Vitamin E Metabolism in Humans - UWA Research …...i Vitamin E Metabolism in Humans Michael William...
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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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Page
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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Page
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
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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
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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
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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
* 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
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
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.
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
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
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.
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.
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-
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.
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,
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
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
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
14
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
15
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
16
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
17
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
18
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.
19
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)
20
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
21
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
22
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
23
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
24
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
25
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
26
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
27
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
28
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
29
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
30
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.
31
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
32
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
33
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
34
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
35
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
36
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.
37
1.13 References
[1] Evans HM and Bishop KS. On the existence of a hitherto unrecognised dietary
factor essential for reproduction. Science 1922; 56: 650.
[2] Maras JE, Bermudez OI, Qiao N, Bakun PJ, Boody-Alter EL, and Tucker KL.
Intake of alpha-tocopherol is limited among US adults. J Am Diet Assoc 2004;
104: 567-575.
[3] Roberts DCK. Vitamin E. Sydney: Australian Professional Publications, 1990.
[4] Pryor WA. Vitamin E and Carotenoids: Abstracts. Lagrange, Illinois: Veris,
1996.
[5] Young IS and Woodside JV. Antioxidants in health and disease. J Clin Pathol
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97: 7500-7502.
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CE, and Williams DE. Alpha-tocopherol modulates Cyp3a expression, increases
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mice fed high gamma-tocopherol diets. Free Radic Biol Med 2005; 38: 773-785.
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receptor. Biochem Pharmacol 2003; 65: 269-273.
[154] Mustacich DJ, Leonard SW, Devereaux MW, Sokol RJ, and Traber MG. alpha-
Tocopherol regulation of hepatic cytochrome P450s and ABC transporters in
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[155] Mustacich DJ, Vo AT, Elias VD, Payne K, Sullivan L, Leonard SW, and Traber
MG. Regulatory mechanisms to control tissue alpha-tocopherol. Free Radic Biol
Med 2007; 43: 610-618.
[156] Lake KD, Aaronson KD, Gorman LE, Pagani FD, and Koelling TM. Effect of
oral vitamin E and C therapy on calcineurin inhibitor levels in heart transplant
recipients. J Heart Lung Transplant 2005; 24: 990-994.
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anti-oxidants Vitamin C and E decreases cyclosporine A trough-levels in renal
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cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta
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Association between serum alpha-tocopherol and serum androgens and
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Virtamo J, and Albanes D. Serum and Dietary Vitamin E in Relation to Prostate
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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.
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
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
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
62
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
63
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
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
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.
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)
67
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
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
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.
70
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.
71
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).
72
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
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
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
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.
76
2.5 References
[1] Schonfeld G. Familial hypobetalipoproteinemia: a review. J Lipid Res 2003; 44:
878-883.
[2] Whitfield AJ, Barrett PH, van Bockxmeer FM, and Burnett JR. Lipid disorders
and mutations in the APOB gene. Clin Chem 2004; 50: 1725-1732.
[3] Hooper AJ, van Bockxmeer FM, and Burnett JR. Monogenic hypocholesterolaemic
lipid disorders and apolipoprotein B metabolism. Crit Rev Clin Lab Sci 2005; 42: 1-31.
[4] Linton MF, Farese RV, Jr., and Young SG. Familial hypobetalipoproteinemia. J
Lipid Res 1993; 34: 521-541.
[5] Granot E and Kohen R. Oxidative stress in abetalipoproteinemia patients
receiving long-term vitamin E and vitamin A supplementation. Am J Clin Nutr
2004; 79: 226-230.
[6] Kayden HJ and Traber MG. Absorption, lipoprotein transport, and regulation of
plasma concentrations of vitamin E in humans. J Lipid Res 1993; 34: 343-358.
[7] 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.
[8] 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.
[9] 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.
77
[10] Bieri JG, Tolliver TJ, and Catignani GL. Simultaneous determination of -
tocopherol and retinol in plasma or red cells by high pressure liquid
chromatography. Am J Clin Nutr 1979; 32: 2143-2149.
[11] Su Q, Rowley KG, and O'Dea K. Stability of individual carotenoids, retinol and
tocopherols in human plasma during exposure to light and after extraction. J
Chromatogr B Biomed Sci Appl 1999; 729: 191-198.
[12] Galli F, Lee R, Dunster C, and Kelly FJ. Gas chromatography mass
spectrometry analysis of carboxyethyl-hydroxychroman metabolites of - and -
tocopherol in human plasma. Free Radic Biol Med 2002; 32: 333-340.
[13] Pope SA, Clayton PT, and Muller DP. A new method for the analysis of urinary
vitamin E metabolites and the tentative identification of a novel group of
compounds. Arch Biochem Biophys 2000; 381: 8-15.
[14] Mori TA, Croft KD, Puddey IB, and Beilin LJ. An improved method for the
measurement of urinary and plasma F2-isoprostanes using gas chromatography-
mass spectrometry. Anal Biochem 1999; 268: 117-125.
[15] Friedewald WT, Levy RI, and Fredrickson DS. Estimation of the concentration
of low-density lipoprotein cholesterol in plasma, without use of the preparative
ultracentrifuge. Clin Chem 1972; 18: 499-502.
[16] Innis-Whitehouse W, Li X, Brown WV, and Le NA. An efficient
chromatographic system for lipoprotein fractionation using whole plasma. J
Lipid Res 1998; 39: 679-690.
