cardiovascular disease, antioxidant enzyme systems, and selenium

246 Pages·2008·3.68 MB·English

CARDIOVASCULAR DISEASE,
ANTIOXIDANT ENZYME SYSTEMS,
AND SELENIUM

Robyn S. Lymbury
B.Biomed.Sci (Hons)

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

January 2008

Heart Foundation Research Centre

School of Medical Science

Griffith University
ABSTRACT

Globally, cardiovascular disease (CVD) is the leading cause of mortality,
representing 30 % of all deaths in 2005. The prevalence of CVD is predicted to
increase steadily over the coming years and this has been attributed to a number of
factors including an increase in overall life expectancy, as well as a projected
increase in the number of people with common risk factors. As in most developed
countries, Australia is experiencing a rise in the proportion of aged population, with
current predictions estimating that by 2051, the number of people aged 65 years and
over will increase from the current 2.5 million to 7.2 million. This is of particular
concern considering the strong correlation between CVD risk and age.

Reactive oxygen species (ROS) and a lowered antioxidant status have been heavily
implicated in the pathogenesis of several CVD states including hypertension, vascular
disease, ischemic heart disease and chronic heart failure. Deficiencies in the essential
micronutrient, selenium (Se), have been associated with an increased incidence and a
worse prognosis of a range of CVDs. The biological function of Se is mediated
through the selenoproteins, of which 35 have been identified to date. Although the
selenoproteins have been implicated in multiple metabolic pathways, they are
primarily recognised for their role in endogenous antioxidant defence. This thesis
aimed to further evaluate the significance of Se and the selenoenzyme antioxidant
systems in CVD pathogenesis and prevention, with particular emphasis on the Se
status of the Australian population.

The first study of this thesis explored the benefits of acutely-administered Se in
myocardial ischaemia-reperfusion (I-R) injury. I-R injury is an unavoidable risk of
clinical interventions for CVD such as coronary angioplasty, by-pass surgery or
transplantation. The role of oxidative stress has been intrinsically linked to damage
incurred during I-R, with antioxidant agents often added to cardioplegic solutions in
order to minimise tissue injury. In this study, an isolated rat heart model was
employed, in which varying concentrations of sodium selenite were added to the
perfusion buffer. The major finding of this study was that Se, at a concentration of
0.5 µM, improved cardiac functional recovery following I-R injury. Notably,
beneficial effects were not observed in hearts perfused with lower levels of sodium
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selenite, whereas higher concentrations were associated with poorer functional
recovery. Biochemical analysis of cardiac tissue revealed no change in selenoenzyme
activity, suggesting that the protection conferred by Se was mediated through some
other mechanism. Further experimentation demonstrated that both inorganic and
organic forms of Se possessed direct free radical scavenging properties in vitro. This
inherent antioxidant property of Se may explain the observed protective effects of Se
when administered in an acute setting.

The second and third studies presented in this thesis investigated the significance of
long-term dietary Se in hypertension and heart failure. The biochemical mechanisms
of the afforded protection were also explored. The spontaneously hypertensive rat
(SHR) was used as an animal model of hypertension induced-heart failure, with the
Wistar-Kyoto (WKY) rat as a normotensive control. Three experimental diet groups
were assessed: 1) Se-free; 2) Normal Se (50 µg Se/kg feed); and 3) High Se
(1000 µg Se/kg feed). Modification of dietary Se was found to influence the
development of both genetic hypertension, as seen in the SHR, and aged-related
hypertension, as seen in the WKY rat, with chronic Se deficiency augmenting
hypertension in both strains. The most striking finding, however, was that a diet high
in Se significantly reduced heart failure-related mortality in the SHR (70 % mortality
for Se-free, 22 % for Normal Se, and 0 % mortality for High Se). Analysis of cardiac
and liver tissue revealed that whilst dietary Se was able to effectively modulate the
activity of the selenoenzymes, there was strong evidence for differential regulation in
selenoprotein activity. This was most evident in the SHR strain, with tissue analysis
revealing that the levels of Se and selenoenzymes were depleted in liver tissue but
maintained in cardiac tissue. Considering the high degree of cardiac oxidative stress
present in the SHR, it is more than likely that the observed depletion of liver Se and
liver selenoenzymes was a compensatory response, enabling maximum selenoprotein
activity where it is most required. These findings reinforce a role for Se in
cardiovascular health, as well as providing evidence of differential regulation of liver
and cardiac selenoproteins under conditions of increased oxidative stress.

