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Title:

OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION IN DIABETIC


SUBJECTS UNDERGOING CORONARY ARTERY BY-PASS GRAFT (CABG)

By

Fredrick Ochieng Owala


(0609996o)

SUPERVISOR:

DR. CARLENE A. HAMILTON

A DISSERTATION

Submitted to the Faculty of Medicine’s Graduate School of the University of Glasgow


in partial fulfilment of the requirement for the award of degree in
Master of Science (MSc. Med. Sc.) in Clinical Pharmacology

Session 2006/2007
Copyright© 2007, Fredrick Ochieng Owala

ii
Dedication

To all the patients who accepted to participate in this study.

iii
Acknowledgement

I hereby with utmost pleasure, sincerely express my appreciation, acknowledgement and


heartfelt gratitude to my university supervisor, Dr Carlene A. Hamilton for her support,
diligent supervision and constructive guidance throughout the project work. This
dissertation would not have been possible without her guidance and constant
encouragement. Her vast wealth of knowledge and dedication to teaching and research
has been a great inspiration to me. Most importantly, I feel greatly indebted to her for
giving me an opportunity to work under her direction as a graduate student.

I indeed thank Dr. Jane Dymott for generously and exclusively availing the clinical data
on the study subjects. She was always readily available and provided constant and
invaluable support. To Mr. S. Miller and Mrs. C. Hawksby, I thank them for teaching me
basic laboratory techniques. I extend my profound appreciation to the Department of
Medical Sciences in the Faculty of Medicine at University of Glasgow and the entire staff
for equipping me with the appropriate knowledge and practical skills in Clinical
Pharmacology. Such knowledge has been used to conduct research upon which this
dissertation is based. Special gratitude is extended to my advisor of studies, Dr. Nicklin
A. Stuart for his invaluable advice, great ideas, encouragement and motivation, during
both difficult and good times throughout my studies. I owe him significantly for that.

I also extend an arm of appreciation to the Commonwealth Scholarship Commission (on


behalf of the British Government) and the University of Glasgow for awarding me this
prestigious scholarship to undertake postgraduate studies in Great Britain. In addition, I
would like to express my heartfelt gratitude to the Postgraduate Secretary of International
and Postgraduate Service, Mr. Brian Cherry for excellently facilitating my stay in the
United Kingdom and providing a lot support and advice. Last but not least, I
acknowledge my dearest parents, David and Elizabeth Owala for their inspiration and
moral support. Their respect and love of knowledge, as well as having confidence in me,
has been instrumental in my quest to be a better person in all aspects of life.

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List of Figures and Tables

List of Figures

Figure 1.0: Sources of oxidative stress and their potential links to endothelial dysfunction
in diabetes. ............................................................................................................................... 5
Figure 2.0: A chart showing the flow of patients during the study................................... 16
Figure 3.0: A box plot of LDL levels in diabetic and nondiabetic subjects ..................... 22
Figure 4.0: HDL levels in diabetic and nondiabetics ......................................................... 23
Figure 5.0: A comparison of fruit and green leafy vegetable intake between diabetic and
nondiabetic subjects .............................................................................................................. 24
Figure 6.0: Superoxide production in diabetic and nondiabetic subjects ......................... 25
Figure 7.0: Inhibition of superoxide production by rotenone in nondiabetic and diabetic
saphenous veins ..................................................................................................................... 27
Figure 8.0: Calcium ionophore induced concentration-relaxation curve in 3.0 µmol/L
preconstricted diabetic and nondiabetic saphenous veins................................................... 29
Figure 9.0: Maximal relaxation of diabetic and nondiabetic saphenous veins to calcium
ionophore ............................................................................................................................... 29

List of Tables

Table 1.0: Clinical characteristics and demographics of the patients................................ 21


Table 2.0: Inhibition of superoxide production by rotenone in diabetic and nondiabetic
saphenous veins ..................................................................................................................... 26
Table 3.0: Mean EC50 of calcium ionophore and maximal relaxation in diabetic and non-
diabetic saphenous veins....................................................................................................... 28

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List of Abbreviations

ROS Reactive Oxygen Species


AGE Advanced Glycation End-products
GTPCH GTP-cyclohydrolase-I
BH4 Tetrahydrobiopterin
PKC Protein kinase C
DAG Diacyglycerol
XO Xanthine oxidase
NK-kB Nuclear Factor kB
VCAM Vascular cellular adhesion molecule-1
ICAM Intracellular adhesion molecule-1
SOD Superoxide Dismutase
GSH Reduced Glutathione
GSSH Oxidized Glutathione
NO Nitric Oxide
OxLDL Oxidized LDL
eNOS Endothelial Nitric Oxide Synthase
NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate
ACEIs Angiotensin Converting Enzyme Inhibitors
ARBs Angiotensin II Receptor Blockers
CABG Coronary Artery By-pass Graft
HDL High Density Lipoprotein Cholesterol
EDHF Endothelial dependent Hyperpolarizing Factor
DPI Diphyleneiodinium
RAGE Receptor for Advanced Glycation End-products
IRS-1 Insulin Receptor Substrate-1
MitoQ Mitoquinone
LNAME Nw-nitro-L-arginine- methyl ester

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Table of Contents

Title ........................................................................................................................................... i
Dedication .............................................................................................................................. iii
Acknowledgement ................................................................................................................ iv
List of Figures and Tables.....................................................................................................v
List of Abbreviations............................................................................................................ vi
Abstract.................................................................................................................................. ix

CHAPTER ONE.................................................................................................................... 1

1.1 Background ............................................................................................................. 1


1.2 Introduction ............................................................................................................. 2
1.2.1 ROS production in Hyperglycaemic states ................................................... 3
1.2.2 Endothelial dysfunction and oxidative stress in diabetes ............................ 6
1.3 Problem Statement and Justification of the Study.............................................. 11
1.4 Objectives of the study ......................................................................................... 12
1.4.1 General Objective......................................................................................... 12
1.4.2 Specific Objectives ....................................................................................... 12
1.5 Hypotheses ............................................................................................................ 12

CHAPTER TWO................................................................................................................. 13

2.1 Methods and Materials ......................................................................................... 13


2.2 Inclusion and exclusion criteria ........................................................................... 14
2.2.1 Inclusion criteria .......................................................................................... 14
2.2.2 Exclusion criteria ......................................................................................... 14
2.3 Sample size considerations................................................................................... 15
2.4 Assay of Superoxide Production Using Lucigenin Chemiluminescence.......... 17
2.5 Measurement of Nitric Oxide Bioavailability..................................................... 19
2.6 Statistical Analyses............................................................................................... 20

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CHAPTER THREE ............................................................................................................ 21

3.1 Results ................................................................................................................... 21


3.1.1 Patient characteristics ................................................................................. 21
3.1.2 Vascular superoxide generation.................................................................. 25
3.1.3 Sources of superoxide production ............................................................... 26
3.1.4 Assessment of the endothelial function ....................................................... 28
3.2 Discussion ............................................................................................................. 30
3.3 Conclusion............................................................................................................. 35

References............................................................................................................................. 36
Appendix 1.0: A Sample Questionnaire.............................................................................. 46

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Abstract

Background: Diabetic patients have an increased risk of cardiovascular morbidity and


mortality. An understanding of the mechanisms of superoxide production and
pathogenesis of endothelial dysfunction as well as their relationship in diabetes will
facilitate the development and application of more effective antioxidant therapeutic
strategies.

Objectives: To compare the superoxide levels, sources of superoxide production and


endothelial dependent relaxation in the saphenous veins from diabetic and nondiabetic
patients undergoing coronary artery by-pass graft (CABG).

Materials and methods: A total of sixty six diabetic (n=23) and nondiabetic (n=43)
patients were studied (mean age, 66.2 + 1.4 years). With the aid of a questionnaire,
clinical characteristics and demographics were collected from the patients. Vascular
superoxide levels were assayed using lucigenin chemiluminescence method. To
investigate the sources of superoxide production, vessel homogenates were incubated
with different inhibitors of oxidative stress pathways and then superoxide levels
measured. To assess the endothelial function between the two groups, relaxation of the
saphenous veins to calcium ionophore was investigated and compared.

Results: Basal superoxide generation was significantly reduced in saphenous veins from
diabetic than from nondiabetic subjects. (323.6 + 10.2 (n=9) versus 691.8 + 11.5 (n=17),
pmol/min/mg, 95% CI, 117 to 383 pmol/min/mg, P= 0.017). Mitochondrial mediated
superoxide production was more enhanced in nondiabetics (mean difference 214.0
pmol/mg/min; 95% CI, 71 to 593 pmol/mg/min; P = 0.003 ), compared to diabetics (mean
difference 50.9; 95% CI, -34 to 134 pmol/mg/min; P = 0.093). An investigation into the
clinical characteristics of the study subjects revealed that the proportion of diabetic
subjects on ACEIs/ARBs was significantly greater compared to nondiabetics (86% versus
53%; P= 0.009).

