EVALUATION OF CLINICAL EFFICACY OF SOME ANTIOXIDANTS …vinayakamission.com/userfiles/phd/O863600015.pdf · 5 METHODOLOGY 55-80 5.1 Permission from Institutional Human Ethical Committee

EVALUATION OF CLINICAL EFFICACY OF

SOME ANTIOXIDANTS IN DIABETIC

NEPHROPATHY

Thesis submitted in Partial Fulfillment

for the award of Degree of

Doctor of Philosophy in Pharmacy

By

VVIITTTTHHAALL GGAAJJAANNAANNRRAAOO KKUUCCHHAAKKEE

Under the guidance of

DDrr.. CC.. DD.. UUPPAASSAANNII

VINAYAKA MISSIONS UNIVERSITY SALEM, TAMILNADU, INDIA

December 2013

VVIINNAAYYAAKKAA MMIISSSSIIOONNSS UUNNIIVVEERRSSIITTYY

DECLARATION

I, Vitthal Gajananrao Kuchake declare that the thesis entitled

‘Evaluation of Clinical Efficacy of Some Antioxidants in Diabetic

Nephropathy’. submitted by me for the Degree of Doctor of Philosophy

in Pharmacy is the record of research work carried out by me during the

period from October 2008 to December 2013 under the guidance of

Dr. C. D. Upasani and has not formed the basis for the award of any

degree, diploma, associate-ship, fellowship, titles in this or any other

University or other similar institutions of higher learning.

Place : Nashik, Maharashtra. Vitthal Gajananrao Kuchake

Date :

VVIINNAAYYAAKKAA MMIISSSSIIOONNSS UUNNIIVVEERRSSIITTYY

CERTIFICATE

I, Chandrashekhar Devidas Upasani certify that the thesis entitled

‘Evaluation of Clinical Efficacy of Some Antioxidants in Diabetic

Nephropathy’. submitted for the Degree of Doctor of Philosophy in

Pharmacy by Mr. Vitthal Gajananrao Kuchake is the record of

research work carried out by him during the period from October 2008

to December 2013 under my guidance and supervision and that this

work has not formed the basis for the award of any degree, diploma

associate-ship, fellowship or other titles in this University or any other

University or Institution of higher learning.

Place : Nashik, Maharashtra. Dr. C. D. UPASANI

Date : PRINCIPAL

SNJB’s SSDJ College of Pharmacy,

Chandwad, Nashik

Acknowledgement

I would like to express my deep and sincere gratitude to my supervisor,

Professor Dr. Chandrashekhar D. Upasani, Principal, SNJB’s Shriman

Sureshdada Jain College of Pharmacy, Neminagar, Chandwad, Nashik. His

understanding, encouraging and personal guidance have provided a good

basis for the present thesis.

I owe my most sincere gratitude to Professor Dr. Sanjay J. Surana, Principal,

R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur and

Management of Shirpur Education Society who gave me the opportunity to

work and providing excellent facilities.

My sincere thanks are due to Prof. Dr. K. Rajendran, Dean (Research) and Dr.

B. Jaykar (Principal, Faculty of Pharmacy), Vinayaka Missions University, Salem

Tamilnadu, for their valuable advice and support during this work.

I warmly thank t o Dr. P. H. Patil, and Dr. A. A. Shirkhedkar, Vice-

Principal for their valuable advice.

My warm thanks are due to Professor Dr. C. R. Patil, Dr. H . S.

Mahajan, Head Department of Pharmacology and Pharmaceutics for their

extensive discussions around my work.

I warmly thank t o Dr. S . B. Bari, Principal, H. R. Patel Institute of

Pharmaceutical Education & Research, Shirpur for their valuable advice.

My warm thanks are due to Dr. P. N. Dighore, General Physician,

Department of Endocrinology Indira Gandhi Memorial Hospital, Shirpur for

extensive discussions around research work.

I wish to thanks D r . P . V . I ng l e , D r . S a me e r G oy a l , D r . A. R.

Tekade, Dr. V. A. Chatpalliwar, Dr. S. G. Gattani, Dr. R. B. Jadhav for

their friendly support and valuable advice during this research study.

During this work I have collaborated with many colleagues,

Dr. A. U. Tatiya, , Dr. A. P. Gorle, Mr. S. S. Chalikwar, Dr. P. P. Ige,

Mr. P. P. Nerkar, Dr. P. S. Jain, Mr. R. R. Patil, Dr. S. C. Khadse,

Mr. Kapil Agrawal, Dr. Haroon Patel, Mr. Malesh Thakare, Mr.

Pankaj Jain, Mr. Manish Gagrani, Mr. V. G. Ugale, Mr. P. J.

Chaudhari, Mr. G. G. Tapadiya, Mr. S. V. Girase, Mr. D. P. Dhakad for

whom I have great regard, and I wish to extend my warmest thanks to all

those who have helped me with my work in the R. C. Patel Institute of

Pharmaceutical Education and Research, Shirpur.

My sincere thanks are due to Mr. Anuj Khadelwal (Biorad India Pvt. Ltd.)

for their co-operation during the study by providing HbA1c kit timely and Dr.

Ambadasaji Chukewar RMO, Hata for time to time moral support.

I warmly thanks to Mr. Nitin Mali, for their excellent photographic work,

and Mr. Jitesh Jadhav, Mr. Hari Patel, Mr. Rajesh Gujar, Mr.

Dnyaneshwar Patil, Mr. Sunil Asapure, Mr. Dhiraj Mali, Mr.

Prashant Lokhande, Mr. Bhoge Nana Mr. Sanjay Pawara, Mr. Pathan

for their sympathetic help in secretarial work.

I’d like to thank to Mr. Massod Siddiqui, Mr. Sumit Chaudhari, Mr.

Shekhar Athare, Mr. Deshraj Chumble, Mr. Nitin Jain, and Nripendra

Singh for their friendly support.

Finally I’d like to thank to all participants of this research for their valuable

participations.

Last, but not least, I owe my loving thanks to my wi fe N il ima , for your

love, endless support and great sense of humour, you have made all of this

possible. Also thank to my cute little daughter Nidhi, for her smiles and words

gives encouragement. My special gratitude is due to my Fa ther; Mother,

Brother, Sister and extend ed family, thanks for a l l o f the love ,

unconditional support and encouragement that you have given me over the years.