The fourth study of this thesis evaluated the clinical benefits of administering pre-
operative metabolic therapy, including Se, to cardiac surgery patients. A double-
blinded, placebo controlled clinical trial was conducted at The Alfred Hospital,
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Victoria. The metabolic substances trialed included coenzyme Q , alpha-lipoic acid,
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omega-3 polyunsaturated fatty acids, orotic acid and Se. These substances have
proven cardiovascular-protective properties when administered individually, however
the combined effect has not been assessed. As expected, cardiac surgery was
associated with an increase in oxidative injury, as well as a concomitant reduction in
plasma antioxidant levels including Se and glutathione peroxidase (GSH-Px), a key
selenoprotein. Metabolic therapy was found to enhance endogenous antioxidant
reserves prior to surgery, providing a better defence against surgery-induced
oxidative stress. Post-operative levels of plasma Se were found to be significantly
higher for patients who received metabolic therapy compared to those who received
placebo therapy. The biological effect of this was reflected in the activity levels of
plasma GSH-Px, with the placebo group showing a significant reduction in activity
following surgery, whereas the metabolic group demonstrated an increase in activity.
Furthermore, metabolic therapy was associated with lower levels of oxidative injury
markers following surgery, as well as a significant reduction in the degree of post-
operative myocardial damage.

The final study of this thesis investigated the Se status in a cohort of the Australian
population, and examined the effect of age, sex and cardiovascular disease. Blood
plasma levels of Se and GSH-Px activity were used as indicators of Se status. Analysis
of the healthy population (140 samples, age range: 20 – 86 years) revealed a mean
plasma Se level of 100.2 ± 1.3 µg/L and a mean plasma GSH-Px activity of 108.8 ±
1.7 U/L. Although the mean value for plasma Se reached the level required for
optimum plasma GSH-Px activity (i.e. 100 µg Se/L), 47 % of healthy Australians fell
below this level. Further evaluation revealed that certain age groups were more at
risk of a lowered Se status than others, in particular the 20 – 30 years and 81 years
and over. Although Se status was not significant influenced by sex, values for males
were often lower than those for females. Analysis of patients with cardiovascular
disease (112 samples, age range 40 – 87 years) revealed that while the mean level of
plasma Se was not significantly lower than healthy age-matched controls (99.6 ±
1.7 µg Se/L for cardiac patients versus 101.2 ± 1.5 µg Se/L for healthy controls),
there was a noticeably higher percentage of CVD patients with a sub-optimal Se
status (57 % for CVD patients versus 44 % for healthy controls). Again, certain age
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groups were identified as being most at risk, with patients in the 81 years and over
age group showing the lowest levels of plasma Se (88.2 ± 5.3 µg Se/L).

Collectively, the studies presented within this thesis demonstrate the antioxidant
benefits of Se therapy, both in an acute and long-term setting. These findings
highlight the importance of an adequate supply of dietary Se and justify further
investigation into the Se status of the Australian population, especially in terms of the
potential health benefits for our ageing population or those more at risk of CVD.
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DECLARATION

This is to certify that:

i) This thesis comprises only my original work towards the PhD except
where indicated in the Preface.
ii) Due acknowledgement has been made in the text to all other material
used.
iii) This thesis is less than 100, 000 words in length, exclusive of tables,
figures, references and appendices.

Robyn S. Lymbury

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PREFACE
Pursuant to the regulations governing the degree of Doctor of Philosophy at
Griffith University, I hereby submit that all work presented in this thesis is my
original research except for the collection of the following data which was
performed as part of a clinical trial conducted at The Alfred Hospital, Melbourne,
Victoria, and is presented in Chapter 6. Professor Franklin Rosenfeldt, Dr Jee-
Yoong Leong, Dr Salvatore Pepe and Ms Alicia Calderone were primarily
responsible for trial design and conduct.
• Plasma and urine sample collection (The Alfred Cardiothoracic Surgical
Unit).
• Quantification of plasma CoQ10 (ARL pathology, Melbourne), plasma
cardiac troponin (The Alfred Hospital Pathology Department), and urinary
isoprostanes and creatinine (The Baker Heart Research Institute, Melbourne).
• Collection of clinical data including cardiac pump function, post-operative
complications and hospital statistics (The Alfred Cardiothoracic Surgical
Unit).

This work has not previously been submitted for a degree or diploma in any
university. To the best of my knowledge and belief, the thesis contains no material
previously published or written by another person except where due reference is
made in the thesis itself.

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ACKNOWLEDGEMENTS

There are a number of people who I would like to acknowledge for their invaluable
contributions, collaborations and support, without which, the work presented in this
thesis would not have been possible.

I wish to express my sincere gratitude to my supervisors, Professor Tony Perkins and
Professor John Headrick. Thank you for your continual support and guidance, and for
providing the funding and facilities for me to complete this project. Tony, as my
principal supervisor, I am deeply indebted for all that you have done throughout the
past four years. Thank you for sharing your wide knowledge and for your continual
understanding and encouragement, particularly in the writing of this thesis. Thank you
also for establishing the many fundamental collaborations which have enabled me to
complete this project and for providing me with the opportunity to work on a project
of this scope. This has undoubtedly enabled me to develop more fully as a scientist.

