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Title: OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION IN DIABETIC SUBJECTS UNDERGOING CORONARY ARTERY BY-PASS GRAFT (CABG)

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

Dedication

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

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

Acknowledgement

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

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.

List of Figures and Tables

List of Figures and Tables List of Figures Figure 1.0 : Sources of oxidative stress and

List of Figures

Figure 1.0: Sources of oxidative stress and their potential links to endothelial dysfunction

in

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

27

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

29

saphenous veins

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

26

saphenous veins

Table 3.0: Mean EC 50 of calcium ionophore and maximal relaxation in diabetic and non-

28

diabetic saphenous veins

List of Abbreviations

List of Abbreviations ROS Reactive Oxygen Species AGE Advanced Glycation End-products GTPCH

ROS

Reactive Oxygen Species

AGE

Advanced Glycation End-products

GTPCH

GTP-cyclohydrolase-I

BH 4

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

N w -nitro-L-arginine- methyl ester

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

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

Abstract

Abstract Background : Diabetic patients have an increased risk of cardiovascular morbidity and mortality. An understanding

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).

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 (EC 50 ) 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.

CHAPTER ONE

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.

1.2 Introduction

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.

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]

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 (H 2 O 2) 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]

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

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

ROS, Reactive oxygen species; AGE, advanced glycation

end-products; GTPCH, GTP-cyclohydrolase-I; BH 4 , 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.

Modified from references [8, 15]

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

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 A 2 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 BH 4 , thereby reducing eNOS availability. [34] In the presence of reduced concentrations of BH 4 , 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, N G -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 BH 4 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]

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 Q 10

Coenzyme Q 10 (CoQ 10 ), an endogenous enzyme cofactor produced in most of the human cells is an important component of the mitochondrial respiratory chain. CoQ 10 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 CoQ 10 in patients with diabetes, suggesting a marked increase in oxidative stress.

Moreover, studies in experimental diabetic models have suggested that a significant decrease in CoQ 10 may be responsible for an enhanced vulnerability of diabetic heart

mitochondria to oxidative damage. [44] However, it is widely known that the conventional antioxidants (reduced CoQ 10 ) 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 AT 1 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.

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.

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.

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 EC 50 (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 (EC 50 ) of the saphenous veins to calcium ionophore in diabetic and nondiabetic subjects.

CHAPTER TWO

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 -4 U/ml)

Allopurinol (10mM)

NADH (100 µM)

Rotenone (1 mM)

Diphyleneiodinium (DPI) (10 -4 M)

L-NAME (N w -nitro-L-arginine methyl ester, 10 -4 M)

Krebs-HEPES buffer (composition in mM: NaCI, 130; KCI, 4.7; NaHCO 3 , 14.9; KH 2 PO4, 1.2; Glucose, 5.5; MgSO 4 .7H 2 O, 1.2; CaCI.2H 2 O, 1.6; CaNa 2 EDTA, 0.027; and 10 -5 mole 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.

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.

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.

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

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

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 (N w -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.

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; NaHCO 3 , 14.9; KH 2 PO4, 1.2; Glucose, 5.5; MgSO 4 .7H 2 O, 1.2; CaCI.2H 2 O, 1.6; CaNa 2 EDTA, 0.027; and 10 -5 mole of indomethacin dissolved in 1ml dimethyl sulfoxide, DMSO). The Krebs solution was constantly aerated with a gas mixture of 95% O 2 plus 5% CO 2 and maintained at 37 o C.

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 -5 M) 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.

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, EC 50 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 EC 50 . 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.

CHAPTER THREE

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 Medications, n (%)

1.041 + 0.001(19)

1.201 + 0.001(37)

0.048*

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 Diet , median value (n)

20 (91)

39 (95)

0.606 ¶

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

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

0.010 to 0.40 mmol/L; P = 0.04, figure 4.0) Figure 3.0: A box plot of LDL

Figure 4.0: HDL levels in diabetic and nondiabetics

Figure 4.0: HDL levels in diabetic and nondiabetics The Hb1ac levels were significantly higher in the

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).

