).154

This study did not relate this effect to

drug metabolism directly, although they suggested that α-TOH might increase CYP3A

in humans and may potentially affect the metabolism of certain drugs.154

Another

consideration is the differential effects of vitamin E forms on PXR mediated drug

transporters within different tissues. For example, rats given subcutaneous α-TOH

injections showed significant increases in MDR1, but no effect on CYP3A protein

expression in lung tissue.155

The use of combined antioxidants RRR-α-TOH with vitamin C in humans showed

a significant reduction in cyclosporin trough levels.156-158

It is unclear which

antioxidant is having the effect or which drug metabolising pathway is being affected,

given cyclosporine is metabolised by both CYP3A enzymes and MDR1.159

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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, due to differences in the amino

acid sequences in the ligand binding domain for this receptor.160

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 takes several weeks to occur.145

This is an important consideration in the

design of any study examining the potential interactions following induction of

CYP3A4 or studies 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

specifically determined.

1.10.3 PXR Activation and Hormone Synthesis

Activation of PXR in humans may have undesirable effects in certain populations. A

recent study examined the effects of altered xenobiotic receptor activity on adrenal

steroid homeostasis in transgenic mice that had the liver-specific expression of the

activated human PXR.161

An increase in corticosterone and aldosterone output was

observed, causing disrupted circadian rhythm and increased expression of steroidogenic

enzymes involved in the production of these steroids.161

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 α-TOH, β-Carotene Prevention Study

(ATBC) study examined the effect of long term α-TOH supplementation (50 mg dl-α-

TOH acetate/day for 5 to 8 years) on prostate cancer incidence and observed a 32%

reduction compared to placebo.162

A follow-up study of 200 men participating in the

ATBC trial observed a significant reduction in both androstenedione and testosterone in

the TOH group compared to placebo.163

There was a significant negative correlation

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between serum α-TOH and androgens in this group of men.163

The investigators

suggest that the reduction in hormone production is one mechanism that TOHs may

reduce the incidence and mortality from prostate cancer. A recent publication from the

ATBC study found that high serum α-TOH concentrations were associated with a

reduced risk of prostate cancer.164

Studies of the association between serum α-TOH

concentrations and all-cause mortality found a significant reduction in risk which was

greater with increasing concentrations of α-TOH, but no additional benefit beyond 13 to

14 mg/L (30 μmol/L).164

Whether vitamin E supplementation can alter the production

of hormones in-vivo across different populations remains an important question for

further investigation, both in relation to potential benefits, but also possible adverse

effects.

1.11 Vitamin E Supplementation and All-cause Mortality

Studies have investigated the metabolism and efficacy of α-TOH in the prevention of

sequelae associated with CVD. Some have been promising in secondary prevention in

conditions associated with oxidative stress. However, the results from large primary

prevention clinical trials with α-TOH have been largely negative.165

A meta-analysis of > 130,000 participants from 19 clinical trials across a wide

range of vitamin E intakes (16.5 to 2000 IU/day) concluded that high dose vitamin E

supplements (≥ 400 IU/day as α-TOH) increases all-cause mortality.22

This conclusion

was challenged in a subsequent analysis 166

suggesting the adverse effect on mortality is

only significant at doses above 2000 IU/day, a dose much higher than the recommended

upper limit of 1600 IU/day.21

This conclusion is in contrast to recent data on the long

term effects of lower dose α-TOH supplementation in subjects from the HOPE trial 23

and the GISSI-Prevenzione trial 24

with subjects receiving 400 IU/day with 7 year

follow up and 300 mg/day with 3.5 year follow up, respectively. These studies reported

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an increase in heart failure in the HOPE trial and heart failure in subjects with left

ventricular dysfunction in the GISSI trial. The relationship between antioxidant

vitamins and blood pressure was examined as a part of the Third National Health and

Nutrition Examination Survey (NHANES).167

This study 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.167

The relationship between α-TOH supplementation and blood pressure

is supported by our own findings, showing that RRR-α-TOH (750 IU/day)

supplementation for 6 weeks increased systolic and diastolic blood pressure in well-

treated diabetic subjects.144

The mechanism(s) for any adverse effect of high dose α-

TOH is unknown. One possible explanation is that α-TOH may affect drug metabolism

and thereby the efficacy of drugs used to treat subjects at risk of sequelae associated

with CVD.

The most comprehensive analysis to date examined antioxidant supplementation

for primary and secondary prevention of disease and included 68 randomised trials with

232,606 participants.140

For vitamin E they examined 26 trials with 105,065

participants in which vitamin E was given either alone or in combination (following

exclusion of trials with a high bias risk or use of selenium) and observed an increase

relative risk on all-cause mortality compared to placebo.140

Whether this risk equates to

healthy populations has not been determined and it is clear that this remains an

important question for future research.

1.12 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. Given the disappointing results from clinical

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trials and if one accepts that the primary role of α-TOH to prevent atherosclerosis is

via the inhibition of in-vivo LDL oxidation, then supplementation of α-TOH in healthy

individuals to prevent atherosclerosis may not be warranted. However, α-TOH is

clearly required by groups of individuals with clinical deficiency in the vitamin, such as

in ABL, who require supplementation to maintain normal neurological function.

However the supposition that antioxidants are safe in all populations is probably not

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 γ-TOH is established and the precise role

of α-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. The potential

drug interactions must also be further explored to examine if any significant clinical

drug interactions result from co-supplementation with vitamin E.

The observation that individuals within a higher baseline serum vitamin E

concentration have a reduced risk of all-cause mortality 168

probably reflects 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 α-TOH intake 2 and it has been suggested this might be attained through the diet as

opposed to supplementation.169

Given the use of vitamin E supplements is high in US

adults,170

it is possible that many individuals have either too much, or too little vitamin

E intake and this may be deleterious to health in both cases.

As more trials are conducted examining supplementation of the TOHs in humans,

the questions arise as to whether an appropriate dose of one or more vitamin E forms,

given singularly or with other compounds, has a role in the prevention and treatment of

disease.

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* A version of this chapter has been published in Clin Chem 2006; 52:1339-45

58

CHAPTER 2

TOH METABOLISM AND OXIDATIVE STRESS IN

FAMILIAL HYPOBETALIPOPROTEINAEMIA

2.1 Introduction

FHBL (OMIM 107730) is a rare autosomal co-dominant disorder of lipoprotein

metabolism characterised by decreased plasma concentrations of total cholesterol, LDL

cholesterol and apolipoprotein (apo) B (< 5th

percentile for age and sex) caused by

mutations in the APOB gene.1

2

3 About 60 nonsense, frameshift, and splicing

mutations in the APOB gene leading to the formation of prematurely truncated apoB

forms have been reported in FHBL subjects. There is some evidence that molecular

changes other than truncations of APOB can cause FHBL.1 3

Heterozygotes for FHBL are usually asymptomatic, but have plasma LDL

cholesterol and apoB concentrations that are one quarter to one third of normal (Figures

1a & 1b). Moreover, they have low to low-normal serum -TOH concentrations and

can display abnormal erythrocyte morphology with characteristic acanthocytes, as seen

in subjects with vitamin E deficiency 4 (Figure 2).

The clinical and biochemical features in homozygous and compound

heterozygous FHBL can include acanthocytosis, deficiencies of fat-soluble vitamins

secondary to malabsorption, an atypical form of retinitis pigmentosa, and

neuromuscular abnormalities. Retinitis pigmentosa and neuromuscular abnormalities

are primarily a result of deficiencies in fat-soluble vitamins, especially vitamins E and

A, due to their impaired absorption and transport.

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59

Abetalipoproteinaemia (ABL; OMIM 200100) is a very rare autosomal recessive

disorder of lipoprotein metabolism caused by mutations in the microsomal triglyceride

transfer protein (MTP) gene characterised by virtually undetectable plasma levels of

apoB-containing lipoproteins. MTP is a molecular chaperone that facilitates the

assembly and secretion of triglyceride-rich apoB-containing lipoproteins, namely

chylomicrons and VLDL. Patients often present in childhood or early infancy with

failure to thrive, fat malabsorption, and low plasma cholesterol and vitamin E

concentrations.

Vitamin E is essential for neurological function and is transported in plasma in

association with the apoB-containing lipoproteins. Acanthocytes typically comprise 50

to 100% of erythrocytes in ABL and may be the result of either vitamin E deficiency, or

an altered membrane lipid composition. Long-term high-dose vitamin E and A

supplementation should prevent or slow progression of the neuromuscular and retinal

degenerative disease.5 Subjects with compound heterozygous FHBL are often clinically

indistinguishable from those with ABL.1 2 3

TOH delivery to tissues in FHBL subjects occurs primarily through chylomicron

and high density lipoprotein (HDL) metabolism.6 Given that vitamin E

supplementation is thought to be safe, it has been recommended that FHBL subjects

receive vitamin E supplementation.4 However, a recent meta-analysis has suggested

that supplementation with ≥ 400 IU of -TOH per day may increase the risk of heart

failure 7 or all-cause mortality

8 in certain population groups. It is not known whether

this risk equates to subjects with low plasma cholesterol, nor is it known whether

subjects with heterozygous FHBL transport sufficient TOH isomers to prevent vitamin

E deficiency or complications associated with oxidative stress.

The studies herein were designed to examine the effects of truncated apoB

variants causing FHBL on vitamin E metabolism, by exploring the relationship between

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60

plasma and cellular TOH concentrations and urinary markers of TOH intake and

oxidative stress.

2.2 Subjects and Methods

2.2.1 Subjects

The primary study included nine subjects with heterozygous FHBL and seven

normolipidaemic individuals. The heterozygous FHBL subjects had molecularly

characterised truncating apoB species ranging from apoB-6.9 to apoB-80.5 and were not

receiving vitamin E supplements. For comparison, two children with homozygous

FHBL (apoB-30.9) and one with ABL receiving -TOH supplementation were also

studied. The baseline characteristics comparing the heterozygous FHBL and the normal

subjects are provided in Table 1. Blood and spot morning urine samples were collected

from each subject after a 10 h overnight fast. Venous blood was collected into a 3 mL

serum and a 9 mL EDTA tube and was processed immediately. The serum tube was

centrifuged at 2000 x g for 10 min at 4°C and sample frozen at -80°C until analysis.

The EDTA tube was used for platelet and erythrocyte analyses. The urine sample was

aliquoted into 5 mL tubes without a preservative and the sample stored at -80°C until

analysis.

2.2.2 Platelet Preparation

Platelets were prepared for TOH analysis as described 9 with minor modifications. In

brief, the sample was centrifuged at 200 x g for 15 min at 4C, the platelet-rich plasma

(PRP) fraction removed, and platelets counted using an ABBOTT CELLDYN 4000

Haematology analyser. 500 μL aliquots of the PRP fraction were dispensed into

Eppendorf microfuge tubes and then spun at 2000 x g for 10 min at 4°C. Platelets

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61

were washed three times in a citrate saline solution (Na3Citrate.2H20 1.7 g/L, NaCl 8.7

g/L). 100 μL of high performance liquid chromatography (HPLC) grade ethanol was

added to the remaining pellet and the sample stored at -80°C until analysis.

Table 1. Characteristics for the normal and heterozygous FHBL subjects.

Normal

Subjects

FHBL

Subjects

P

N 7 9 NS

Age (year)

41 5 40 5 NS

Gender (M/F) 6/1 7/2 NS

BMI (kg/m2)

25 2 27 1 NS

Total Cholesterol (mmol/L) 4.7 0.2 2.4 0.2 < 0.001

HDL Cholesterol (mmol/L) 1.61 0.14 1.52 0.14 NS

LDL Cholesterol (mmol/L) 2.8 0.3 0.7 0.1 < 0.001

Triglyceride (mmol/L) 0.87 0.10 0.52 0.1 0.01

ApoB (g/L) 0.84 0.08 0.23 0.02 < 0.001

ApoA-I (g/L) 1.61 0.14 1.49 0.10 NS

Values given are mean SEM

2.2.3 Erythrocyte Preparation

Erythrocytes were prepared for TOH analysis as described.10

In brief, erythrocytes were

washed three times in NaCl (9 g/L containing 10 g/L pyrogallol) and re-suspended in

this solution to give a haematocrit of about 50%. The exact haematocrit was then

determined and then 500 μL aliquots of washed erythrocytes were stored at -80°C for

analysis.

2.2.4 HPLC Analysis of TOHs

Serum TOHs were extracted as described 11

with minor modifications. In brief, 200 μL

of serum in a borosilicate glass tube was spiked with 50 μL of tocol (10 μg/mL), mixed

for 20 s then ethanol (200 μL) added and mixed again for 60 s and left in the dark for 5

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min. The sample was extracted with 1 mL of hexane and 600 μL of hexane removed,

evaporated under nitrogen, and reconstituted in methanol (200 μL). One μL of sample

was injected onto the column.

Platelet TOHs were extracted as described 9 with minor modifications. In brief,

the prepared platelet pellets were sonicated using a Branson Sonifier 150 for five

quick pulses with the power setting at three. 800 μL of hexane was added followed by

50 μL of tocol (1 μg/mL). The samples were mixed for 60 s then 600 μL of hexane was

removed, evaporated under nitrogen and reconstituted in 150 μL of methanol. 15 μL

was injected onto the column.

Erythrocyte TOHs were extracted as described 10

with minor modifications. In

brief, to 500 μL of washed erythrocytes 500 μL of ethanol was added and the samples

mixed for 20 s. The samples were spiked with 50 μL of tocol (10 μg/mL) and mixed for

20 s. Two mL of hexane was added and the samples mixed for 60 s followed by

centrifugation at 2700 x g for 10 min at 4°C. 1 mL of hexane was removed, dried under

nitrogen and reconstituted in 300 μL of methanol. 15 μL of sample was injected onto

the column.

TOHs were separated by HPLC on a Lichrospher 100 RP-18 (5 μmol/L)

reverse-phase column using a mobile phase of 99% methanol and 1% water containing

10 μmol/L of lithium acetate. Tocol was used as an internal standard. Detection was

performed on an ESA Coulochem III coulometric electrochemical detector set at 600

V and 1 μA for each assay and the flow rate was 1 mL per min using an Agilent 1100

HPLC. All reagents were HPLC grade and Calbiochem supplied the standards.

In-house quality control samples were assayed in duplicate at the beginning,

middle and end of each assay. The inter- and intra-assay coefficient of variation’s (CV)

for the TOH methods were less than 10%. All measurements were performed in

duplicate and samples with a duplicate CV of greater then 10% were re-assayed. The

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recovery for and -TOHs was between 95 and 105% for serum, erythrocytes, and

platelets. The minimum limit of detection for and -TOH in serum, packed

erythrocytes and platelets was 1.0 μmol/L, 0.3 μmol/L and 0.03 nmol/109

cells,

respectively. These values were determined by assaying multiple samples across the

linear range for the assay and were defined as the lowest reportable concentration of

analyte giving CV% < 10, when assayed in triplicate. Repeat analyses showed that

and -TOH was stable for at least six months at -80°C and all samples were assayed

within four months of collection.

2.2.5 GCMS Analysis of TOH Metabolites

The urinary TOH metabolites were measured using gas chromatography mass

spectrometry (GCMS) as described 12

with minor modifications. In brief, 5 nmol of

internal standard d9--CEHC in ethanol was added to 1 mL of freshly thawed urine

followed by 1 mL of 0.33 mol/L potassium phosphate buffer (pH 7.4) containing 350 U

of E. coli -glucuronidase (Type IX-A). The urine was incubated for 3.5 h at 37°C in

the dark then acidified with 0.25 mL of 6 mmol/L HCl and extracted with 8 mL of

hexane/tert-butyl methyl ether (4:1 vol/vol). Urine extracts were dried under nitrogen

and sialated with 50 L of anhydrous pyridine and 50 L of (BSTFA (N, O-

bis(trimethylsilyl)trifluoroacetamide) + 1% TMCS (trimethylchlorosilane)), then heated

at 60°C for 1 h. Samples were injected directly onto GC-MS (Agilent 5973) for

analysis using a DB-5MS column (25 m x 0.2 mm (internal diameter); 0.33 m film

thickness). Initial oven temperature of 160°C was held for 0.5 min, and increased at

20°C/min to 300°C with a final hold at 300°C for 10 min. The carrier gas was helium.

Selected ion monitoring (SIM) of the molecular ion and one major fragment ion (as a

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64

qualifying ion) of each metabolite was as follows, internal standard, d9--CEHC, m/z

431, 246; -CEHC, m/z 422, 237; -CEHC, m/z 408, 223 and δ-CEHC, m/z 394,

209 13

. Quality control samples were ran with each assay and the inter- and intra-assay

coefficient of variation’s (CV) were less than 10%.

2.2.6 F2-isoprostanes

Free F2-isoprostanes were measured in spot urine samples by GC-MS as described.14

Quality control samples were run with each assay and the inter- and intra-assay CV

were less than 10%.

2.2.7 Lipid Analysis

Total cholesterol [CHOD-PAP (HiCo)], triglyceride (GPO-PAP), and HDL cholesterol

(direct method HDL-Plus) were measured using the enzymatic, colourimetric assays

using reagents obtained from Roche Diagnostics on a Hitachi 917 chemistry

analyser. LDL cholesterol was calculated according to the Friedewald equation.15

ApoA-I and apoB concentrations were measured using reagents obtained from Behring

on a Behring BN-II nephelometer. Lipid and apoprotein assays had inter- and intra-

assay CV’s of less than 6% and 10%, respectively.

2.2.8 FPLC Analysis of lipoproteins

Fast Performance Liquid Chromatography (FPLC) analysis of lipoproteins was

performed as described 16

with minor modifications. Prior to injection of sample EDTA

plasma was diluted ½ in mobile phase. The mobile phase consisted of (50 mM PBS,

0.1 M NaCl, 0.001M EDTA, 0.02% NaN3, pH 7.4). Separation of lipoprotein fractions

was performed on a Pharmacia Superose 6HR 10 x 300 mm column. The flow rate was

set at 0.3 mL/min, 200 L of diluted sample was injected onto the column and 30 x 0.6

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65

mL fractions were collected. Collected fractions were assayed for cholesterol,

triglyceride, apoB and apoA-1 using the same reagents described above, but with

modifications described below.