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In addition, the diabetics consumed more fruits and green leafy vegetables as calculated
and determined by the total portions taken per week (22.5 versus 10.5; P=0.0147; 95% CI
1.0 to 18.0). The plasma LDL levels in the diabetics was lower than controls (1.57 + 0.18
versus 2.16 + 0.15 mmol/L; 95% Cl, 1.061 to 0.113 mmol/L; P = 0.017). Interestingly,
there was insignificant variation in the proportion of patients taking HMG CoA reductase
inhibitors between the two groups (91% versus 95%; P=0.606). The HDL levels were
higher in the nondiabetics than diabetics. However, the oxLDL/LDL ratio was weakly
insignificant between the two groups. Relaxation of saphenous veins to calcium
ionophore was decreased in the diabetics compared to controls (34.2 + 3.2% versus 48.9
+ 4.9%; 95%CI, 2.3 to 27.0%; P=0.022) while the sensitivity (EC50) to calcium
ionophore was insignificant between the two groups (164.8 + 12.6 versus 178.6 + 13.5
nmol/L in diabetics and nondiabetics respectively; 95%CI, -42.0 to 201.1 nmol/L; P =
0.832).

Conclusion: There is significantly decreased superoxide production in saphenous veins


from diabetic patients taking ACEIs/ARBs, higher fruits and green leafy vegetable intake
and having lower plasma LDL levels. Mitochondrial mediated superoxide production is
more enhanced in non-diabetics and its role in diabetic vasculature warrants further
investigations in studies with larger sample size. The endothelium dependent relaxation is
attenuated in the diabetic vessels. In consistent with previous studies, the reduced plasma
HDL levels and potentially enhanced effects of oxLDL in the diabetics may be a possible
explanation to the observed endothelial dysfunction.

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CHAPTER ONE

1.1 Background

It is a well established fact that patients with diabetes have an increased risk of
cardiovascular morbidity and mortality. [1] Cardiovascular disease is the leading cause
of death in the industrialized nations and has also been claimed to be responsible for
approximately 70-80% of diabetic deaths. [2] Although it is also an important cause of
mortality in the developing nations, the recent proliferation of cardiovascular disease
[3], as well as diabetes in these countries, threatens devastating effects on the already
overburdened health care system. For example, it has been projected that in the near
future, diabetic-related cardiovascular disease complications, which were initially
considered rare in Sub-Sahara Africa, will overtake infectious diseases as the most
common cause of mortality. [4] Therefore, a detailed comprehension of the mechanisms
of cardiovascular complications as characterized by oxidative stress and endothelial
dysfunction in diabetes will continue to draw much attention.

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1.2 Introduction

Oxidative stress plays an important role in the onset of diabetes mellitus and
development of both microvascular (retinopathy, neuropathy and nephropathy) and
macrovascular (myocardial infarction, stroke and peripheral vascular disease) diabetic
complications. [5] Numerous studies have implicated oxidative stress as an important
pathogenic factor in diabetic complications in both type I and type II diabetes mellitus.
[6, 7] The drivers of diabetic oxidative stress are lipids, elevated free fatty acids
(FFAs), hyperglycaemia and hyperinsulinaemia.

Endothelial dysfunction is also a key feature of diabetes and alongside oxidative stress
is thought to play an important role in diabetic vascular complications. Endothelium
controls the tone of the underlying vascular smooth muscle through the production of
vasodilator mediators such as nitric oxide (NO), prostacyclin and endothelium derived
hyperpolarisaton factor (EDHF). Impaired endothelium dependent vasodilation has been
demonstrated in various animal models of diabetes and in humans with type I and type
II diabetes. [4]

Oxidative stress causes decreased bioavailability of NO and subsequent impairment of


the endothelial dependent vasodilation. [9] Clinical trials have shown that antioxidants
improve endothelial function in diabetic patients, suggesting that oxidative stress plays
an important role in the pathogenesis of endothelial dysfunction in diabetes. [10, 11]
These findings imply that the vulnerability of diabetic patients to vascular complications
may be a function of oxidative stress. Therefore, a thorough understanding of the
pathogenesis of endothelial dysfunction as well as mechanisms of oxidative stress at the
cellular level in the diabetes and diabetic complications is important in the identification
of potential pharmacologic targets of therapy.

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1.2.1 ROS production in Hyperglycaemic states

Reduced antioxidant defences and increased levels of reactive oxygen species (ROS)
have been demonstrated in diabetic patients. [12] Hyperglycaemia, which is a
characteristic feature of diabetes mellitus, can induce oxidative stress by various
mechanisms, including: the stimulation of the polyol pathway, the formation of
advanced glycation end-products (AGE) as well as autoxidation of glucose. FFAs and
hormones such as leptin and insulin have also been implicated in oxidative stress in
diabetic patients.

The Polyol Pathway

The enzymes Aldose reductase and sorbitol dehydrogenase contribute to ROS


generation. Aldose reductase utilizes NADPH for the reduction of glucose to sorbitol.
Although under normal circumstances, this pathway does not constitute a major
chemical process, during hyperglycaemia, a significant amount of glucose is
metabolized via this pathway, thereby resulting in reduced bioavailability of
NADPH.[13] This leads to decreased glutathione regeneration and nitric oxide synthase
activity, thus increased oxidative stress.[14] Sorbitol dehydrogenase oxidizes sorbitol to
fructose with concomitant NADH production, which in turn is utilized by NADPH to
produce superoxide. [15]

Advanced Glycation End-products (AGEs)

AGEs are formed as result of non-enzymatic covalent bonding of aldehyde or ketone


groups of reducing sugars to the free amino groups of proteins. AGEs have been
proposed to contribute atherosclerotic lesions in diabetic patients, by modifying
lipoproteins and the extracellular matrix and activation of the receptor of AGE (RAGE).
Stimulation of RAGE causes production of ROS probably via an NADPH oxidase. [16]
Pharmacological agents that block AGE-RAGE interactions such as the soluble RAGE
or RAGE specific IgG have shown great prospects in diabetic experimental models.
[49]

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Autoxidation of Glucose

Hyperglycaemia may result in increased glucose metabolism and consequently lead to


increased production of Nicotinamide Adenine Dinucleotide (NADH). [14] Excess
NADH levels cause increased mitochondrial proton gradient and electrons are
transferred to oxygen, thereby producing superoxide. [17] The NADH dehydrogenase of
complex I and the interface between ubiquinone and complex III constitute the two
main sites of the superoxide production by the electron transport chain.[18]
Mitochondrial–derived superoxide has been demonstrated to cause increased
diacyglycerol (DAG) synthesis and subsequent protein kinase C (PKC) activation.[14,
15]

Leptinaemia and Insulinaemia

Leptin is a hormone produced by adipocytes. Other than acting on the CNS to reduce
food intake, it also exerts effects on the endothelial cells. [19] The plasma levels of
leptin are increased in type 2 diabetes. [20] Endothelial cells incubated with leptin
produce increased levels of ROS. However, the exact mechanism of ROS production
still remains elusive. [21] Hyperinsulinaemia has been shown to stimulate oxidative
stress. Insulin induces the production of hydrogen peroxide (H2O2) when activating its
receptors, which in turn can indirectly activate oxidative reactions. Insulin also activates
the sympathetic nervous system, thereby leading to the activation of the
neurotransmitters and their enzymatic systems, several of which induce oxidative stress.
[22]

4
Figure 1.0: Sources of oxidative stress and their potential links to endothelial
dysfunction in diabetes.

Modified from references [8, 15] ROS, Reactive oxygen species; AGE, advanced glycation
end-products; GTPCH, GTP-cyclohydrolase-I; BH4, tetrahydrobiopterin; PKC, protein kinase
C; DAG, diacyglycerol; XO, xanthine oxidase; NK-kB, nuclear factor kB; VCAM, vascular
cellular adhesion molecule-1; ICAM, intracellular adhesion molecule-1; SOD, superoxide
dismutase; GSH, reduced glutathione; GSSH, oxidized glutathione; NO, nitric oxide; oxLDL,
oxidized LDL; eNOS, endothelial nitric oxide synthase.