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

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

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

117 to 383 pmol/min/mg, P= 0.017, figure 6.0). Figure 6.0: Superoxide production in diabetic and nondiabetic

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 (N w -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)

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

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 (EC 50 ) to calcium

ionophore between the two groups was not significant (table 3.0).

Table 3.0: Mean EC 50 of calcium ionophore and maximal relaxation in diabetic and non-diabetic saphenous veins

 

EC 50 (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 %)

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

preconstricted diabetic and nondiabetic saphenous veins . Figure 9.0: Maximal relaxation of diabetic and nondiabetic

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

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

3.2 Discussion

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

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.

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 (EC 50 ) to calcium ionophore (an endothelium dependent vasodilator).

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 (BH 4 ) thereby compromising its bioavailability. Low levels of BH 4 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]

Nofer et al, [86] reported that HDL played an important role in the regulation of the vascular tone via lysophospholipid receptor SIP 3 – 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.

3.3 Conclusion

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.

References

References

1. Donnelly, R., Emslie-Smith A.M., Gardner, I.D., and Morris, A.D. ABC of arterial and venous disease: Vascular complications of diabetes. BMJ 2000; 320;

1062-1066.

2. The International Diabetes Federation. Diabetes Atlas. Executive Summary. Second Edition, pp 1-58. http//www.eatlas.ids.org. Accessed on 19 th August,

2007.

3. Steyn K, Sliwa K, Hawken S, Commerford P, Onen C, Damasceno A, Ounpuu S, Yusuf S. Risk factors associated with myocardial infarction in Africa: the INTERHEART Africa Study. Circulation. 2005;112: 3554–3561.

4. Kengne, A.P., Albert, G., Amoah, B., and Mbanya, J.C. Cardiovascular complications of diabetes mellitus in Sub-Saharan Africa. Circulation 2005; 112: 3592-3601.

5. Rosen, P., Nawroth, P.P., King, G., Moller, W., Tritschler, H.J., Packer. The role of oxidative stress in the onset and progression of diabetes and its complications. Diabetes Metab. Res. Rev. 2001; 17: 189-212.

6. Martin-Gallan, P., Carrascosa, A., Gussinye, M., Dominguez, C. Biomarkers of diabetes associated oxidative stress and anti-oxidant status in young diabetic patients with or without sub-clinical complications. Free Radic. Biol. Med. 2003; 34:1563-1574.

7. Evans, J.L., Goldfine, I.D., Maddux, B.A. Grodsky, G.M. Oxidative stress and stress-activated signalling pathways: a unifying hypothesis of type 2 diabetes. Endocr. Rev. 2002; 23:599-622.

8. Vriese, A.S.D, Verbeuren, T.J., Voorde, J.V.D., Lameire, N.H. Vanhoutte, P.M. Endothelial dysfunction in diabetes. British Journal of Pharmacology 2000; 130: 963-974.

9.

Guzik, T.J., Mussa, S., Gastaldi, D., et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: the role of NADPH oxidase and endothelial nitric oxide synthase. Circulation 2002; 105: 1656-1662.

10. Skyrme-Jones, R.A., O’Brien, R.C., Berry, K.L., Meredith, I.T. Vitamin E supplementation improves endothelial function in type I diabetes mellitus: a randomized, placebo-controlled study. J. Am. Coll. Cardiol. 2000; 36: 94-102.

11. Pieper, G.M., Siebeneich, W. Oral administration of the antioxidant, N- acetylcysteine abrogates diabetes-induced endothelial dysfunction. J. Cardiovasc. Pharmacol. 1998; 32: 101-105.

12. Seghrouchni, I., Drai, J., Bannier, E. et al. Oxidative stress parameters in type I, type II and insulin-treated type 2 diabetes mellitus: insulin treatment efficiency. Clin. Chim. Acta. 2002; 321:89-96.

13. Ramana, K. V., Chandra, D., Srivastava, S., Bhatnagar, A., Srivastava, S.K. Nitric oxide regulates the polyol pathway of glucose metabolism in vascular smooth muscles cells. FASEB J. 2003; 17: 417-425.