December 2013 Vitthal Gajananrao Kuchake

AFFECTIONALY

DEDICATED

TO GOD

And MY

BELOVED

LATE Grand Father

INDEX

Sr. No. Chapter Page

Number

List of Tables I

List of Figures II-III

List of Abbreviations IV-V

1 INTRODUCTION 1-12

1.1 Rational and Motivation for the Study 1

1.2 Research Problem and Questions 4

1.3 Free Radicals 5

1.4 Antioxidant Defense Systems 6

1.5 Free Radical-Mediated Oxidative Tissue Damage 8

1.6 The Role of Free Radicals and Oxidative Stress in the

Pathogenesis of Diabetes

9

1.7 Role of Antioxidants and Antidiabetic Agents 10

2 REVIEW OF LITERATURE 13-51

2.1 Prevalence of Diabetic Nephropathy in India 15

2.2 Symptoms of Diabetic Nephropathy 17

2.3 Clinical Stages in Progression of Diabetic Nephropathy 18

2.4 Clinical Diagnosis of Diabetic Nephropathy 21

2.5 Major Risk Factors for Diabetic Nephropathy 22

2.6 Mechanisms Involved in Pathology of Diabetic

Nephropathy

23

2.7 Approaches In the Treatment of Diabetic Nephropathy 40

2.8 Role of Antioxidant in Diabetic Nephropathy 42

2.9 Clinical Studies on Use of Antioxidants in Treatment of

Diabetic Nephropathy

44

2.10 Drugs Used in Study 46

3 NEED FOR THE STUDY 52-53

4 OBJECTIVES & HYPOTHESIS 54

5 METHODOLOGY 55-80

5.1 Permission from Institutional Human Ethical

Committee & Hospital Authority

55

5.2 Patient Data Collection 56

5.3 Informed Consent Form 56

5.4 Study Design 57

5.5 Study Site 59

5.6 Inclusion Criteria 59

5.7 Exclusion Criteria 59

5.8 Biochemical and Hematological Parameters 59

5.8.1 Specimen Collection and Storage 59

5.8.2 Determination of Blood Glucose Level (Fasting and

Postprandial)

60

5.8.3 Micral (Urine Albumin and Serum Albumin) 63

5.8.4 Serum Creatinine 66

5.8.5 Urine Creatinine 68

5.8.6 Blood Urea Nitrogen 70

5.8.7 Total Protein 72

5.8.8 Determination of HbA1c Level 74

5.8.9 Blood Pressure Estimation 80

5.9 Statistical Analysis 80

6 RESULTS & DISCUSSION 81-102

6.1 Effect of Antioxidants on Microalbuminuria in DN 86

6.2 Effects of Antioxidants on Fasting and Postprandial

Blood Glucose in DN

88

6.3 Effects of Antioxidants on Urine Creatinine & Serum

Creatinine in DN

88

6.4 Effect of Antioxidants on Blood Urea Nitrogen (BUN) in

DN

91

6.5 Effect of Antioxidants on Serum Albumin and Globulin in

DN

92

6.6 Effect of Antioxidants on (Glycosylated Hemoglobin)

HbA1c in DN

95

6.7 Effect of Antioxidants on Total Protein in DN 98

6.8 Effect of Antioxidants on Diastolic and Systolic BP in DN 99

7 CONCLUSION 103-104

8 REFERENCES 105-119

9 ANNEXURES I-XIII

I Institutional Human Ethical Committee (IHEC) Approval

Letter I

II Proforma II-VI

III Informed Consent Form (ICF) VII-IX

IV List of Publications X-XIII

List of Tables

I

Table No.

Table Legends Page No.

Chapter 2 Review of Literature

2.1 Clinical Stages of Diabetic Nephropathy 19

2.2 Direct & Indirect Effect on Development of DN 24

Chapter 5 Methodology

5.1 Assay Parameters for Blood Glucose Determination 62

5.2 Procedure for Blood Glucose Test 62

5.3 Assay Parameters for Blood Albumin Determination 65

5.4 Procedure for Blood Albumin Test 65

5.5 Assay Parameters for Serum Creatinine Determination 67

5.6 Procedure for Serum Creatinine Test 67

5.7 Assay Parameters for Urine Creatinine Determination 69

5.8 Procedure for Urine Creatinine Test 69

5.9 Assay Parameters for BUN Determination 71

5.10 Procedure for BUN Test 71

5.11 Assay Parameters for Total Protein Determination 73

5.12 Procedure for Total Protein Test 73

Chapter 6 Results and Discussion

6.1 Effect of Antioxidants on Renal and Hematological

Parameters in DN

85

List of Figures

Figure No.

Figure Legends Page No.

