UNIVERSITI PUTRA MALAYSIA

OXIDATIVE DNA DAMAGE IN LEUKOCYTES AND ITS ASSOCIATION WITH METABOLIC CONTROL IN DIABETIC

PATIENTS WITH AND WITHOUT MICROALBUMINURIA

SIM SZE KIAT

FPSK (M) 2001 8

OXIDATIVE DNA DAMAGE IN LEUKOCYTES AND ITS ASSOCIATION WITH METABOLIC CONTROL IN DIABETIC PATIENTS WITH AND

WITHOUT MICROALBUMINURIA

By

SIM SZE KIAT

Thesis Submitted i n Fulfi lment of the Requirement for the Degree of Master of Science in the Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

June 2001

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science.

OXIDATIVE DNA DAMAGE IN LEUKOCYTES AND ITS ASSOCIATION WITH M ETABOLIC CONTROL IN DIABETIC PATIENTS WITH AND

WITHOUT MICROALBUM INURIA

By

SIM SZE KIAT

June 2001

Chairman: Associate Professor Zarida Hambali, MO, DCP, Ph.D.

Faculty: Medical and Health Sciences

Type 2 diabetes mellitus is the predominant type of diabetes mel li tus

and the type l ikely to go undiagnosed. Oxidative damage was suggested in

the development of diabetic microangiopathic and macroangiopathic

complications in these patients. Since the long-term compl ications are the

main cause of morbidity and mortality in diabetic patients, a quantitative

determination of the level of oxidative stress is a valuable indicator of the

degree of the severity of the disease and of the effectiveness of treatment.

The main aim of this study is to evaluate the extent of oxidative DNA damage

with different severity of microalbuminuria in diabetic patients and its

associations with other metabolic controls. The comet assay was adopted to

measure the level of oxidative DNA damage in leukocytes. One hundred and

twenty seven (127) Type 2 diabetic patients with- and without

microalbuminuria were recruited from the Outpatients Department and

ii

Medical Outpatients Department, Kajang Hospital. Selection was based on

the appearance of urine albumin in the spot urine test by using urine test strip

(Micral Test II, Roche). Patients were grouped into four groups, based on the

findings in the urine test: N1 (Omg/dl) and N2 (20mg/dl) as

Normoalbuminuria, M1 (50mg/dl) and M2 ( 1 00mg/dl) as Microalbuminuria.

Fasting blood samples were collected for the determination of fasting blood

glucose (FBG), glycated haemoglobin (HbA1 c), lipoprotein(a) , total

cholesterol, triglycerides, low density lipoprotein-cholesterol (LDL-Chol) and

high density lipoprotein-cholesterol (HDL-Chol), and were done on an

automated chemistry analyzer (Cobas Miras, Roche). Low DNA damage

(type 0 & 1 ) was seen in normal subjects and patients with Omg/dl and

20mg/dl urine microalbumin test. High and complete DNA damage (type 2, 3

& 4) were detected at microalbuminuria marked 50 mg/dl and more. The

Microalbuminuria group had shown a significant elevation of DNA damage

compared to Normoalbuminuria group ( 1 8 .54 ± 3. 1 7% Vs 1 2.00 ± 1 .37%,

P<0.01 ) . There was a significant difference in the level of DNA damage

between the M2 and the M1 (20.32 ± 2.82% Vs 1 6. 1 9 ± 1 .78%, P<0.01 ), and

between M1 and N2 ( 1 6.1 9 ± 1 .78% Vs 1 1 .88 ± 1 .46%, P<0.01 ) . However,

there was no difference in level of DNA damage between the N2 and N 1

group. As expected, there were a significant correlation (p<0.01 ) between

FBG and HbA 1 c in both Normo- and Microalbuminuria groups. But no

significant difference was detected in the mean values of FBG and HbA 1 c

between all diabetic groups. The level of oxidative DNA damage was

increased significantly after the concentration of 20 mg/dl microalbuminuria.