Sensitive assays were established using the Cobas Mira automated chemistry

analyser with sample volumes changed from 2 L to 50 L for cholesterol and

triglyceride and reagent volumes changed from 250 L to 180 L from traditional

serum assays. These assays were linear across the ranges required and had functional

sensitivities of 0.005 mM and 0.004 mM for cholesterol and triglyceride respectively.

Sensitive assays were also established for apoA-1 and apoB using 50 L fraction, 10 L

n-diluent, 150 L reaction buffer, 10 L supplementary reagent and 40 L of antiserum

(all provided by Behring). These assays were linear across the ranges required and had

functional sensitivities of 4.1 mg/L and 8.6 mg/L for apoA-1 and apoB respectively.

TOH analysis was performed on 200 L of the collected fraction using the serum

assay protocol, but with a modified low standard curve. Linearity and recovery studies

in the mobile phase demonstrated linearity across the range required, with 99% and 98%

recovery for α and γ-TOH respectively.

Appropriate quality controls were included in all assays and all had inter and

intra-assays CV < 5%.

2.2.9 Statistical Analysis

All data are presented as mean SEM. Mean comparisons were performed using SPSS

for Windows version 12 (SPSS Inc, Chicago). A P value <0.05 was considered

significant.

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66

2.3 Results

When compared to controls, heterozygous FHBL subjects had significantly decreased

fasting plasma total cholesterol (-49%), triglyceride (-40%), LDL-cholesterol (-75%),

and apoB (-73%) concentrations (Table 1). Plasma HDL cholesterol and apoA-I

concentrations were not different between the two groups. Serum - (-53%), and -

TOH (-44%) concentrations were significantly lower in heterozygous FHBL subjects

when compared to controls (all P<0.03) (Table 2). Although erythrocyte -TOH

concentrations were decreased (-17%) in heterozygous FHBL subjects, no differences

were observed with lipid-adjusted serum TOHs, erythrocyte -TOH, platelet - or -

TOH, or urinary F2-isoprostanes and TOH metabolites.

Table 2. TOH, TOH metabolite and F2-isoprostane concentrations in normal and

heterozygous FHBL subjects.

Normal

Subjects

(n=7)

FHBL

Subjects

(n=9)

P

Serum (μmol/L) 28.7 1.4 13.6 1.0 <0.001

Serum (μmol/L) corrected * 5.2 0.4 4.6 0.1 NS

Serum (μmol/L) 1.8 0.3 1.0 0.1 0.03

Serum (μmol/L) corrected * 0.3 0.1 0.3 0.1 NS

Red cell 6.0 0.3 5.0 0.2 <0.005

(μmol/L packed RBC’s)

Red cell

0.44 0.06 0.42 0.03 NS

(μmol/L packed RBC’s)

Platelet 1.28 0.10 1.30 0.06 NS

(nmol/109 platelets)

Platelet 0.10 0.03 0.10 0.01 NS

(nmol/109 platelets)

Urinary -CEHC (μmol/mmol)

(μmol/mmol)Creatinine)

0.25 0.08 0.28 0.05 NS

Urinary -CEHC (μmol/mmol)

Creatinine)

0.69 0.25 0.66 0.13 NS

Urinary -CEHC (μmol/mmol)

Creatinine)

0.06 0.02 0.04 0.03 NS

Urinary F2-isoprostanes

863 113 1024 215 NS

(pmol/mmol creatinine)

Values given are mean SEM. *Corrected values obtained by dividing total by

(total cholesterol plus total triglycerides)

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0.000

0.050

0.100

0.150

0.200

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Fraction

Co

nce

ntr

atio

n m

mo

l/L

Cholesterol

Triglyceride

Figure 1a. FPLC chromatograms of a normal subject. VLDL and LDL peaks for

cholesterol, triglyceride and the TOHs are normal.

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Fraction

α-TOH

γ-TOH

Conce

ntr

atio

n u

mol/

L

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68

0.000

0.050

0.100

0.150

0.200

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Fraction

Co

nce

ntr

atio

n m

mo

l/L

Cholesterol

Triglyceride

Figure 1b. FPLC chromatograms from a heterozygous FHBL subject. VLDL and

LDL peaks for cholesterol, triglyceride and the TOHs with peak areas lower than a

normal subject, whereas the HDL is comparable.

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Fraction

Conce

ntr

atio

n u

mol/

L

um

ol/

L

α-TOH

γ-TOH

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69

Normal Vitamin E deficient

Heterozygous FHBL

Acanthocytes

Marked Acanthocytosis

Abetalipoproteinaemia

Acanthocytes

Figure 2. Blood film morphologies of human subjects with normal and abnormal

cell membranes, characterised by the presence of acanthocytes. The heterozygous

FHBL subject is one of the subjects from this study.

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The characteristics and analytical results from two children with

homozygous FHBL and one with ABL receiving α-TOH supplementation are shown in

Table 3. Homozygous FHBL subject 1 had similar plasma lipid parameters to

homozygous FHBL subject 2, but was receiving a two-fold higher daily dose of -TOH

reflected in increased platelet -TOH and urinary -CEHC concentrations. This might

in part explain the low concentration of urine F2-isoprostanes levels in this subject.

Homozygous FHBL subject 2 received the lowest dose of -TOH supplement and this

was reflected in lower platelet -TOH and urinary -CEHC concentrations. This

subject had the highest F2-isoprostanes of all the subjects and also had very low levels

of urinary -CEHC, possibly reflecting a low dietary intake of -TOH.

The ABL subject had a similar lipid profile to the two homozygous FHBL

subjects, but as predicted low serum and cellular TOH concentrations. The increased

urine metabolites might reflect high-dose -TOH supplementation in this subject.

Moreover, the urinary F2-isoprostanes output was higher than that of normal subjects,

but lower than that of the homozygous FHBL subject taking the lower dose supplement.

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Table 3. Lipid, apoprotein, TOH, TOH metabolite, and F2-isoprostane concentrations

in homozygous FHBL and ABL subjects.

HoFHBL/1 HoFHBL/2 ABL

Age (year/month)

2/4 0/6 1/2

Gender (M/F)

M/F

M F M

Treatment (-TOH U/day) 1,000 450 10,000

Total cholesterol (mmol/L) 0.6 0.7 0.7

HDL cholesterol (mmol/L) 0.6 0.7 0.6

Triglyceride (mmol/L) 0.2 0.2 0.04

LDL cholesterol (mmol/L) <0.01 0.01 0.02

ApoA-I (g/L) 0.56 0.64 0.42

ApoB (g/L) <0.05 <0.05

<0.05

Serum (μmol/L) 9.2

6.7 2.8

Serum (μmol/L) corrected * 11.5

7.4 3.8

Serum (μmol/L) <1 <1 <1

Serum (μmol/L) corrected * <1 <1 <1

Red cell 7.1 7.2 3.1

(μmol/L packed RBC’s)

Red cell

<0.3 <0.3 <0.3

(μmol/L packed RBC’s)

Platelet 2.97 2.01 0.85

(nmol/109 platelets)

Platelet 0.04 <0.03 <0.03

(nmol/109 platelets)

Urinary -CEHC (μmol/mmol)

Creatinine)

2.51 1.65 3.51

Urinary -CEHC (μmol/mmol)

Creatinine)

0.51 0.04 0.84

Urinary -CEHC (μmol/mmol)

Creatinine)

0 0 0

Urinary F2-isoprostanes 311 5594 2803

(pmol/mmol creatinine)

Values given are mean SEM.

* Corrected values obtained by dividing total by (total cholesterol plus total

triglycerides).

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2.4 Discussion

The present studies were designed to examine the effects of truncated apoB variants

causing FHBL on vitamin E metabolism, by exploring the relationship between plasma

and cellular TOH concentrations and urinary markers of TOH intake and oxidative

stress. The major findings were that when compared to controls, heterozygous FHBL

subjects had significantly decreased total cholesterol, triglyceride, LDL-cholesterol,

apoB, - and -TOH concentrations in the circulation. Although erythrocyte -TOH

concentrations were decreased in heterozygous FHBL subjects, no differences were

observed with lipid-adjusted serum TOHs, erythrocyte -TOH, platelet - or -TOH, or

urinary F2-isoprostanes and TOH metabolites. Taken together, our findings do not

support the recommendation that heterozygous FHBL subjects require vitamin E

supplementation.

The search for an appropriate marker of TOH status in-vivo in subjects with low

cholesterol has proved difficult. The use of plasma to assess TOH status is a common

approach and it has been suggested that both plasma and adipose tissue TOHs are

equally good markers of intake.17

The challenge arises when plasma lipid levels are

low, lipoprotein transport pathways are saturated and serum TOH levels are decreased.

One approach has been to measure cellular TOH concentrations to assess TOH

transport in-vivo. Erythrocytes have the advantage in that they are in the circulation for

about 120 days and therefore might reflect long-term intake. However, erythrocyte

TOH concentrations have been shown to passively reflect lipid concentrations.9 This

could explain the lower erythrocyte -TOH concentrations observed with heterozygous

FHBL subjects. Erythrocyte to HDL exchange of -TOH has been described in-vitro 18

and might also contribute to the reduction in erythrocyte -TOH concentrations

observed. Although it has not been demonstrated that platelet TOHs correlate with

peripheral nervous tissue concentration, they might better reflect dietary vitamin E

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73

intake as they are not passively influenced by lipid concentrations.19

Our preference,

therefore, was to use platelet TOH concentrations to assess TOH transport in-vivo.

Platelet TOHs were not different between the FHBL heterozygotes and normal

subjects.

The assessment of TOH metabolites in urine provides a marker of short-term

TOH intake.20

12

The measurement of F2-isoprostanes, a marker of in-vivo lipid

peroxidation,21

22

in spot urine samples also provides an assessment of daily isoprostane

excretion in human subjects.23

TOH metabolites were not different between the

heterozygous FHBL subjects and normal controls, consistent with a similar dietary

intake of TOHs between the two groups. The lack of difference in urinary F2-

isoprostane excretion between heterozygous FHBL and normal subjects indicates the

lack of significant oxidative stress in heterozygous FHBL subjects.

Recent studies have demonstrated the potential importance of -TOH in the

regulation of specific genes,24

so it follows that a reduction in cellular -TOH may

have clinical consequences. Although erythrocyte -TOH was decreased, no

differences were observed with lipid-adjusted serum TOHs, erythrocyte -TOH, platelet

-TOH or -TOH, or urinary F2-isoprostanes and TOH metabolites. These findings

suggest that adequate delivery of TOHs to cells and the inhibition of oxidative stress in-

vivo occurs via the chylomicron remnant and HDL metabolic pathways.

We also examined TOH status and oxidative stress in two subjects with

homozygous FHBL and one subject with ABL on vitamin E supplementation.

Compared to normal controls, we observed moderately elevated erythrocyte -TOH

concentrations in the homozygous FHBL subjects and there was no difference between

the two subjects. However, there were two-three fold elevations in platelet -TOH

concentrations in these subjects compared to normal controls. This was particularly

evident for homozygous FHBL subject 1 taking the higher dose, consistent with the

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74

notion that platelet TOH concentrations provide the best measure of cellular TOH

transport and reflect intake. The measurement of urine metabolites also provides

important information regarding compliance to therapy for these subjects, and as can be

seen the highest dose of -TOH giving the highest output for urine -CEHC.

Recent studies have examined the efficacy of vitamin supplementation in subjects

with ABL and homozygous FHBL.25

5 Retinal changes were evident in ABL and

homozygous FHBL subjects, despite early treatment with high-dose oral vitamin A and

E,25

however, oxidative stress was not assessed in this study. Oxidative stress was

measured in subjects with ABL subjects supplemented with high dose vitamin E and A

since infancy, with no evidence found for oxidative stress using plasma carbonyls and

the lag phase for the oxidization of plasma and HDL.5

In our study one of the FHBL homozygotes had low urinary F2-isoprostanes,

whereas the other had a marked increase. This could be due to the age difference

between the two subjects, the dose of -TOH used, or possibly the contribution from

dietary sources of vitamin E. It has been shown that babies have highly elevated

urinary isoprostanes,26

possibly due to the oxidative stress at birth and the low

efficiency of natural antioxidant systems in the newborn.27

Of note, the homozygous

FHBL subject with elevated urinary F2-isoprostanes was only six months of age and

had very low levels of the -CEHC metabolite of -TOH, indicating that dietary intake

of this nutrient is decreased.

There has been considerable interest in the potential benefits of -TOH for human

health 28

29

and a recent analysis suggests that various forms of vitamin E may be

important in the preservation of cognition in subjects with Alzheimer’s disease.30

Clearly, further investigation is needed to assess the efficacy of the different forms of

vitamin E and their relative effects on oxidative stress in subjects with very low

cholesterol. Amid recent interest in the potential benefits of tocotrienols and

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75

neurological function,30

there remains potential for different forms of vitamin E to

further reduce sequelae in conditions associated with low cholesterol and oxidative

stress.

In summary we have examined serum and cellular TOH levels in-vivo and

oxidative stress in subjects with heterozygous FHBL. Taken together, in the absence of

clinical signs associated with vitamin E deficiency, our findings do not support the

recommendation that heterozygous FHBL subjects require vitamin E supplementation.

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[10] Bieri JG, Tolliver TJ, and Catignani GL. Simultaneous determination of -

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[18] Simon E, Paul JL, Atger V, Simon A, and Moatti N. Study of vitamin E net

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[19] Lehmann J. Comparative sensitivities of tocopherol levels of platelets, red blood

cells and plasma for estimating vitamin E nutritional status in the rat. Am J Clin

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[20] Swanson JE, Ben RN, Burton GW, and Parker RS. Urinary excretion of 2,7, 8-

trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of

elimination of -tocopherol in humans. J Lipid Res 1999; 40: 665-671.

[21] Morrow JD. Quantification of isoprostanes as indices of oxidant stress and the

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[22] Meagher EA, Barry OP, Lawson JA, Rokach J, and FitzGerald GA. Effects of

vitamin E on lipid peroxidation in healthy persons. JAMA 2001; 285: 1178-

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[23] Cracowski JL, Durand T, and Bessard G. Isoprostanes as a biomarker of lipid

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[24] Azzi A, Gysin R, Kempna P, Munteanu A, Villacorta L, Visarius T, and Zingg

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[25] Chowers I, Banin E, Merin S, Cooper M, and Granot E. Long-term assessment

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[26] Barden AE, Mori TA, Dunstan JA, Taylor AL, Thornton CA, Croft KD, Beilin

LJ, and Prescott SL. Fish oil supplementation in pregnancy lowers F2-

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[27] Frank L and Sosenko IR. Development of lung antioxidant enzyme system in

late gestation: possible implications for the prematurely born infant. J Pediatr

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[28] Jiang Q, Christen S, Shigenaga MK, and Ames BN. -Tocopherol, the major

form of vitamin E in the US diet, deserves more attention. Am J Clin Nutr 2001;

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[29] Christen S, Woodall AA, Shigenaga MK, Southwell-Keely PT, Duncan MW,

and Ames BN. -Tocopherol traps mutagenic electrophiles such as NO(X) and

complements -tocopherol: physiological implications. Proc Natl Acad Sci USA

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* A version of this chapter has been published in Am J Clin Nutr. 2006 Jan 83(1):95-

102.

80

CHAPTER 3

EFFECT OF MIXED TOHs ON SERUM γ-TOH, BLOOD

CELL γ-TOH AND BIOMARKERS OF PLATELET

ACTIVATION IN SUBJECTS WITH TYPE 2 DIABETES

3.1 Introduction

A number of large clinical trials and smaller supplementation studies have investigated

the link between -TOH and CVD risk. The results from large clinical trials with -

TOH have been largely negative.1-4

Other studies have been more promising in subjects

with enhanced oxidative stress associated with end-stage renal disease,5 in subjects with

elevated cholesterol,6 and in combination with vitamin C in transplant subjects.

7

-TOH is the other major form of TOH in the diet. Recent studies have suggested

a potential role for -TOH in preventing complications associated with diseases

involving oxidative stress and inflammation.8 However, it has been shown that

supplementation with -TOH can significantly reduce the plasma concentrations of -

TOH.9, 10

Despite this, there is little information comparing changes in both plasma and

cellular TOH concentrations following supplementation with either and -TOH.

Individuals with type 2 diabetes have abnormal platelet function. This is

characterised by increased interaction of platelets and von Willebrand factor (vWf) and

platelets and fibrin.11

Coagulation factors are increased and anticoagulant factors

decreased, leading to increased platelet activation and risk of thrombosis.11

Platelet

activation is associated with plaque instability and represents an important therapeutic

target in subjects at risk of thrombosis.12

The contribution of platelet derived factors to

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monocyte recruitment and inflammation within the vessel wall is depicted in Figure 1.

Supplementation with -TOH has been shown to inhibit platelet function in

subjects with type 1 diabetes,13

type 2 diabetes14

and subjects with

hypercholesterolaemia.15

In healthy subjects, a mixed TOH supplement comprised

predominantly of -TOH inhibited ex-vivo ADP-induced platelet aggregation and

increased ecNOS activation and NO release, whereas synthetic -TOH did not alter

these endpoints.16

In this study our objective was to examine the relative serum and cellular uptake

of the TOH isomers following supplementation with RRR--TOH or mixed TOHs

comprised predominantly of -TOH. The excretion of vitamin E metabolites α, γ, and δ-

CEHC was also determined to assess in-vivo TOH status. We also sought to examine

the effects of each supplement on in-vivo markers of platelet activation in type 2

diabetic subjects, a group at increased risk of CVD.