Chapter 1 Introduction

1.1 Complications of Diabetes 2

Chapter 2 Review of Literature

2.1 Prevalence of Diabetic Nephropathy in India 16

2.2 Differences in Mortality Rates Among Diabetic and Non-

Diabetic Individuals

17

2.3 Mechanism of Diabetic Nephropathy 23

2.4 Potential Interaction Between Metabolic and

Haemodynamic in Pathogenesis of Diabetic Nephropathy

25

2.5 Formation of AGE’s (Advanced Glycation End Products) 27

2.6 Mechanism Involved in Production of Oxidative Stress 28

2.7 Polyol Pathway 29

2.8 PKC Activation Pathway 30

2.9 Mechanisms Involved in Glucose Induced Oxidative

Stress

31

2.10 Mechanisms Leading to Oxidative Stress in

Hyperglycemia

32

2.11 Role of Hypertension in Diabetic Nephropathy 35

2.12 Role of Hyperlipidemia in Progression of Diabetic

Nephropathy

36

2.13 Endogenous Stimuli Leading to ROS Generation 40

2.14 Vitamin E 46

2.15 Vitamin C 48

2.16 Interaction Between Antioxidants in the Process of

Detoxifying Lipid Peroxides

49

2.17 Reduced Glutathione 50

Chapter 5 Methodology

5.1 Screening of Diabetic Nephropathy Patients 58

5.2 Design of Study 58

II

III

5.3 Diabetes Control Card ( HbA1c) 76

5.4 Bio-rad Micromat II HbA1c Monitoring Instrument 80

Chapter 6 Results and Discussion

6.1 Origin of Microalbuminuria 82

6.2 Effect of Antioxidants on Microalbuminuria in DN 87

6.3 Effects of Antioxidants on Urine Creatinine in DN 90

6.4 Effects of Antioxidants on Serum Creatinine in DN 91

6.5 Effects of Antioxidants on Blood Urea Nitrogen in DN 92

6.6 Working Model of Albuminuria, Particularly for Albumin

Excretion in Humans

93

6.7 Effects of Antioxidants on Serum Albumin in DN 94

6.8 Effects of Antioxidants on Serum Globulin in DN 95

6.9 Effects of Antioxidants on HbA1c in DN 98

6.10 Effect of Antioxidants on Total Protein in DN 99

6.11 Effect of Antioxidants on Systolic BP in DN 101

6.12 Effect of Antioxidants on Diastolic BP in DN 102

List of Abbreviations

Abbreviations Full Form

ACEI Angiotensin Converting Enzyme Inhibitors

ADP Adenosine Diphosphate

AER Albumin Excretion Rate

AGE’s Advanced Glycation End Products

AO Antioxidants

ARBs Angiotensin Receptors Blockers

ATP Adenosine Triphosphate

BMI Body Mass Index

BP Blood Pressure

CAPD Chronic Ambulatory Peritoneal Dialysis

CML Carboxyl Methyl Lysine

CRF Chronic Renal Failure

CuZn Copper and Zinc

DAG Diacylglycerol

DCCT Diabetes Control & Complications Trial’s

DHA Dehydroascorbate Radical

DN Diabetic Nephropathy

DNA Deoxyribonucleic Acid

ECM Extra Cellular Matrix

EDRF Endothelium Derived Relaxing Factor

eNOS Endothelial Nitric Oxide Synthase

ESRD End Stage Renal Disease

ESRF End Stage Renal Failure

FPG Fasting Plasma Glucose

GFR Glomerular Filtration Rate

GSH Glutathione

GST Glutathione S-Transferases

H2O2 Hydrogen Peroxide

HbA1c Glycosylated Hemoglobin

HDL High Density Lipoproteins

IV

ICMR Indian Council of Medical Research

IDDM Insulin Dependent Diabetes Mellitus

IDF International Diabetes Federation

IgG Immunoglobulin

LDL Low Density Lipoproteins

MCs Mesangial Cells

MDA Malonyldialdehyde

Mn-SOD Manganese Superoxide Dismutase

NAD Nicotinamide Adenine Dinucleotide

NADPH Nicotinamide Adenine Dinucleotide Phosphate

NIDDM Non Insulin Dependent Diabetes Mellitus

OHAs Oral Antihyperglycemic Agents

PKC Protein Kinase-C

PPG Postprandial Plasma Glucose

PUFA Polyunsaturated Fatty Acids

RAGE Receptor for Advanced Glycation End Products

RNS Reactive Nitrogen Species

ROS Reactive Oxygen Species

SOD Superoxide Dismutase

SREBP Sterol Regulatory Element-Binding Protein

TGF-β Transforming Growth Factor

UAER Urinary Albumin Excretion Rate

WHO World Health Organization

V

CHAPTER 1 INTRODUCTION

1

1 INTRODUCTION

1.1 Rational and Motivation for the Study

The aim of present study was to perceive the effects of antioxidant

vitamins in patients with diabetes mellitus. India leads the world with

largest number of diabetic subjects earning the dubious distinction of

being termed the “diabetes capital of the world”. According to the

Diabetes Atlas 2011 published by the International Diabetes Federation

(IDF), the number of people living with diabetes is expected to rise from

366 million in 2011 to 552 million by 2030, unless urgent preventive

steps are taken. This equates to approximately three new cases every

ten seconds or almost ten million per year. IDF also estimates that as

many as 183 million people are unaware that they have diabetes1.

The manifestations of the disorder cause considerable human

sufferings and massive economic cost, despite of the enormous

facilities available to control its growth rate. The alarming spread and

rising incidence prompted the formulation of guidelines by a reputed

organization like the Indian Council of Medical Research (ICMR) in

collaboration with WHO and ratified by a team of experts in the field.

Diabetes mellitus is a chronic disorder that is growing in prevalence

worldwide. Aggressive glycemic control has been demonstrated to

decrease microvascular and macrovascular complications, although the

latter claim remains controversial2.


2

Figure1.1 Complications of Diabetes

The Canadian Diabetes Association 2003 Clinical Practice Guidelines

for the Prevention and Management of Diabetes recommends a target

hemoglobin A1c concentration of 7.0 % or less for all patients with

diabetes and, for those in whom it can be safely achieved, a target

hemoglobin A1c concentration in the normal range (usually ≤ 6.0 %)3.

Although nonpharmacologic therapy (e.g., diet, exercise and weight

loss) remains a critical component in the treatment of diabetes,

pharmacologic therapy is also necessary to achieve optimal glycemic

control but managing the disorder and controlling its associated

complications require correct diagnosis, self-care, exercise and sticking

to the strict drug-dose-food-intake regimen because the progression of

disorders cause prolonged exposure of vascular tissues to


3

hyperglycemia resulting in long-term microvascular/macrovascular

complications as cardiovascular diseases, renal disease, cerebro-

vascular diseases, etc.; these are the prime causes of morbidity,

disability and premature death4 . Any irregularities in dosage regimen

can definitely invite severe complications in the health; and nephropathy

is one of them.

By convention, diabetic nephropathy is a clinical syndrome

characterized by persistent albuminuria (> 300 mg/24 hours), on at least

two occasions separated by 3-6 months5. This process is often

associated with rising blood pressure6. Conventionally, nephropathy is

divided into two types based on the urinary albumin excretion rate

(UAER): incipient and overt incipient nephropathy is manifested as

microalbuminuria (UAER 20 -200 mg/24 hours) and usually occurs after

6-15 years of diabetes7, although microalbuminuria is an indicator of

nephropathy8. Roughly 25 % of patients will regress to normal albumin

excretion and 40 % will remain microalbuminuric9.

Diabetic nephropathy is a common complication of diabetes associated

with oxidative stress and reduced levels of antioxidants because

oxidative stress is hypothesized to play an important part in the

development of late diabetes complications because chronic

hyperglycemia increases oxidative stress and considerably modifies the

structure and function of proteins and lipids due to glycoxidation and

peroxidation. These modified products could contribute to the


4

morphological and functional abnormalities seen in the kidney of

patients with diabetes as a result of development of proteinuria,

culminating in end-stage renal disease with a particular high risk of

cardiovascular morbidity and mortality in diabetic patients4. It affects

more than one-third of patients with type 1 diabetes and an ever

increasing proportion of patients with type 2 diabetes10.

For these reasons, there has been need in the use of externally

administered antioxidants to attenuate diabetic nephropathy as well as

to control the worsen condition of the patients because supplementation

of antioxidants when it comes to treat patients with diabetes which is

claimed to be increases the effectiveness of main therapies and devoid

of side-effects.

1. 2 Research Problem and Questions

The two principal defects in diabetes are insulin deficiency and insulin

resistance. The ultimate or primary goal of therapy for diabetes is to

prevent the mortality and morbidity related to the microvascular and

macrovascular complications11,12. Since these diseases are lifelong

disorders, reduction in the number of tablets and daily doses is a very

important consideration from the patient’s point of view13,14.

It is increasingly obvious that to achieve this on a global perspective we

will need to identify better and more effective treatment strategies to

maintain tight glycemic control. The current practice of starting therapy

with one agent and increasing to maximum dosage before adding a


5

second agent, rather than there is a need to add supplementary therapy

like antioxidants along with antidiabetic agents, also needs to be

addressed15.

There is much evidence to suggest that initiating therapy with

antioxidants which lowers doses of OHA which leads to have

complementary effects and can increase the overall efficacy and

decreases the incidence of further complications and control the worsen

condition of patients and decrease the adverse effects16. Therefore,

combining effect of antioxidants along with OHA will augment the

efficacy of current antihyperglycemic agents.17-19.Because

complementary mechanisms of action of orally administered

antioxidants in combination with antidiabetic agents is associated with

additive beneficial effect on the glycemic control20,21.