I nterestingly, the FBG and HbA 1 c results did not follow with the severity of

iii

microalbuminuria. A significant correlation (P<0.05) was detected between

the level of DNA damage with FBG and HDL-Chol in both Normo- and

Microalbuminuria groups. Concentration of HDL-Chol in Microalbuminuria

group (1.06 ± 0.49 mmolll) was lower compared to Normoalbuminuria group

(1.16 ± 0.44 mmolll). Total cholesterol , triglyceride, LDL-Chol and

concentration of Lp(a) did not show any association with the level of oxidative

DNA damage. In M2 group, BMI and HbA 1 c were significantly associated

(p<0.01 and p<0.05 respectively) with the level of DNA damage. In this

study, comet assay had shown that cellular DNA damage occurred in the

diabetic patients. This study also suggests that oxidative stress occurred in

the early stage of complication and even before overt microalbuminuria was

detected. Furthermore, FBG and HbA 1 c did not show any relation with the

severity of microalbuminuria indicating that conventional markers do not

portray the true condition of the oxidative stress in diabetic patients.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains.

KEROSAKAN DNA SECARA OKSIDATIF PADA LEUKOSIT DAN PERKAITANNYA DENGAN KAWALAN M ETABOLIK 01

KALANGAN PESAKIT KENCING MAN IS YANG M EMPUNYAI MIKROALBUMINURIA AT AU SEBALlKNYA

Oleh

SIM SZE KIAT

Jun 2001

Pengerusi: Profesor Madya Zarida Hambali, Mo, OCP, Ph.D.

Fakulti: Perubatan dan Sains Kesihatan

Type 2 diabetes mellitus merupakan jenis penyakit kencing manis

yang utama dan selalunya tidak dikesan awa!. Kerosakan oksidatif tetah

disyaki dalam pembentukan komplikasi mikroangiopati dan makroangiopati di

kalangan pesakit kencing manis ini . Memandangkan komplikasi ini dalam

jangka panjang merupakan punca utama kepada kecederaan dan kematian

pad a pesakit kencing manis, maka satu penentuan kuantitatif untuk tahap

stres oksidatif in i adatah penting sebagai satu petunjuk kepada tahap

keterukan penyakit serta keberkesanan sesuatu rawatan. Tujuan utama

kajian in i adalah untuk meni lai kerosakan oksidatif pada DNA dengan

kepekatan albuminuria yang berbeza pada pesakit kencing manis serta

perkaitannya dengan kawalan metabolik yang lain . Assai Comet telah

digunakan untuk menentukan kerosakan oksidatif DNA pada se! darah putih .