3.2 Subjects and Methods

3.2.1 Study Protocol

Fifty-eight men and women with type 2 diabetes were recruited from the Perth general

population to the School of Medicine and Pharmacology at the University of Western

Australia. All participants had a previous diagnosis of type 2 diabetes. All medication

was taken as normal on the morning of each study visit. Exclusion criteria included:

BMI >35 kg/m2, use of insulin, NSAIDs or nitrate medication, smoking, use of

vitamin E supplements, recent coronary or cerebrovascular event (< 6 months),

impaired renal function (serum creatinine > 110 μmol/L for men and > 100 μmol/L for

women) and alcohol intake > 40 g/day men and > 30 g/day women. All volunteers

ceased any vitamin, antioxidant and fish oil supplements for at least 3 weeks prior to

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Figure 1. Platelet-Derived Mediators of the Inflammatory Response.

Activated platelets release inflammatory and mitogenic substances into the

microenvironment, primarily altering the chemotactic, adhesive, and proteolytic

properties of the endothelium. Preformed platelet mediators, stored in α-granules, can be

released immediately after platelet activation through a process of exocytosis triggered

by increased intracellular calcium levels. Activated platelets are also capable of time-

dependent synthesis of protein mediators, such as tissue factor and interleukin-1β. CD40

ligand is stored in the cytoplasm of resting platelets and rapidly presents on the surface

after platelet activation. After cleavage, to generate a soluble, functional fragment

(soluble CD40 ligand), the mediator is released into the extracellular environment,

inducing inflammatory responses in the endothelium by binding CD40 on endothelial

cells. P-selectin is released from platelet granules and binds to the P-selectin

glycoprotein ligand 1 (PSGL-1) receptor on monocytes, enhancing the adhesion of the

monocytes to vascular-cell adhesion molecule-1 (VCAM-1) and the other adhesins

expressed on activated endothelial cells and inducing the production of tissue factor by

monocytes. Activated platelets also release chemokines that trigger the recruitment of

monocytes (e.g., regulated on activation normal T-cell expressed and secreted

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83

[RANTES]) or promote the differentiation of monocytes into macrophages (e.g.,

platelet factor 4), as well as matrix-degrading enzymes such as matrix metalloproteinase

(MMP) 2 or 9. Interleukin-1β is a major mediator of platelet-induced activation of

endothelial cells, causing enhanced chemokine release and up-regulation of endothelial

adhesion molecules to promote the adhesion of neutrophils and monocytes to the

endothelium. ICAM denotes intracellular adhesion molecule, mRNA messenger RNA,

MCP-1 monocyte chemoattractant protein 1, and OH− hydroxyl radical.

Taken from 17

and reproduced with permission.

study entry. Following washout, subjects were randomised to receive either (i) 500

mg/day RRR-α-TOH (749 IU), (ii) 500 mg/day mixed TOHs (315 mg -, 75 mg -, 110

mg -TOH), or (iii) placebo (soybean oil containing < 1 mg TOHs). 250 mg tablets

were taken twice daily at breakfast and dinner. All volunteers provided written

informed consent prior to inclusion in the study. The study was approved by the

University of Western Australia Human Ethics Committee.

3.2.2 Sample Preparation

Blood and 24 h urine samples were collected from each subject at baseline and at the

completion of the six week intervention. The urine samples were aliquoted into 5 mL

tubes without a preservative and frozen at -80C until analysis. Blood was collected

into 3 mL serum and citrate tubes and was processed immediately. Serum samples for

sCD40L and TXB2 determination were allowed to clot for 1 h at 37C before being

separated.18

The remaining serum and citrate samples were spun at 2000 x g for 10 min

at 4C and samples were then frozen at -80C prior to analysis. One 9 mL EDTA

sample was also collected for erythrocyte and platelet TOH concentrations and was

processed within 4 h of collection. It was determined that both erythrocyte and platelet

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TOHs were stable for at least 24 h post collection (unpublished data). The preparation

of platelets and erythrocytes for TOH analysis is described in Chapter 2.

3.2.3 HPLC analysis of TOHs with Electrochemical Detection

HPLC analysis of TOHs is described in Chapter 2.

3.2.4 Urinary TOH Metabolites

GCMS analysis of TOH metabolites is described in Chapter 2.

3.2.5 ELISA Assays

Serum and plasma concentrations of sCD40L were determined using the BMS239MST

Module kit supplied by (Bender MedSystems, Vienna, Austria) according to the

manufacturer’s instructions. For the citrate plasma samples the assay was modified to

detect low concentrations of CD40L.19

Plasma soluble P-selectin (sP-selectin) was

measured using the R&D kit by (R&D systems, Minneapolis, USA). Serum TXB2

and urinary 11-dehydro-TXB2 were measured using EIA kits supplied by (Cayman

Chemical, Michigan, USA). vWf was measured using the Stago Kit latex

immunoassay on the STA-R automated coagulation analyser (Diagnostica Stago,

Asnieres, France). All assays had inter and intra-assay CV’s less than 10% and all of

the manual assay samples were assayed in duplicate.

3.2.6 Lipid Analysis

Total cholesterol was measured using the enzymatic method of CHOD-PAP (HiCo),

triglyceride using the enzymatic GPO-PAP assay and HDL by the direct method HDL-

Plus. The kits were supplied by Boehringer Mannheim and the assays performed on

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the Hitachi 917 chemistry analyser. All assays had inter and intra-assay CV’s less

than 6%.

3.2.7 Statistical Analysis

Analyses were performed using SPSS for Windows version 12 (SPSS Inc, Chicago). All

data are presented as mean SEM. Values not normally distributed were log-

transformed before analysis. Baseline characteristics were compared using analysis of

variance. General linear models were used to examine differences in post-intervention

values after adjustment for baseline values used as covariates. Post-hoc comparisons

were performed using Bonferroni correction. P < 0.5 was considered significant.

3.3 Results

3.3.1 Serum and Cellular TOH Analysis

55 subjects completed the study. Three withdrew due to changes in treatment by their

General Practitioner during the trial. The subjects were well matched at baseline and

their characteristics are presented in Table 1.

Serum, erythrocyte and platelet TOH concentrations post intervention are

presented in Figures 1, 2 and 3 respectively. Supplementation with -TOH significantly

increased serum, red cell and platelet -TOH concentrations. Supplementation with

mixed TOHs increased serum -TOH concentration but the increase in platelet -TOH

and red cells did not reach statistical significance. Supplementation with mixed TOHs

gave a 4-fold increase in serum, erythrocyte and platelet -TOH concentrations.

Conversely, supplementation with -TOH led to a significant decrease in erythrocyte -

TOH while the decrease in serum and platelet -TOH concentrations did not reach

statistical significance.

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Table 1. Baseline characteristics for all subjects involved in the study separated by

treatment group.

Treatment

RRR-

TOH

Mixed

TOHs Placebo

n 18 19 18

Age (y) 64 7 58 5 62 7 Gender Male/Female 13/5 12/7 16/2

BMI (kg/m2) 29 0.5 28 0.6 28 1.0

Hypertension 9 13 12

Statin use 11 11 7

Aspirin use 8 5 8

Total Cholesterol (mmol/L) 4.71 0.23 4.70 0.25 4.62 0.16 LDL (mmol/L) 2.67 0.17 2.64 0.23 2.63 0.18 HDL (mmol/L) 1.27 0.08 1.35 0.07 1.35 0.11 Triglyceride (mmol/L) 1.70 0.16 1.50 0.16 1.38 0.18

Values given are mean SEM. There were no significant differences between the

groups.

There was no significant change in serum or cellular TOH concentrations in the placebo

group. There were no changes in serum cholesterol, HDL or triglyceride concentrations

during the 6 week intervention (post supplementation values for -TOH, mixed TOH

and placebo groups respectively for total cholesterol were 4.94 0.2, 4.84 0.3 and

4.71 0.2 mmol/L, HDL cholesterol 1.29 0.08, 1.28 0.06 and 1.33 0.10 mmol/L,

triglyceride 1.87 0.22, 1.76 0.17 and 1.59 0.24 respectively).

3.3.2 Urinary TOH Metabolite Excretion

The excretion of the three major TOH metabolites α, γ, and δ-CEHC, corrected for

creatinine excretion are given in Table 2. At baseline low levels of α-CEHC could be

detected in most subjects, possibly reflecting reasonably high serum α-TOH

concentrations. Very low concentrations of δ-CEHC were present at baseline and

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87

excretion of this metabolite increased significantly after supplementation with mixed

TOHs (containing 110 mg of δ-TOH). Major increases in the excretion of α-CEHC and

γ-CEHC were observed following supplementation with α-TOH and mixed TOHs

respectively. Interestingly, excretion of γ-CEHC increased significantly after

supplementation with α-TOH. This suggests that the decrease in plasma and cellular γ-

TOH seen with α-TOH supplementation may be the result of increased γ-TOH

metabolism to γ-CEHC.

Table 2. Urinary excretion of TOH metabolites α, γ and δ – CEHC before and after

treatment.

Treatment

RRR-α-TOH Mixed TOHs Placebo

n 18 19 18

Pre Post Pre Post Pre Post

α-CEHC 0.21 ± 0.04 7.84 ± 1.4* 0.22 ± 0.03 1.29 ± 0.21* 0.19 ± 0.06 0.17 ± 0.03

γ-CEHC 0.65 ± 0.09 0.88 ± 0.11# 0.63 ± 0.07 10.46 ± 1.61* 0.51 ± 0.08 0.52 ± 0.06

δ-CEHC 0.07 ± 0.01 0.08 ± 0.01 0.08 ± 0.01 3.49 ± 0.48* 0.06 ± 0.01 0.06 ± 0.01

Values are presented as mean ± SEM (μmol/mmol creatinine). Baseline-adjusted post

intervention differences analysed using general linear models and Bonferroni correction.

* P<0.001, #

P=0.01

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-TOH Mixed TOH Placebo

0

20

40

60

80

P < 0.001

P = 0.02

-t

oco

ph

ero

lm

mo

l/L

-TOH Mixed TOH Placebo

0

5

10

P < 0.001

P = 0.09

-t

oco

ph

ero

l

mo

l/L

Figure 2. Post intervention serum concentrations of and -TOH, corrected for

baseline values.

Supplementation was for six weeks with either -TOH, mixed TOHs or placebo. Data

presented are mean SEM. General linear models were used to examine differences in

post-intervention values after adjustment for baseline values and post-hoc comparisons

were performed using Bonferroni correction. P values represent comparison to placebo.

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- TOH Mixed TOH Placebo

0

5

10

15

20

P < 0.001

-t

oco

ph

ero

lm

mo

l/L

- TOH Mixed TOH Placebo

0

1

2

3

P = 0.004

P < 0.001

-t

oco

ph

ero

l

mo

l/L

Figure 3. Post intervention red cell concentrations of and -TOH, corrected for

baseline values.

Supplementation was for six weeks with -TOH, mixed TOHs or placebo. Data is

quoted per litre of packed red cells and presented as mean SEM. General linear

models were used to examine differences in post-intervention values after adjustment

for baseline values and post-hoc comparisons were performed using Bonferroni

correction. P values represent comparison to placebo.

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- TOH Mixed TOH Placebo

0

1

2

3

4P < 0.001

P = 0.08

-T

oc n

mo

l/1

09 c

ells

- TOH Mixed TOH Placebo

0.0

0.2

0.4

0.6

0.8

P < 0.001

-T

oc n

mo

l/1

09 c

ells

Figure 4. Post intervention platelet concentrations (per 109 cells) of and -TOH,

corrected for baseline values.

Supplementation was for six weeks with either -TOH, mixed TOHs or placebo. Data

presented are mean SEM. General linear models were used to examine differences in

post-intervention values after adjustment for baseline values and post-hoc comparisons

were performed using Bonferroni correction. P values represent comparison to placebo.

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3.3.3 Markers of Platelet and Endothelial Function

As shown in Table 3, there was no effect from any TOH treatment on sCD40L, serum

TXB2, urinary 11-dehydro TXB2, sP-selectin and vWf. As expected baseline values for

both serum TXB2 and urine 11-dehydro-TXB2 concentrations were lower in aspirin

(serum TXB2 5.2 ± 1.8 ng/mL, urinary 11-dehydro-TXB2 838 ± 217 ng/day) versus non-

aspirin treated subjects (serum TXB2 59.6 ± 6 ng/mL, urinary 11-dehydro-TXB2 1018 ±

264 ng/day). Neither aspirin nor statin use was significantly different between the

treatment groups (P = 0.421 (aspirin) P = 0.351 (statin)). In addition, adjustment for

aspirin and statin use did not influence the results.

3.4 Discussion

The present controlled intervention trial demonstrates that high dose -TOH

supplementation over 6 weeks caused -TOH concentrations in serum and cells to more

than double and -TOH concentrations in red cells to decrease. Previous studies have

examined the effects of -TOH supplementation on serum and cellular TOH

concentrations showing significant increases in -TOH with a corresponding decrease

in -TOH.9, 20

Our study in a diabetic population resulted in similar changes in TOH

concentrations. Taken together, these studies conclusively demonstrate that serum and

cellular concentrations of -TOH are reduced following -TOH supplementation.

Loss of -TOH from serum or cells may have adverse health consequences.8 Low

serum concentrations of -TOH, but not -TOH have been observed in subjects with

CHD.21, 22

Therefore, supplementation with -TOH alone may have disadvantages over

mixed TOHs rich in -TOH. The present study is the first to examine changes in serum

and cellular TOHs following supplementation with -enriched TOHs. Previous studies

have examined the effect of deuterated -TOH on γ-CEHC production 23

and the effects

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Table 3. Effects of treatment on biomarkers of platelet function

Treatment

RRR--TOH Mixed TOHs Placebo

n 18 19 18

Pre Post P Pre Post P Pre Post

Citrate CD40L

(ng/mL)

0.5.0.1 0.59.0.1 1.0 0.40.1 0.5.1 1.0 0.70.1 0.70.1

Serum CD40L

(ng/mL)

3.10.4 3.40.6 0.32 4.30.7 2.60.3 1.0 4.80.7 3.40.4

Serum CD40L †

(ng/mL)

6.41.0 6.40.8 1.0 6.41.0 6.30.8 1.0 6.41.0 6.80.9

Serum TXB2 †

(ng/mL)

3910 33 8 1.0 419 478 0.22 369 319

Urine 11-

dehydro TXB2

(ng/day)

757227 617186 0.54 1008212 886203 1.0 1111469 673264

Citrate P-selectin

(ng/mL)

504 565 1.0 503 524 1.0 564 553

vWf (%) 15210 15510 1.0 1219 12711 1.0 13812 13411

Values presented are mean SEM. P values presented are for baseline-adjusted post

intervention differences analysed using general linear models and Bonferroni correction.

Data log-transformed for statistical analysis.

† Samples clotted for 1 h at 37C.

of a mixed TOH supplement in subjects with end-stage renal disease 24

and in normal

subjects to examine platelet function.16

Supplementation with mixed TOHs in our study resulted in a significant increase

in serum -TOH, together with 4 -fold increases in -TOH in serum, erythrocytes and

platelets. Interestingly, the response to supplementation with mixed TOHs appeared

greater for platelets than in serum and erythrocytes. Relatively higher uptake by

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platelets of -TOH during supplementation has been observed previously.20

Our study

showed a higher uptake of -TOH by platelets after mixed TOH supplementation and

suggest that platelets provide an excellent marker of intake. Importantly, all our

subjects had normal fasting plasma cholesterol concentrations. This may be important

in TOH transport given that absorption of -TOH into plasma and cells is inhibited in

hyperlipidaemic subjects.25

The excretion of TOH metabolites in urine provide a good marker of vitamin E

status. They are formed via cytochrome P450-mediated ω-oxidation followed by β-

oxidation in the liver.26-28

The excretion of each metabolite strongly reflected the

composition of the TOH supplement. It is interesting to note that supplementation with

-TOH caused an increase in the excretion of γ-CEHC together with decreased -TOH

in plasma and red cells. This result suggests that the decrease in cellular -TOH is due

to increased metabolism to γ-CEHC, possibly reflecting displacement of -TOH by α-

TOH from incorporation into lipoproteins in the liver. This is in contrast to the finding

of Bruno et.al. who saw no correlation between plasma -TOH and γ-CEHC following

supplementation with a lower dose of 75 mg/day of -TOH for 6 days.29

Since subjects with type 2 diabetes are thought to have increased platelet

activation, we examined the potential effects of TOH supplementation on biomarkers of

platelet activation. CD40 ligand is a transmembrane protein primarily found within

platelets 30

and has the capacity to initiate inflammatory reactions when bound to CD40

present on a number of cells including endothelial cells.31

The soluble form, sCD40

ligand can be detected in plasma, and its importance in thrombosis was recently

highlighted in subjects with acute coronary syndromes 32

and in healthy women at risk

of cardiovascular events.33

Soluble CD40L concentrations are also elevated in diabetic

patients 34, 35

and have been reduced in type 2 diabetics following treatment with PPAR

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agonists.34, 36

Statin therapy can reduce the elevated sCD40L concentrations in

hypercholesterolemic subjects,37, 38

independent of cholesterol lowering.39

Treatment

with aspirin has also led to reductions in sCD40L in stroke patients.40

It has been

demonstrated that CD40L expression in primary human T cells is regulated by the NF-

B transcription factor.41

The activation of this transcription factor can be induced in

the retina of diabetic rats and it can be inhibited by supplementation with antioxidants

such as dl--TOH acetate.42

Given that -TOH can inhibit the oxidised LDL mediated

activation of the NF-B transcription factor in endothelial cells,43

we hypothesised that

one or both of our TOH supplements could inhibit the production and hence release of

sCD40L into the circulation. However, we saw no effect with either TOH supplement

on sCD40L release in serum or plasma in subjects with type 2 diabetes. We cannot rule

out a treatment effect on platelet CD40L expression, however this seems unlikely given

that platelet CD40L has been shown to significantly correlate with sCD40L.39

Platelet activation and subsequent adhesion to dysfunctional endothelium is an

important step in thrombus formation and the formation of thromboxane (TX) A2.12

A

metabolite of TXA2 , 11-dehydro TXB2 , can be measured in urine and provides a good

indication of in-vivo TXA2 production.44

Neither treatment affected 11-dehydro TXB2

excretion nor whole blood TXB2 production.