As discuss earlier diabetic nephropathy is associated with oxidative

stress as a results of abnormal generation of free radicals.

1.3 Free Radicals

In 1956, Denham Harman, father of the free radical theory postulated

that free radicals produced during aerobic respiration cause cumulative

oxidative damage, resulting in aging and death. Free radicals are

generally considered harmful byproducts of oxidative metabolism22,

causing molecular damage in living systems. This concept has

implications in numerous biological phenomena such as cellular aging,

mutagenesis, inflammation, and other pathologies. Furthermore, it has


6

been suggested that free radicals are implicated in the process in part

for the development of diabetic microangiopathy and

macroangiopathy23, and excessive free radical production has been

reported in diabetics with chronic renal failure treated by

haemodialysis24. Consequently, free radical mechanisms have been

implicated in the pathogenesis of tissue damage in diabetes25. The term

"free radical" can defined as any atoms or molecules that contain an

unpaired electron in its outer obit that can exist independently. As a

result, they can be highly reactive, although this varies from radical to

radical, reacting locally to accept or donate electrons to other molecules

to achieve a more stable state. Ground state 02 (302) has two unpaired

electrons each located in a different antibonding orbital. An oxidizing

agent, such as 02 is effective at absorbing electrons from the molecule it

oxidizes 26,27. The collective terms reactive oxygen species (ROS) or

active oxygen species have been applied for a variety of free radicals

and non-radicals intermediates.

1.4 Antioxidant Defense Systems

Antioxidants are defined as any substance that when present at low

concentrations, compared with those of the oxidative substrate

considerably delays or inhibits oxidation of the substrate. Antioxidants

can act at many different stages in an oxidative sequence including

removing oxygen or decreasing local oxygen concentrations, removing

catalytic metal ions, removing key ROS such as oxygen and hydrogen


7

peroxide, scavenging initiating free radicals, breaking the chain of an

initiated sequence, and quenching or scavenging singlet oxygen specie.

Furthermore, a variety of antioxidant defense systems operates,

including enzymatic and nonenzymatic antioxidants. Enzymatic

antioxidants directly involved in the detoxification of ROS are super

oxide disputes (SOD) and hydroxyperoxidases such as catalase (CAT)

and glutathione peroxidase (GSHPx) a selenium-containing enzyme

glutathione (GSH)26,28. Cells have formidable defense mechanisms

against oxidative damage of which some may not be readily

recognisable as antioxidants. Enzyme such as SOD rapidly promotes

the dismutation of superoxide into hydrogen peroxide and oxygen at a

rate considerably faster than it occurs uncatalyzed. Two different

superoxide dismutases are found in mammalian tissue, namely a

Cu/Zn-containing enzyme which is found in the cytoplasm of most cells,

and a further Mn-containing enzyme present within the mitochondrial

compartment29. Both enzymes catalase the same reaction as shown

below

202* + 2H+ H202+02

Hydrogen peroxide, a product of the dismutation reaction, can be

destroyed by two enzymes, catalase and glutathione peroxidase.

Glutathione peroxidase can metabolise hydrogen peroxide, generated

by SOD, by oxidizing the tripeptide glutathione into its oxidized from


8

(GSSG). In addition, catalase transforms hydrogen peroxide into water

and oxygen as shown below.

2GSH + H202 GSSG + 2H20

2H202 2H20 + 02

Apart from these endogenous antioxidants, an important source of

antioxidants is in the diet, which contains numerous compounds

exhibiting antioxidant activity. The most prominent dietary antioxidants

are tocopherols, the fat-soluble vitamin (vitamin E), ascorbate water-

soluble vitamin (vitamin C) and carotenoids. Furthermore, other

antioxidants such as albumin and other proteins including ceruloplasmin

and transferin also protect against oxidative injury by binding the

transition metals Fe+2 and Cu+2 thereby preventing generation of the

hydroxyl radical via the Fenton reaction28.

1.5 Free Radical-Mediated Oxidative Tissue Damage

The human body has a multiplicity of different antioxidant defense

mechanisms25. If the defensive processes are overwhelmed, free

radicals can then become highly destructive to cells and tissues. During

oxidative stress, the prooxidant-antioxidant balance is tipped in favor of

the former, and this may be due to exogenous sources of free radicals

or other endogenous stresses29. However, oxidative stress can produce

major interrelated derangements of cell metabolism, including DNA

damage, protein damage and peroxidation of lipids. The relative

importance of damage to different molecule as targets in producing cell


9

injury or death by improving oxidative stress depends on duration,

degree of stress underlying mechanism and the nature of the system

stressed25.

1.6 The Role of Free Radicals and Oxidative Stress in the

Pathogenesis of Diabetes

There is emerging evidence suggesting that subjects with diabetes have

concomitant increased free radicals production and depletion of cellular

antioxidant defense systems. It is well established that alloxan and

streptozotocin induced diabetic animals become hyperglycaemic as the

result of destruction of ß-cells of the pancreas by free radicals22. It is

probable that in certain genotypes, glycation and glycoxidation lead to

an increased susceptibility to oxidative stress than in other genotypes.

This would be the genotypes in which ß-cell destruction leads to the

development of type 2 diabetes30. Pancreatic ß-cell are especially

vulnerable to oxidative stress, probably because of their low free radical

scavenging enzyme capacity reflected in low SOD, catalase and

glutathione peroxidase activities. Recent studies have reported a direct

link between the imbalance of oxidative stress and antioxidants leading

to impaired glucose uptake. In adiposities, glucose uptake was rapidly

decreased when they were incubated with glucose oxidise which

resulted in a steady production of hydrogen peroxide. The reduction of

insulin-dependent 2-deoxyglucose uptake was consequently

accompanied by decreased P13 kinase activity and GLUT4


10

translocation. These observations support the suggestion that free

radicals and antioxidant depletion could impair insulin-mediated PI3

kinase activity, which results in impaired GLUT4 translocation and

defective insulin mediated glucose uptake. Increased oxidative stress,

in addition to antioxidant depletion, leads to decreased glucose uptake

were also observed in muscle cells. Furthermore, depletion of

antioxidants accompanied by decreased glucose uptake has also been

observed in subjects with type 2 diabetes. These observations lead to

the hypothesis that the imbalance of free radicals and antioxidants is an

important pathogenic factor affecting insulin-signaling pathways.

However, clinical and experimental studies have demonstrated that

supplementation with antioxidants such as vitamin E and a-lipoic acid

stimulate glucose uptake through activation of the insulin-signaling

pathway and provide protective effects to diabetic state31.

So we have selected antioxidants to assess its effects on glycemic

control, renal parameters and HbA1c in patients with diabetes

nephropathy. We thought this might contribute to existing knowledge

and aid and assist the people with diabetes.