5
1.2.2 Endothelial dysfunction and oxidative stress in diabetes

Studies have shown that patients with either type I [26] or type 2 [27] exhibit
endothelial dysfunction. Moreover, the endothelial function in the diabetic patients can
be improved with antioxidants implying that oxidative stress plays an essential role in
endothelial dysfunction. [10, 11] Increased production of superoxide via NAD(P)H
oxidase and uncoupled eNOS has been shown to be a major contributor to endothelial
oxidative stress in the diabetics.

NADPH oxidase

High endothelial NADPH oxidase activity is attributed to oxLDL, AGE, FFA and
hyperglycemia and its stimulation has been shown to be mediated by PKC. [28]
Hyperglycaemia causes de novo synthesis of diacyglycerol (DAG), leading to the
activation of PKC. [29] The prevention of diacyglycerol-protein kinase C mediated
vascular dysfunction in diabetes by vitamin E supports the linkage between oxidative
stress and PKC pathway. [30] Moreover, incubation of endothelial cells and smooth
muscle cells with high glucose increases mitochondrial ROS and intracellular DAG
levels, consequently leading to PKC activation. [31]

Incubation of the tissue with the PKC inhibitor resulted in improved endothelial
function as well as abrogation of the hyperglycaemia-induced NF-kB activation and
VCAM-I expression in human aortic endothelial cells. [32, 50] Blood vessels from
diabetics and nondiabetics exhibit increased superoxide production, which is inhibited
by diphenylene iodinium, thereby demonstrating that NADPH oxidases are active in
these two groups, but may be more enhanced in the diabetics. [9]

Low Density Lipoprotein cholesterols

High levels of free fatty acids have been reported in diabetic patients. [23] Excess FFAs
enter the Krebs cycle and generate acetyl CoA. This causes excess production of NADH
which results in increased mitochondrial superoxide production. Lopes et al. [24]
demonstrated that acute infusions of FFA in humans caused elevations in isoprostanes
which are markers of lipid peroxidation. The import of oxLDL or their local formation
in the vessel walls has been reported to be an important mechanism involving oxidative

6
stress in the atherosclerotic process in diabetes. Oxidized LDL produces oxidative stress
in the endothelial cells via activation of a NADPH oxidase through a Phospholipase A2
signalling mechanism. [25]

Increased oxidative stress and altered plasma lipid composition have been implicated in
macrovascular endothelial dysfunction in the diabetics. HMG-CoA reductase inhibitors
(statins) have demonstrated beneficial effects in large clinical trials involving the
diabetic patients. [48] Consequently, statin therapy has been recommended in all
patients at high risk of any type of major cardiovascular event, including the diabetics.
[65]

Uncoupled eNOS

Superoxide can also react with NO to produce peroxynitrite [33] which in turn can
oxidize BH4, thereby reducing eNOS availability. [34] In the presence of reduced
concentrations of BH4, eNOS becomes uncoupled and transfers electrons to molecular
oxygen instead of L-Arginine to produce superoxide rather than NO. [35] Incubation of
the diabetic vessels with nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl
ester resulted in reduced superoxide production, thus supporting the presence of
uncoupled eNOS in the diabetic as well as non-diabetic vasculature. [9]

Moreover clinical studies have demonstrated that BH4 supplementation given to diabetic
patients improves their endothelium dependent vasodilation thus supporting the notion
that uncoupled eNOS plays a role in diabetic endothelial dysfunction. [36] Therefore,
the reduced bioavailability of NO due to oxidative stress caused by diabetes results in
impairment of endothelial-dependent vasodilation. Hyperglycemia-induced
mitochondrial production and activation of hexosamine pathway, may also lead to
reduced availability of eNOS. [36]

Hyperglycaemia causes O-linked N-acetylglucosamine modification of serine 1177 on


eNOS, the Akt activation site, thereby preventing its phosphorylation. [37] Akt activity
is inhibited by diabetic oxidative stress, which basically acts by inducing serine
phosphorylation of insulin receptor substrate-1 (IRS-1), and then targeted for
degradation. The resultant reduction in IRS-1 leads to the impaired activation of the
phosphatidylinositol-3-kinase/Akt pathway. [38]

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Xanthine Oxidase

In patients with diabetes mellitus (and mild hypertension), it has been shown that
allopurinol; an inhibitor of xanthine oxidase improved endothelial function suggesting
that xanthine oxidase plays a role in diabetic endothelial dysfunction. [39] Superoxide
production via xanthine oxidase has been reported to be significantly enhanced in
diabetic subjects and this is consistent with beneficial effects of allopurinol on their
endothelial function. [40] A recent study by Inkster et al. [41] demonstrated that
xanthine oxidase contributes to neurovascular dysfunction in experimental diabetes.
Treatment of the diabetic rats with allopurinol resulted in improved nerve and vascular
function.

Some studies in human beings have reported no beneficial effects of allopurinol in


diabetic patients. For example, a randomized, double-blind placebo controlled trial
reported that allopurinol was ineffective in the reduction of oxidative stress in the
diabetic subjects. [51] However, the sample size in this study was too small hence
possibly not powered enough to detect any significant effects, if present. Some authors
have also claimed that allopurinol induces diabetes. [52, 53] These inconsistent reports
therefore necessitate the execution of sufficiently powered and well designed studies
aimed at unravelling the sources of oxidative stress and their potential association with
endothelial dysfunction in diabetes.

Coenzyme Q10

Coenzyme Q10 (CoQ10), an endogenous enzyme cofactor produced in most of the


human cells is an important component of the mitochondrial respiratory chain. CoQ10
exists in the oxidized (ubiquinone) and reduced (ubiquinol) forms. The reduced form is
a potent lipophilic antioxidant. Studies have suggested that mitochondrial dysfunction
induced by oxidative stress plays a pivotal role in the pathogenesis of insulin resistance
and vascular disease in subjects with diabetes. [42] Lim et al [43] reported a remarkable
change in CoQ10 in patients with diabetes, suggesting a marked increase in oxidative
stress.

Moreover, studies in experimental diabetic models have suggested that a significant


decrease in CoQ10 may be responsible for an enhanced vulnerability of diabetic heart

8
mitochondria to oxidative damage. [44] However, it is widely known that the
conventional antioxidants (reduced CoQ10) have got limited efficacy due to their
impermeability to mitochondrial membrane. Mitoquinone (MitoQ), recently developed
by conjugating the lipophilic triphenylphosphonium cation to ubiquinol, is a
mitochondrion targeted antioxidant that can permeate biological membranes and
consequently accumulate within the mitochondria. [54] Therefore, there is great
prospect in the future use of MitoQ in protection against mitochondrial oxidative
damage in numerous diseases including diabetes.

Renin Angiotensin System

Angiotensin II generates oxidative stress in vasculature by stimulating NADH oxidase


and is also said to mimic the effects of insulinaemia. [22] ACE inhibition has been
shown to decrease angiotensin II–induced NADPH oxidase activity, hence reducing
vascular production of superoxide [45] and consequently improving endothelium
dependent vasodilation in diabetes mellitus. [46] Similarly in experimental models,
angiotensin AT1 receptor antagonism has been shown to ameliorate diabetes–generated
oxidative stress, indicating an important role of the renin–angiotensin system in the
development of diabetic complications. [49]

To further support this, the Heart Outcomes Prevention Evaluation (HOPE) study on the
effects of Ramipril on cardiovascular events, reported marked reduction in the incidence
of complications related to diabetes and new diabetic cases. [55] However, the HOPE
trial failed to clearly report on the dose-response relation of ramipril regarding the end-
points in the diabetic subjects, despite being tested at two dose levels, 2.5mg and
10.0mg. Another clinical trial, the Captopril Prevention Project Study (CPPS) group
demonstrated a lower rate of newly diagnosed diabetes in patients who were assigned to
receive captopril than in those patients who were receiving a diuretic or beta-blocker.
[56] ACE inhibition and angiotensin II antagonism as antioxidant mechanisms have
been suggested as part of the explanation for these findings.

Dietary Antioxidants

Although there is substantial evidence of the antioxidant effects of vitamin C and E in


experimental models and man, inconsistent results have been reported in clinical trials.

9
Some studies have demonstrated positive results [57, 58] while others have failed to
show any benefits.[59, 60] The possible reasons for these inconsistent results may be
due to variations in administered dosages, study designs, combinations of vitamins as
well as differences in the status of oxidative stress in the study subjects. [61]

However, despite all these controversies, the importance of a healthy diet in the
attenuation and prevention of cardiovascular disease is widely acknowledged. In the
Oxford Fruit and Vegetable Study, increased fruit and vegetable consumption in the
intervention group, resulted in a significant decrease in the both systolic and diastolic
pressure. [62] These findings suggest that a healthy diet that is rich in fruit and
vegetables may offer cardiovascular protection due to increased anti-oxidant capacity,
and possibly provide beneficial effects in susceptible diabetic subjects as well.