Membrane bound P-selectin is a cell adhesion molecule that mediates the

adhesion of leucocytes to endothelial cells and platelets and subsequently has an

important role in thrombosis45

with the soluble form readily detectable in plasma. vWF

is stored with P-selectin in Weibel-Palade bodies and both are released during

stimulation with prothrombotic stimuli.46

-TOH has been shown to inhibit P-selectin

expression in human platelets.47

A small uncontrolled study in subjects with

hypercholesterolaemia reported significant reductions in sP-selectin, vWf and urinary

11-dehydro-TXB2 following supplementation with 600 mg -TOH acetate per day for 2

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weeks.15

A subsequent study also in subjects with hypercholesterolaemia observed a

significant reduction in vWf following supplementation with -TOH (400 IU and 800

IU).48

High dose (1200 IU/day) RRR--TOH given to diabetic subjects for 3 months

was also shown to reduce sP-selectin concentrations.14

The lack of treatment effect on

sP-selectin and vWf observed in our study may relate to circulating cholesterol and

hence background oxidised LDL concentrations. Twenty-nine of our subjects were

taking statins as lipid lowering therapy for cholesterol management and 21 were taking

aspirin. Although adjusting for statin or aspirin use did not affect our results after

adjusting for baseline values, it is possible that our study lacked sufficient power to

establish whether the TOH treatments have any potential therapeutic benefits in subjects

not taking either treatment.

The aim of our study was to examine subjects in a realistic clinical setting, in an

attempt to examine the potential benefits of vitamin E supplements. Amid recent

concern that high dose -TOH ( 400 IU/day) may increase all-cause mortality 49

and

the risk of heart failure 50

there seems little benefit in taking high dose -TOH

supplements to prevent CVD and inhibit platelet function. As for a mixed TOH

supplement enriched in -TOH, data regarding safety in large doses and any potential

benefit on CVD are lacking at this time. Our findings suggest -TOH supplements do

not inhibit platelet function or sCD40L release in well controlled type 2 diabetic

subjects. However, mixed TOH treatment does result in increased concentrations of

both and -TOH in serum without the reduction in red cell -TOH seen with high dose

-TOH supplementation. Future studies should determine if this supplement can

prevent sequelae associated with inflammation and oxidative stress.

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3.5 References

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[2] Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after

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per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet 1999; 354:

447-455.

[3] Collins R, Armitage J, Parish S, Sleight P, and Peto R. MRC/BHF Heart

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individuals: a randomised placebo-controlled trial. Lancet 2002; 360: 23-33.

[4] Vivekananthan DP, Penn MS, Sapp SK, Hsu A, and Topol EJ. Use of

antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis

of randomised trials. Lancet 2003; 361: 2017-2023.

[5] Boaz M, Smetana S, Weinstein T, Matas Z, Gafter U, Iaina A, Knecht A,

Weissgarten Y, Brunner D, Fainaru M, and Green MS. Secondary prevention

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[6] Salonen RM, Nyyssonen K, Kaikkonen J, Porkkala-Sarataho E, Voutilainen S,

Rissanen TH, Tuomainen TP, Valkonen VP, Ristonmaa U, Lakka HM,

Vanharanta M, Salonen JT, and Poulsen HE. Six-year effect of combined

vitamin C and E supplementation on atherosclerotic progression: the

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Circulation 2003; 107: 947-953.

[7] Fang JC, Kinlay S, Beltrame J, Hikiti H, Wainstein M, Behrendt D, Suh J, Frei

B, Mudge GH, Selwyn AP, and Ganz P. Effect of vitamins C and E on

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progression of transplant-associated arteriosclerosis: a randomised trial. Lancet

2002; 359: 1108-1113.

[8] Jiang Q, Christen S, Shigenaga MK, and Ames BN. -Tocopherol, the major

form of vitamin E in the US diet, deserves more attention. Am J Clin Nutr 2001;

74: 714-722.

[9] Huang HY and Appel LJ. Supplementation of diets with alpha-tocopherol

reduces serum concentrations of gamma- and delta-tocopherol in humans. J Nutr

2003; 133: 3137-3140.

[10] Handelman GJ, Epstein WL, Peerson J, Spiegelman D, Machlin LJ, and Dratz

EA. Human adipose alpha-tocopherol and gamma-tocopherol kinetics during

and after 1 y of alpha-tocopherol supplementation. Am J Clin Nutr 1994; 59:

1025-1032.

[11] Creager MA, Luscher TF, Cosentino F, and Beckman JA. Diabetes and vascular

disease: pathophysiology, clinical consequences, and medical therapy: Part I.

Circulation 2003; 108: 1527-1532.

[12] Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999; 340:

115-126.

[13] Colette C, Pares-Herbute N, Monnier LH, and Cartry E. Platelet function in type

I diabetes: effects of supplementation with large doses of vitamin E. Am J Clin

Nutr 1988; 47: 256-261.

[14] Devaraj S, Chan AV, Jr., and Jialal I. alpha-Tocopherol supplementation

decreases plasminogen activator inhibitor-1 and P-selectin levels in type 2

diabetic patients. Diabetes Care 2002; 25: 524-529.

[15] Davi G, Romano M, Mezzetti A, Procopio A, Iacobelli S, Antidormi T,

Bucciarelli T, Alessandrini P, Cuccurullo F, and Bittolo Bon G. Increased levels

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of soluble P-selectin in hypercholesterolemic patients. Circulation 1998; 97:

953-957.

[16] Liu M, Wallmon A, Olsson-Mortlock C, Wallin R, and Saldeen T. Mixed

tocopherols inhibit platelet aggregation in humans: potential mechanisms. Am J

Clin Nutr 2003; 77: 700-706.

[17] Davi G and Patrono C. Platelet Activation and Atherothrombosis. N Eng J Med

2007; 357: 2482-2494.

[18] Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F,

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endoperoxide synthases. J Pharmacol Exp Ther 1994; 271: 1705-1712.

[19] Thom J, Gilmore G, Yi Q, Hankey GJ, and Eikelboom JW. Measurement of

soluble P-selectin and soluble CD40 ligand in serum and plasma. J Thromb

Haemost 2004; 2: 2067-2069.

[20] Lehmann J, Rao DD, Canary JJ, and Judd JT. Vitamin E and relationships

among tocopherols in human plasma, platelets, lymphocytes, and red blood

cells. Am J Clin Nutr 1988; 47: 470-474.

[21] Kontush A, Spranger T, Reich A, Baum K, and Beisiegel U. Lipophilic

antioxidants in blood plasma as markers of atherosclerosis: the role of -

carotene and -tocopherol. Atherosclerosis 1999; 144: 117-122.

[22] Ohrvall M, Sundlof G, and Vessby B. , but not tocopherol levels in serum are

reduced in coronary heart disease patients. J Intern Med 1996; 239: 111-117.

[23] Leonard SW, Paterson E, Atkinson JK, Ramakrishnan R, Cross CE, and Traber

MG. Studies in humans using deuterium-labeled alpha- and gamma-tocopherols

demonstrate faster plasma gamma-tocopherol disappearance and greater gamma-

metabolite production. Free Radic Biol Med 2005; 38: 857-866.

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[24] Himmelfarb J, Kane J, McMonagle E, Zaltas E, Bobzin S, Boddupalli S,

Phinney S, and Miller G. Alpha and gamma tocopherol metabolism in healthy

subjects and patients with end-stage renal disease. Kidney Int 2003; 64: 978-991.

[25] Hall WL, Jeanes YM, and Lodge JK. Hyperlipidemic subjects have reduced

uptake of newly absorbed vitamin E into their plasma lipoproteins, erythrocytes,

platelets, and lymphocytes, as studied by deuterium-labeled alpha-tocopherol

biokinetics. J Nutr 2005; 135: 58-63.

[26] Schultz M, Leist M, Petrzika M, Gassmann B, and Brigelius-Flohe R. Novel

urinary metabolite of -tocopherol, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-

hydroxychroman, as an indicator of an adequate vitamin E supply? Am J Clin

Nutr 1995; 62: 1527S-1534S.

[27] Birringer M, Drogan D, and Brigelius-Flohe R. Tocopherols are metabolized in

HepG2 cells by side chain omega-oxidation and consecutive beta-oxidation.

Free Radic Biol Med 2001; 31: 226-232.

[28] Sontag TJ and Parker RS. Cytochrome P450 omega-hydroxylase pathway of

tocopherol catabolism. Novel mechanism of regulation of vitamin E status. J

Biol Chem 2002; 277: 25290-25296.

[29] Bruno RS, Leonard SW, Li J, Bray TM, and Traber MG. Lower plasma {alpha}-

carboxyethyl-hydroxychroman after deuterium-labeled {alpha}-tocopherol

supplementation suggests decreased vitamin E metabolism in smokers. Am J

Clin Nutr 2005; 81: 1052-1059.

[30] Inwald DP, McDowall A, Peters MJ, Callard RE, and Klein NJ. CD40 is

constitutively expressed on platelets and provides a novel mechanism for platelet

activation. Circ Res 2003; 92: 1041-1048.

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[31] Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus

G, and Kroczek RA. CD40 ligand on activated platelets triggers an

inflammatory reaction of endothelial cells. Nature 1998; 391: 591-594.

[32] Heeschen C, Dimmeler S, Hamm CW, van den Brand MJ, Boersma E, Zeiher

AM, and Simoons ML. Soluble CD40 ligand in acute coronary syndromes. N

Engl J Med 2003; 348: 1104-1111.

[33] Schonbeck U, Varo N, Libby P, Buring J, and Ridker PM. Soluble CD40L and

cardiovascular risk in women. Circulation 2001; 104: 2266-2268.

[34] Varo N, Vicent D, Libby P, Nuzzo R, Calle-Pascual AL, Bernal MR, Fernandez-

Cruz A, Veves A, Jarolim P, Varo JJ, Goldfine A, Horton E, and Schonbeck U.

Elevated plasma levels of the atherogenic mediator soluble CD40 ligand in

diabetic patients: a novel target of thiazolidinediones. Circulation 2003; 107:

2664-2669.

[35] Lim HS, Blann AD, and Lip GY. Soluble CD40 ligand, soluble P-selectin,

interleukin-6, and tissue factor in diabetes mellitus: relationships to

cardiovascular disease and risk factor intervention. Circulation 2004; 109: 2524-

2528.

[36] Marx N, Imhof A, Froehlich J, Siam L, Ittner J, Wierse G, Schmidt A, Maerz W,

Hombach V, and Koenig W. Effect of rosiglitazone treatment on soluble CD40L

in patients with type 2 diabetes and coronary artery disease. Circulation 2003;

107: 1954-1957.

[37] Cipollone F, Mezzetti A, Porreca E, Di Febbo C, Nutini M, Fazia M, Falco A,

Cuccurullo F, and Davi G. Association between enhanced soluble CD40L and

prothrombotic state in hypercholesterolemia: effects of statin therapy.

Circulation 2002; 106: 399-402.

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[38] Kinlay S, Schwartz GG, Olsson AG, Rifai N, Sasiela WJ, Szarek M, Ganz P,

and Libby P. Effect of atorvastatin on risk of recurrent cardiovascular events

after an acute coronary syndrome associated with high soluble CD40 ligand in

the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering

(MIRACL) Study. Circulation 2004; 110: 386-391.

[39] Sanguigni V, Pignatelli P, Lenti L, Ferro D, Bellia A, Carnevale R, Tesauro M,

Sorge R, Lauro R, and Violi F. Short-term treatment with atorvastatin reduces

platelet CD40 ligand and thrombin generation in hypercholesterolemic patients.

Circulation 2005; 111: 412-419.

[40] Serebruany VL, Malinin AI, Oshrine BR, Sane DC, Takserman A, Atar D, and

Hennekens CH. Lack of uniform platelet activation in patients after ischemic

stroke and choice of antiplatelet therapy. Thromb Res 113: 197-204.

[41] Srahna M, Remacle JE, Annamalai K, Pype S, Huylebroeck D, Boogaerts MA,

and Vandenberghe P. NF-kappaB is involved in the regulation of CD154 (CD40

ligand) expression in primary human T cells. Clin Exp Immunol 2001; 125: 229-

236.

[42] Kowluru RA, Koppolu P, Chakrabarti S, and Chen S. Diabetes-induced

activation of nuclear transcriptional factor in the retina, and its inhibition by

antioxidants. Free Radic Res 2003; 37: 1169-1180.

[43] Li D, Saldeen T, and Mehta JL. -Tocopherol decreases ox-LDL-mediated

activation of nuclear factor-kappaB and apoptosis in human coronary artery

endothelial cells. Biochem Biophys Res Commun 1999; 259: 157-161.

[44] Davi G, Catalano I, Averna M, Notarbartolo A, Strano A, Ciabattoni G, and

Patrono C. Thromboxane biosynthesis and platelet function in type II diabetes

mellitus. N Engl J Med 1990; 322: 1769-1774.

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[45] Vandendries ER, Furie BC, and Furie B. Role of P-selectin and PSGL-1 in

coagulation and thrombosis. Thromb Haemost 2004; 92: 459-466.

[46] Wagner DD and Burger PC. Platelets in inflammation and thrombosis.

Arterioscler Thromb Vasc Biol 2003; 23: 2131-2137.

[47] Murohara T, Ikeda H, Otsuka Y, Aoki M, Haramaki N, Katoh A, Takajo Y, and

Imaizumi T. Inhibition of platelet adherence to mononuclear cells by alpha-

tocopherol: role of P-selectin. Circulation 2004; 110: 141-148.

[48] Desideri G, Marinucci MC, Tomassoni G, Masci PG, Santucci A, and Ferri C.

Vitamin E supplementation reduces plasma vascular cell adhesion molecule-1

and von Willebrand factor levels and increases nitric oxide concentrations in

hypercholesterolemic patients. J Clin Endocrinol Metab 2002; 87: 2940-2945.

[49] Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, and

Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase

all-cause mortality. Ann Intern Med 2005; 142: 37-46.

[50] Lonn E, Bosch J, Yusuf S, Sheridan P, Pogue J, Arnold JM, Ross C, Arnold A,

Sleight P, Probstfield J, and Dagenais GR. Effects of long-term vitamin E

supplementation on cardiovascular events and cancer: a randomized controlled

trial. JAMA 2005; 293: 1338-1347.

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103

CHAPTER 4

VITAMIN E SUPPLEMENTATION AND DRUG

METABOLISM IN HUMANS

4.1 Introduction

During the last 20 years a number of studies have investigated the potential for -TOH

supplementation to prevent CVD. Experiments performed in animal models have

shown both inhibition of atherosclerosis and prevention of oxidative stress.1 2 However,

results from large human intervention trials examining -TOH supplementation (50-800

IU/day) on CVD mortality and morbidity have been disappointing.3 Furthermore,

recent meta-analysis have concluded that -TOH supplementation in high risk

individuals is associated with an increase in all-cause mortality.4, 5

The mechanism for any possible adverse effect of high dose -TOH is unknown.

The Third National Health and Nutrition Examination Survey (NHANES) in the United

States showed a higher odds ratio for hypertension in subjects with higher serum

vitamin E concentrations.6 This is supported with our own previous study showing an

increase in blood pressure in subjects with type 2 diabetes following vitamin E

supplementation.7 One possibility is that vitamin E may affect antihypertensive drug

efficacy through altered drug metabolism.

4.1.1 Vitamin E, Drug Metabolism and PXR

Drug metabolism by the cytochrome P450 (CYP) enzymes is probably the most

important means to metabolise and eliminate potentially toxic compounds, with the

human CYP3A isoforms the most significant enzymes for drug metabolism in both the

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liver and intestine.8 Activation of CYP3A4 and many other enzymes involved in the

metabolism of xenobiotics is mediated by the pregnane X receptor (PXR).8 PXR is a

transcription factor that is activated by specific ligands resulting in expression of genes

involved in drug metabolism and the metabolism of endogenous compounds.9 PXR is

expressed in a number of human tissues including the liver, intestine, kidney and

adrenal gland.10

Activated PXR forms a heterodimer with the retinoic acid receptor

(RXR) and this in turn activates a number of enzymes involved in xenobiotic and

endobiotic metabolism, as shown in Figure 1.

The activation of CYP450 microsomal hydroxylation by vitamin E in rats was

first reported in 1972, where the capacity for drug hydroxylation was decreased in the

vitamin E deficient rat, but restored with α-TOH supplementation and this appeared to

be independent of any antioxidant action.11

Subsequently, a study in the cebus monkey

demonstrated that 75 IU of dl-α-tocopheryl acetate for 43 days significantly increased

the clearance of antipyrine compared to controls,12

however this study lacked a control

group. α-TOH was shown to significantly increase the activities of phase 1 and phase 2

drug metabolising enzymes following deoxycholate administration in rats,13

with

induction of these enzymes known to be mediated by PXR.9 Sidorova et al. have shown

an increase in liver phase 1 enzyme levels in rats following treatment with α-TOH.14, 15

A number of different forms of vitamin E have been shown to activate gene

expression through activation of PXR in HepG2 cells in culture.16

α-TOH can increase

PXR activity 2-3 fold following incubation for 48 h, but activation was higher with

rifampicin (a known inducer) and also with α and γ-tocotrienol.16

This activation of

PXR could lead to an increase in TOH metabolism following supplementation and

potentially increase metabolism of other compounds by the same CYP3A enzyme. It

has been suggested that high dose α-TOH supplementation might interfere with drug

metabolism through activation of CYP3A4, whereas γ-TOH and the tocotrienols might

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not, due to increased metabolism and excretion of these compounds by the liver.17

However, this potential interaction has not been adequately tested in human subjects.

4.1.2 Assessment of PXR mediated CYP3A4 activity

A number of methods have been employed to evaluate CYP3A activity in-vivo. An

important consideration in any study evaluating the metabolism of a xenobiotic

compound is the known overlap between CYP3A and P-Glycoprotein in relation to

substrate specificity. P-Glycoprotein is a phase III efflux membrane pump expressed in

a number of cells, including enterocytes and hepatocytes and is involved in the

removal of drugs including calcium-channel blockers and some benzodiazepines.18

Drugs are commonly used to probe CYP450 enzyme activity in humans.