1.7 Role of Antioxidants and Antidiabetic Agents

Decreased antioxidant status has an important role in development of

diabetic nephropathy, so antioxidant treatment could become a key

element in prevention and reversal of diabetic nephropathy. This study

undertaken to demonstrate the possessions of orally administered


11

antioxidants on the renal function of the patients who have been

diagnosed to have microalbuminuria. It was found that chronic blockade

of the oxidative stress pathway in renal artery stenosis using oral

antioxidant vitamin supplementation improves renal hemodynamics and

decreases oxidative stress, intrarenal inflammation and tubulointerstitial

fibrosis in the kidney32.These underscore the role of increased oxidative

stress in the pathogenesis of ischemic nephropathy and suggest a role

for antioxidant vitamins in preserving the function and structure of the

stenotic kidney. Short duration of vitamin C and E treatment with

pharmacological doses in type 2 diabetic patients with

microalbuminuria/ macroalbuminuria significantly lowers AER.

Glutathione deficiency contributes to oxidative stress, which plays a key

role in aging and the pathogenesis of diabetic nephropathy. The primary

role of glutathione is to protect cells from oxidative stress. GSH levels

reflect a reduced oxidative stress and improved insulin sensitivity33-35.

Managing the disorder and to control its associated complications

requires tight blood glucose control because the DCCT confirmed the

significance of tight blood glucose control in slowing the improvement of

proteinuria in diabetic patients. As concern with self-care and exercise,

the blood pressure control is one of the most important to prevent the

growth of DN to manage hypertension and thereby slow the decline in

the GFR at various levels36. The ACE inhibitor in diabetic nephropathy

showed the beneficial effect in the treatment of diabetic


12

glomerulosclerosis; Subsequent studies have confirmed this

observation for both ACE inhibitors and ARBs37. Most agree that ACEI

are first line therapy for diabetic glomerulosclerosis, but ARBs are

regarded by some as equivalent38. The beneficial effect of angiotensin II

inhibition may result from decline in glomerular hypertension with

slowing of mesangial expansion, a reduction in proteinuria with an

expected decrease in proteinuria-associated prosclerotic events and

decrease in angiotensin II stimulated TGF-ß synthesis37. Dietary protein

restriction has been shown to slow the loss of GFR in proteinuric

diabetics. Protein restricted diets (0.6 -0.8 g/kg body wt/day) decrease

glomerular hypertension, the production of prosclerotic cytokines,

proteinuria, and glomerulosclerosis and remain a viable therapeutic

option for compliant patients38,39. Early and aggressive therapy for

microalbuminuria taken along with antioxidant vitamins is expected to

slow disease progression39.

Heavy proteinuria is a risk factor for progressive renal failure, including

diabetic nephropathy. There is abundant evidence that abrogating

proteinuria with dietary and antihypertensive interventions and /or ACE

inhibitors, or ARBs, results in a slower loss of GFR in proteinuric

states37. In this regard, the supplementation of antioxidant vitamins in

combination with antidiabetic therapy slows the progression of diabetic

nephropathy40.

CHAPTER 2 REVIEW OF LITERATURE

13

2 REVIEW OF LITERATURE

Diabetic nephropathy is the major cause of end-stage renal disease

(ESRD) in the industrialized world. It is the medical term for kidney

disease caused by diabetes and most common cause of kidney failure

in developed and developing countries. All people with diabetes are

susceptible to kidney disease but those who have type 1 diabetes or

who develop type 2 diabetes at an early age are at particular risk.

Recent research indicates that nephropathy can even affect people with

prediabetics. The high glucose levels in the blood can damage the

membranes within the kidney‘s nephrons that are responsible for

filtering the blood and forming urine. Hyperglycemia is a necessary

prerequisite but genetic susceptibility is also crucial for the development

of diabetic nephropathy including familial aggregation; suggest the

existence of genes where allelic variation contributes to risk of diabetic

nephropathy. For instance, the diabetic patient with proteinuria has a

two- to four fold increased risk of morbidity and mortality from

cardiovascular diseases even with chronic dialysis, the cardiac death

rate of diabetic patients is 50% higher than nondiabetic patients41.

Various studies have shown that diabetes mellitus is associated with

increased formation of free radicals and decrease in antioxidant

potential. Due to these events, the balance normally present in cells

between radical formation and protection against them is disturbed

leads to oxidative damage of cell components such as proteins, lipids,


14

and nucleic acids. In both insulin dependent (type 1) and non-insulin-

dependent diabetes (type 2) there is increased oxidative stress mean

excess formation and/or insufficient removal of highly reactive

molecules such as reactive oxygen species (ROS) and reactive nitrogen

species (RNS). ROS include free radicals such as superoxide, hydroxyl,

peroxyl, hydroperoxyl as well as nonradical species such as hydrogen

peroxide and hydrochlorous acid. RNS include free radicals like nitric

oxide and nitrogen dioxide, as well as nonradical such as peroxynitrite,

nitrous oxide, alkyl peroxynitrates, these reactive molecules are the

most widely studied species and play important roles in the diabetic

nephropathy42. DN is characterized by accumulation of extracellular

matrix (ECM) in the kidney. Glomerular mesangial expansion and

tubulointerstitial fibrosis eventually leads to renal failure. The mediators

of renal injury in this disease have not been fully identified43.The peptide

angiotensin-II (Ang-II) has many hemodynamic and biochemical effects

that could contribute to DN. A prominent role for Ang-II has been

suggested by experimental and clinical evidence indicating that

angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin

receptor blockers (ARBs) have renoprotective effects and these agents

can attenuate the progression of glomerulosclerosis44.In clinical studies,

as well as studies conducted in experimental diabetic animals, it is

difficult to separate hemodynamic from non hemodynamic effects of

Ang-II45. On the other hand, in vitro studies using cultured cells allow


15

study of the specifically nonhemodynamic effects of Ang-II and its

inhibition. These nonhemodynamic effects of Ang-II include stimulation

of transforming growth factor (TGF)-β1, activation of matrix protein

synthesis and inhibition of matrix degradation. Ang-II also increases

generation of reactive oxygen species (ROS) in mesangial cells (MCs)

and may contribute to oxidant-induced renal injury46.

2.1 Prevalence of Diabetic Nephropathy in India

Diabetic nephropathy develops in about one third of patients with

diabetes, and its incidence is sharply increasing in the developing world,

with the Asia - Pacific region being the most severely affected. It was

the most common cause of end-stage renal disease in 9 of 10 Asian

countries, with an incidence that had increased from 1.2 % of the overall

population with end-stage renal disease in 1998 to 200047.

According to the most recent estimates published in the Diabetes Atlas

2006, India has the largest number of diabetic patients in the world,

estimated to be 40.9 million in the year 2007 and expected to increase

to 69.9 million by the year 202548.


16

Figure 2.1 Prevalence of diabetic nephropathy in India

The National Urban Diabetes Survey (NUDS), a population based study

was conducted in six metropolitan cities across India and recruited

11,216 subjects aged 20 yr and above representative of all socio-

economic strata. This study also revealed that the prevalence in the

southern part of India to be higher-13.5 % in Chennai, 12.4%, in

Bangalore, and 16.6 % in Hyderabad; compared to eastern India

Kolkata, 11.7 %; northern India i.e. New Delhi, 11.6 %; and western

India i.e. Mumbai, 9.3 %49.