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1.3 Problem Statement and Justification of the Study

From the reviewed literature it is quite apparent that the mechanisms of superoxide
production in the diabetics are not completely understood. This has been exemplified by
the inconsistent findings on the role of antioxidants in reducing oxidative stress and
improving endothelial function in the diabetics. Moreover, there is limited number of
reports in the diabetic population with a view of assessing mechanisms of superoxide
production and endothelial dysfunction. Most of the reported findings are just as a result
of sub-group analyses from huge studies with inadequately and/or poorly defined
diabetic populations. A better understanding of the relationship between mechanisms of
superoxide production and endothelial dysfunction in the diabetes will facilitate the
development and application of more effective antioxidant therapeutic strategies.

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1.4 Objectives of the study

1.4.1 General Objectives

This study was therefore aimed at comparing superoxide production and endothelial
function in saphenous veins, obtained from diabetic and non-diabetic patients
undergoing coronary artery by-pass graft (CABG).

1.4.2 Specific Objectives

2. To compare superoxide production and their sources in the diabetic and non-
diabetic vessels.
3. To compare EC50 (the effective concentration of calcium ionophore that caused
50% of maximal relaxation) and maximal relaxations of the vessels in the two
groups.

1.5 Hypotheses

2. There is no difference in superoxide levels between diabetic and nondiabetic


saphenous veins.
3. There is no variation in the source of superoxide generation between diabetic
and nondiabetic vasculature.
4. There is no difference in the maximal relaxation and sensitivity (EC50) of the
saphenous veins to calcium ionophore in diabetic and nondiabetic subjects.

12
CHAPTER TWO

2.1 Methods and Materials

Materials

 Saphenous veins
 Lucigenin (5 µmol/L)
 Xanthine (0.1 to 1.0 µmol/L )
 Xanthine oxidase (10-4U/ml)
 Allopurinol (10mM)
 NADH (100 µM)
 Rotenone (1 mM)
 Diphyleneiodinium (DPI) (10-4 M)
 L-NAME (Nw-nitro-L-arginine methyl ester, 10-4M)
 Krebs-HEPES buffer (composition in mM: NaCI, 130; KCI, 4.7; NaHCO3, 14.9;
KH2PO4, 1.2; Glucose, 5.5; MgSO4.7H2O, 1.2; CaCI.2H2O, 1.6; CaNa2EDTA,
0.027; and 10-5mole of indomethacin dissolved in 1ml Dimethyl Sulfoxide,
DMSO)
 Phenylephrine ( 3 µmol/L )
 Potassium chloride (KCI) (100mM)
 Calcium ionophore (10-8 to 10-5 M)
 Liquid scintillation counter (Hewlett Parkard Model 2100TR)
 Organ bath chambers

Source of the materials

Xanthine, xanthine oxidase, rotenone, diphyleneiodinium (DPI), lucigenin and


indomethacin were purchased from Sigma-Aldrich Co., St. Louis, USA. Allopurinol
was bought from ICN biomedical In., Aurora, Ohio, USA.

13
Methods

Diabetic and non-diabetic patients with coronary artery disease (CAD), undergoing
coronary artery bypass graft surgery (CABG) were recruited. The subjects attended
BHF Glasgow Cardiovascular Research Centre prior on the day prior to surgery. With
the aid of a questionnaire (appendix 1.0), information on the diet, smoking, age, and
exercise were collected from the patients. Blood samples from the study subjects were
assayed for LDL and oxLDL levels in various laboratory units within the research
centre. The study was approved by the local ethics committee and all the patients gave
written informed consent.

2.2 Inclusion and exclusion criteria

2.2.1 Inclusion criteria

1. Patients with known coronary artery disease as diagnosed on angiography and


presenting for CABG surgery.
2. Patients who are clinically stable as is usual prior to a planned CABG operation.

2.2.2 Exclusion criteria

1. Patients presenting for valvular operations or repeat CABG surgery.

14
2.3 Sample size considerations

In a similar study by Guzik and others [9], differences in vascular superoxide


production in saphenous veins (37.9 + 4.9 versus 21.6 + 1.4 relative light units per mg;
P< 0.01) was shown when comparing 45 diabetics with 45 nondiabetics respectively. A
previous study in our laboratory by Al-Benna et al. [40] demonstrated a difference in
endothelial function (maximal relaxation to calcium ionophore, 26 + 2% versus 60 +
1%; P < 0.001) and vascular superoxide production ( 890 + 90 versus 560 + 60
pmol/mg/min; P = 0.008) when comparing 51 patients with coronary artery disease with
51 controls. We expected similar numbers in order to show differences in superoxide
levels and endothelial function between diabetic and nondiabetic saphenous veins.

However, due to time constraints and other factors as shown in figure 2.0, we managed
to study 23 diabetics and 43 nondiabetics (N = 66). Although it was also desired (in a
worst case scenario) to have at least one-third of the subjects to be diabetic, which in
our case was realized, the total number of patients in the present study was relatively
small.

15
Figure 2.0: A chart showing the flow of patients during the study

16
Vessels preparation

The saphenous veins collected at the time of CABG surgery were stored in krebs-
HEPES buffer overnight and assayed the next day. The vessels were then carefully
dissected free of loose connective and fatty tissues.

2.4 Assay of Superoxide Production Using Lucigenin Chemiluminescence

Xanthine/ Xanthine Oxidase Calibration Curve

Lucigenin, which acts as a chemilumigenic probe is first reduced by one electron to


produce the lucigenin cation radical. [63] The lucigenin cation radical then reacts with
the biologically derived superoxide to yield an unstable dioxetane intermediate. The
lucigenin dioxetane decomposes to produce two molecules of N-methylacridone, one of
which is in an electronically excited state, which upon relaxation to the ground state
emits a photon. [63] Therefore, the biological production of superoxide is assayed
through the measurement of the photon emission or chemiluminescence.

In order to assay for superoxide production in the sample vessels, a xanthine/ xanthine
oxidase calibration curve was prepared. Xanthine oxidase catalyses the oxidation of
xanthine in the presence of molecular oxygen (which acts as an electron acceptor) to
produce uric acid and superoxide anion. [64]

In our calibration experiments lucigenin and xanthine oxidase were added into vials
containing 2ml of buffer resulting in final concentrations of 5 µmol/L and 10-4 U/ml
respectively. Counts were then obtained at 3 minute intervals after the introduction of
xanthine (which was added in varying concentrations of 0.1 to 1.0 µmol/L). 0.1 µmol/L
xanthine and 10-4 U/ml xanthine oxidase generated 28nmol of superoxide, which then
reacted with 5 µmol/L lucigenin. The resultant chemiluminescence was detected with a
liquid scintillator counter (Hewlett Parkard Model 2100TR) set in non-coincidence
mode.

The combinations of xanthine oxidase and varying concentrations of xanthine produced


chemiluminescent signals in a manner dependent on the concentration of the xanthine,
which was quantified by the integration of the Areas Under the Curve (AUC) generated

17
in the experiments. Therefore, plots of AUC against superoxide concentration (in
nmoles) yielded calibration curves that were used to quantify the concentration of the
superoxide in the saphenous veins.

Assaying of superoxide in the vessels

By taking care not to damage the endothelium, 2- to 3 mm segments/rings were sliced


and weighed. The vessels were then randomly placed and incubated into the vials
containing 2ml of Krebs-HEPES buffer. The rings were incubated at room temperature
in the absence (control) or presence of an inhibitor of xanthine oxidase, allopurinol; a
non-specific inhibitor of NADPH oxidase, diphenylene iodinium, DPI; an inhibitor of
endothelial nitric oxide synthase (eNOS), LNAME (Nw-nitro-L-arginine-methyl ester)
or an inhibitor of mitochondrial respiratory chain, rotenone, for 1 hour before the
quantification of superoxide.

In order to assess the effects of a positive control of whether the vessels were functional,
one of the rings from each vessel was treated with 100 µmol/L of NADH. In order to
allow for uptake or absorption, chemiluminescence was measured 6 minutes after the
exposure to lucigenin. The basal rate of superoxide production was measured and
expressed in nanomoles per milligram dry weight of tissue per minute. Further
conversions were made to facilitate appropriate statistical analysis.

18
2.5 Measurement of Nitric Oxide Bioavailability

Vessel preparation

The freshly prepared rings of saphenous veins were suspended on wire hooks, under one
gram tension in individual organ baths containing krebs buffer of the following
composition (mM); NaCI, 130; KCI, 4.7; NaHCO3, 14.9; KH2PO4, 1.2; Glucose, 5.5;
MgSO4.7H2O, 1.2; CaCI.2H2O, 1.6; CaNa2EDTA, 0.027; and 10-5mole of indomethacin
dissolved in 1ml dimethyl sulfoxide, DMSO). The Krebs solution was constantly
aerated with a gas mixture of 95% O2 plus 5% CO2 and maintained at 37oC.