Examples include the cardiac glycoside digoxin for P-Glycoprotein 19

and the

benzodiazepine midazolam for CYP3A4.20

Midazolam is a short acting central nervous

system depressant, a known CYP3A4 substrate and the metabolism of midazolam is one

of the most widely used in-vivo drug probes to study CYP3A activity.18, 21-23

The aim of this study was to establish whether supplemental -TOH given to

humans can alter the metabolism of intravenously administered midazolam, through

induction of hepatic CYP3A4. Although some in-vitro studies have shown that some

drugs may increase hepatic CYP3A concentrations within a few days,18

our aim was to

supplement healthy human volunteers with moderate dose (750 IU/day) and high dose

(1500 IU/day) over a 3 and 6 week period respectively, to measure any effect on liver

CYP3A4 activity.

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Figure 1. Molecular mechanism of PXR action.

After binding xenobiotic (e.g. drugs) or endobiotic ligands, PXR is activated and binds

to the regulatory region of target genes as a heterodimer with RXRα. In the case of

CYP3A4 these elements are located in the proximal promoter and the XREM region

located 8 kb upstream of the CYP3A4 gene. Other target genes include multiple CYP

enzymes, dehydrogenases, carboxylesterases; phase II enzymes such as glutathione-S-

transferases, UDP-glucuronosyl transferases and sulfotransferases; and transporters p-

glycoprotein/multi-drug resistance protein (MDR1), multi-drug drug resistance

associated proteins (Mrps), organic anion transporting peptide 2. CYPs carry out

oxidative reactions making lipophilic substrates more water-soluble by addition of

hydroxyl groups, while phase II enzymes perform conjugating reactions [e.g. addition of

glutathione (GSH)] further increasing water solubility and reducing toxicity. Finally,

PXR increases the expression of several transporters, thereby increasing uptake of

substances from blood into cells and elimination into bile depending on the

import/export properties of the transporter and location within sinusoidal or bile

canalicular membranes of hepatocytes.

*Adapted with permission from Matic et al.9

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4.2 Subjects and Methods

4.2.1 Subjects

12 Healthy subjects were recruited from the Perth general population to the School of

Medicine and Pharmacology at the University of Western Australia. Subjects underwent

a clinical examination and fasting blood samples taken for routine biochemical analysis

(Department of Clinical Biochemistry, Royal Perth Hospital). Subjects were included

in the study if they met the following criteria; free from diabetes, liver, renal disease and

hypertension; had a normal lipid profile; non smokers; consumed < 2 standard alcohol

drinks/day and were not taking any vitamin supplements, herbal remedies or any other

drugs. All subjects were asked to refrain from any alcohol consumption the evening

prior to a visit and were instructed to avoid grapefruit and grapefruit juice before and

during the study due to the known inhibition of CYP3A4.8 The study was approved by

the Royal Perth Hospital Ethics committee.

4.2.2 Study Protocol

After recruitment, the subjects were stratified according to age and then randomised to

receive either α-TOH or placebo. The baseline characteristics for the subjects are

provided in Table 1. There were no significant differences between the groups.

Table 1. Baseline characteristics for subjects for the TARDIS study.

TOH Placebo

Age (years) 49.3 ± 10.5 46.7 ± 12.5

Sex M/F 2/4 0/6

BMI (kg/m2) 24.7 ± 3.0 27.3 ± 5.2

Total cholesterol (mmol/L) 4.7 ± 1.3 4.9 ± 0.6

Triglycerides (mmol/L) 0.8 ± 0.1 0.8 ± 0.1

* Values are mean ± SD.

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This study was a double-blind, randomised, placebo controlled clinical trial consisting

of 3 repeated measures over 2 phases;

Phase 1: Following an overnight fast, the subjects provided an early morning spot urine

sample for TOH metabolite analysis. A serum sample was also collected for TOH

analysis. Subjects were supine for 15 min and then given a 1 mg bolus of intravenous

midazolam. Plasma samples were then collected to determine estimates of Area Under

Curve (AUC) for midazolam (see below for midazolam AUC). Subjects were then

randomised into two groups of six and received either RRR-α-TOH capsule (750 IU

weighing 500 mg) or matching placebo capsule (soybean oil 500 mg) per day. This

treatment continued for 3 weeks. The subjects returned following treatment and the

same sample collection protocol was carried out.

Phase 2: At the end of the first phase the subjects ceased all supplements for 8 weeks.

At the end of this period the subjects were asked to resume the same treatment as before

without re-randomisation, but at a higher dose of 1500 IU of either RRR-α-TOH or a

matching placebo capsule (soybean oil 1000 mg) for a period of 6 weeks. Blood and

urine samples were again collected at the end of phase 2. During phase 2, 2 subjects

from each group withdrew from the study due to personal or work commitments.

Compliance with treatment in this study was assessed by pill counting and by serum

TOH and urine TOH metabolite analysis (see results).

4.2.3 Midazolam Area Under Curve (AUC)

Studies have been performed to evaluate limited sampling protocols to estimate AUC in

comparison with standard AUC measurements of midazolam transport and clearance.

An early study examined data from 17 studies involving 224 healthy volunteers and

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found that a single 4 h sample provided excellent agreement with an AUC

measurement for both 2 mg oral (r2

= 0.91) and 1 mg intravenous administration (r2 =

0.80).21

However, a later study that examined CYP3A activity using midazolam

concentrations following both inhibition and induction of CYP3A, found that a 6 h

measure provided better correlations (r2

= 0.94 for 3 mg oral and 0.89 for I.V. 1 mg).23

These findings are supported by a recent study that concluded that 0.5 and 6 h samples

or 0.5, 2 and 6 h samples gave unbiased AUC estimates, assessed by correlation, for

AUC calculated from a full x time point sampling protocol over 6 h in healthy adults

given midazolam.24

For our study a single 1 mg dose of intravenous midazolam was given to subjects

to avoid any contribution from intestinal CYP3A4. The midazolam was administered

by an anaesthetist, subjects were monitored for any adverse effects and samples were

collected at 0.5 and 6 hour time points. To evaluate this limited sampling protocol we

collected extra samples during the study with timepoints at 5, 30, 180 and 360 min.

AUC was calculated from all of these points and compared to AUC with the 30 and 360

min timepoints. The two sample AUC compared well with the four sample AUC (see

Figure 2) and for this study the two sample protocol was used to estimate AUC for

midazolam and hydroxymidazolam.

4.2.4 Power Calculations to Determine Minimum Subject Numbers

Based on previously published data we expect an AUC of approximately 28 ± 4

following intravenous administration of 1mg midazolam to healthy subjects. 23

For our

study, we predict a 30% reduction in AUC. Using a parallel designed study with

significance set at 0.05 and 80% power, we needed five subjects per group to see a

statistically significant change.

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110

Figure 2. Correlation between the 4 sample AUC and 2 sample AUC used in this study.

4.2.5 Materials

Midazolam and alprazolam standards in methanol were both purchased from Sigma and

hydroxymidazolam from Toronto Research Chemicals. Ethyl acetate, heptane and

acetonitrile were of HPLC grade (AJAX Chemicals). The MTBSTFA was from Sigma.

The TOH and placebo capsules were provided by Cognis and the I.V. midazolam

was provided by Roche as Hypnovel (Roche Products Pty Ltd, Dee Why, NSW,

Australia). Tablet content was confirmed by analysis using HPLC with ECD.

4.2.6 Midazolam measurement using GCMS

Measurement of midazolam and 1-hydroxymidazolam was performed using GCMS

with selected ion monitoring and alprazolam as an internal standard according to the

y = 0.776x + 220.96

r 2 = 0.9842

0

500

1000

1500

2000

2500

3000

3500

4000

0 1000 2000 3000 4000 5000

AUC estimated from 2 samples (ng*min/mL)

AU

C f

rom

4 s

ample

s (n

g*m

in/m

L)

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111

method of Thummel et al,20

with minor modifications. In brief, 1.5 mL of heparinsed

plasma was dispensed into glass tubes then 100 L of alprazolam was added (250

ng/mL in methanol). This was mixed for 5 s, left for 5 min and then mixed again. The

samples were alkalised using 0.5 mL of 1M NaOH and mixed again. The analytes were

extracted into a 3 mL solution of ethyl acetate:heptane (1:1 v/v), then mixed thoroughly

on a multi-tube vortexor for 5 min. The samples were centrifuged for 5 min at 1400 g

and the organic phase retained, dried down under vacuum in a centrifugal evaporator for

30 min. The samples were then derivatised for 2 h at 70C using 50 L of 20%

MTBSTFA in acetonitrile. This solution was transferred into autosampler vials and 3

L was injected onto the column.

GCMS separation was performed on an Agilent HP6890 gas chomatograph

coupled to an Agilent HP5973 detector, using a DB5MS column supplied by Agilent

(25 m, I.D. 0.2 mm, film 0.33 m). The initial temperature was 200C and this was

increased up at 10C/min to 280C at 9 min. The temperature was then increased at a

rate of 1C/min up to 300C. The total run time per sample was 29 min. The retention

times for alprazolam, midazolam and hydroxymidazolam were 16.20, 11.03, 16.47 min,

respectively. The ions monitored were m/z 308 (alprazolam), m/z 310 (midazolam) and

m/z 398 (hydroxymidazolam) see Figure 3. The assay had a functional sensitivity of 0.5

ng/mL for both midazolam and hydroxymidazolam and was linear across the measured

range. In-house quality controls were assayed with each run at the beginning and end of

each assay. The inter- and intra-assay CV were less than 5% for both analytes.

4.2.7 Serum TOH and Urine TOH Metabolites

The collection of samples and assaying of serum TOH and urine TOH metabolites is

described in chapter 2.

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112

Alprazolam

Midazolam

Hydroxymidazolam

Figure 3. A representative chromatogram from a human subject receiving 1mg I.V.

midazolam.

4.2.8 Statistics

Plasma midazolam concentration, midazolam AUC, TOH and TOH metabolite

concentrations were analysed using mixed models repeated measures analysis of

variance (PROC MIXED, version 9.1; SAS Institute Inc). Treatment, study visits and

their interaction (treatment x time) were modelled as fixed factors, whereas subjects

were treated as random. The treatment x time interaction allowed the assessment of

whether the slope over time during the intervention period differed between the TOH

and the placebo groups. A P value <0.05 was considered significant.

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113

4.3 Results

4.3.1 Serum α and γ-TOH Concentrations

Serum α and γ-TOH concentrations (corrected for plasma cholesterol + triglyceride) are

shown in Figure 4. There was a significant 2-fold increase in serum α-TOH

concentrations after supplementation for either 3 weeks with 750 IU or 6 weeks with

1500 IU, with no apparent difference between the two treatments suggesting the

lipoproteins were saturated with α-TOH with the lower dose.

4.3.2 Urine TOH Metabolites

Urine α-CEHC concentrations are shown in Figure 5. There was a significant increase

following supplementation for either 3 weeks with 750 IU or 6 weeks with 1500 IU,

but no additional increase in α-CEHC output with the higher dose of α-TOH.

4.3.3 Effect of TOH Supplementation on Midazolam AUC

There was no effect of α-TOH treatment for either 3 weeks with 750 IU or 6 weeks with

1500 IU on midazolam AUC (Figure 6), consistent with a lack of effect of α-TOH

treatment on PXR-mediated liver CYP3A4 activity.

4.3.4 Hydroxymidazolam AUC

There was no effect of treatment on the AUC for the metabolite of midazolam, 1-

hydroxy-midazolam (data not shown).

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114

TOH Placebo TOH Placebo TOH Placebo0

5

10

15Visit 1

Visit 2

Visit 3

Treatment

*A

dju

sted

-T

OH

(u

mo

l/m

mo

l)

TOH Placebo TOH Placebo TOH Placebo0.0

0.1

0.2

0.3

0.4

0.5Visit 1

Visit 2

Visit 3

Treatment

*A

dju

sted

-T

OH

(u

mo

l/m

mo

l)

Figure 4. Serum α and γ-TOH concentrations for visit 1 (baseline), visit 2 (following α-

TOH, 750 IU for 3 weeks), visit 3 (following α-TOH, 1500 IU for 6 weeks).

1 Statistical analysis was performed using mixed models repeated measures ANOVA.

The group*time interaction is significant for α-TOH (P <0.0001) but not for γ-TOH (P

= 0.1339).

2 Data provided are mean ± SEM

* Calculated as TOH / (cholesterol + triglycerides)

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TOH Placebo TOH Placebo TOH Placebo0

2

4

6

8Visit 1

Visit 2

Visit 3

Treatment

Ad

juste

d

-CE

HC

(u

mol/

mm

ol)

Figure 5. Urine α-CEHC concentrations for visit 1 (baseline), visit 2 (following α-TOH,

750 IU for 3 weeks), visit 3 (following α-TOH, 1500 IU for 6 weeks).

1 Statistical analysis was performed using mixed models repeated measures ANOVA.

The group*time interaction is significant for α-CEHC (P = 0.0006)

2 Data provided are mean ± SEM

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TOH Placebo TOH Placebo TOH Placebo0

1000

2000

3000

4000Visit 1

Visit 2

Visit 3

Treatment

Mid

azo

lam

AU

C n

g*

min

/mL

Figure 6. Midazolam AUC separated by treatment group and visit 1 (baseline), visit 2

(following α-TOH, 750 IU for 3 weeks) and visit 3 (following α-TOH, 1500 IU for 6

weeks). 1 Statistical analysis was performed using mixed models repeated measures

ANOVA. There was no effect of TOH treatment on AUC compared to placebo.

2 Data provided are mean ± SEM

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4.4 Discussion

4.4.1 CYP3A4 and α-TOH Metabolism

The lack of treatment effect on midazolam AUC following α-TOH supplementation

suggests that liver CYP3A4 activity was not altered. The observation that typical doses

of both 750 IU and 1500 IU with α-TOH led to similar serum TOH concentrations, with

comparable urine α-CEHC output between the doses supports our finding that liver

CYP3A4 expression was not increased following treatment, as this enzyme is thought to

be involved in hydroxylation of α-TOH leading to the eventual formation of α-CEHC.17

An alternative explanation is that α-TOH is not a significant substrate for CYP3A4 in

humans and may not induce its expression.

Induction of CYP3A4 by rifampicin has been shown to induce the metabolism of

α-TOH and increase the formation of the α-CEHC in HepG2 cells.25

However, it has

been shown that rifampicin can induce other enzymes such as CYP2C9.26

Studies

examining the role of CYP3A4 in α-TOH metabolism used ketoconozole to inhibit

metabolite formation, implicating CYP3A4 in TOH metabolite production.27

Ketoconozole is not a specific inhibitor of CYP3A4 28

so it is possible that other CYP

enzymes can be involved in the metabolism of α-TOH and these enzymes may be

induced through supplementation. It is also possible that excess α-TOH was either not

absorbed or was metabolised by the intestine before it reached the liver, which might

explain the observation that the increase in dose did not affect urine metabolite

production.

The increase in serum α-TOH and urine α-CEHC in our subjects following

supplementation is consistent with other studies. Kelly et al. showed comparable serum

α-TOH concentrations and urine α-CEHC production with 100, 200 and 400 mg/day

given to the same healthy individuals at 6 week intervals.29

A subsequent study in

hypercholesterolaemic subjects examined the relationship between the dose of α-TOH

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and oxidative stress and found a 2-fold change in plasma α-TOH concentrations with

800 IU/day, which is consistent with our findings.30

Clearly, the doses used in our

study were sufficient to saturate plasma lipoproteins and lead to high α-CEHC output.

4.4.2 PXR Activation by Vitamin E in Human cell Cultures

Recent cell culture studies have evaluated the efficacy of vitamin E forms to activate

PXR and thereby increase CYP3A4 expression.16, 31

One study examined the effects of

TOHs and tocotrienols on the expression of a PXR gene reporter assay in HepG2 cells

incubated for 48 h.16

The greatest increase in PXR activation occurred with tocotrienols

(~ 10-fold) and was similar to the known inducer rifampicin. This coincided with a 2-

fold increase in CYP3A4 mRNA expression. The response with RRR-α-TOH was

modest in comparison with only a 2-fold increase in measured PXR activity.16

A

second study showed activation of PXR by tocotrienols (10 M) in cell culture

experiments using both HepG2 cells and LS180 cells performed over a 24 hour period,

whereas the TOHs (10 M) did not.31

Importantly, these studies in cell cultures may

not necessarily reflect vitamin E metabolism and PXR activation in-vivo, suggesting the

necessity for appropriately controlled human intervention trials.

4.4.3 Effect of Vitamin E on CYP450 mediated Drug Metabolism in Animals

In a mouse model α-TOH (20 mg/day) for 3 months caused a 2-3 fold increase in

CYP3a11 mRNA (murine equivalent to human CYP3A4) expression and a similar

induction was obtained with 200 mg/kg dosing.32

Importantly γ-tocotrienol did not

induce CYP3a11 mRNA abundance. A second study examined the effects of high dose

γ-TOH supplementation in mice for 5 weeks and observed a correlation of CYP3a

protein concentrations with hepatic α-TOH, but not with hepatic γ-TOH

concentrations.33

Recently, subcutaneous α-TOH injections given to rats increased

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hepatic α-TOH levels (12 ± 1 nmol/g) at time 0, up to (338 ± 37 nmol/g) at day 18.

There was also a significant increase in liver microsomal CYP3A protein (168 ± 3%)

and P-glycoprotein (189 ± 7%) from day 0 to day 18.34

The same group subsequently

found that α-TOH supplementation in rats significantly increased lung P-glycoprotein

levels within 9 days 35

suggesting that this increase is not mediated by PXR, as PXR

mRNA is not expressed in rat lung. 36

It is possible that some other unknown pathway

leads to the increase in P-glycoprotein levels.