A recent population based study reported that the prevalence of overt

nephropathy was 2.2 % in Indians while microalbuminuria was present

in 26.9 %. Glycated hemoglobin, duration of diabetes and systolic blood

pressure were independently associated with diabetic nephropathy.


17

Figure 2.2 Differences in mortality rates among diabetic and non-

diabetic individuals

2.2 Symptoms of Diabetic Nephropathy

Diabetes-related kidney disease tends to be asymptomatic until the later

stages of the disease as a result of excretion of high amounts of protein

in the urine or due to renal failure: Once the symptoms occur, kidney

function tends to be 25 % lower than normal, and the damage is

irreversible. When the symptoms appear49, they may include:

Edema: Swelling, usually around the eyes in the mornings; later,

general body swelling may result, such as swelling of the legs

Foamy appearance or excessive frothing of the urine

Unintentional weight gain (from fluid accumulation)

Anorexia (poor appetite)


18

Nausea, vomiting, Fatigue, Headache

Frequent hiccups

Generalized itching

Renal failure is one of the possible complications of diabetes. Patients

with renal failure require dialysis or a kidney transplant. Patients with

terminal kidney disease often have other complications50 these includes

skin problems such as sores, eye problems (diabetic retinopathy),

neuropathy (numbness and tingling, generally in the hands and feet)

and atherosclerosis (coronary artery disease and peripheral vascular

disease) which increases the risk of heart attack, stroke and vascular

disease in the arms and legs. Peripheral vascular disease could entail

the risk of amputation due to complications from poor circulation50.

2.3 Clinical Stages in Progression of Diabetic Nephropathy

There are five clinical stages characterize the progression of diabetic

nephropathy on the basis of the values of the glomerular filtration rate

(GFR), urinary albumin excretion (UAE), and systemic blood pressure.

Silent phase: Very few patients develops microalbuminuria during the

first ten years of their diabetes (type 2 diabetes may remain

undiagnosed for many years and present with advanced disease) so-

called silent phase, early histological abnormalities in hypertrophy and

subtle thickening of the glomerular basement membrane are detected in

the electron microscopy of kidney sections51.


19

Table 2.1 Clinical Stages of Diabetic Nephropathy

Stage-1 Hyperfiltration-hypertrophy stage: The earliest renal

manifestations in type 1 diabetes are nephromegaly and glomerular

hypertrophy, which are accompanied by afferent arteriolar vasodilation,

renal hyper perfusion and glomerular hyper filtration. Microscopically,

there is thickening of the glomerular and tubular basement membranes.

The UAE in this stage is normal (approx 30 mg/d) but occasionally a

transient increase in UAE (“transient microalbuminuria”) is present

secondary to poor glycemic control or infection. Typically the blood

pressure is below the hypertensive range (140/90 mm Hg). The GFR is

increased by 20% to 40% above normal values, with higher levels being

frequently achieved when glycemic control is poor. Some studies

suggest that patients with the highest degrees of hyperfiltration (GFR

150 ml/min) are at increased risk for the future development of overt

nephropathy41.

Stages GFR UAE BP Years after

Diagnosis Hyperfiltration Supernormal < 30 mg/d Normal 0

Microalbuminuria High-normal 30-300 mg/d Rising 5-15

Overtproteinuria Normal-

decreasing > 300 mg/d Elevated 10-20

Progressive Decreasing Increasing Elevated 15-25

ESRD < 15 ml/min Massive Elevated 20-30


20

Stage-2 Incipient or latent nephropathy: It is defined by the

appearance of microalbuminuria (UAE of 30–300 mg/d or 20–200

g/min). Without intervention, UAE increases at the rate of 10% to 20%

per year and is almost always accompanied by a steady rise in blood

pressure. Hypertension (140/90 mm Hg) is typically diagnosed 1 to 2

years after the appearance of microalbuminuria in type 1diabetes.

Microalbuminuria rarely develops before 5 years of disease duration

(median, 10 years) in type 1 diabetes, but in type 2 diabetes

microalbuminuria may be present at the time of diagnosis of

hyperglycemia in up to 20% of patients with as many as 40% of patients

having elevated blood pressure as well51.

Stage-3 Overt clinical nephropathy: This stage is characterized by

the development of overt proteinuria (total protein excretion 500 mg/dL)

or macroalbuminuria (UAE 300 mg/d). In type 1 patients this occurs

after an average of 15 years of diabetes. Hypertension is almost always

present. If a renal biopsy were to be performed, the glomeruli would

typically demonstrate diffuse glomerulosclerosis and/or nodular

glomerulosclerosis. Hypertension and subsequent fall in GFR,

retinopathy is almost always present. Once significant proteinuria

develops, further progression of renal failure is fairly rapid and normally

occurs in 5 years51.

Stage-4 Progressive nephropathy: After approximately 5 years of

overt nephropathy, untreated patients progress to advanced


21

nephropathy, as characterized by nephrotic-range proteinuria (3.5 g/d),

worsened hypertension that becomes difficult to control, and a

progressive decline in GFR. The rate of decline in GFR is steady over a

period of months but is variable from patient to patient and depends on

the degree of elevation of blood pressure as well as the amount of UAE.

Stage-5 End-stage renal failure (ESRF): This stage is characterized

by the development of overt proteinuria (total protein excretion 500

mg/d) or macroalbuminuria (UAE 300 mg/d). In type 1 patients this

occurs after an average of 15 years of diabetes. Hypertension is almost

always present. Management of diabetic patients with ESRF is

complicated by the frequent co-existence of complications affecting

other organ systems including proliferative retinopathy, cardiovascular

disease, peripheral neuropathy or autonomic neuropathy. Renal

replacement therapy is the choice of treatment for these patients. If

contraindications to transplantation are present, a decision must be

made regarding Chronic Ambulatory Peritoneal Dialysis (CAPD) verses

haemodialysis51.

2.4 Clinical Diagnosis of Diabetic Nephropathy

The diagnosis of diabetic glomerulosclerosis can be made with a renal

biopsy. Light microscopic findings include increased mesangial matrix

and thickening of the glomerular basement membrane52.

Immunofluorescence is characterized by increased staining of the

glomerular and tubular basement membrane and Bowman’s capsule for


22

IgG and albumin in a linear pattern. The presence of diabetic

glomerulosclerosis can also be inferred from the clinical presentation.