Seramai seratus dua puluh tujuh (127) pesakit kencing manis Type 2 dengan

v

mikroalbuminuria atau sebal iknya telah diambi l dari Jabatan Pesakit Luar,

Hospital Kajang. Pemil ihan adalah berpandukan kepada kewujudan

albuminuria dalam ujian kencing rawak dengan menggunakan Micral Test /I

(Roche). Pesakit telah dibahagikan kepada empat kumpulan berasaskan

keputusan ujian kencing: N 1 (Omg/dl) dan N2 (20mg/dl) sebagai

Normoalbuminuria, M 1 (50mg/dl) dan M2 (100mg/dl) sebagai

Microalbuminuria. Sampel darah berpuasa telah diambi l untuk penentuan

g lukos darah berpuasa (FBG), glycated haemoglobin (HbA1c), /ipoprotein(a),

kolesterol total , triglisarid, l ipoprotein-kolesterol ketumpatan rendah (LDL­

Chol) dan l ipoprotein-kolesterol ketumpatan tinggi (HDL-Chol). Ujian

tersebut telah dijalankan dengan menggunakan penganalisa kimia automatik

(Cobas Miras, Roche). Kerosakan DNA peringkat rendah (kelas 0 & 1) telah

ditemui pada subjek normal serta pesakit dengan mikroalbuminuria

berkepekatan Omg/dl dan 20mg/dl . Kerosakan DNA peringkat tinggi dan

sepenuhnya (kelas 2, 3 & 4) telah dikesani pada kepekatan mikroalbuminuria

50 mg/dl dan lebih. Kumpulan Mikroalbuminuria tetah menunjukkan

peningkatan kerosakan DNA yang signifikan berbanding dengan kumpulan

Normoalbuminuria (18.54 ± 3.17% Vs 12.00 ± 1.37%, P<0.01). Terdapat

perbezaan yang signifikan pada tahap kerosakan DNA antara M2 dan M1

(20.32 ± 2.82% Vs 16.19 ± 1.78%, P<0.01), dan antara M1 dan N2 (16. 19 ±

1.78% Vs 11 .88 ± 1.46%, P<0.01 ). Walau bagaimanapun, tidak ada

perbezaan yang signifikan pada tahap kerosakan DNA antara kumpulan N2

dan N 1. Seperti yang dijangka, terdapat perkaitan yang signifikan (p<0.01)

antara FBG dengan HbA 1 c pada kedua-dua kumpulan Normo- dan

Microalbuminuria. Tetapi tidak ada perbezaan yang signifikan dikesan untuk

vi

nilai min F8G dan HbA1c antara semua kumpulan pesakit. Tahap kerosakan

D NA oksidatif telah meningkat dengan signifikan pada kepekatan albuminuria

20 mg/dl dan seterusnya. Keputusan F8G dan HbA 1 c tidak mengikut

peringkat kepekatan mikroalbuminuria. Korelasi signifikan (P<0.05) telah

dikesan antara tahap kerosakan D NA dengan F8G dan HDL-Chol pada

kedua-dua Normo- dan Microalbuminuria. Kepekatan HDL-Chol pada

kumpulan Microalbuminuria (1.06 ± 0.49 mmolll) adalah rendah berbanding

kumpulan Normoalbuminuria (1.16 ± 0.44 mmolll). Tahap kolesterol total,

triglisarid, LDL-Chol dan kepekatan Lp(a) tidak mempamerkan apa-apa

perkaitan dengan tahap kerosakan oksidatif D NA. Dalam kumpulan M2,

indeks jisim tubuh (8MI) dan HbA 1 c berkaitan secara signifikan (p<0.01 dan

p<0. 05) dengan tahap kerosakan DNA. Dalam kajian ini, assai Comet telah

menunjukkan kerosakan sel DNA berlaku di kalangan pesakit kencing manis.

Kajian juga mencadangkan stres oksidatif berlaku pada peringkat awal

komplikasi, dan mungkin sebelum mikroalbuminuria yang jelas dapat

dikesan. Malahan, F8G dan HbA 1 c tidak mempamerkan perkaitannya

dengan peringkat kepekatan mikroalbuminuria. Ini menunjukkan petanda­

petanda biasa tidak dapat mempamerkan keadaan stres oksidatif yang

sebenamya berlaku pada pesakit kencing manis.

vii

ACKNOWLEDGEMENTS

Deepest appreciation to:

My respected and beloved Father, Mother and Brother for their love, care and

support throughout the duration of my rigorous study in Universiti Putra Malaysia.

Deepest gratitude and thankful to:

Associate Professor Dr. Zarida Hambali, Dr. Rozita Rosli, Professor Dr. Abdul Salam

Abdullah and Dr. Um Seh Kin (Kajang Hospital, Selangorj for their constructive

criticisms, patience and effort, wise guidance and suggestions during the supervision

of this thesis.