14. Bonnefont-Rousselot, D. Glucose and reactive oxygen species. Curr. Opin. Clin. Ntri. Metab. Care. 2002; 5: 561-568.

15. Jay, D., Hitomi, H., Griendling, K.K. Oxidative stress and diabetic cardiovascular complications. Free Radic. Biol. Med. 2006; 40:183-192.

16. Wautier, M., Chappey, O., Corda, S. et al. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE. Physiol. Endocrinol. Metab. 2001; 280: E685-E694.

17. Ceriello, A., Motz, E., Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes and cardiovascular disease? The common soil hypothesis revisited. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 816-823.

18.

Nikishawa, T., Edelstein, D., Du, X., et al. Normalizing oxidative superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404:

787-790.

19. Peelman, F., Waelput, W., Iserentant, H. Leptin: Linking adipocyte metabolism with cardiovascular and autoimmune diseases. Prog. Lipid Res. 2004; 43: 283-

301.

20. Wauters, M. Considine, R.V., Yudkin, J.S., Peiffer, F., De Leeuw, I., Van Gaal, L.F. Leptin levels in type 2 diabetes: associations with measures of insulin resistance and insulin secretion. Horm. Metab. Res. 2003; 35: 92-96.

21. Yamagishi, S.I., Edelstein, D., Du., X., Kaneda, Y., Guzman, M., Brownlee, M. Leptin induces mitochondrial superoxide production and monocyte

chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A. J. Biol. Chem. 2001; 276: 25096-

25100.

22. Wiernsperger, N.F. Oxidative stress as a therapeutic target in diabetes: revisiting the controversy. Diabetes Metab. 2003; 29: 579-585.

23. Steinberg, H., Baron, A. Vascular function, insulin resistance and fatty acids. Diabetologia 2002; 45: 623-634.

24. Lopes HF., Morrow, J.D., Stojiljkovic, M.P, Goodfriend, T.L., Egan, B.M. Acute hyperlipidaemia increases oxidative stress more in African Americans than in White Americans. Am. J. Hypertens. 2003; 16(5 pt 1): 331-336.

25. O’Donnel, R.W., Johnson, D.K., Ziegler, L.M., DiMattina, A.J., Stone, R., Holland, J.A. Endothelial NADPH oxidase: mechanism of activation by low- density lipoprotein. J. Endothelial. Cell. Res. 2003; 10: 291-297.

26. Jarvisalo, M.J., Raitakari, M., Toikka, J.O. et al. Endothelial dysfunction and increased arterial intima-media thickness in children with type I diabetes. Circulation 2004; 109: 1750-1755.

27. Rizzoni, D., Porteri, E., Guelfi, D. et al. Endothelial dysfunction in small resistance arteries of patients with non-insulin-dependent diabetes mellitus. J. Hyperts. 2001; 19: 913-919.

28. Inoguchi, T., Li, P.,Umeda, F., et al. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C- dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 2000; 49: 1939-1945.

29. Koya, D., King, G.L. Protein kinase C activation and the development of diabetic complications. Diabetes 1998; 47: 859-866.

30. Kunisaki, M., Bursell, S.E., Clermont, A.C. et al. Vitamin E prevents diabetes- induced abnormal retinal blood flow via the diacyglycerol-protein kinase C pathway. Am. J. Physiol. 1995; 269: E239-E246.

31. Ishii, H., Koya, D., King, G.L. Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J. Mol. Med. 1998; 76: 21-31.

32. Hink, U., Li, H., Mollnau, H., et al. Mechanisms underlying endothelial dysfunction in Diabetes Mellitus. Circ. Res. 2001; 88: E14-E22.

33. Milstien, S., Katusic, Z., Oxidation of tetrahydrobiopterin by peroxynitrite:

implications for vascular endothelial dysfunction. Biochem. Biophys. Res. Commun. 1999; 263: 681-684.

34. Kossenjans, W., Eis, A., Sahay., R., Brockman, D., Myatt, L. Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia. Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H1311-H1319.

35. Xia, Y., Tsai, A., Berka, V., Zweier, J. Superoxide generation from endothelial nitric–oxide synthase: a Ca2+/calmodulin-dependent and Tetrahydrobiopterin regulatory process. J. Biol. Chem. 1998; 273: 25804-25808.

36. Heitzer, T., Krohn, K., Albers, S., Meinertz, T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia 2000; 43: 1453-1438.