For example, in the first placebo controlled, double blind study of the

effect of ACE inhibitors in diabetic nephropathy a diagnosis of diabetic

glomerulosclerosis was inferred if a patient had diabetes at least 7-10

years, exhibited demonstrable diabetic retinopathy, and had

macroscopic proteinuria (albuminuria>300 mg/d). Diabetics with heavy

proteinuria, but lacking the disease for a sufficient period of time and/or

retinopathy, may require renal biopsy. These patients may suffer from

primary glomerulopathies such as membranous nephropathy or other

glomerular diseases. Diabetic glomerulopathy is the most common

cause of nephrotic syndrome. Thus, early in the course of the disease,

the serum creatinine is normal despite heavy proteinuria (>3 gm/24

hours). In this regard, a diabetic patient presenting with elevated serum

creatinine in the absence of macroscopic proteinuria should suggest

additional diagnostic possibilities (such as other glomerulopathies)53.

2.5 Major Risk Factors for Diabetic Nephropathy

Genetic susceptibility as evidenced by diabetic nephropathy in a

sibling

Increased GFR

Hypertension or high-normal BP

Worse glycemic control

Increased sodium-lithium and sodium hydrogen counter transport54.


23

2.6: Mechanisms Involved In Pathology of Diabetic Nephropathy

Nephropathy develops in about 20% to 40% of all diabetic

patients, although a somewhat lower percentage of those with type

2 diabetes progresses to ESRD, perhaps in part because these

patients are older and die from cardiovascular complications55.

Hyperlipidemia has been considered to be a major determinant of

progression of nephropathy in patients with diabetes. The experimental

evidence suggests that hyperlipidemia may mediate renal injury by

increased the expression of sterol regulatory element-binding protein

(SREBP), which is responsible for increasing the synthesis of

cholesterol and triglycerides in the kidney. Hyperglycemia mediated

production of ROS in diabetes will play a major role in the pathogenesis

of diabetic nephropathy56.

Figure 2.3 Mechanism of Diabetic Nephropathy


24

Key to the development of diabetic nephropathy is the

hyperglycemic state, Its affects directly or indirectly. Those direct and

indirect effects are

Table 2.2 Direct & Indirect Effect on Development of DN

Direct Effect Indirect Effect

Glucose in sustained high

concentration may be directly

toxic to cells.

Altering cell growth and protein

expression and increasing

extracellular matrix and growth

factor production.

Glucose may induce its effect

indirectly through the formation of

metabolic derivatives such as

oxidants, AGE’s.

AGE’s may damage the cells by

modification to extracellular matrix

protein.

The sustained production of such metabolites may results in continuous

activation of different pathways, involving phospholipids kinase45.

Pathophysiology of diabetic nephropathy involves an interaction of

metabolic and haemodynamic factors. It is probable that glucose-

dependent processes are involved in diabetic nephropathy57.


25

Figure 2.4 Potential interaction between metabolic and haemodynamic

in pathogenesis of diabetic nephropathy

2.6.1 Hemodynamic Pathway

The hemodynamic changes (flow/pressure) alleviate albumin leakage

from the glomerular capillaries and overproduction of mesangial cell

matrix as well as thickening of glomerular basement membrane and

injury to podocytes. These hemodynamic changes can induce localized

release of certain cytokines and growth factors. The action of

vasoactive hormone such as angiotensin II and endothelin is mediator

of renal hemodynamic changes. Glomerular hypertension and hyper

filtration contribute to the development of diabetic nephropathy because

the use of rennin-angiotensin blockers preserves kidney function and

morphology. Blockade of rennin angiotensin aldosteron system


26

antagonizes the profibrotic effect of angiotensin II by reducing its

stimulation of TGF‐β58.

2.6.2 Metabolic pathway

A] Advanced glycation end products

AGEs are a chemically heterogeneous group of compounds formed as

a result of the ‘‘Millard reaction’’ when reducing sugars react non-

enzymatically with amine residues, predominantly lysine and arginine,

on proteins, lipids and nucleic acids. The initial stage of the reaction

leading to the formation of reversible glycosylation proteins termed

Schiff bases is rapid and glucose dependent, a much slower reaction

over a period of day’s results in the formation of the more stable

Amadori product. These early glycosylation products accumulate

predominantly on long lived proteins such as vessel wall collagen and

crystalline undergoing a series of in vivo rearrangements to form

irreversible complex compounds and cross-links, termed AGEs. Binding

of AGEs to the RAGE receptor activates a number of pathways

implicated in the development of diabetic complications58.


27

Figure 2.5 Formation of AGE’s

B] Mechanism

Capillary basement membrane thickening and hypertrophy of

extravascular matrix are common features of diabetic microvascular

complications. AGEs accumulate in extracellular matrix proteins as a

physiological process during aging. This accumulation happens earlier

and with an accelerated rate in diabetes mellitus than in non-diabetic

individuals. Increased serum and tissue levels of AGEs, due to a

reduced removal by kidney have been evidenced in end-stage renal

failure58.

C] Glucose auto-oxidation

Glucose can be auto-oxidized in a cell-free system under physiological

conditions which generates hydrogen peroxide; reactive intermediate

such as hydroxyl and superoxide radicals, and ketoaldehydes. Several


28

studies have reported that glucose auto-oxidation can actually occur

and could be responsible for increased oxygen radicals in diabetes58.

Figure 2.6 Mechanism involved in production of oxidative stress

2.6.3 Polyol pathway

Excessive entry of glucose into this pathway results in tissue

accumulation of sorbitol which leads to cataract formation and

possibly osmotic vascular damage. Aldose reductase, the first and

rate-limiting enzyme in this pathway, catalyzes the NADPH-dependent

reduction of hexose or pentose sugars to their corresponding sugar

alcohols, or polyols. Tissues that do not require insulin for glucose

uptake (kidney, lens, retina, and peripheral nerves) become subject to

relatively greater loads of intracellular glucose. Increased oxidation of


29

sorbitol to fructose via fructose dehydrogenase is coupled to reduction

of NAD to NADH, and a more reduced cytosolic ratio of NADH/NAD

may result in abnormalities of cellular function, including myoinositol

depletion, ROS generation, PKC stimulation, and even TGF59

production activation of this pathway may also alter various

enzyme activities and thus contribute to the pathologic changes.

Sustained hyperglycemia may also result in increased diacylglycerol

levels with activation of protein kinase C. This kinase has been

implicated as a cause of altered RBF, vascular permeability, and

increased growth factor and extracellular matrix production in the

diabetic kidney55. These effects include the generation of reactive

oxygen species, increased extracellular matrix accumulation,

stimulation of transforming growth factor-p (TGF-P), connective

tissue growth factor and platelet-derived growth factor production

and macrophage activation57.

Figure 2.7 Polyol Pathway


30

Figure 2.8 PKC activation pathways

Oxidative stress has been considered to be a common pathogenic

factor of diabetic complications including nephropathy. Oxidative stress

is increased in the diabetic kidney preferably before clinical signs of

nephropathy60. Oxidative stress is caused by a relative overload of

oxidants, i.e., reactive oxygen species. This impairs cellular functions

and contributes to the pathophysiology of many diseases. Evidence has

accumulated suggesting that diabetic patients are under oxidative

stress and that complications of diabetes seem to be partially mediated

by oxidative stress.