There are limitations when comparing findings in animal studies to those in

humans with induction or inhibition of drug metabolism mediated by PXR. The DNA

binding domain for PXR is 95% homologous between humans, mice, rats and rabbits

and yet they only share between 75-80% of amino acids in the ligand binding domain.37

This may lead to differences in the expression of genes controlled by PXR between

humans and animal models and provides a possible reason why we did not see an

increase in PXR-mediated liver CYP3A4 activity in our study.

4.4.4 Vitamin E and Drug Metabolism in Humans

A paucity of human data exhists on the potential effects of vitamin E forms to affect

PXR mediated drug metabolism. Several studies have examined the effect of vitamin E

supplementation on cyclosporine clearance given alone 38

or in combination with

vitamin C 39-41

with all showing a reduction in cyclosporine concentrations. Given that

cyclosporine is metabolised by both CYP3A enzymes and P-glycoprotein,42

it is not

known which elimination pathways are being affected. A recent study has examined the

effect of 400 IU/day α-TOH given to human subjects who were also taking statins.43

To

evaluate CYP3A activity the ratio of urinary cortisol to its metabolite 6β-

hydroxycortisol was measured and showed no change with TOH supplementation. It

has been suggested that this ratio is not a sensitive indicator of CYP3A activity due to

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metabolism of cortisol within the kidney and may not be an appropriate marker of

CYP3A activity in-vivo.18

To examine liver CYP3A4 activity we used the pharmacokinetics of the

benzodiazepine midazolam and observed no effect on midazolam AUC following

supplementation with α-TOH. Midazolam has a short plasma half-life (~ 3 hours)

which makes it an ideal drug to measure AUC for CYP3A4 phenotyping studies.44

The

hydroxylation of midazolam has been examined in human liver microsomes in which a

number of different polymorphisms for CYP3A4 and CYP3A5 were present.45

These

genetic differences had minimal effect on midazolam metabolism and there was no

effect from age, gender or smoking.45

Another study examined the influence of race

and CYP3A5 genotype and observed no difference with midazolam clearance.46

The

expression of CYP3A4 tends to be higher in women than in men 47

and midazolam

pharmacokinetics are not affected by the menstrual cycle.48

The hydroxylation of

midazolam meets the criteria for a liver CYP3A probe given that liver microsomal

midazolam hydroxylation correlates well with the clearance of the drug in-vivo 20

and

that inhibition and induction of CYP3A leads to an expected change in midazolam

metabolism.18

It takes two to three weeks for steady state levels of CYP3A to increase in humans

after typical induction by a number of agents and the reversal of this effect takes several

weeks to occur.8 This is an important consideration in the design of any study

examining the potential interactions following induction of CYP3A4 or studies 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 specifically determined.

Furthermore, in any study designed to examine PXR activation in-vivo it is relevant to

note that different drugs activate CYP3A4 and MDR1 to different extents in the one

tissue and that the degree interaction of the ligand with the promoter region for PXR

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directly affects transcription of CYP3A4 and MDR1.49

This could explain the

differences observed in CYP3A4 expression with different vitamin E forms. It has been

observed that large variation exists in response to vitamin E supplementation and a

recent study involved an in-silico search to find important polymorphisms in genes

involved in vitamin E metabolism.50

Both CYP3A4 and CYP4F2 are highly

polymorphic and could account for the differences in CEHC metabolite production

following vitamin E supplementation.50

More than 30 single nucleotide polymorphisms

(SNP) have been identified in the CYP3A4 gene with allelic frequencies in the coding

region of the gene occurring in <5% subjects.51

Mutations in the promoter region have

also been found 52

and another study demonstrated that allelic variation was a major

determinant of CYP3A4 expression and activity.53

Although the induction of CYP3A4

in response to drugs like rifampicin may be consistent in humans, taken together this

suggests that any PXR activation and CYP3A4 expression with α-TOH may differ

between populations. Our finding that there was no effect on hepatic CYP3A4

following α-TOH supplementation may not apply to all individuals.

4.4.5 PXR and NF- κB

Further consideration must also be given to the role of the transcription factor NF-κB on

PXR expression, given that NF-κB p65 fragment has been shown to interfere with the

binding of the PXR:RXR heterodimer to its response element.54

Studies have shown

that PXR agonists, including rifampicin, can inhibit the expression of inflammatory

genes controlled by NF-κB p65.55

Vitamin E forms can potentially inhibit NF-κB

activation possibly through an antioxidant action 56

so any effect on PXR may involve

NF-κB p65 and be dependant upon intracellular oxidative stress. The subjects in our

study were healthy individuals replete with vitamin E, so it is possible that individuals

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with low vitamin E status and/or high oxidative stress may respond differently to high

dose RRR-α-TOH supplementation and activate PXR.

4.5 Conclusion

We did not observe any effect of either 750 IU RRR-α-TOH for 3 weeks, or 1500 IU

RRR-α-TOH for 6 weeks on liver CYP3A4 activity, assessed by the clearance of 1 mg

of intravenous midazolam in healthy human subjects. Compliance with treatment was

confirmed by serum TOH and urine TOH metabolite analysis. We cannot rule out any

effect of higher dose of TOHs nor of the same doses given for longer periods, nor can

we conclude that RRR-α-TOH supplements will not increase CYP3A4 expression

within the intestine. A limitation of the second phase was that only 4 subjects per group

completed the study and therefore we had limited power to detect a 30% change in

midazolam AUC. However, we did not observe any trend for a reduction in midazolam

AUC with the 1500 IU treatment or α-CEHC production, so it is unlikely that this dose

has affected liver CYP3A4 activity.

It is possible that a longer human intervention trial may lead to a measurable

increase in PXR mediated gene expression given that an increase in CYP3a11 mRNA

expression with α-TOH has been observed from 3 to 9 months in a murine model.32

Given that some of the larger clinical trials of α-TOH supplementation reporting

negative findings ran for many years,57, 58

we can not rule out a long term effect on drug

metabolism from our findings.

In conclusion, our study does not support the hypothesis that RRR-α-TOH

supplementation in healthy humans with typical doses for up to 6 weeks up-regulates

liver CYP3A4 activity in-vivo. Further studies are required to evaluate the potential for

other forms of vitamin E to activate this pathway, particularly given the increasing

availability of supplements containing other vitamin E forms.

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rate in renal transplant recipients. Nephrol Dial Transplant 2005; 20: 1970-

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* A version of this chapter will be submitted to the American Journal of Clinical

Nutrition.

131

CHAPTER 5

THE EFFECTS OF SESAME INGESTION ON TOH

METABOLISM AND PLATELET FUNCTION IN HUMAN

SUBJECTS WITH CHARACTERSTICS OF THE

METABOLIC SYNDROME

5.1 Introduction

The metabolism and transport of vitamin E forms has received considerable attention

during the last decade with the identification of the water soluble conjugated

metabolites of α-TOH (α-CEHC) and γ-TOH (γ-CEHC) being described.1, 2

The

formation of these metabolites is shown in Chapter 1, Figure 3. These water soluble

metabolites are excreted mainly as glucuronide or sulfate conjugates in the urine and are

readily detected using GCMS.3

A number of studies have evaluated the contribution of cytochrome P450 enzymes

in the formation of vitamin E metabolites.4 The enzyme CYP4F2 has been implicated

in the ω-hydroxylation of γ-TOH in rat and human liver microsomes, leading to the

subsequent formation of the water soluble metabolites, and this process was inhibited by

sesamin, a lignan found in sesame seeds.5 It was shown that rifampicin could induce

the metabolism of α-TOH and increase the formation of the α-CEHC in HepG2 cells,

presumably through induction of CYP3A4.6 However rifampicin lacks specificity for

CYP3A4 and can also induce CYP2C9,7 so it is possible that other CYP enzymes can

be involved in TOH metabolism.

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The inhibition of CYP mediated vitamin E metabolism represents one potential

mechanism by which an increase in plasma and tissue vitamin E might occur, leading to

an alteration of vitamin E status and function in-vivo. Although the exact mechanism

for the metabolism and degradation of all of the forms of vitamin E have not been fully

elucidated, the CYP family of enzymes are the most likely candidates.8

5.1.1 -TOH and its Metabolite γ-CEHC

-TOH differs from α-TOH in that the 5 position is un-substituted whereas with α-TOH

the chromanol ring is fully substituted (see chapter 1, Table 3). It is the major form of

TOH in the U.S. diet and recent studies have suggested a potential role for -TOH in

preventing complications associated with diseases involving oxidative stress and

inflammation.9 There is in-vitro evidence that -TOH is superior to -TOH at

detoxifying NO2 10

and this inhibition occurs because reactive NOx species can be

trapped by γ-TOH, which leads to the formation of 5-nitro--TOH.11

The presence of 5-

nitro--TOH has been found in subjects with CHD. 12

Other potential benefits of -TOH relate to its superiority over α-TOH in

suppressing inflammation in both rats 13

and humans,14

inhibition of cylooxygenase

activity in-vitro 15

and inhibition of platelet aggregation.16

The metabolite γ-CEHC has

been shown to possess natriuretic properties 17, 18

which could potentially lead to a

reduction in blood pressure. However, we have previously shown an increase in blood

pressure following supplementation with a mixed TOH supplement high in -TOH,19

so

this hypothesis remains controversial.

A potential negative effect of α-TOH supplementation is the reduction in serum γ-

and δ-TOHs seen following high dose α-TOH supplementation, possibly through

displacement of these TOHs in the liver.20

In contrast, treatment of humans with statins

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has been shown to increase serum γ-TOH concentrations, possibly through inhibition

of cytochrome P450 mediated γ-TOH metabolism.21

5.1.2 Sesame Ingestion, TOH Metabolism and Risk Factors for Cardiovascular

Disease

Sesame seeds and oil derived from sesame seeds have been used in foods for thousands

of years and continue to be a nutrient source throughout the world. The highest

production of sesame occurs in India, with Japan being the highest importer.22

Sesame

seeds and sesame oil contain a number of constituents including protein, carbohydrates,

vitamins (including vitamin E forms) and lignans.

Sesame lignans are natural components of sesame seed oil with the major lignans

being sesamin and sesamolin (0.4% and 0.5% v/v, respectively),22

see Figure 1. Sesame

lignans and their metabolites have been studied to determine their potential effects as

antioxidants, ability to lower cholesterol and reduce blood pressure, however most of

this work is limited to animal models.23

Sesamin has been shown to lower LDL

cholesterol rats 24

and in humans with hypercholesterolaemia.25

Sesame lignans have

been shown to increase antioxidant capacity of TOHs in rat liver microsomes, 26

suggesting additional benefits from sesame aside from the inhibition of TOH

metabolism. A recent pilot study has reported benefit with sesame oil on blood pressure

in hypertensive subjects,27

however this finding awaits confirmation in an appropriate

placebo controlled clinical trial.

The aim of this study was to determine the effects of sesame ingestion on lipid

metabolism, TOH metabolism and platelet function in subjects with some of the risk

factors associated with the metabolic syndrome. This syndrome is characterised by

dyslipidaemia, increased platelet activation and endothelial dysfunction leading to an

increased risk of thrombosis. 28, 29

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Figure 1. The chemical structures of 2 major lignans found in sesame seeds; sesamin

and sesamolin.

Taken from 30

with permission.

5.2 Subjects and Study Design

5.2.1 Subjects

Men and post-menopausal women were recruited via newspaper advertisement from the

Perth general population to the School of Medicine and Pharmacology at the University

of Western Australia. Potential volunteers were invited for an interview during which a

fasting blood sample for routine biochemical measurements and clinic blood pressure

readings were taken (measured oscillometrically in a sitting positioning in the right arm

after a 10 min rest. Repeated three times every two minutes and the average of the three

readings were used). Exclusion criteria included: BMI > 35 kg/m2, recent coronary or

cerebrovascular events <6 months, type 1 diabetes, use of insulin, arthritis or chronic

inflammation, smoking, taking lipid lowering medication, prior or current use of pure

vitamin E supplement, use of non-steroidal inflammatories, use of oral contraceptive,

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alcohol intake > 40 g/day men or > 30 g/day women, elevated serum creatinine (> 110

mol/L men or > 100 mol/L women) or gamma-glutamyl transferase (-GT) (> 60 U/L

for men or > 40 U/L women).

Volunteers were included if they had at least one of the risk factors for the

metabolic syndrome as defined by the American Heart Association Adult Treatment

Panel III (AHA ATP-III) criteria, or if they had elevated LDL cholesterol (>3.4

mmol/L). After screening, 38 volunteers were eligible and participated in the study. All

volunteers gave informed written consent and the study was approved by the University

of Western Australia Human Ethics Committee. The study is registered at the Australian

New Zealand Clinical Trials Registry (http://www.anzctr.org.au/), registration number

ACTRN12607000158460. Five subjects withdrew from the study before completion,

one because of a change to prescribed medication, two moved away from Western

Australia and two due to personal reasons (undisclosed).

5.2.2 Study Design

This was a randomised, placebo-controlled cross-over study. During an initial four week

wash out period after recruitment, the volunteers stopped all multi-vitamin, fish oil, any

other non-prescribed supplements and were advised to avoid intake of foods containing

sesame seeds. After the washout period, one half of the volunteers replaced their usual

breakfast with the sesame bars (SB) and the other half with placebo bars (PB) for 5

weeks. After another 4 week wash out (during which time the volunteers reverted to

their usual breakfast intake), the volunteers commenced a second five week treatment

period where they replaced their breakfast with the alternative bar. The order of

treatment was randomised via permutated block randomisation. During each treatment,

volunteers were provided with a batch of bars for two weeks then given a second lot of

freshly made bars for the remaining three weeks.

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Volunteers were contacted mid-way through each treatment period by phone to

ensure treatment compliance, and the actual compliance was checked by counting the

number of bars returned at the end of each period.

The treatment bars were distinguishable by appearance and taste, but the

volunteers were not aware of the possible different health benefits of the two treatment

bars, and were advised to maintain their lifestyle including diet and physical activity

throughout the study period. Volunteers were instructed on the accurate recording of

three days of food intake by the study dietician (two weekdays and one weekend) during

the last week of each treatment period. The diet records were used to calculate average

macronutrient intake using the FoodWorks nutrient analysis program (version 5, based

on the 2007 Australian Nutrient File, Xyris software, Qld, Australia).

Volunteers visited the study centre on the morning before the start of each

treatment period as well as the morning after the end of each treatment period, where

fasting blood and a 24h urine sample was collected. The demographic of the subjects is

provided in Table 1.

5.2.3 Power Calculation

A previous study in our group suggested that LDL cholesterol in subjects with

metabolic syndrome was 3.5 ± 0.7 (mean ± SD). With an of 0.05 (P-value) and power

of 0.8, a 2 tailed t-test of paired comparisons, a sample size of 33 was determined to be

needed to detect a minimum change of 10% in LDL cholesterol. To allow for a 10%

drop-out rate we aimed to recruit 38 subjects.

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Table 1. Demographic profile at baseline1

Characteristic

Age (years) 54.7 ± 8.6

BMI (kg/m2) 30.8 ± 3.8

Fasting serum lipids

Total cholesterol (mmol/L)

LDL cholesterol (mmol/L)

HDL cholesterol (mmol/L)

Triglycerides (mmol/L)

5.53 ± 0.76

3.58 ± 0.70

1.32 ± 0.36

1.39 ± 0.49

SBP (mmHg) 124 ± 14.2

DBP (mmHg) 72.7 ± 6.3

Glucose (mmol/L) 4.81 ± 0.54

Gender (M/F) 21 / 17

Taking BP medication 5

Elevated waist circumference

> 102cm in men

> 88cm in women

33

Elevated fasting glucose > 5.5 mmol/L 4

Elevated clinic blood pressure 2 10

Reduced fasting HDL cholesterol

<1.03 mmol/L in men

<1.3 mmol/L in women

10

Elevated fasting triglycerides > 1.7mmol/L 9

Volunteer with metabolic syndrome 8

Elevated fasting LDL cholesterol > 3.4 mmol/L 27

1 Data are presented as mean ± SD.

2 Untreated Hypertension - SBP > 130 mmHg, DBP > 85 mmHg.

5.2.4 Supplement Composition

The sesame and placebo bars were prepared by a commercial bakery (Bodhi’s bake

house, Fremantle, Australia). The composition of the bars is shown in Table 2. The

ingredients were blended, divided into bar format, and heated at 140C for 18 min in an

oven. The bars were cooled and sealed into individual polypropylene packs weighing

60 g (each sesame bar pack contained 26 g sesame seeds) before delivery to study

volunteers.

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The sesame bars and placebo bars had a similar energy content and

macronutrient compositions (Table 3). Sesamin and vitamin E contents in sesame seeds

and vegetable oils were analysed by HPLC as described previously.31

32

The minor

differences in - and -content between the two treatment bars (due to differing vitamin

E contents in the ingredients) were corrected for by the addition of purified TOHs (all

natural RRR- isomers, supplied by Cognis Ltd, Australia) to the ingredient mixture

prior to formulation of the bars.

Table 2. Composition of sesame and placebo bars

Placebo Bar Sesame Bar

g/100 g

Sesame seeds - 43.6

Sugar 14.5 14

Honey 14.5 14

Rice bubbles 13.5 28

Salt - 0.4

Canola oil 9.7 -

Sunflower oil 13.5 -

Rice flour 10.8 -

Wheat gluten protein 9.8 -

Wheat bran 12.3 -

Butter 1.3 -

Table 3. Contents of nutrients in sesame and placebo bars

Placebo Bar Sesame Bar

Unit/60 g

Total energy (kJ) 1213 1171

Protein (g) 7.2 6.9

Total Fat (g) 15.4 14.9

Carbohydrate (g) 30.3 29.3

Dietary Fiber (g) 3.2 3.0

-TOH (mg) 5.6 5.6

-TOH (mg) 4.4 4.4

Sesamin (mg) - 39.5

Sesamolin (mg) - 12.2

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5.3 Methods

Samples from the same individual were always analysed in the same run except for

fasting lipids, plasma glucose and urinary creatinine, which were obtained immediately

after each volunteer’s visit by The Department of Core Clinical Biochemistry at Royal

Perth Hospital, using Roche reagents on a Hitachi 917 analyser ). All biochemical

measurements were carried out in a blinded fashion.