[17] El-Sohemy A, Baylin A, Ascherio A, Kabagambe E, Spiegelman D, and
Campos H. Population-based study of alpha- and gamma-tocopherol in plasma
and adipose tissue as biomarkers of intake in Costa Rican adults. Am J Clin Nutr
2001; 74: 356-363.
78
[18] Simon E, Paul JL, Atger V, Simon A, and Moatti N. Study of vitamin E net
mass transfer between -tocopherol-enriched HDL and erythrocytes: application
to asymptomatic hypercholesterolemic men. Free Radic Biol Med 2000; 28:
815-823.
[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
Nutr 1981; 34: 2104-2110.
[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
risk of atherosclerosis in humans. Arterioscler Thromb Vasc Biol 2005; 25: 279-
286.
[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-
1182.
[23] Cracowski JL, Durand T, and Bessard G. Isoprostanes as a biomarker of lipid
peroxidation in humans: physiology, pharmacology and clinical implications.
Trends Pharmacol Sci 2002; 23: 360-366.
[24] Azzi A, Gysin R, Kempna P, Munteanu A, Villacorta L, Visarius T, and Zingg
JM. Regulation of gene expression by alpha-tocopherol. Biol Chem 2004; 385:
585-591.
[25] Chowers I, Banin E, Merin S, Cooper M, and Granot E. Long-term assessment
of combined vitamin A and E treatment for the prevention of retinal
degeneration in abetalipoproteinaemia and hypobetalipoproteinaemia patients.
Eye 2001; 15: 525-530.
79
[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-
isoprostanes in neonates at high risk of atopy. Free Radic Res 2004; 38: 233-
239.
[27] Frank L and Sosenko IR. Development of lung antioxidant enzyme system in
late gestation: possible implications for the prematurely born infant. J Pediatr
1987; 110: 9-14.
[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;
74: 714-722.
[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
1997; 94: 3217-3222.
[30] Sen CK, Khanna S, and Roy S. Tocotrienol: the natural vitamin E to defend the
nervous system? Ann N Y Acad Sci 2004; 1031: 127-142.
* 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
81
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
82
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
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
84
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
85
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.
86
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
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
88
-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.
89
- 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.
90
- 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.
91
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
92
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
93
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
94
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
95
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.
96
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[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.
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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
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[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.
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[28] Sontag TJ and Parker RS. Cytochrome P450 omega-hydroxylase pathway of
tocopherol catabolism. Novel mechanism of regulation of vitamin E status. J
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[29] Bruno RS, Leonard SW, Li J, Bray TM, and Traber MG. Lower plasma {alpha}-
carboxyethyl-hydroxychroman after deuterium-labeled {alpha}-tocopherol
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[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.
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[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.
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[37] Cipollone F, Mezzetti A, Porreca E, Di Febbo C, Nutini M, Fazia M, Falco A,
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101
[38] Kinlay S, Schwartz GG, Olsson AG, Rifai N, Sasiela WJ, Szarek M, Ganz P,
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[39] Sanguigni V, Pignatelli P, Lenti L, Ferro D, Bellia A, Carnevale R, Tesauro M,
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[42] Kowluru RA, Koppolu P, Chakrabarti S, and Chen S. Diabetes-induced
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[43] Li D, Saldeen T, and Mehta JL. -Tocopherol decreases ox-LDL-mediated
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* A version of this chapter will be submitted for publication in JAMA 2008
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
104
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
105
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.
106
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
107
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.
108
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
109
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.
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)
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.
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.
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).
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)
115
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
116
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
117
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
118
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
119
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
120
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
121
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
122
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.
123
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[40] Blackhall ML, Fassett RG, Sharman JE, Geraghty DP, and Coombes JS. Effects
of antioxidant supplementation on blood cyclosporin A and glomerular filtration
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[41] de Vries AP, Oterdoom LH, Gans RO, and Bakker SJ. Supplementation with
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GP, Zhou G, and Zhou HH. Effects of genetic polymorphisms of CYP3A4,
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[44] Chung E, Nafziger AN, Kazierad DJ, and Bertino JS, Jr. Comparison of
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[45] He P, Court MH, Greenblatt DJ, and von Moltke LL. Factors influencing
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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.
132
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
133
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
134
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,
135
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.
136
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.
137
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.
138
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
139
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
140
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.
141
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).
142
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.
143
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.
144
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).
145
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.
146
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.
147
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.
148
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)
149
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
150
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
151
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.
152
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.
153
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Yamada H. Inhibition of cholesterol absorption and synthesis in rats by sesamin.
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Hypocholesterolemic effect of sesame lignan in humans. Atherosclerosis 1996;
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[28] Nieuwdorp M, Stroes ES, Meijers JC, and Buller H. Hypercoagulability in the
metabolic syndrome. Curr Opin Pharmacol 2005; 5: 155-159.
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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
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[36] Lemcke-Norojarvi M, Kamal-Eldin A, Appelqvist LA, Dimberg LH, Ohrvall M,
and Vessby B. Corn and sesame oils increase serum gamma-tocopherol
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sex hormones, antioxidant status, and blood lipids in postmenopausal women. J
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1. Biochimi Biophys Acta 2001; 1534: 1-13.
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160
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
161
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
162
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
163
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.
i
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.
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