I would like to thank the staff at the Baker Heart Research Institute and The Alfred
Hospital (Melbourne, Victoria) who were involved in the many aspects of the ‘Pre-
operative metabolic therapy for cardiac surgery’ trial. In particular I would like to
thank Professor Franklin Rosenfeldt, Dr Jee-Yoong Leong, Dr Salvatore Pepe and
Ms Alicia Calderone for the coordination and provision of plasma samples and
clinical data. I would also like to acknowledge Professor Lyn Griffiths, Mr Micky
Ovcaric, Ms Laura Newey and Ms Sharon Quinlon at the Genomics Research Centre,
Griffith University, for the provision of plasma samples from healthy controls.

Many thanks to the staff at the Prince Charles Hospital, Brisbane: Dr Kathy Brown,
Mr Jason Brightwell and Mr Ben Bolton. Their talent for rat echocardiography was
remarkable, especially considering it was following a long day performing human
sonography. I would also like to thank Professor Lindsay Brown at the University of
Queensland for facilitating collaborations with the Prince Charles staff.

I wish to gratefully acknowledge Dr Ujang Tinggi at the Queensland Health Scientific
Services, Brisbane, for performing sample Se analysis.

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A very big thank you is extended to the past and present members of the Heart
Foundation Research Centre for providing a stimulating and cheerful work
environment. In particular I would like to sincerely thank Dr Kylie Venardos for all of
her guidance and support in the initial stages of this project, especially the sharing of
her Langendorff expertise.

I would also like to acknowledge several of the staff at Griffith University for their
technical advice throughout this project including Dr Glenn Harrison,
Ms Suzzie Owens, Dr Darren Grice, Ms Emma Swettenham and Dr Jessica
Vanderlelie. Thank you also to the wonderful staff at the Griffith University Animal
facility: Corinne, Simone, Anna and Carmen. The contributions of the research
students to this project was much appreciated – thank you to Theodore Jeffreys,
Matthew Marino and Adrian Castelli for their help with animal transport and enzyme
assays.

A very special thank you for my dear friends (you know who you are ☺). Thank you
for always being there and for providing inspiration, adventures and sanity outside of
the scientific world. And to my family – words cannot express my gratitude for their
love and never-ending support. The past four years have certainly been eventful for all
of us, and I feel so blessed to have such special people in my life with whom to share
the journey.

“Writing is a solitary occupation. Family, friends, and society are the natural enemies of the writer. He
must be alone, uninterrupted, and slightly savage if he is to sustain and complete an undertaking”
Jessamyn West (1902 – 1984)

For my natural enemies …..

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TABLE OF CONTENTS

ABSTRACT....................................................................................................................i
DECLARATION...........................................................................................................v
PREFACE....................................................................................................................vi
ACKNOWLEDGEMENTS.........................................................................................vii
TABLE OF CONTENTS.............................................................................................ix
LIST OF FIGURES....................................................................................................xii
LIST OF TABLES ....................................................................................................xvi
PUBLICATIONS AND PRESENTATIONS...........................................................xvii
LIST OF ABBREVIATIONS....................................................................................xix
LIST OF COMPOUND ABBREVIATIONS...........................................................xxv

CHAPTER 1 INTRODUCTION.................................................................................1
1.1 Impact and Significance of Cardiovascular Disease............................................2
1.2 Pathophysiology of Cardiovascular Disease.........................................................3
1.2.1 Atherosclerosis.................................................................................................................3
1.2.2 Ischemic Heart Disease.....................................................................................................5
1.2.3 Chronic Heart Failure.......................................................................................................6
1.3 Current Treatment Options for Cardiovascular Disease..................................10
1.4 Myocardial Ischaemia-Reperfusion Injury........................................................13
1.4.1 Myocardial Ischaemia.....................................................................................................13
1.4.2 Reperfusion Injury..........................................................................................................16
1.4.3 Mechanisms of Ischaemia-Reperfusion Injury...............................................................17
1.5 Myocardial Oxidative stress - Sources of Reactive Oxygen Species.................21
1.6 Myocardial Antioxidant Defence Systems..........................................................24
1.7 Selenium and Antioxidant Enzyme Systems.......................................................27
1.7.1 Glutathione System: Glutathione Peroxidase.................................................................29
1.7.2 Thioredoxin System: Thioredoxin Reductase.................................................................31
1.7.3 Thyroid Hormone Deiodinases.......................................................................................32
1.8 Biological Significance of Selenium.....................................................................33
1.8.1 Selenium Deficiency.......................................................................................................33
1.8.2 Selenium Toxicity...........................................................................................................35
1.8.3 A Safe Dietary Intake.....................................................................................................36
1.8.4 Selenium and Cardiovascular Disease............................................................................39
1.9 Research Aims.......................................................................................................44

CHAPTER 2 MATERIALS AND METHODS........................................................45
2.1 Materials................................................................................................................46
2.2 Animals..................................................................................................................46
2.3 Sample Preparation..............................................................................................46
2.3.1 Tissue Extract Preparation..............................................................................................46
2.3.2 Plasma Preparation.........................................................................................................47
2.4 Protein Estimation................................................................................................48
2.5 Biochemical Analysis............................................................................................48
2.5.1 Glutathione Peroxidase Assay........................................................................................49
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