37. Du, X.L., Edelstein, D., Dimmer, S., Ju, Q., Sui, C., Brownlee, M.

Hyperglycaemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J. Clin. Invest. 2001; 108: 1341-

1348.

38. Potashnik, R., Bloch-Damti, A., Bashan, N., Rudich, A. IRSI degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress. Diabetologia. 2003; 46: 639-448.

39. Butler, R., Morris, A.D., Belch, J.J.F., Hill, A., Struthers, A.D. Allopurinol normalizes endothelial dysfunction in type 2 diabetes with mild hypertension. Hypertension 2000; 35: 746-751.

40. Al-benna, S., Hamilton, C.A., McClure, J.D., et al. Low-Density lipoprotein cholesterol determines oxidative stress and endothelial dysfunction in saphenous veins from patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 218-223.

41. Inkster, M.E., Cotter, M.A., Cameron, N.E. Treatment with the xanthine oxidase inhibitor, allopurinol, improves nerve and vascular function in diabetes rats. European Journal of Pharmacology 2007; 561:63-71.

42. Schrauwen,

P.,

Hasselink

MKC.

Oxidative

capacity,

lipotoxicity

and

mitochondrial damage in type 2 diabetes. Diabetes 2004; 53: 1412-1417.

43. Lim, S.C., Tan, H.H., Goh, S.K., Subramaniam, T., et al. Oxidative burden in prediabetic and diabetic individuals: evidence from plasma coenzyme Q10. Diabet. Med. 2006; 23:1344-1349.

44.

Santos, D.L., Palmeira, C.M., Seica, R., et al. Diabetes and mitochondrial oxidative stress: A study using heart mitochondria from the diabetic Goto- Kakizaki rat. Mol Cell Biochem. 2003; 246: 163-170.

45. Vanhoutte, P.M., Boulanger, C.M., Momboulli, J.V. Endothelial derived relaxing factors and angiotensin converting enzyme inhibition. Am. J. Cardiol. 1995; 76: E3-E12.

46. O’Driscol, G., Green, D., Maiorana, A., Stanton, K., Colreavy, F., Taylor, R. Improvement in endothelial function by angiotensin converting enzyme inhibition in non-insulin dependent diabetes mellitus. J. Am. Coll. Cardiol. 1999; 33:1506-1511.

47. Dorenkamp, M., Riad,A., Stiehl, S., et al. Protection against oxidative stress in diabetic rats: Role of angiotensin AT1 receptor and beta 1-adrenoceptor antagonism. European Journal of Pharmacology 2005; 520: 179-187.

48. Collins, R., Armitage, J., Parish, S., Sleigh, P., Peto, R. Heart protection study of cholesterol-lowering with simvastatin in 5963 people with diabetes: a randomized placebo-controlled trial. Lancet 2003; 361: 2005-2016.

49. Bucciarrelli, L., Wendt, T., Qu, W., et al. RAGE blockade stabilizes established atherosclerosis in diabetic apolipoprotein E-null mice. Circulation 2002; 106:

2827-2835.

50. Kouroedov, A., Eto, M., Joch, H. et al. Selective inhibition of protein kinase Cβ2 prevents acute effects of high glucose on vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 2004; 110:91-96.

51. Afshari, M., Larijani, B., Rezaie, A. et al. Ineffectiveness of allopurinol in reduction of oxidative stress in diabetic patients; a randomized, double blind- controlled clinical trial. Biomedicine and Pharmacotherapy 2004; 58: 546-550.

52.

Ohashi, K., Ishibashi S., Yakazi, Y., Yamada, N. Improved glycaemic control in a diabetic patient after discontinuation of allopurinol. Diabetes Care 1998; 21:

192-193.

53. Sommers, L.M., Schoene R.B. Allopurinol hypersensitivity syndrome associated with pancreatic exocrine abnormalities and new-onset diabetes mellitus. Arch. Intern. Med. 2002; 162: 1190-1192.

54. Victor, V.M and Rocha, M. Targeting antioxidants to mitochondria: a potential new therapeutic strategy for cardiovascular diseases. Curr. Pharm. Des. 2007; 13: 845-63.