Vascular reactivity studies

The eight organ chambered arrangements were run concurrently. Changes in the
isometric tension were detected by a force transducer and recorded by a personal
computer by using application software. The rings were allowed to equilibrate for about
30-60 minutes. In order to get rid of any traces of anesthetics, the Krebs buffer was
washed from the organ chambers. Tension adjustments were also done as required
during the course of equilibration.

The rings were constricted with 100 mM Potassium Chloride (KCI) twice at different
times and the response monitored. This was meant for standardization and allowing for
the differences in ring sizes as well as confirmation of whether the vessels were
functional. The rings were washed repeatedly every 5 minutes until the tone fell towards
the baseline, after which they were allowed to equilibrate for about 20-30 minutes. The
rings were then constricted with 3 µmol/L phenylephrine. After a stable contraction
plateau was reached, all the rings were exposed cumulatively to calcium ionophore (10-8
to 10-5M) and changes in tension were read from the computer. Phenylephrine and
calcium ionophore were washed out thoroughly and the vessels allowed to equilibrate.
The relaxant responses to calcium ionophore were expressed as a percentage of the
contraction to phenylephrine.

19
2.6 Statistical Analyses

The levels of superoxide production, sources of superoxide and endothelium dependent


relaxation to calcium ionophore in diabetic and nondiabetic patients were compared.
Data of clinical characteristics and demographics (given in table 1.0) are expressed as
mean + SEM or n (%), unless stated otherwise. All variables were tested for normal
distribution. Mann-Whitney test was used to compare non-normally distributed data
between the diabetic and non-diabetic groups.

Because the values of vascular superoxide production, EC50 and HDL levels were
skewed, they were log-transformed to improve normality for statistical testing. They
were then analyzed using parametric tests such as unpaired t-tests and back-transformed
for clear interpretation. Relaxant responses to calcium ionophore between diabetic and
nondiabetic groups were compared using unpaired t-test. Where appropriate, the paired
t-test was used. The chi-square test was used for categorical variables. The effective
concentration of calcium ionophore that caused 50% of maximal relaxation was defined
as the EC50. The results are shown as mean + SEM, or as median, including 95% CIs
where appropriate. All the analyses were performed using Minitab version 13 (Minitab
Inc.). The value of P< 0.05 was considered statistically significant.

20
CHAPTER THREE

3.1 Results

3.1.1 Patient characteristics

The study population consisted of 66 patients. The clinical characteristics and


demographics of the patients are shown in the table 1.0. These include data on the age,
sex, risk factors, medications taken as well as fruits and green leafy vegetable intake.

Table 1.0: Clinical characteristics and demographics of the patients

Diabetics Non-diabetics P value


Age(yrs), (n) 67.35 + 2.21(23) 65.38 + 1.85(43) 0.542
Sex, M/F, n (%) 21/1 (95/5) 37/6) (86/14) 0.227
Risk Factors
Hypertension, n (%) 19 (83) 16 (37) < 0.001*
Hb1ac, % (n) 7.69 + 0.32 (20) 5.50 + 0.06 (35) < 0.001*
LDL cholesterol, mmol/L (n) 1.57 + 0.18 (15) 2.16 + 0.15 (35) 0.017*
oxLDL cholesterol, mmol/L (n) 60.75 + 4.25 (13) 67.30 + 4.10 (30) 0.275
oxLDL/LDL ratio ,median value (n) 36.2 (12) 33.0 (30) 0.0583
HDL cholesterol, mmol/L 1.041 + 0.001(19) 1.201 + 0.001(37) 0.048*
Medications, n (%)
Aspirin 19 (86) 36 (87) > 0.990 ¶
ACE Inhibitors/ARBs 19 (86) 23 (53) 0.009*
Calcium Channel Blockers 10 (45) 15 (36) 0.448
Nitrates 11 (50) 22 (54) 0.782
Beta blockers 17 (77) 29 (70) 0.577
HMG CoA reductase Inhibitors 20 (91) 39 (95) 0.606 ¶
Diet , median value (n)
Fruits & Vegetables (total portions/week) ◄ 22.5 (20) 10.5 (34) 0.0147*

Total portions/week: ◄ - Calculated as follows: = (Number of days in a typical week a


patient eats fruits, multiplied by the number of pieces or servings of fruits he/she takes
on those days) + (Number of days in a typical week a patient eats green leafy
vegetables, multiplied by the number servings or meals having the vegetables that
he/she would take on those days).
¶: The obtained P values after performance of Fisher’s Exact Test. (*): P value is < 0.05

21
The mean age of the patients studied was 66.2 + 1.4 years, with no significant
difference between the diabetic and nondiabetic groups (67.3 + 2.2 versus 65.6 + 1.8
years, 95% CI -4.02 to 7.55, P= 0.542). The proportion of males was higher than
females in both groups. The plasma levels of LDL were significantly lower in the
diabetics compared to non-diabetics (1.57 + 0.18 versus 2.16 + 0.15 mmol/L; 95% Cl,
0.113 to 1.061 mmol/L; P = 0.017, figure 3.0). However, there was no significant
difference in the oxLDL/LDL ratio in the two groups (36.2 versus 33.0; 95% CI, -1.1
to15.6; P = 0.0583). Interestingly, despite lower levels of LDL levels in the diabetic
group, there was no significant variation in the proportion of subjects taking HMG CoA
reductase inhibitors in diabetic and nondiabetic subjects ( 91% versus 95%; P = 0.513).
The HDL levels were higher in the nondiabetics than diabetics (1.201 + 0.001 versus
1.041 + 0.001 mmol/L; 95% CI, 0.010 to 0.40 mmol/L; P = 0.04, figure 4.0)

Figure 3.0: A box plot of LDL levels in diabetic and nondiabetic subjects

22
Figure 4.0: HDL levels in diabetic and nondiabetics

The Hb1ac levels were significantly higher in the diabetes as compared to nondiabetic
patients (7.69 + 0.32% versus 5.50 + 0.06%, respectively; 95% Cl, 1.51 to 2.85 %; P <
0.001). Hypertension was significantly more frequent among the diabetic subjects (83%
versus 37% in the nondiabetics; P < 0.001). Consequently, the proportion of the patients
taking ACE inhibitors and ARBs in the diabetic subjects was greater compared to the
nondiabetics (86% versus 53% respectively; P = 0.009).

23
Another important finding based on self-reporting, was that the diabetic subjects
consumed more fruits and vegetables than the nondiabetics, as calculated and
determined by the total portions taken per week (22.5 versus 10.5; P = 0.0147; 95% CI,
1.0 to 18.0, figure 5.0).

Figure 5.0: A comparison of fruit and green leafy vegetable intake between
diabetic and nondiabetic subjects

24
3.1.2 Vascular superoxide generation

Basal superoxide generation from saphenous veins was determined using the lucigenin
chemiluminescence method from intact rings from diabetic and nondiabetic subjects.
Superoxide levels were significantly elevated in vessels from non-diabetics than from
diabetic patients (691.8 + 11.5 (n=17) versus 323.6 + 10.2 (n=9), pmol/min/mg, 95%
CI, 117 to 383 pmol/min/mg, P= 0.017, figure 6.0).

Figure 6.0: Superoxide production in diabetic and nondiabetic subjects

25
3.1.3 Sources of superoxide production

In order to investigate the possible sources of superoxide production in diabetic and


nondiabetic vessels, we assayed superoxide generation in response to allopurinol, an
inhibitor of xanthine oxidase; diphenylene iodinium (DPI), a non-specific inhibitor of
NADPH oxidase; LNAME (Nw-nitro-L-arginine-methyl ester), an inhibitor of
endothelial nitric oxide synthase (eNOS), and/or rotenone, an inhibitor of mitochondrial
respiratory chain.

However, due to the unavailability of sufficient experimental data on the inhibition of


other oxidative stress pathways owing to limited harvested vessels, we managed only to
document superoxide production in response to inhibition of mitochondrial respiratory
chain by rotenone from diabetic and nondiabetic patients (table 2.0). Although rotenone
inhibited superoxide production in the two groups of subjects, its effects in the diabetics
was minimal compared to nondiabetics. In the nondiabetic vessels, superoxide
production was significantly inhibited by rotenone, suggesting that mitochondrial
respiratory chain played an important role in oxidative stress in this group (table 2.0 and
figure 7.0).