Sincere thanks to:

Dr. John W Baynes (University of South Carolina, Columbia), Dr. Andrew Col/ins

(Rowett Research Institute, Scotland), Marie Vasquez (Integrated Laboratory

Systems Inc.), Dr. Mohsen Meydeni (Tufts University) Dr. Narendra P. Singh

(University of Washington), Stefan Eriksson (Stockholm University, Sweden), Sian

Astley (Norwich Research Park, u.K.), Dr.S.J.L.Bakker (University Hospital

Groningen, Netherlands) for all their help, opinions and suggestions that they had

offered and given to me.

Deepest Gratitude to:

Abang Syahril, Kak Nurma, Kak Shariza and Thila for their assistance throughout my

work in the Molecular Biology Laboratory; Alis for her assistance in my data

collection; Haiza for her assistance in running the biochemical tests for me; and all

roy friends around me who have brought me the cheers and supports ..... .

vill

I certify that an Examination Committee met on 18th June 2001 to conduct the final examination of Sim Sze Kiat on his Master of Science thesis entitled "Oxidative DNA Damage in Leukocytes and Its Association with Metabolic Control in Diabetic Patients With and Without M icroalbuminuria" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as fol lows:

Abdu l Manan Mat Jais, Ph.D. Associate Professor Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (Chairman)

Zarida Hambal i , M.D. , DCP, Ph.D. Associate Professor Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (Member)

Rozita Rosl i , Ph.D. Lecturer F acuity of Medicine and Health Sciences, Universiti Putra Malaysia (Member)

Abdul Salam Abdul lah, Ph.D. Professor Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (Member)

Lim Seh Kin, M.D. , M .Med. Physician Hospital Kajang, Selangor (Member)

Professor HAZAU MOHAYIDIN, Ph.D.

Deputy Dean of Graduate School, Universiti Putra Malaysia Date: 2 2 JUN 2001

ix

This thesis s ubmitted to the Senate of Univers iti P utra Malaysia has been accepted as the f ulfilment of the req uirement for the degree of Master of Science .

x

AIN I IDERIS , Ph. D. Professor Dean of Grad uate School, Universiti Putra Malaysia Date :

f1 2 JUL 2001'

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. J also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

Sim Sze Kiat Date: 22. 0 6 . 2 00 [

xi

TABLE OF CONTENTS

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL SHEETS DECLARATION FORM LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

1

2

INTRODUCTION Problem Statement Importance and Validity of Research Aim of Study Objectives Null Hypothesis

LITERATURE REVIEW Diabetes Mellitus

New D iagnosis Criteria for D iabetes Mellitus Type 2 Diabetes Mellitus

D iabetic Complications Molecular Basis of Diabetes Complications

Free Radicals Hyperglycemia Increases Oxidative Stress

Increased Oxidative Stress in Diabetic Patients Microalbuminuria

Role of Microalbuminuria M icroalbuminuria and Metabolic Control Practical Value of Albuminuria

Glycated Haemoglobin Lipid Profiles in Diabetic Patients

Total Cholesterol Triglycerides Low Density Lipoprotein High Density Lipoprotein Lipoprotein(a)

General Approaches to Detect Oxygen Free Radicals and Assess Oxidative Stress D NA Damage as a Screening Marker Comet Assay

xii

Page i i v

viii ix xi xv xvi xvii

1 3 4 5 5 6

7 7 8 10 11 14 14 16 20 21 23 24 25 25 28 29 29 30 33 34

37 38 39

3 METH ODOLOGY 41 S tudy Design 41

Diabe tic Pa tien ts 41 Con trol G roup 42 Collection of Clini cal Da ta 42

Blood Sampling 43 Ma te rials 44 Me thod 45

Come t Assay 45 Evalua tion of DNA Damage 48 P re tes t on H202 Induc ed DNA Damage 50 Bio chemical Tes ts 51 Fas ting Blood Glucos e 51 Glyca ted Haemoglobin 52 Lipop ro tein (a ) 53 To tal Choles te rol 54 T rigly cerides 55 LDL -Choles te rol 57 HDL- Choles te rol 58