55. Yusuf, D., Sleight, P., Pogue, J. Effects of an angiotensin-converting-enzyme inhibitor, Ramipril, on cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N. Engl. J. Med. 2000; 342: 145-153.

56. Hansson, L., Lindholm, L.H., Niskanen, L., et al. Effect of angiotensin- converting-enzyme inhibition campared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPP) randomised trial. Lancet 1999; 353:611-616.

57. Stephens, N.G., Parsons, A., Schofield, P. et al. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Anti-oxidant Study (CHAOS). Lancet 1996; 347:781-786.

58. Salonen, R.M., Nyyssonen, K., Kaikoomen, J. et al. Six year effect of combined vitamin C and E supplementation on atherosclerotic progression : the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) Study. Circulation 2003; 107: 947-953.

59. Yusuf, S., Dagenais, G., Pogue, J. et al. Vitamin supplementation and cardiovascular events in high risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N. Engl. J. Med. 2000; 342: 154-160.

60.

Heart Protection Study Collaborative Group. MRC/BHF Heart Protection study of anti-oxidant vitamin supplementation in 20536 high-risk individuals: a randomised placebo-controlled trial. Lancet 2002; 360:7-22.

61. Jialal, I., and Devaraj, S. Anti-oxidants and atherosclerosis: don’t throw out the baby with the bath water. Circulation 2003; 107: 926-928.

62. John, J.H., Ziebland, S., Yudkin, P., Roe, L.S. and Neil, H.A.W. for the Oxford Fruit and Vegetable Study Group. Effects of fruit and vegetable consumption on plasma anti-oxidant concentrations and blood pressure: a randomised controlled trial. Lancet 2002; 359:1969-1974.

63. Faulkner, K., and Fridovich, I. Luminol and lucigenin as detectors for superoxide Free Radical Biol. Med. 1993; 15: 447–451.

64. Chung, H.Y, Baek, B.S, Song, S.H., et al. Xanthine dehydrogenase/xanthine oxidase and oxidative stress. AGE 1997; 20(3): 127-140.

65. Cholesterol Treatment Trialists’ (CTT) Collaborators. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90056 participants in 14 randomized trials of statins. The Lancet 2005; 366:1267-1278

66. Arzamastseva, N.E, Lankin, V.Z., Konovalova, G.G., Tikhaze, A.K., Ageev, F.T., Lapina, Y. V., Narusov, O.Y., Mareev, V., Belenkov, Y. N. Oxidative Stress in Patients with Chronic Heart Failure and Type 2 Diabetes Mellitus. Bulletin of Experimental Biology and Medicine 2007; 143 (2): 207-209.

67. Aliciguzel, Y., Ozen, I., Aslan, M. and Karayalcin, U. Activities of xanthine oxidoreductase and antioxidant enzymes in different tissues of diabetic rats. J. Lab. Clin. Med. 2003; 142:172-177.

68. Guzik, T.J., West, N.E., Black, E., McDonald, D., Ratnatunga, C., Pillai, R. and Channon, K.M. Vascular superoxide production by NAD (P) H oxidase:

association with endothelial dysfunction and clinical risk factors. Circ. Res. 2000; 86:E85-90.

69. Lankin, V.Z., Lisina, M.O., Arzamastseva, N.E., Konovalova, G.G., Nedosugova, L,V., Kaminnyi, A.I., Tikhaze, A.K., Ageev, F.T., Kukharchuk,V.V., and Belenkov, Y. N. Oxidative Stress in Atherosclerosis and Diabetes. Bulletin of Experimental Biology and Medicine 2005; 140(1): 41-43.

70. Suzumura, K., Yasuhara M, Tanaka K, et al. Protective effect of fluvastatin sodium (XU-62–320), a 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on oxidative modification of human low-density lipoprotein in vitro. Biochem Pharmacol. 1999; 57: 697–703.

71. Aviram, M., Hussein, O., Rosenblat, M., et al. Interactions of platelets, macrophages, and lipoproteins in hypercholesterolemia: antiatherogenic effects of HMG-CoA reductase inhibitor therapy. J Cardiovasc Pharmacol. 1998; 31:

39–45.