Table 2.0: Inhibition of superoxide production by rotenone in diabetic and


nondiabetic saphenous veins

Diabetics Nondiabetics
(n=8) (n=15)

Basal Superoxide, 333.4 + 28.1 744.7 + 19.2


(pmol/mg/min)
+ Rotenone, 282.5 + 32.3 530.9 + 14.1
(pmol/mg/min)
Mean difference 50.9 213.8
(pmol/mg/min)
P = 0.093 *P = 0.003
(95% CI, -34 to 134 (95% CI, 71 to 593
pmol/mg/min) pmol/mg/min)

26
Figure 7.0: Inhibition of superoxide production by rotenone in nondiabetic
and diabetic saphenous veins

27
3.1.4 Assessment of the endothelial function

To assess the endothelial function in vessels of the study subjects, nitric oxide
bioavailability was investigated by cumulative exposure of the vessels to calcium
ionophore (0.01 to 10 µmol/L), after prior precontraction with 3 µmol/L Phenylephrine.
Therefore, the relaxant responses to calcium ionophore were expressed as a percentage
of the contraction to phenylephrine. Calcium ionophore induced relaxations in a
concentration-dependent manner in both diabetic and nondiabetic saphenous veins, with
the relaxant effect being maximal at about 10 µmol/L (figure 8.0). The maximal
relaxation to calcium ionophore was significantly greater in the nondiabetics compared
to diabetics (table3.0, figure 8.0 and 9.0). However, the sensitivity (EC50) to calcium
ionophore between the two groups was not significant (table 3.0).

Table 3.0: Mean EC50 of calcium ionophore and maximal relaxation in


diabetic and non-diabetic saphenous veins

EC50 (nmol/L) Max. relaxation to


calcium ionophore (%)
Diabetics 164.8 + 12.6 34.2 + 3.4
(n = 10)
Non-diabetics 178.6 + 13.5 48.9 + 4.9
(n = 15)
P = 0.832 *P = 0.022
(95%CI, -42.0 to 201.1 nmol/L) (95%CI, 2.3 to 27.0 %)

28
Figure 8.0: Calcium ionophore induced concentration-relaxation curve in 3.0
µmol/L preconstricted diabetic and nondiabetic saphenous veins.

Figure 9.0: Maximal relaxation of diabetic and nondiabetic saphenous veins to


calcium ionophore

29
3.2 Discussion

The present study has shown that basal superoxide levels in the saphenous veins from
the diabetic patients undergoing coronary artery by-pass graft (CABG) are lower
compared to nondiabetics. This is contrary to what has been reported in various studies
that have otherwise demonstrated that superoxide levels in blood vessels from
nondiabetics are lower compared to diabetics. [6, 7, 9] However, in a study by
Arzamastseva et al [66], the degree of oxidative stress in subjects with type II diabetes
was reported to be lower compared to those with type II diabetes plus congestive heart
failure (CHF) and also when compared to those with CHF only. With a view to find a
possible explanation for these interesting results in our study, the characteristics of the
patients in terms of their hypertensive and biochemical status, therapeutic profiles and
dietary intake were investigated and compared in the two groups as outlined in table 1.0.

Vascular sources of superoxide include NADPH oxidase, eNOS, lipid radicals,


mitochondrial respiratory chain and xanthine oxidase. [40, 67, 68] In diabetes, NADPH
oxidase and eNOS have been demonstrated to be the major sources of superoxide
production. [9] Guzik et al reported that in the saphenous veins from both diabetic and
non-diabetic subjects, mitochondrial respiratory chain did not significantly contribute to
superoxide production. [9]

In contrast to Guzik’s findings, the current study has demonstrated enhanced


mitochondrial mediated superoxide production, particularly in the nondiabetics as
reflected by the significant inhibitory effects of rotenone (table 2.0, figure 7.0). Our
diabetic subjects were relatively fewer in number hence possibly justifying the
insignificant rotenone inhibitory effects obtained in this group. These results therefore,
emphasize the need for future studies to focus on the role of mitochondrial mediated
ROS generation and the potential application of recently developed Mitoquinone
(MitoQ) in the protection against mitochondrial oxidative damage in both diabetics and
nondiabetics.

Several studies have demonstrated the role of LDL and oxLDL in oxidative stress,
which is a well-recognized phenomenon in the progression of diabetic complications.
[25, 69] In the present study, consistent with lower superoxide levels in the diabetic

30
subjects, there was significant reduction in plasma LDL levels in the diabetics compared
to nondiabetics. Surprisingly, the difference in oxLDL levels in the two groups was not
significant. From the weakly insignificant difference in the oxLDL/LDL ratio, there
seems to be a trend suggesting an increased oxidation of LDL in the diabetics compared
to the nondiabetics. An interesting observation worth noting is that there was almost the
same proportion of subjects in the two groups taking HMG-CoA reductase inhibitors
(statins). It is important to acknowledge a limitation based on our study design, that we
failed to provide the dosages of statin therapy in the two groups, and consequently
recommend the inclusion of dosages in future study designs and not merely the number
of subjects only.

However, from the observed results we possibly suspect that the diabetic patients on
average, may have been on higher doses of statin therapy, compared to the non-
diabetics. Other than reversing the inhibitory effect of oxidized LDL on eNOS, statins
have also been shown to have direct antioxidant effects on LDL in vitro and ex vivo [70,
71]. The hydroxy metabolites of atorvastatin inhibit oxidation of LDL and very-low-
density lipoprotein. [72]

A recent study by Vecchione et al [73] revealed a novel mechanism of action for statins
against diabetes-induced oxidative stress. In human blood vessels exposed to high
glucose, atorvastatin prevented oxidative stress and this protective effect was associated
with impairment of Rac-1 activation [73]. Statins may also have indirect effects against
oxidative mechanisms by attenuating the ability of macrophages to oxidize LDL.
Consistent with above mentioned benefits and also with those of larger clinical trials
[48], the present study indirectly support possible pleiotropic advantages of statins in
the diabetic subjects.

Diabetes and hypertension are essential independent risk factors for increased oxidative
stress. The coexistence of hypertension and diabetes results in remarkable increase in
vascular complications [74]. In this study, the proportion of hypertensive patients was
significantly greater in the diabetic compared to the nondiabetic group. Consequently,
the number of subjects taking angiotensin converting enzyme (ACE) inhibitors and/or
angiotensin II receptor blockers (ARBs) was significantly greater in the diabetic than in
the nondiabetic group.

31
It is well recognized that renin-angiotensin system plays an essential role in the
generation of ROS in the diabetics [22, 45, 49 ] and nondiabetics [75]; and that ACE
inhibitors and ARBs provide beneficial effects in reducing the incidence of
complications related to diabetes [55, 56]. The current study indirectly support the
above mentioned benefits since, in the diabetic group who actually had the higher
proportion of subjects on ACE/and or ARBs, registered lower levels of superoxide
production than the nondiabetic group.

Another interesting finding in the present study was the inverse relationship between
superoxide levels and the weekly consumption of fruits and green leafy vegetables
between the two groups. The diabetic patients who apparently registered lower levels of
superoxide, appeared to consume more fruits and green leafy vegetables as determined
by the total portions eaten per week, than the non-diabetics who, on the other hand had
higher levels of superoxide. This relationship may be due to the presence of antioxidants
which are widely known to be important components in a rich diet of fruits and green
leafy vegetables such as vitamin C, beta-carotene and vitamin E; and have been
suggested to play a protective role in cardiovascular disease. [76, 77]

It important to note however, that the credibility of these observations cannot be wholly
ascertained, because the data on fruits and vegetable intake was purely based on self-
reporting by the patients. Moreover, the patients were not under supervision from a
professional nutritionist or a dietician. Nevertheless, the present study supports the
notion that a healthy diet consisting of fruits and green leafy vegetables in patients at
risk of cardiovascular disease (such as diabetics), may offer beneficial effects due to
increased anti-oxidant capacity. [62, 78, 79]

Impaired vascular relaxation is a recognized marker of endothelial dysfunction in


diabetes. Vasodilation of the saphenous veins is equally essential since many CABG
patients require vasodilator treatment. In the current study, it has been demonstrated that
endothelium-dependent vasorelaxation of saphenous veins initially preconstricted with
phenylephrine is impaired in the diabetic subjects. Maximum relaxation was
significantly higher in the vessels obtained from nondiabetics, compared to diabetics
(table 3.0, figures 8.0 and 9.0), without any significant difference in the sensitivity
(EC50) to calcium ionophore (an endothelium dependent vasodilator).