5.3.1 HPLC Analysis of TOHs with Electrochemical Detection

HPLC analysis of TOHs is described in chapter 2.

5.3.2 Plasma F2-isoprostanes

Plasma F2-isoprostanes analysis is described as for urine in chapter 2.

5.3.3 Urinary TOH Metabolites

GCMS analysis of TOH metabolites is described in chapter 2.

5.3.4 ELISA Assays

Serum IL-6, TNF-, sVCAM-1, sICAM-1 and citrated plasma sP-selectin were all

measured using the R&D high-sensitivity ELISA kits by (R&D systems, Minneapolis,

USA), all according to the manufacturers instructions. All assays had intra- and inter-

assay CVs <10%.

5.3.5 Platelet PFA 100 analysis

Performed on the Platelet Function 100 analyser from Dade Behring (Miami, FL, USA),

using epinephrine/ADP cartridges on citrated whole blood. Whole blood flows through

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a membrane coated with epinephrine and ADP at high shear rate and the platelets

become activated and start to adhere to the membrane. The system detects the time

required for the closure of a small aperture in the membrane.

5.3.6 Routine Biochemistry

High sensitivity CRP was assayed using the particle-enhanced immunonephelometry

system on the Dade Behring BNII analyser with an inter-assay CV of 3% (Siemens

Diagnostics, Deerfield, USA). Measurement of cholesterol and triglyceride is described

in chapter 2.

5.3.7 Statistical Analysis

Data are expressed as mean ± SD. Non-normally distributed data was log-transformed

prior to analysis, and presented as geometric mean (95% confidence interval). The SAS

software (version 9.1; SAS Institute Inc, Cary, NC) was used for all statistical analysis

(PROC MIXED procedures). Subjects were included as a random factor nested under

treatment sequence within a linear mixed model. The following factors were always

included as fixed effects in the model; baseline values, treatment, period and treatment

sequence. Differences were considered significant when P 0.05.

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5.4 Results

5.4.1 Serum TOH and Urine CEHC concentrations

The effect of sesame treatment versus placebo of serum TOH concentrations is shown in

Figures 2 and 3. Urine CEHC concentrations are provided in Figure 4. Sesame

supplementation had no effect on serum -TOH concentrations but increased serum -

TOH by 17% (corrected for plasma lipids) and decreased urine -CEHC production by

31%. There was also a 40% reduction in urine -CEHC content. In the placebo group

there was a 34% increase in urine -CEHC production.

5.4.2 Subject food intake during study period

Daily nutrient intake of subjects during the study is shown in Table 4. This

demonstrates that nutrient intake was similar between the treatment groups except for

fibre intake.

Table 4. Macronutrient intake of volunteers per day during food bar supplementation1

Nutrient Sesame bars Placebo bars P2

Energy (kJ) 9250 ± 2766 9677 ± 2868 0.293

Protein (g) 103.3 ± 35.2 106.2 ± 35.2 0.693

Fat

Total (g)

Saturated (g)

Poly-unsaturated (g)

Mono-unsaturated (g)

94.2 ± 33.1

34.3 ± 15.2

17.4 ± 5.3

34.8 ± 13.2

96.7 ± 39.3

34.4 ± 15.6

16.5 ± 6.3

37.8 ± 19.9

0.696

0.989

0.446

0.319

Carbohydrate (g) 227.7 ± 77.4 242.8 ± 80.0 0.159

Alcohol3 (g) 11 ( 7.4, 16.4) 13 ( 7.9, 21.3) 0.305

Dietary fiber3 (g) 22.4 ( 19.5, 25.7) 27.8 ( 23.5, 32.9) 0.01

1 Data are presented as mean ± SD except where indicated.

2 Comparison of after treatment means by mixed model analysis.

3 Data were log-transformed

prior to statistical analysis, and presented as geometric mean (95% confidence interval).

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Pre Post0

10

20

30

40

50Treatment

Placebo

-TO

H (

M)

Figure 2a. Serum α-TOH concentrations comparing treatment with sesame versus

placebo.

1 Data are presented as mean ± SD.

2 Comparison of after treatment means by mixed model analysis P = 0.179.

Pre Post0

1

2

3

4Treatment

Placebo

-T

OH

(

M)

Figure 2b. Serum γ-TOH concentrations comparing treatment with sesame versus

placebo.

1 Data are presented as mean ± SD.

2 Comparison of after treatment means by mixed model analysis P = 0.011.

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Pre Post0

2

4

6Treatment

Placebo

-T

OH

/Lip

ds

(m

ol/m

mol

)

Figure 3a. Serum α-TOH concentrations corrected for plasma lipids comparing

treatment with sesame versus placebo.

1 Data are presented as mean ± SD.

2 Comparison of after treatment means by mixed model analysis P = 0.423.

3 Serum TOH expressed normalised to lipids, which are calculated as total cholesterol

plus triglycerides.

Pre Post0.0

0.2

0.4

0.6Treatment

Placebo

-T

OH

/Lip

ids(

mol/

mm

ol)

Figure 3b. Serum γ-TOH concentrations corrected for plasma lipids comparing

treatment with sesame versus placebo.

1 Data are presented as mean ± SD.

2 Comparison of after treatment means by mixed model analysis P = 0.012.

3 Serum TOH expressed normalised to lipids, which are calculated as total cholesterol

plus triglycerides.

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Pre Post0.0

0.1

0.2

0.3Treatment

Placebo

-C

EH

C/c

reat

inin

e (

mm

ol/

mm

ol)

Figure 4a. α-CEHC urine metabolites comparing treatment with sesame versus placebo.

1 Comparison of after treatment means by mixed model analysis P = 0.058.

2 Urinary vitamin E metabolites are expressed normalised to creatinine excretion.

3 Data were log-transformed prior to statistical analysis, and presented as geometric

mean (95% confidence interval).

Pre Post0.0

0.2

0.4

0.6

0.8

1.0Treatment

Placebo

-C

EH

C/c

reat

inin

e (

mm

ol/

mm

ol)

Figure 4b. γ-CEHC urine metabolites comparing treatment with sesame versus placebo.

1 Comparison of after treatment means by mixed model analysis P = 0.0001.

2 Urinary vitamin E metabolites are expressed normalised to creatinine excretion.

3 Data were log-transformed prior to statistical analysis, and presented as geometric

mean (95% confidence interval).

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Pre Post0.00

0.05

0.10

0.15Treatment

Placebo

-C

EH

C/c

reat

inin

e (

mm

ol/

mm

ol)

Figure 4c. δ-CEHC urine metabolites comparing treatment with sesame versus placebo.

1 Comparison of after treatment means by mixed model analysis P = 0.0001.

2 Urinary vitamin E metabolites are expressed normalised to creatinine excretion.

3 Data were log-transformed prior to statistical analysis, and presented as geometric

mean (95% confidence interval).

5.4.3 Effects of sesame on body weight, and biochemical parameters

Effects of treatment versus placebo of weight, lipids, glucose, creatinine and -GT are

shown in Table 5. There was no effect of sesame on any of the parameters measured,

compared to placebo.

5.4.4 Effect of treatment on platelet and endothelial activation

The effects of treatment versus placebo on markers of platelet and endothelial activation

are shown in Table 6. There was a modest increase in ICAM with sesame ingestion.

There was no effect of sesame on any of the parameters measured, compared to placebo.

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Table 5. Subjects body weight, plasma lipids, creatinine and -GT.1

Sesame Placebo

Pre Post Pre Post

Weight (kg) 91.9 ± 18.5 92.3 ± 18.1 93.4 ±17 93.2 ± 17.7

Total cholesterol

(mmol/L)

5.84 ± 0.84 5.86 ± 0.95 5.94 ± 0.82 5.83 ± 0.80

LDL-C

(mmol/L)

3.75 ± 0.76 3.82 ± 0.84 3.84 ± 0.75 3.81 ± 0.72

HDL-C

(mmol/L)

1.37 ± 0.38 1.37 ± 0.39 1.36 ± 0.38 1.35 ± 0.37

Triglycerides

(mmol/L)3

1.47 (1.27, 1.69) 1.35 (1.17, 1.56) 1.51 (1.34, 1.7) 1.35 (1.16, 1.57)

Glucose

(mmol/L)

5.21 ± 0.54 5.19 ± 0.59 5.15 ± 0.54 5.10 ± 0.44

Creatinine

(mol/L)

75.7 ± 13.7 75.7 ± 14.3 76.7 ± 15.4 77.8 ± 14.7

-GT (U/L)3 22.0 (18.0, 26.8) 21.1 (17.1, 26.0) 23.9 (19.7, 29.0) 20.8 (17.3, 25.1)

1 Data are presented as mean ± SD except where indicated.

2 Comparison of after treatment means by mixed model analysis.

None of the parameters

were statistically different.

3 Data were log-transformed prior to statistical analysis, and presented as geometric mean

(95% confidence interval).

Table 6. Platelet and endothelial function markers.1

Sesame Placebo

Pre Post Pre Post

ICAM (ng/mL) 242.9 ± 39.9 247.8 ± 46.13 251.0 ± 54.7 244.6 ± 43.7

VCAM (ng/mL) 604.6 ± 145.2 626.1 ± 190.8 616.0 ± 154.4 620.0 ± 166.4

P-selectin (ng/mL) 46.3 ± 11.4 44.1 ± 10.5 47.3 ± 12.7 46.2 ± 12.4

Platelet function (s) 88.7 ± 18.0 85.9 ± 17.1 88.3 ± 22.4 88.4 ± 24.3

1 Data are presented as mean ± SD.

2 Comparison of after treatment means by mixed model analysis.

3 P = 0.024. No other parameter was statistically significant.

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5.4.5 Effect of treatment on systemic inflammation and oxidative stress

The effects of sesame versus placebo on markers of inflammation and oxidative stress

are shown in Table 7. There was no effect of treatment compared to placebo except for

F2-isoprostanes, but this is probably to the post placebo value dropping compared to

treatment.

Table 7. Serum inflammatory and oxidative stress biomarkers1

Sesame Placebo

Pre Post Pre Post

IL-6 (pg/mL) 2.08

(1.74, 2.48)

2.05

(1.74, 2.40)

2.14

(1.77, 2.60)

2.25

(1.87, 2.72)

TNF- (pg/mL) 1.85

(1.70, 2.02)

1.83

(1.65, 2.02)

1.83

(1.64, 2.03)

1.80

(1.62, 2.0)

Hs-CRP (mg/L) 2.10

(1.45, 3.04)

1.89

(1.35, 2.66)

1.98

(1.38, 2.84)

2.02

(1.39, 2.93)

F2-isoprostanes

(pmol/L)

4118

(3568, 4753)

40833

(3555, 4689)

4201

(3654, 4830)

3683

(3199, 4242)

1 Data were log-transformed prior to statistical analysis, and presented as geometric

mean (95% confidence interval).

2 Comparison of after treatment means by mixed model analysis.

3 P = 0.047. No other parameter was statistically significant.

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5.5 Discussion

5.5.1 Sesame and TOH metabolism

The metabolism of vitamin E isomers involves -hydroxylation followed by -

oxidation of the phytyl side chain leading to formation of the CEHC metabolites.8

There has been considerable interest in the role of cytochrome P450 enzymes in the

vitamin E metabolite formation. Studies have used ketoconozole to inhibit metabolite

formation implicating CYP3A4 in α-TOH metabolism,33

however ketoconozole is not a

specific inhibitor of CYP3A4.34

Induction of CYP3A4 by rifampicin has been shown

to induce the metabolism of α-TOH and increase the formation of the α-CEHC in

HepG2 cells.6 The role of the CYP3A forms in TOH metabolism has been questioned

following in-vitro studies with human microsomes expressing CYP3A4 and CYP3A7

showing no TOH metabolising capacity.5 Furthermore CYP4F2 did catalyse TOH

hydroxylation, with preference for -TOH and that this was inhibited by sesamin.5

The potential for sesame lignan ingestion to inhibit γ-TOH metabolism and

increase serum γ-TOH concentrations has been studied. Feeding rats with γ-TOH and

sesame in combination led to an increase in serum and tissue γ-TOH and a decrease in

urine γ-CEHC excretion.35

Sesamin oil ingestion with ~ 100 mg sesame lignans/day for

four weeks in humans has been shown to increase serum -TOH concentrations by ~

30%, however the sesame oil group also received ~ 10 mg/day of -TOH, so it is

unclear if the other components of sesame oil could have been responsible for the

effect.36

Another recent study in postmenopausal women examined the effect of 50

g/day of sesame seeds for five weeks and observed an increase in α-TOH (18%

corrected by total cholesterol) and γ-TOH (73% corrected by total cholesterol).37

In

comparison our study involved supplementation with 39.5 mg of sesamin and 12.2 mg

of sesamolin per day. There was no effect on serum α-TOH or urine α-CEHC output,

but we did observe an increase in serum -TOH by 17% (corrected for plasma lipids)

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and a decrease in urine -CEHC production by 31%. This was accompanied by a 40%

reduction in urine -CEHC output. The change in δ-CEHC output provides support for

inhibition of δ-TOH metabolism by sesame lignans. In the placebo group there was a

34% increase in urine -CEHC production, but no effect was observed on serum γ-TOH

concentrations suggesting the 4.4 mg (content of -TOH in placebo) was not sufficient

to increase lipoprotein γ-TOH content.

Our results are consistent with previous findings that sesame lignan ingestion in

humans can inhibit the metabolism of γ-TOH and δ-TOH, possibly through inhibition of

CYP4F2.

5.5.2 Sesame, lipid metabolism, inflammation and oxidative stress

An important regulator of hepatic and vascular wall inflammation and the regulation of

target genes involved in lipid metabolism is the transcription factor peroxisome

proliferator-activated receptor (PPAR-).38

This transcription factor regulates gene

expression through heterodimer formation with the RXR and subsequent binding to

DNA gene promoter regions and gene transcription. PPAR- is activated by a number

of ligands including fatty acids and drugs such as fibrates. This activation leads to

reduced inflammation within the artery wall, decreased expression of inflammatory

cytokines such as IL-1 and IL-6 and reduced CRP and fibrinogen from the liver. 38

Fibrate drugs are PPAR-α agonists and represent an important means to treat subjects

with the metabolic syndrome where dyslipidaemia and increased coagulation are central

components.29

The potential mechanisms by which sesame lignans might affect lipid metabolism

and reduce LDL cholesterol have been investigated in the rat model.24, 39-42

These

studies suggest that sesame lignans might increase the expression of genes involved in

hepatic fatty acid oxidation and decrease expression of enzymes involved in fatty acid

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synthesis, in particular PPAR-α and the sterol regulatory element binding protein 1

(SREPB–1).43

Sesamin ingestion in the rat model, in combination with either

docosahexanoic acid (DHA) or eicosapentanoic acid (EPA), leads to enhanced

expression of genes involved in hepatic fatty acid oxidation and this effect was

increased in combination with fish oil.39

These effects are different in rats, mice and

hamsters fed the same doses of sesamin and episesamin, which may be due to the

different metabolism and bioavailability of these compounds within each species.40

There is a paucity of data on the effects of sesame ingestion on lipid metabolism

in humans. Sesame supplementation (32 mg/d sesame vs placebo for eight weeks) in

subjects with type IIa and type IIb hypercholesterolaemia and resulted in reductions in

total cholesterol, LDL cholesterol and apoB.25

A subsequent placebo controlled study

in healthy women examined the effects of high dose (~ 380 mg/day of sesame lignans

for five weeks) and led to a significant 10% reduction in LDL cholesterol.37

In our study we observed no effect on lipid parameters, markers of either

inflammation or oxidative stress after treatment with sesame seeds. In subjects with at

least one of the risk factors for the metabolic syndrome or elevated LDL cholesterol.

This may in part relate to possibility that individuals with higher cholesterol or

increased background inflammation might respond differently to sesame treatment. The

small significant reduction in F2-isoprostanes is of borderline significance and the

placebo group actually had lower isoprostanes post treatment.

5.5.3 Sesame, γ-TOH, platelet and endothelial function

Platelet TOH concentrations are a marker of both α and γ-TOH intake in human

subjects.44, 45

There has been considerable interest in the potential for vitamin E isomers

to inhibit platelet activation using both α-TOH 46, 47

and γ-TOH. 16, 48

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VCAM and ICAM are both important adhesion molecules found on the surface

of endothelial cells and are involved in monocyte adhesion to the endothelial wall.49

The transcription of VCAM and ICAM are under the control of NF-B 50

and this redox

sensitive transcription factor can be inhibited by vitamin E isomers.51

-TOH has also

be shown to inhibit oxidised LDL mediated activation of the NF-B transcription factor

in endothelial cells.52

PPAR-α agonists also inhibit NF-B transcription 38

so there may

be a synergistic inhibition of NF-B mediated gene transcription with γ-TOH and

sesame lignans, leading to a reduction in the release of VCAM and ICAM from

endothelial cells.

Membrane bound P-selectin is a cell adhesion molecule that mediates the

adhesion of leucocytes to endothelial cells and platelets and subsequently has an

important role in thrombosis 53

with the soluble form readily detectable in plasma. α-

TOH has been shown to inhibit human platelet P-selectin expression and platelet-

leukocyte interaction 54

and reduce the release of sP-selectin into the plasma.55

Given

that platelet activation is a feature of the metabolic syndrome and that we expected an

increase in plasma and platelet γ-TOH following sesame ingestion, we hypothesised that

treatment with sesame lignans would inhibit the release of sVCAM-1, sICAM-1, sP-

selectin into the plasma and reduce platelet aggregation. However, we observed no

effect on any of these parameters, despite the fact that serum -TOH was increased by

17% (corrected for plasma lipids), indicating that subtle dietary changes that increase -

TOH to a modest extent are unlikely to lower these indices of platelet activation and

that a greater change might be required achieve this outcome. It is possible that sesame

seed ingestion given in combination with greater levels of -TOH might further increase

γ-TOH and decrease these parameters.