72. Aviram, M., Rosenblat, M., Bisgaier, C.L., et al. Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation. Atherosclerosis. 1998; 138: 271–280.

73. Vecchione, C., Gentile, M., Aretini, A., Marino, G., Poulet, R., Maffei, A., Passarelli, F., Landolfi, A., Vasta, A., Lembo, G. A novel mechanism of action for statins against diabetes-induced oxidative stress. Diabetologia 2007; 50(4):

874-880.

74. Epstein M. Diabetes and hypertension: the bad companions. J Hypertens 1997;

15(Suppl):S55–S62.

75. Berry, C., Hamilton, C.A., Brosnan, M.J., Magill, F.G., Berg, G.A., McMurray, J.J.V., and Dominczak A.F. Investigation into the sources of superoxide in human blood vessels: Angiotensin II increases superoxide production in human internal mammary arteries. Circulation 2000; 101:2206-2212.

76. Kaur, C., Kapoor H.C., Antioxidants in fruits and vegetables: The millenium’s health. International Journal of Food Science and Technology 2001; 36: 703-

725.

77. Hamilton, C.A., Miller, W.H., Al-Benna, S, Brosnan, M.J., Drummond, R.D.,

, cardiovascular disease. Clinical Science 2004; 106: 219-234.

Mcbride, M.W., Dominiczak, A.F

Strategies to reduce oxidative stress in

78. Bazzano, L.A., Serdula, M.K., Liu, S. Dietary intakes of fruits and vegetables and risk of cardiovascular disease. Curr. Atheroscl. Rep. 2003; 5: 492–499.

79. Panagiotakos, D.B., Pitsavos, C., Kokkinos, P., Chrysohoou, C., Vavuranakis, M., Stefanadis, C., Toutouzas, P. Consumption of fruits and vegetables in relation to the risk of developing acute coronary syndromes; the CARDIO2000 case-control study. Nutr. J. 2003; 2:2.

80. Ito, A., Tsao, P.S., Adimoolam, S., Kimoto, M., Ogawa T. and Cooke, J.P. Novel Mechanism for Endothelial Dysfunction: Dysregulation of Dimethyl- arginine Dimethylaminohydrolase. Circulation 1999; 99; 3092-3095.

81. Shaul P.W. Regulation of endothelial nitric oxide synthase: location, location, location. Annu. Rev. Physiol. 2002: 64: 749-774.

82. Blair A., Shaul, P.W., Yuhanna, I.S., Conrad, P.A., Smart, E.J. Oxidized LDL displaces eNOS from plasmalemmal caveolae and impairs eNOS activation. J. Biol. Chem. 1999; 274: 32512– 32519.

83. Uittenbogaard, A., Shaul, P.W., Yuhanna, I.S., Blair, A., Smart, E.J. HDL prevents oxidized LDL-induced inhibition of eNOS localization and activation in caveolae. J. Biol. Chem. 2000; 275: 11278–11283.

84. Mineo, C., Yuhanna, I.S., Quon, M.J., Shaul, P.W. HDL-induced eNOS activation is mediated by Akt and MAP kinases. J. Biol. Chem. 2003; 278: 9142 – 9149.

85. Mineo, C., Shaul., P.W. HDL Stimulation of Endothelial Nitric Oxide Synthase A Novel Mechanism of HDL Action. Trends. Cardiovasc. Med. 2003; 13: 226–

231.

86. Nofer, J.R., Van Der Giet, M., Tölle, M., Wolinska, I., Lipinski, K.V.W., Baba, H.A., et al. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J. Clin. Invest. 2003; 113: 569–581.

87. Bisoendial, R.J., Hovingh, G.K., Levels, J.H., Lerch, P.G., Andresen, I., Hayden, M.R, et al. Restoration of endothelial function by increasing HDL in subjects with isolated low HDL. Circulation 2003; 107: 2944– 2948.