S ta tis tical Analysis 61

4 RESULTS 62 Level of Oxida tive DNA Damage 83 Associa tions of Oxida tive DNA Damage and Me tabolic Co ntrols 86

5 DIS CUSS ION 90 DNA Damage as a Sc reening Mark er in Diabe tic Pa tien ts 91 Measu remen t of Oxida tive S tress using Come t Assay 93 Level of Oxida tive DNA Damage in Diabe tic Pa tie nts 96 Associa tions be tween Oxida tive DNA Damage and Me tabolic Con trols 100

Fas ting Blood Glu cose 100 Gly cated Haemoglobin 101

Lp (a ) and Oxida tive DNA Damage 102 Lipid p ro file and Oxida tive DNA Damage 103 Mic roalbumin uria and Oxida tive DNA Damage 105

6 CON CLUS ION 108 Sugges tions to Fu tu re S tudies 110

BIBLIOGRAPHY 111

xiii

APPENDIX A B C D E F G

Ethical Clearance for Research Proposal Patient Consent Form Clinical Data Collection Form Sampling Tubes Used for Different Blood Tests Chemicals Used in Comet Assay Reagent Kits for Biochemical Tests Equipment Used in Laboratory Tests

BIODATA OF AUTHORS

xiv

1 21 1 23 1 25 1 27 1 29 1 31 1 33

1 35

Table

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

LIST OF TABLES

Values for Diagnosis of Diabetes Mellitus and Other Categories of Hyperglycaemia.

Classifications of Comets.

Ethnic and Sex Distributions of Study Groups.

Ethnic and Sex Distributions of Diabetic Patients.

Clinical Characteristics of Study Groups.

Clinical Data of Patients as In Concentration of Albuminuria.

Mean Level of Oxidative DNA Damage in Control, Normo- and Microalbuminuria Groups.

Mean Level of Oxidative DNA Damage in Different Concentrations of Albuminuria Groups.

Associations between Level of DNA Damage and Metabolic Controls In Diabetic Patients.

Association between Metabolic Controls and Level of DNA Damage as in Different Groups of Albumin Concentration Diabetic Patients.

xv

Page

9

49

63

64

71

73

83

84

87

89

LIST OF FIGURES

Figure Page

1. Generation of Free Radicals and Mechanism Proposed in 17 Diabetic Patients.

2. The Main Steps in Comet Assay. 47

3. Sex Distributions among Different Study Groups. 66

4. Ethnic Distributions among Different Study Groups. 67

5. Comparisons of Lipid Profiles in Diabetic Groups and Control 74 Group.

6. Comparisons of Lp(a) Concentrations in Diabetic Groups and 75 Control Group.

7a. Classifications of Comets [Type 0, 1, 21- 77

7b. Classifications of Comets [Type 3, 4]. 78

8. Comets Induced by H202 [A: 20 liM; B: 501iM]. 79

9. Statistics Distribution of the Extent of Oxidative DNA Damage. 81

10. Statistic Distributions of the Extent of Oxidative DNA Damage 82 Among Different Albuminuria Subgroups.

11. Relative Means of FBG, HbA 1 c and Level of Oxidative DNA 85 Damage among the Different Albuminuria Subgroups.

OM

ROS

O2-

·OH

H202

AGE

DNA

SSB

ALS

FBG

HbA1 c

TG

LDL-Chol

HDL-Chol

Lp(a)

8-0HdG

ROOHs

TBARS

SCGE

EtBr

HCL

DMSO NaCI

EDTANa2

PBS

NaOH

LIST OF ABBREVIATIONS

Diabetes Mellitus

Reactive oxidation species

Superoxide Anion

Hydroxyl Radical

Hydrogen Peroxide

Advanced Glycosylation End Products

Deoxyribonucleic Acid

Single Strand Break

Alkali Labile Site

Fasting Blood Glucose

Glycated Haemoglobin

Triglycerides

Low Density Lipoprotein Cholesterol

H igh Density Lipoprotein Cholesterol

Lipoprotein (a)