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5.6 Conclusion

In conclusion we observed no effect on lipid parameters, markers of inflammation,

oxidative stress nor platelet function after treatment with ~ 25g/day sesame seeds for

five weeks given on the background of normal dietary TOH intake in volunteers with at

least one of the risk factors for the metabolic syndrome as defined by the AHA ATP

(III) criteria, or if they had elevated LDL cholesterol.

Treatment of subjects with higher doses of sesame seeds or lignans either given

alone, in combination with TOH supplements may have had a greater effect on PPAR-α

and affect lipid metabolism or suppress inflammation through inhibition of NF-B.

However the dose of sesame seeds given in our study was considered realistic to

achieve as part of a daily food supplement. We are therefore unable to support the

hypothesis that reasonable amounts of sesame are able to improve plasma lipids,

suppress inflammation and platelet activation in subjects with at least one

cardiovascular risk factor. This group was used for this study to evaluate the potential

benefits of sesame lignans in individuals at risk of developing the full metabolic

syndrome. We can not equate our findings to other populations, in particular

individuals with overt hypercholesterolaemia or diabetes and await further studies in

these patient groups.

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5.7 References

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Nutr 1995; 62: 1527S-1534S.

[2] Stahl W, Graf P, Brigelius-Flohe R, Wechter W, and Sies H. Quantification of

the - and -tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-

hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in

human serum. Anal Biochem 1999; 275: 254-259.

[3] Galli F, Lee R, Dunster C, and Kelly FJ. Gas chromatography mass

spectrometry analysis of carboxyethyl-hydroxychroman metabolites of - and -

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[4] Brigelius-Flohe R and Traber MG. Vitamin E: function and metabolism. FASEB

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HepG2 cells by side chain omega-oxidation and consecutive beta-oxidation.

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Quantitative analysis of constitutive and inducible CYPs mRNA expression in

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tocopherols inhibit platelet aggregation in humans: potential mechanisms. Am J

Clin Nutr 2003; 77: 700-706.

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WH. A new endogenous natriuretic factor: LLU-alpha. Proc Natl Acad Sci USA

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precursor. J Nutr Sci Vitaminol (Tokyo) 2004; 50: 277-282.

[19] Ward NC, Wu JH, Clarke MW, Puddey IB, Burke V, Croft KD, and Hodgson

JM. The effect of vitamin E on blood pressure in individuals with type 2

diabetes: a randomized, double-blind, placebo-controlled trial. J Hypertens

2007; 25: 227-234.

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2003; 133: 3137-3140.

[21] Werba JP, Cavalca V, Veglia F, Massironi P, De Franceschi M, Zingaro L, and

Tremoli E. A new compound-specific pleiotropic effect of statins: Modification

of plasma gamma-tocopherol levels. Atherosclerosis 2007; 193: 229-233.

[22] Namiki M. Nutraceutical functions of sesame: a review. Crit Rev Food Sci Nutr

2007; 47: 651-673.

[23] Nakai M, Harada M, Nakahara K, Akimoto K, Shibata H, Miki W, and Kiso Y.

Novel Antioxidative Metabolites in Rat Liver with Ingested Sesamin. J Agric

Food Chem 2003; 51: 1666-1670.

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[24] Hirose N, Inoue T, Nishihara K, Sugano M, Akimoto K, Shimizu S, and

Yamada H. Inhibition of cholesterol absorption and synthesis in rats by sesamin.

J Lipid Res 1991; 32: 629-638.

[25] Hirata F, Fujita K, Ishikura Y, Hosoda K, Ishikawa T, and Nakamura H.

Hypocholesterolemic effect of sesame lignan in humans. Atherosclerosis 1996;

122: 135-136.

[26] Ghafoorunissa, Hemalatha S, and Rao MV. Sesame lignans enhance antioxidant

activity of vitamin E in lipid peroxidation systems. Mol Cell Biochem 2004; 262:

195-202.

[27] Sankar D, Rao MR, Sambandam G, and Pugalendi KV. A pilot study of open

label sesame oil in hypertensive diabetics. J Med Food 2006; 9: 408-412.

[28] Nieuwdorp M, Stroes ES, Meijers JC, and Buller H. Hypercoagulability in the

metabolic syndrome. Curr Opin Pharmacol 2005; 5: 155-159.

[29] Kakafika AI, Liberopoulos EN, Karagiannis A, Athyros VG, and Mikhailidis

DP. Dyslipidaemia, hypercoagulability and the metabolic syndrome. Curr Vasc

Pharmacol 2006; 4: 175-183.

[30] Davin LB and Lewis NG. Dirigent proteins and dirigent sites explain the

mystery of specificity of radical precursor coupling in lignan and lignin

biosynthesis. Plant Physiol 2000; 123: 453-462.

[31] Penalvo JL, Heinonen SM, Aura AM, and Adlercreutz H. Dietary sesamin is

converted to enterolactone in humans. J Nutr 2005; 135: 1056-1062.

[32] Ruperez FJ, Martin D, Herrera E, and Barbas C. Chromatographic analysis of

alpha-tocopherol and related compounds in various matrices. J Chromatogr A

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[33] Parker RS, Sontag TJ, and Swanson JE. Cytochrome P4503A-dependent

metabolism of tocopherols and inhibition by sesamin. Biochem Biophys Res

Commun 2000; 277: 531-534.

[34] Parker RS, Sontag TJ, Swanson JE, and McCormick CC. Discovery,

characterization, & significance of the cytochrome P450 & -hydroxylase

pathway of vitamin E catabolism. Ann N Y Acad Sci 2004; 1031: 13-21.

[35] Ikeda S, Tohyama T, and Yamashita K. Dietary sesame seed and its lignans

inhibit 2,7,8-trimethyl- 2(2'-carboxyethyl)-6-hydroxychroman excretion into

urine of rats fed gamma-tocopherol. J Nutr 2002; 132: 961-966.

[36] Lemcke-Norojarvi M, Kamal-Eldin A, Appelqvist LA, Dimberg LH, Ohrvall M,

and Vessby B. Corn and sesame oils increase serum gamma-tocopherol

concentrations in healthy Swedish women. J Nutr 2001; 131: 1195-1201.

[37] Wu WH, Kang YP, Wang NH, Jou HJ, and Wang TA. Sesame ingestion affects

sex hormones, antioxidant status, and blood lipids in postmenopausal women. J

Nutr 2006; 136: 1270-1275.

[38] Zambon A, Gervois P, Pauletto P, Fruchart J-C, and Staels B. Modulation of

hepatic inflammatory risk markers of cardiovascular diseases by PPAR-

activators: clinical and experimental evidence. Arterioscler Thromb Vasc Biol

2006; 26: 977-986.

[39] Arachchige PG, Takahashi Y, and Ide T. Dietary sesamin and docosahexaenoic

and eicosapentaenoic acids synergistically increase the gene expression of

enzymes involved in hepatic peroxisomal fatty acid oxidation in rats.

Metabolism 2006; 55: 381-390.

[40] Kushiro M, Takahashi Y, and Ide T. Species differences in the physiological

activity of dietary lignan (sesamin and episesamin) in affecting hepatic fatty acid

metabolism. Br J Nutr 2004; 91: 377-386.

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[41] Ashakumary L, Rouyer I, Takahashi Y, Ide T, Fukuda N, Aoyama T,

Hashimoto T, Mizugaki M, and Sugano M. Sesamin, a sesame lignan, is a potent

inducer of hepatic fatty acid oxidation in the rat. Metabolism 1999; 48: 1303-

1313.

[42] Ide T, Ashakumary L, Takahashi Y, Kushiro M, Fukuda N, and Sugano M.

Sesamin, a sesame lignan, decreases fatty acid synthesis in rat liver

accompanying the down-regulation of sterol regulatory element binding protein-

1. Biochimi Biophys Acta 2001; 1534: 1-13.

[43] Ide T, Hong DD, Ranasinghe P, Takahashi Y, Kushiro M, and Sugano M.

Interaction of dietary fat types and sesamin on hepatic fatty acid oxidation in

rats. Biochimi Biophys Acta 2004; 1682: 80-91.

[44] Lehmann J, Rao DD, Canary JJ, and Judd JT. Vitamin E and relationships

among tocopherols in human plasma, platelets, lymphocytes, and red blood

cells. Am J Clin Nutr 1988; 47: 470-474.

[45] Clarke MW, Hooper AJ, Headlam HA, Wu JHY, Croft KD, and Burnett JR.

Assessment of tocopherol metabolism and oxidative stress in familial

hypobetalipoproteinemia. Clin Chem 2006; 52: 1339-1345.

[46] Freedman JE, Farhat JH, Loscalzo J, and Keaney JF, Jr. Tocopherol inhibits

aggregation of human platelets by a protein kinase C-dependent mechanism.

Circulation 1996; 94: 2434-2440.

[47] Devaraj S and Jialal I. The effects of -tocopherol on critical cells in

atherogenesis. Curr Opin Lipidol 1998; 9: 11-15.

[48] Saldeen T, Li D, and Mehta JL. Differential effects of - and -tocopherol on

low-density lipoprotein oxidation, superoxide activity, platelet aggregation and

arterial thrombogenesis. J Am Coll Cardiol 1999; 34: 1208-1215.

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[49] Galkina E and Ley K. Vascular Adhesion Molecules in Atherosclerosis.

Arterioscler Thromb Vasc Biol 2007; 27: 2292-2301.

[50] de Winther MP, Kanters E, Kraal G, and Hofker MH. Nuclear factor kappaB

signaling in atherogenesis. Arterioscler Thromb Vasc Biol 2005; 25: 904-914.

[51] Glauert HP. Vitamin E and NF-kappaB activation: a review. Vitam Horm 2007;

76: 135-153.

[52] Li D, Saldeen T, and Mehta JL. -Tocopherol decreases ox-LDL-mediated

activation of nuclear factor-kappaB and apoptosis in human coronary artery

endothelial cells. Biochem Biophys Res Commun 1999; 259: 157-161.

[53] Vandendries ER, Furie BC, and Furie B. Role of P-selectin and PSGL-1 in

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[54] Murohara T, Ikeda H, Otsuka Y, Aoki M, Haramaki N, Katoh A, Takajo Y, and

Imaizumi T. Inhibition of platelet adherence to mononuclear cells by alpha-

tocopherol: role of P-selectin. Circulation 2004; 110: 141-148.

[55] Davi G, Romano M, Mezzetti A, Procopio A, Iacobelli S, Antidormi T,

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CHAPTER 6

CONCLUSION AND FUTURE DIRECTIONS

6.1 Summary

In nearly 100 years of research and interest in the metabolism and function of vitamin E,

much still remains to be discovered about the effects associated with consumption of

these essential nutrients. The important role of vitamin E in the prevention of

neurological disease has been clearly established, with deficiency in vitamin E being

treated successfully with -TOH. The bodies’ preference for this form of vitamin E

within the liver and the incorporation into lipoproteins has led to the hypothesis that any

benefit from supra-nutritional doses of vitamin E should be found from -TOH.

Vitamin E supplements clearly have a role in disease prevention in individuals

with a deficiency, which can lead to neurological disease. However, there are many

instances where the recommendation for supplementation may be controversial. In our

study, subjects who were heterozygous for FHBL had abnormal erythrocyte

morphology with slightly lower erythrocyte TOH concentrations. The presence of

abnormal red cell morphology may indicate a modest deficiency in membrane TOHs,

but could also be a function of a reduction in membrane cholesterol. However, there

was no difference in platelet TOHs or F2-isoprostanes between FHBL and normal

subjects which suggests there is no significant difference in cellular TOHs or systemic

oxidative stress. Given recent concern over negative findings from -TOH, we could

not recommend supplementation in this group, particularly due to the lack of any

clinical signs of deficiency.

Research in recent years has centred on the capacity for -TOH to protect LDL

from oxidative modification in-vivo and prevent its accumulation in atherosclerotic

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plaques. Although high doses of -TOH given to humans may lead to a reduction in

systemic oxidative stress and platelet aggregation, clinical trials using this supplement

in high risk individuals have failed to show any benefit for CVD. Results from our own

study do not support the recommendation that -TOH, or a mixed TOH supplement

high in -TOH given to individuals with type 2 diabetes mellitus provides any

additional benefit in combination with normal treatment options.

There has been considerable interest in the potential for supplementation with -

TOH given to humans to induce CYP3A4 and alter the metabolism of clinically

important drugs. We examined two doses of -TOH (750 IU/day and 1500 IU/day)

over a three and six week period in healthy human subjects to measure any effect on

liver CYP3A4 activity. Treatment with vitamin E had no effect on the hepatic

metabolism of the benzodiazepine midazolam, consistent with the concept that -TOH

is unable to activate CYP3A4 in the liver and affect drug metabolism by this pathway.

Given the potential adverse effects from high dose TOH supplementation we used

sesame ingestion to affect the metabolism of -TOH. This approach was designed to

provide a dietary means to inhibit the metabolism of -TOH and raise the concentration

in serum and cells without direct supplementation. The lack of treatment effect from

this study may indicate that modest changes in -TOH concentration are not sufficient to

suppress inflammation and platelet aggregation.

6.2 Conclusion

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

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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.

Our findings challenge the hypothesis that TOH supplements are essentially

harmless and provide a means to suppress inflammation, oxidative stress and platelet

aggregation. The potential for any pro-oxidant effects of -TOH must be considered

when designing clinical studies using this supplement, in particular the role of co-

antioxidants and overall oxidation. Given the reported apparent increase in all-cause

mortality following supplementation with -TOH, the exact mechanism for this effect

must be elucidated before any large scale trials with -TOH should continue. At this

juncture, vitamin E supplementation only seems warranted in cases of actual vitamin E

deficiency.

6.3 Future Directions

In this last decade, there has been interest in other forms of vitamin E, namely -TOH

and the tocotrienols in disease prevention and treatment. However, in the case of -

TOH, our in-vivo studies have failed to confirm findings from in-vitro and experimental

studies showing anti-inflammatory, anti-platelet and blood pressure lowering effects of

this substance, thus, complicating any interpretation of data using supplements

containing this form of vitamin E. The production of pure -TOH is an expensive

process, however, to adequately address this question pure supplements containing only

-TOH would need to be used.

Recent studies have suggested that tocotrienols may have a potential use in

treating neurological disease and reducing cholesterol. Their considerable capacity to

activate PXR and suppress inflammation in-vitro requires confirmation in an

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appropriately controlled human intervention trial. Although plasma concentrations of

tocotrienols tends to be low, supplementation with sesame or other inhibitors of TOH

metabolism might raise the amount of tocotrienols in-vivo to sufficient levels to provide

benefit in individuals with neurological disease or hypercholesterolaemia. Any benefit

must also be balanced against any harmful effects such as the potential to raise blood

pressure or affect drug metabolism. However, based on our study, it would seem that

-TOH may not activate PXR and lead to significant drug interactions in the liver,

effects on CYP3A4 or other enzymes in intestinal cells have not been tested in-vivo and

this question should be further addressed to confirm that clinically significant drug

interactions are unlikely with this popular supplement.

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CURRICULUM VITAE

NAME: Michael William Clarke

PLACE OF BIRTH: Bedfordshire, England

YEAR OF BIRTH: 1972

EDUCATION: Bachelor of Science (Medical Science), Curtin University, Western

Australia, 1998

PUBLICATIONS RELATED TO THIS THESIS

1. Clarke MW, Hooper AJ, Headlam HA, Wu JH, Croft KD, Burnett JR.

Assessment of tocopherol metabolism and oxidative stress in familial

hypobetalipoproteinemia.

Clin Chem 2006; 52: 1339-45.

2. Clarke MW, Ward NC, Wu JH, Hodgson JM, Puddey IB, Croft KD.

Supplementation with mixed tocopherols increases serum and blood cell gamma-

tocopherol but does not alter biomarkers of platelet activation in subjects with type 2

diabetes.

Am J Clin Nutr 2006; 83: 95-102.

3. Clarke MW, Burnett JR, Croft KD.

Vitamin E Metabolism in Health and Disease.

Crit Rev Clin Lab Sci 2008 (in press).

4. Ward NC, Wu JH, Clarke MW, Puddey IB, Burke V, Croft KD, Hodgson JM.

The effect of vitamin E on blood pressure in individuals with type 2 diabetes: a

randomized, double-blind, placebo-controlled trial.

J Hypertens 2007; 25: 227-34.

5. Clarke MW, Ledowski T, Wu JH, Hodgson JM, Puddey IB, Burnett JR, Croft KD.

Findings from the Tocopherol And Responsive Drug Interaction Study (TARDIS).

Submitted to JAMA as a brief report 2008.

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ii

6. Wu JH, Hodgson JM, Clarke MW, Puddey IB, Croft KD.

The Effects of Sesame Ingestion on Tocopherol Metabolism and Platelet Function in

Subjects with the Metabolic Syndrome.

Will be submitted to AJCN 2008.

PUBLICATIONS UNRELATED TO THIS THESIS

1. Wu JH, Hodgson JM, Ward NC, Clarke MW, Puddey IB, Croft KD.

Nitration of gamma-tocopherol prevents its oxidative metabolism by HepG2 cells.

Free Radic Biol Med 2005 39: 483-94.

2. Ward NC, Hodgson JM, Croft KD, Clarke MW, Burke V, Beilin LJ, Puddey IB.

Effects of vitamin C and grape-seed polyphenols on blood pressure in treated

hypertensive individuals: results of a randomised double blind, placebo-controlled trial.

Asia Pac J Clin Nutr 2003;12 Suppl:S18.

AWARDS AND PRESENTATIONS RELATED TO THIS THESIS

Presentation of poster for the Australian Atherosclerosis Society conference in

Darwin September 2005

Presentation of poster for the Society for Free Radical Research in the Gold Coast

December 2005

Award for Patient Orientated Research at the University of Western Australia

Research Showcase July 2006

Award for the Young Investigators day at Royal Perth Hospital August 2006 in the

Clinical section

Finalist at the University of Western Australia Research Showcase October 2007

Finalist at the Young Investigators day at Royal Perth Hospital October 2007

Presentation of poster for the Australian Atherosclerosis Society conference in Perth

October 2007