Appendix 1.0: A Sample Questionnaire

Dear Study Participant,

Appendix 1.0: A Sample Questionnaire Dear Study Participant, Vascular function in Coronary Artery Bypass patients –

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 8 / 53 2 Date
For example:
No.
Question
1 Sex
Male
1
Female
2
8
/ 53
2
Date of Birth
/ 5
Day
Month
Year
4
How many children have you ever
had?
(insert number of children)
2
12
Which of the following best describes
your main work status over the last 12
months?
Full-time employee
1
Part-time
2
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 126 University Place, Glasgow G12 8TA, Scotland, UK Telephone: +44 (141) 330-2738 Fax: +44 (141) 330-6997 Email: ad7e@clinmed.gla.ac.uk

46

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 had?

(insert number of children)

 

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 live births)?

(insert number of brothers)

 

8

How

many

brothers

are

alive

 

now?

9

How many sisters do you have? (all live births)

(insert number of sisters)

 

10

How

many

sisters

are

alive

 

now?

Study Number

M / F

No.

Question

 

11

What is the highest level of education you have completed?

Primary school completed

1

Secondary school completed

2

 

Technical college completed

3

University completed

4

Post graduate degree

5

12

Which of the following best describes your main work status over the last 12 months?

Full-time employee

1

Part-time

2

Retired / at home / unemployed

3

13

 

European or Caucasian

1

Which of the following best describes your racial background?

Other: Please

specify:

2

14

Would you say that in general your quality of life is -

Excellent

1

Very Good

2

 

Good

3

Fair

4

Poor

5

15

Would you say that in general your health is -

Excellent

1

Very Good

2

 

Good

3

Fair

4

Poor

5

Study Number

 

M

/ F

The next questions are about your family.

 

No.

Question

   

F1

Have any of your relatives had a heart attack?

   

Yes

1

No (skip the next question and go to question F3)

2

F2

If yes in question F1, who has had a heart attack and how old were they at heart attack?

   

Mother

Yes 1

No 2

Age

Father

Yes 3

No 4

Age

(Please enter "not known" if you are not sure. Please indicate if you know approximate ages but not exact ages.)

Sister

Yes 5

No 6

Age

Brother

Yes 7

No 8

Age

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 question F5)

2

F4

If yes in question F3, who has had a stroke and how old were they at stroke?

   

Mother

Yes 1

No 2

Age

Father

Yes 3

No 4

Age

(Please enter "not known" if you are not sure. Please indicate if you know approximate ages but not exact ages.)

Sister

Yes 5

No 6

Age

Brother

Yes 7

No 8

Age

Son/Daughter

Yes 9

No 10

Age

 

Other

Yes 11 No 12

Age

F5

Is there any relative in your family who has or had high blood pressure?

   

Yes

1

No (skip the next question and go to question F7)

2

F6

If yes in question F5, who has or had high blood pressure and how old were they when high blood pressure was diagnosed?

   

Mother

Yes 1

No 2

Age

Father

Yes 3

No 4

Age

Sister

Yes 5

No 6

Age

(Please enter "not known" if you are not sure. Please indicate if you know approximate ages but not exact ages.)

Brother

Yes 7

No 8

Age

Son/Daughter

Yes 9

No 10

Age

Other

Yes 11 No 12

Age

Study Number

M

/ F

No.

Question

 

F7

Is there any relative in your family who has or had diabetes (high blood sugar)?

   

Yes

1

No (skip the next question and go to the next section)

2

F8

If yes in question F5, who has or had diabetes and how old were they when diabetes was diagnosed?

   

Mother

Yes 1

No 2

Age

Father

Yes 3

No 4

Age

(Please enter "not known" if you are not sure. Please indicate if you know approximate ages but not exact ages.)

Sister

Yes 5

No 6

Age

Brother

Yes 7

No 8

Age

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 contains alcohol?

Yes

1

No (skip this section and go to the next section)

2

A2

Have you consumed alcohol in the past 12 months?

Yes

1

No (skip this section and go to the next section)

2

A3

In

the

past

12

months, how

Daily

1

frequently have you had at least one drink?

3 to 4 days per week

2

Weekly

3

Fortnightly

4

Monthly or on special occasions only

5

A4

When you drink alcohol, on average, how many drinks do you have during one day?

Number of drinks per day:

(A drink is equal to 1 small glass of wine, a half pint of beer, 1 shot of spirits or liqueur.)

These questions are about smoking and use of tobacco.