8-hydroxy-2-deoxyguanosine

hydroperoxides

thiobarbituric acid-reacting substances

single cell gel electrophoresis

Ethidium Bromide

Hydrochloric Acid

Dimethyl Sulfoxide Sodium Chloride

Disodium Ethylenediamine Tetra Acetate

Phosphate Buffered Saline

Sodium Hydroxide

xvii

CHAPTER 1

INTRODUCTION

Diabetes mel l i tus is a disease characterized by derangement of

carbohydrates and lipids. The manifestations of diabetes result from

persistently raised blood glucose level and altered energy metabol ism as a

consequence of reduced production and/or impaired effectiveness of

insul in. It is a common cause of blindness, kidney fai lure, infections

necessitating leg amputations, and birth defects. In addition, people with

diabetes are twice as l ikely to develop cardiovascular problems as those

without diabetes (Cataldo et aI. , 1991).

There are generally two forms of d iabetes mel l itus: Type 1, insulin­

dependent diabetes mellitus ( IDDM) and Type 2, non-insulin-dependent

d iabetes mel l itus (NIDDM). Both are characterized by a systemic defect in

insul in-dependent metabolism but differ in their mechanisms of

pathogenesis, insulin deficiency vs. insul in resistance, respectively

(Anderson et aI. , 1998).

1

2

Production of reactive oxygen species (ROS) and l ipid peroxidation

are increased in d iabetic patients, especially in those with poor diabetic

control and hypertriglyceridaemia (Kitahara et aI., 1 980). Hyperglycemia, a

key biochemical abnormal ity of diabetes mell itus, not only generates more

ROS, but also attenuates antioxidative mechanisms by scavenging

enzymes and substances. Therefore, oxidative stress has been considered

to be one of the common pathogenic factors of diabetic compl ications such

as cardiovascular disease, nephropathy or neuropathy (Baynes, 1 991 ;

Giugl iano et aI. , 1 996). In past few years, increased oxidative stress has

been indicated by elevated concentrations of l ipid peroxidation products

such as thiobarbituric acid-reactive substances in the plasma (Lyons, 1 991 ).

Excess ROS not only accelerate oxidative damage to

macromolecules such as proteins and l ipids, but also to D NA (Dandona et

aI. , 1 996). ROS causes base modifications and strand breaks in DNA

(Ricther, 1 995), and thus result in oxidative damage to DNA. Extended

exposure of plasm ids to sugars wil l increase the degree of damage in

DNA. Recently, a high concentration of 8-hydroxydeoxyguonosine (8-

OhdG), a typical product of DNA oxidation, was reported in the

lymphocytes of IDDM and NIDDM patients (Dandona et ai, 1 996).

Guanine residues, deoxyguanosine (dG) were found to be oxidized to 8-

oxo-dGuo when DNA was exposed to ROS (Richter, 1 995). Besides,

study by Coll ins et a l . (1 998) showed that there was a higher level of DNA

damage in lymphocytes from type I d iabetic patients compared to healthy

3

people. Hence, the DNA damage might be useful as a screening marker

to indicate and/or predict the development of diabetic microangiopathic

and macroangiopathic complications which is caused by oxidative stress.

Problem Statement

Type 2 diabetes mellitus is the predominant type of diabetes mellitus

(90 to 95 percent of all cases), and the type l ikely to go undiagnosed and

most often developed in people over 40 and appears to be associated with

obesity (often of long duration), abdominal fat, and physical inactivity

(Cataldo et aI . , 1 991 ) . Oxidative damage may contribute to the

development of d iabetic microangiopathic and macroangiopathic

complications in these patients. Since the long-term compl ications are the

main cause of morbidity and mortality in d iabetic patients, a quantitative

determination of the level of oxidative stress is a valuable indicator of the

degree of the severity of the disease and of the effectiveness of treatment.