32
It has been shown that oxidative stress caused by diabetes leads to the decreased
bioavailability of NO and the consequent impairment of endothelial directed
vasodilation. Excess superoxide reacts with NO to form peroxynitrite, which then
oxidizes tetrahydrobiopterin (BH4) thereby compromising its bioavailability. Low levels
of BH4 leads to the formation of uncoupled eNOS, which then produces superoxide
rather than NO, via the transfer of electrons to molecular oxygen. [9, 33, 34, 35]

However, in our study the levels of superoxide production were lower in the diabetics
compared to nondiabetics. Surprisingly, the endothelium dependent relaxation was
attenuated in the diabetic saphenous veins, with an insignificant variation in sensitivity
(EC50) to calcium ionophore between two groups. Therefore, there is a likelihood that,
in addition to endothelial dysfunction, there may be increased smooth muscle cell
defects in the diabetic vessels compared to the nondiabetic ones. However, due to
limited tissue segments from the subjects, we could not assess the saphenous veins’
smooth muscles using sodium nitroprusside, an endothelium independent vasodilator.

As initially observed from the weakly insignificant difference in oxLDL/LDL ratio,


there appears to be a trend suggesting increased oxidation of LDL in the diabetics
compared to nondiabetics. This could be a possible contribution to the observed
endothelial dysfunction in the diabetics compared to the nondiabetics as shown by the
relaxation of the vessels to calcium ionophore. Studies have implicated oxLDL in
increasing asymmetric dimethyl arginine (ADMA); an endogenous competitive
inhibitor of eNOS, thereby promoting endothelial dysfunction due to reduced NO
bioavailability. [80] Oxidized LDL has also been shown to cause depletion of caveolae
cholesterol and consequently eNOS redistribution in the endothelial cells, which then
results in reduced eNOS activity. [81, 82]

Another notable observation in the current study was the significantly higher HDL
plasma levels in the nondiabetic subjects compared to the diabetics. Uittenbogaard and
colleagues [83] elegantly demonstrated that HDL attenuated oxLDL-induced inhibition
of eNOS activation as well as localization in endothelial cell caveolae. It has also been
suggested that HDL activates eNOS via Src stimulation, which then leads to the
activation of Akt and MAP kinases and subsequently having modulating effects on the
eNOS. [84, 85]

33
Nofer et al, [86] reported that HDL played an important role in the regulation of the
vascular tone via lysophospholipid receptor SIP3 – mediated NO release. Collectively,
these findings show that HDL plays an important role in enhancing endothelial function.
Consistent with higher levels of HDL and better endothelial function in the nondiabetics
compared to the diabetics, the current study supports the previously reported findings by
Bisoendial and others, [87] that a positive relationship exists between HDL plasma
levels and endothelial dependent vasodilation.

Further limitations of this study must be recognized. Since all the patients had coronary
artery disease (CAD) and that the saphenous veins used in the study were considered
functional and consequently used as bypass grafts, it is difficult to ascertain the impact
of CAD on superoxide production in both groups of our study population. Moreover,
most of the patients were receiving pharmacotherapy for comorbid conditions such as
hypertension, pain etc and therefore complex drug interactions and effects of
comorbidity on superoxide production and endothelial dysfunction remain potential
confounding factors. In addition, whether the levels of superoxide production and the
degree of endothelial dysfunction as compared in the two groups studied, have any
clinically significant effect or not, remains to be established.

Owing to the small sample size as possibly demonstrated by the wider confidence
intervals in most of the variables analyzed, it was not rational to do regression analysis
and consequently our study could not provide evidence for causal-relationships of
various variables of interest. Nonetheless, the current study has convincingly,
demonstrated that diabetic patients taking ACEIs/ARBs, more fruits and vegetables, and
having lower plasma LDL levels have significantly reduced superoxide generation in
their saphenous veins. In addition, it has also been shown that mitochondrial mediated
superoxide is enhanced in nondiabetic vasculature. The endothelial function in diabetics
is compromised and that reduced HDL as well increased LDL oxidation may possibly
be a contributory factor.

34
3.3 Conclusion

The present study has shown that there is significantly decreased superoxide production
in the saphenous veins from diabetic patients taking Angiotensin Converting Enzyme
(ACE) inhibitors and/or Angiotensin II Receptor Blockers (ARBs) medication, higher
fruit and green leafy vegetable intake. Moreover, lower plasma LDL levels in the
diabetics were consistent with lower superoxide levels in their vasculature compared to
the nondiabetics. Although the mitochondrial mediated oxidative stress was more
enhanced in the nondiabetic vasculature, the role of mitochondrial respiratory chain in
superoxide production in the diabetics may also be important and consequently warrants
further investigations.

Despite lower superoxide levels in the diabetics, the endothelial dependent relaxation
was more attenuated in this group compared to the nondiabetics. In addition to
endothelial dysfunction, it is possible that the integrity of the smooth muscles in the
diabetic vasculature had been compromised due to disease. Therefore, we recommend
that further studies should focus on assessing endothelial-independent alongside
endothelial-dependent relaxations in both diabetic and nondiabetic vessels. In consistent
with previously reported findings, lower levels of plasma HDL and potentially enhanced
effects of oxLDL may also be an explanation to the observed attenuation of endothelial
function in the diabetic vasculature.

35
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46
Appendix 1.0: A Sample Questionnaire

Dear Study Participant,

Vascular function in Coronary Artery Bypass patients – VASCAB Study

We would like you to answer a few questions. Ideally you might do this at home before your
appointment visit. However, if you need assistance we will go through the list together at your
appointment visit.

Please read the questions, then look at the options and tick the most appropriate answer in the
answer box. If you are unsure of anything, put a mark beside it and discuss it with us at your
appointment visit. If there is a question you prefer not to answer, please simply put a mark beside it
so that we know.

For example:

No. Question

1 Sex Male 1
Female 2

2 Date of Birth 8 5 53
______/______/______
Day Month Year

4 How many children have you ever (insert number of children)


had? 2
______

12 Which of the following best describes Full-time employee 1


your main work status over the last 12 Part-time 2
months? I don't want to answer this
Retired / at home 3

Remember your name is not recorded on any of the pages of the main questionnaire to help
maintain your privacy.

VASCAB Study Team

BHF Glasgow Cardiovascular Research Centre, University of Glasgow 46


126 University Place, Glasgow G12 8TA, Scotland, UK
Telephone: +44 (141) 330-2738 Fax: +44 (141) 330-6997 Email: ad7e@clinmed.gla.ac.uk
Study Number _____________________M / F

Participant Main Questionnaire

Section 1 - Demographics and Family

The background of a person has a substantial effect on an individual’s risk of heart disease. In this
first section we would like to find out a bit about you, your living circumstances and your family.

No. Question

1 Sex Male 1
Female 2

2 Date of Birth ______/______/______


Day Month Year

3 Marital status Single (never married) 1


Married 2
Living with partner 3
Divorced or separated 4
Widowed 5

4 How many children have you ever (insert number of children)


had? ______

5 How many children are alive (insert number)


now? ______

6 Are you one of a twin? No 1


Yes, identical 2
Yes, non-identical 3

7 How many brothers do you have (all (insert number of brothers)


live births)? ______

8 How many brothers are alive


now? ______

9 How many sisters do you have? (all (insert number of sisters)


live births) ______

10 How many sisters are alive


now? ______

VASCAB Version MQ.2.0 – 22 February 2007 47


Study Number _____________________M / F

No. Question

11 What is the highest level of education Primary school completed 1


you have completed? Secondary school completed 2
Technical college completed 3
University completed 4
Post graduate degree 5

12 Which of the following best describes Full-time employee 1


your main work status over the last 12 Part-time 2
months?
Retired / at home / unemployed 3

13 Which of the following best describes European or Caucasian 1


your racial background? Other: Please specify: 2

___________________________

14 Would you say that in general your Excellent 1


quality of life is - Very Good 2
Good 3
Fair 4
Poor 5

15 Would you say that in general your Excellent 1


health is - Very Good 2
Good 3
Fair 4
Poor 5

VASCAB Version MQ.2.0 – 22 February 2007 48


Study Number _____________________M / F

The next questions are about your family.