Study Number

M / F

No.

Question

   

S1

Have you ever smoked any tobacco products?

Yes, currently smoke

 

1

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 next section)

4

S2

How old were you when you first started smoking daily?

(Give age in years)

 

S3

What is the maximum number you have smoked per day for as long as a year

(insert number of cigarettes / cigars / hand made cigarettes per week / oz. 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 daily?

Months ago

 

2

Weeks ago

3

These questions are about your diet.

No.

Question

 

D1

In a typical week, on how many days do you eat fruit?

(Insert number of days)

D2

Approximately how many pieces/ servings of fruit do you eat on one of those days?

(Insert number of servings/ pieces)

D3

In a typical week, on how many days do you eat green leafy vegetables? (e.g. spinach, salad leaves)

(Insert number of days)

D4

Approximately how many servings/ meals would you have green leafy vegetables on one of those days?

(Insert number of servings/ meals)

These questions are about your regular exercise and physical activity.

Study Number

M / F

No.

Question

 

P1

On average, how much physical activity do you do each day during working hours?

Lots (e.g. heavy lifting, digging, going up & down stairs)

1

Medium (e.g. light lifting, walking, light house-work, shopping, painting)

2

(if retired or at home, this refers to during the day)

Light

activity

(e.g.

standing,

3

occasional working)

 

Almost none (e.g. desk job, sitting, driving)

4

P2

On average, how much physical

Lots (e.g. competitive sports, aerobics, multiple times a week)

1

each day after

activity do you do working hours?

Medium (e.g. Casual sports, going to gym, regular walks 1-2 times per week)

2

(if retired, this refers to evenings and weekends)

Light activity (e.g. occasional working or bowls)

3

Almost none (e.g. Watching TV, listening to music, cooking, driving)

4

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 or other health worker that you have

high blood pressure hypertension?

or

Yes

1

No, my blood pressure was always normal (skip the next question and go to question M3)

2

 

No, I have never had my blood pressure taken (skip the next question and go to question M3)

3

M2

If yes, about how long ago were you first told by a doctor that you had high blood pressure?

(insert number of years)

 

Study Number

M

/ F

No.

Question

   

M3

Have you ever been told by a doctor or other health worker that you have diabetes (high blood sugar)?

   

Yes

1

No, my blood sugar was always normal (skip the next question and go to question M5)

2

 

No, I have never had my blood sugar taken (skip the next question and go to question M5)

3

M4

If yes, about how long ago were you first told by a doctor that you had diabetes (a high blood sugar)?

   

(insert number of years)

 

M5

Have you had a medical diagnosis of a heart attack/ myocardial infarction?

Yes

1

No

2

M6

Have you had a medical diagnosis of a Stroke/ transient ischemic attack

Yes

1

No

2

M7

Have you had a medical diagnosis of blood vessel disease in your legs/ peripheral vascular disease

Yes

1

No

2

M8

Have you had a medical diagnosis heart failure

of a weak heart/

Yes

1

No

2

M9

Have you had a medical diagnosis of kidney disease/ renal failure

Yes

1

No

2

M10

Have you had a medical diagnosis of lung/chest problems? e.g. bronchitis/emphysema/COPD/Asthma

Yes

1

No

2

M11

Do you have or have you ever been given a diagnosis of cancer?

Yes

1

No

2

If yes what type:

 

M12

Do you have rheumatoid arthritis? (inflammation of joints)

Yes

1

No

2

M13

Do you have osteoarthritis ?(wear and tear arthritis)

Yes

1

No

2

Study Number

M / F

No.

Question

 

M14

Do you have any other long standing medical conditions that are not already listed?

 

Yes

1

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 contraceptive pill (OCP) or hormone replacement therapy (HRT)?

Yes currently

1

Yes previously but now stopped

2

 

(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 (retinopathy) from having diabetes?

 

Yes

1

No

2

CD2

Do you have any foot problems due to diabetes (neuropathy)? e.g. ulcers, numbness, have missing /lost toes due to diabetes

 

Yes

1

No

2

CD3

Have you ever been told that your kidneys have been damaged from having diabetes (nephropathy)?

 

Yes

1

No

2

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)