There are a number; of ways to detect oxidative stress. An alternative

method for detecting DNA damage is single cell gel electrophoresis or

comet assay. The comet assay is a sensitive and reliable method for

measuring DNA single-strand breaks and alkali-labile sites. This assay

had been adopted as a useful tool to assess DNA-damage in short-term

genotoxicity and human biomonitoring studies (Rojas et aI . , 1 999).

4

Importance and Validity of Research

In this study, DNA damage wil l be used as a screening marker in the

evaluation of diabetic complications. Although development of oxidative

stress in diabetes is wel l published, the oxidative stress has a primary role

in the pathogenesis of diabetic complications is sti l l questionable. It is

important to know whether oxidative stress occurs at an early stage in

diabetes, preceding the appearance of complications, or whether it is

merely a common consequence of the tissue damage, reflecting the

presence of complications. In future, the effectiveness of therapy used to

treat diabetic patients and its complications can be measured by DNA

damage, which is caused by oxidative stress. Hence the failure of therapy

for diabetic can be detected and complications can be prevented or slowed

down.

This method may be used to monitor levels of DNA damage in

individual patients associated with regimen treatment and that the regimen

could be changed accordingly. Beside that, the level of DNA damage

among some specific groups can be evaluated through this assay and the

risk of getting complications can be determined. Also, the comet assay

can be used in the study of the mechanism of action of new drugs and in

the analYSis of interactions between antidiabetic agents acting at DNA

level. Such studies may lead to therapeutic approaches for l imiting the

damage from g lycation and oxidation reactions and for complementing

existing treatment of the complications of diabetes.

Aim of Study

The main purpose of the study is to compare the extent of oxidative

DNA damage with different levels of microalbuminuria in diabetic patients

and its associations with other metabol ic controls.

Objectives

5

i) To determine the level of oxidative DNA damage in white blood cel ls

among different groups: Normal group (control group), diabetic

patients with- and without microalbuminuria.

i i ) To determine metabolic controls (fasting blood glucose, l ipoprotein

(a) and glycosylated haemoglobin) and l ipid profi les (total cholesterol ,

triglycerides, LDL-cholesterol and HDL-cholesterol) that characterize

diabetic status among different study groups.

iii) To compare the level of oxidative DNA damage in white blood cells

among the study groups.

iv) To compare the measured metabolic controls and l ipid profiles

among the study groups.

6

v) To study the associations between the extent of oxidative DNA

damage and metabolic controls among the study groups.

vi) To develop comet assay as a simple and sensitive tool for measuring

oxidative DNA damage in diabetic cases with different levels of

urinary albumin excretion.

Null Hypothesis

1 . There is no difference between the level of oxidative DNA damage in

white blood cells among the study groups.

2. There is no association between the level of oxidative DNA damage in

white blood cells, metabol ic controls and l ipid profiles among the study

groups.

7

CHAPTER 2

LITERATURE REVIEW

Diabetes Mell itus

The term diabetes mel l itus describes a metabolic disorder of multiple

etiology characterized by chronic hyperglycemia with disturbances of

carbohydrate, fat and protein metabol ism resulting from defects in insulin

secretion, insulin action or both. Hyperglycemia is the cause of the

metabolic syndrome and is the parameter most closely monitored to make

the d iagnosis and to judge therapy (Kaufman and McKee, 1996). Diabetes

mel l itus may present with characteristic symptoms such as thirst, polyuria,

blurring of vision, and weight loss. A diagnosis of diabetes has been defined

by the World Health Organization (1985) as either a fasting plasma g lucose

concentration greater than 7.8mmolll or a 2-hour postprandial value greater

than 1 1. 1 mmolll .

The cause of d iabetes mel l itus is unknown. Many etiologic factors are

suspected, with major d ifferences between those factors that are etiologic for

Type 1 and Type 2 diabetes. Etiologic factors of Type 1 diabetes are