No. Question

F1 Have any of your relatives had a heart Yes 1


attack? No (skip the next question and go to 2
question F3)

F2 If yes in question F1, who has had a Mother Yes 1 No 2 Age ___
heart attack and how old were they at Father Yes 3 No 4 Age ___
heart attack?
Sister Yes 5 No 6 Age ___
(Please enter "not known" if you are
not sure. Please indicate if you know Brother Yes 7 No 8 Age ___
approximate ages but not exact ages.) Son/Daughter Yes 9 No 10 Age ___
Other Yes 11 No 12 Age ___

F3 Have any of your relatives had a Yes 1


stroke? No (skip the next question and go to 2
question F5)

F4 If yes in question F3, who has had a Mother Yes 1 No 2 Age ___
stroke and how old were they at Father Yes 3 No 4 Age ___
stroke?
Sister Yes 5 No 6 Age ___
(Please enter "not known" if you are
not sure. Please indicate if you know Brother Yes 7 No 8 Age ___
approximate ages but not exact ages.) Son/Daughter Yes 9 No 10 Age ___
Other Yes 11 No 12 Age ___

F5 Is there any relative in your family Yes 1


who has or had high blood pressure? No (skip the next question and go to 2
question F7)

F6 If yes in question F5, who has or had Mother Yes 1 No 2 Age ___
high blood pressure and how old Father Yes 3 No 4 Age ___
were they when high blood pressure
was diagnosed? Sister Yes 5 No 6 Age ___
(Please enter "not known" if you are Brother Yes 7 No 8 Age ___
not sure. Please indicate if you know Son/Daughter Yes 9 No 10 Age ___
approximate ages but not exact ages.)
Other Yes 11 No 12 Age ___

VASCAB Version MQ.2.0 – 22 February 2007 49


Study Number _____________________M / F

No. Question

F7 Is there any relative in your family Yes 1


who has or had diabetes (high blood No (skip the next question and go to 2
sugar)? the next section)

F8 If yes in question F5, who has or had Mother Yes 1 No 2 Age ___
diabetes and how old were they when Father Yes 3 No 4 Age ___
diabetes was diagnosed?
Sister Yes 5 No 6 Age ___
(Please enter "not known" if you are
not sure. Please indicate if you know Brother Yes 7 No 8 Age ___
approximate ages but not exact ages.) Son/Daughter Yes 9 No 10 Age ___
Other Yes 11 No 12 Age ___

Section 2 - Life Style Factors

In this section, there are questions about your lifestyle. A person’s lifestyle can give us important
clues as to the cause of their heart disease.

The first questions are about how much alcohol you drink.

No. Question

A1 Have you ever consumed a drink that Yes 1


contains alcohol? No (skip this section and go to the 2
next section)

A2 Have you consumed alcohol in the Yes 1


past 12 months? No (skip this section and go to the 2
next section)

A3 In the past 12 months, how Daily 1


frequently have you had at least one 3 to 4 days per week 2
drink?
Weekly 3
Fortnightly 4
Monthly or on special occasions only 5

A4 When you drink alcohol, on average, Number of drinks per day:


how many drinks do you have during (A drink is equal to 1 small glass of
one day? wine, a half pint of beer, 1 shot of ______
spirits or liqueur.)
These questions are about smoking and use of tobacco.

VASCAB Version MQ.2.0 – 22 February 2007 50


Study Number _____________________M / F

No. Question

S1 Have you ever smoked any tobacco Yes, currently smoke 1


products? Yes, but stopped within past 12 2
months
Yes, but stopped more than 12 3
months ago
No (skip this question and go to the 4
next section)

S2 How old were you when you first (Give age in years)
started smoking daily? ______

S3 What is the maximum number you (insert number of cigarettes / cigars /


have smoked per day for as long as a hand made cigarettes per week / oz.
______
year of tobacco)

S4 PAST SMOKERS – only On doctor's advice 1


Why did you give up smoking? Other reason 2

S5 PAST SMOKERS – only Years ago 1


How long ago did you stop smoking Months ago 2
daily? Weeks ago 3

These questions are about your diet.

No. Question

D1 In a typical week, on how many days (Insert number of days)


do you eat fruit? ______

D2 Approximately how many pieces/ (Insert number of servings/ pieces)


servings of fruit do you eat on one of
those days? ______

D3 In a typical week, on how many days (Insert number of days)


do you eat green leafy vegetables?
(e.g. spinach, salad leaves)
______

D4 Approximately how many servings/ (Insert number of servings/ meals)


meals would you have green leafy
vegetables on one of those days?
______
These questions are about your regular exercise and physical activity.

VASCAB Version MQ.2.0 – 22 February 2007 51


Study Number _____________________M / F

No. Question

P1 On average, how much physical Lots (e.g. heavy lifting, digging, 1


activity do you do each day during going up & down stairs)
working hours? Medium (e.g. light lifting, walking, 2
(if retired or at home, this refers to light house-work, shopping,
during the day) painting)
Light activity (e.g. standing, 3
occasional working)
Almost none (e.g. desk job, sitting, 4
driving)

P2 On average, how much physical Lots (e.g. competitive sports, 1


activity do you do each day after aerobics, multiple times a week)
working hours? Medium (e.g. Casual sports, going to 2
(if retired, this refers to evenings and gym, regular walks 1-2 times per
weekends) week)
Light activity (e.g. occasional 3
working or bowls)
Almost none (e.g. Watching TV, 4
listening to music, cooking, driving)

Section 3 - Current Medical conditions and risk factors

This final section is about your medical conditions and treatments.

No. Question

M1 Have you ever been told by a doctor Yes 1


or other health worker that you have No, my blood pressure was always 2
high blood pressure or normal (skip the next question and
hypertension? go to question M3)
No, I have never had my blood 3
pressure taken (skip the next
question and go to question M3)

M2 If yes, about how long ago were you (insert number of years)
first told by a doctor that you had
high blood pressure? ______

VASCAB Version MQ.2.0 – 22 February 2007 52


Study Number _____________________M / F

No. Question

M3 Have you ever been told by a doctor Yes 1


or other health worker that you have No, my blood sugar was always 2
diabetes (high blood sugar)? normal (skip the next question and
go to question M5)
No, I have never had my blood sugar 3
taken (skip the next question and go
to question M5)

M4 If yes, about how long ago were you (insert number of years)
first told by a doctor that you had
diabetes (a high blood sugar)? ______

M5 Have you had a medical diagnosis of a heart attack/ Yes 1


myocardial infarction? No 2

M6 Have you had a medical diagnosis of a Stroke/ transient Yes 1


ischemic attack No 2

M7 Have you had a medical diagnosis of blood vessel Yes 1


disease in your legs/ peripheral vascular disease No 2

M8 Have you had a medical diagnosis of a weak heart/ Yes 1


heart failure No 2

M9 Have you had a medical diagnosis of kidney disease/ Yes 1


renal failure No 2

M10 Have you had a medical diagnosis of lung/chest Yes 1


problems? e.g. bronchitis/emphysema/COPD/Asthma No 2

M11 Do you have or have you ever been given a diagnosis of Yes 1
cancer? No 2
If yes what type:________________

M12 Do you have rheumatoid arthritis? (inflammation of Yes 1


joints) No 2

M13 Do you have osteoarthritis ?(wear and tear arthritis) Yes 1


No 2

VASCAB Version MQ.2.0 – 22 February 2007 53


Study Number _____________________M / F

No. Question

M14 Do you have any other long standing medical Yes 1


conditions that are not already listed? No 2

If yes what are these conditions?


(you may leave blank if you prefer not to answer)

The next 2 questions are for women only

No. Question

W1 Have you gone through the Yes 1


menopause? i.e. have your periods
No 2
stopped

W2 Have you ever taken the oral Yes currently 1


contraceptive pill (OCP) or hormone
Yes previously but now stopped 2
replacement therapy (HRT)?
(Number of years stopped ____)
No never 3

The next 3 questions are for patients with diabetes only.

No. Question

CD1 Have you ever been told you have damage to your eyes Yes 1
(retinopathy) from having diabetes? No 2

CD2 Do you have any foot problems due to diabetes Yes 1


(neuropathy)? e.g. ulcers, numbness, have missing /lost
No 2
toes due to diabetes

CD3 Have you ever been told that your kidneys have been Yes 1
damaged from having diabetes (nephropathy)?
No 2

VASCAB Version MQ.2.0 – 22 February 2007 54


Study Number _____________________M / F

Please write the name of your current medications as they are labelled from the medicine
box, or your script.

It may be easier for you just to bring a current medication list issued by your doctor or by
your chemist with you. If you have such a list please leave the following box blank.

Name of medication How long have you been taking


this medication?

T1
____________________________ (please insert years/months) ______

T2
____________________________ (please insert years/months) ______

T3
____________________________ (please insert years/months) ______

T4
____________________________ (please insert years/months) ______

T5
____________________________ (please insert years/months) ______

T6
____________________________ (please insert years/months) ______

T7
____________________________ (please insert years/months) ______

T8
____________________________ (please insert years/months) ______

T9
____________________________ (please insert years/months) ______

T10
____________________________ (please insert years/months) ______

T11
____________________________ (please insert years/months) ______

VASCAB Version MQ.2.0 – 22 February 2007 55