31

Figure 2.9 Mechanisms involved in glucose induced oxidative stress

(Mechanisms involved in glucose induced oxidative stress Elevated

extra- and intra-cellular glucose concentrations result in an oxidative

stress. The increased free radical production is associated with a

concomitant increase in intracellular AGE formation. Antioxidants such

as α-tocopherol, desferroxamine or dimethylsulfoxide inhibit both

production of free radicals and AGE formation. AGEs compounds found

in human are: - Pentosidine and (CML) Carboxyl methyl lysine)61.


32

Figure 2.10 Mechanisms leading to oxidative stress in

hyperglycemia

(Mechanisms leading to oxidative stress in hyperglycemia. The normal

end-point of glucose metabolism is the generation of ATP from ADP. In

the presence of excess glucose ADP becomes rate-limiting, and the

pathway becomes clogged. ROS then result at several points, including

escape of electrons in the mitochondrial transfer chain to generate

superoxide, and NADH oxidase is activated also producing superoxide.

When glycolysis slows, fructose 1-6-bisphosphate is shunted into the

hexosamine pathway that produces oxidative stress. Excess glucose is

diverted to the polyol pathway as well as activating intracellular and

extracellular glycation reactions)62.


33

2.6.4 Role of Hypertension in Diabetic Nephropathy

The relationship between hypertension and poor vascular outcomes,

including progression of renal disease, is unequivocal and independent

of other confounding factors63. The impact of hypertension on outcomes

is exponential rather than linear. A sustained reduction in blood

pressure seems to be currently the most important single intervention to

slow progressive nephropathy in type 1 and type 2 diabetes. Long-term

follow-up studies of initially normotensive diabetic subjects without renal

disease demonstrate a blood pressure dependent decline in GFR with

blood pressure levels within the reference range. Patients with a blood

pressure corresponding to 130/80 mmHg rarely develop

microalbuminuria and show an annual decline in GFR close to the age-

matched normal population. Diabetic patients with a blood pressure

between 130/80 and 140/90 mmHg have a greater decline in GFR, with

30 % of patients developing associated microalbuminuria or proteinuria

over the subsequent 12 to 15 years. In microalbuminuric and proteinuric

type 1 and 2 diabetic patients, numerous studies have demonstrated

that treatment of hypertension, irrespective of the agent used produces

a beneficial effect on albuminuria. Aggressive targets for blood pressure

control in diabetic patients have been shown to result in reduced

development and retardation in the progression of incipient and overt

nephropathy, as well as decreasing macrovascular events64. Diabetic

nephropathy includes a progressive increase in urinary albumin


34

excretion and a decline in glomerular filtration rate (GFR) which occurs

in association with an increase in blood pressure, ultimately leading to

end stage renal failure. These renal functional changes develop as a

consequence of structural abnormalities, including glomerular basement

membrane thickening, mesangial expansion with extracellular matrix

accumulation, changes in glomerular epithelial cells (podocytes),

including a decrease in number and/or density, podocyte foot process

broadening and effacement, glomerulosclerosis, and tubulointerstitial

fibrosis64. Glomerular basement membrane thickening and mesangial

expansion with increased extracellular matrix deposition. In type 1

diabetic, there is a direct relationship between the extent of mesangial

expansion and clinical severity of disease. In this regard, Mauer’s

seminal paper demonstrated a direct correlation between the degree of

mesangial expansion and magnitude of proteinuria, severity of

hypertension, and degree of renal impairment. Taken together, these

data suggest that clinical findings predict severity of glomerular damage

and underscore the necessity of understanding the mediators of

mesangial expansion in this disease53.Glomerular hypertension

resulting from afferent vasodilatation and efferent vasoconstriction in

conjunction with altered glomerular permeability causes proteinuria,

activation of proximal tubular epithelial cells, renal fibrosis and ultimately

nephron loss and renal failure63.


35

Figure 2.11 Role of hypertension in diabetic nephropathy

2.6.5 Genetic Factors

Strong evidence for genetic factors in nephropathy come from studies of

families with type 1 diabetes. In families with 2 or more siblings with

type 1diabetes, when 1 sibling has developed nephropathy, the other

has a 4 -fold risk of nephropathy compared with a sibling of a patient

without nephropathy. In a larger study, if the proband had nephropathy,

the cumulative risk of nephropathy to diabetic siblings was 71.5 %, and

the risk decreased to 25.4 % when the proband did not have

nephropathy65. Other factors have been suggested as determinants of

risk for diabetic nephropathy, including a family history of

hypertension65.

2.6.6 Hyperlipidemia

A typical feature of diabetic nephropathy is hyperlipidemia causes

glomerular injury remains unknown, although lipid abnormalities are

already present during microalbuminuria. Serum triglyceride level is an


36

independent risk factor for diabetic nephropathy in type 1 and type 2

diabetes. Lipid lowering treatment in almost totally nephrectomized rats

has been associated with a small reduction in proteinuria and

glomerulosclerosis without any adverse effects on renal function and

plasma lipid levels. In overt nephropathy, hypercholesterolemia has

been associated with cardiovascular mortality and with a rapid decline

in renal function. Raised plasma triglycerides and low level of high

density of lipoproteins (HDL) have been correlated with the

development of diabetic nephropathy as well as with cardiovascular

diabetic complication. Triglyceride and cholesterol reduction, although

important in reducing cardiovascular risk, has not been found to alter

the progression of renal disease and importance of hyperlipidemia

remains to be established in this respect66.

Figure 2.12 Role of Hyperlipidemia in progression of diabetic

nephropathy


37

2.6.7 Smoking

It is associated with the development and progression of diabetic

nephropathy6,7.The risk of smoker to develop microalbuminuria and

acceleration of the progression of nephropathy has been equally well

documented in type 1 and type 2 diabetes. Smoking increase the risk of

develops microalbuminuria, shorten the interval from microalbuminuria

to overt nephropathy and accelerate progression of nephropathy and

losses GFR67.

2.6.8 Gender

Due to the paucity of large prospective studies, the role of gender is not

entirely clear evidence does exist that in males, the prevalence of

diabetic nephropathy is higher and the progression of ESRD is faster 67.

2.6.9 Dietary Protein Intake

High protein diet increases the development and progression of diabetic

nephropathy39,66,68. The long-term effects of protein intake 20% of

calories on diabetes management and its complications are unknown.

Although such diets may produce short-term weight loss and improved

glycemia, it has not been established that these benefits are maintained

long term. Gluconeogenesis, a major biochemical process that

produces glucose from protein is accelerated in diabetes mellitus. So

kind and amount of protein may affect diabetic conditions In individuals

with type 2 diabetes, ingested protein can increase insulin response


38

without increasing plasma glucose concentrations. Therefore, protein

should not be used to treat acute or prevent nighttime hypoglycemia69.

2.6.10 Role of Free Radicals

Since numerous studies demonstrated that oxidative stress, mediated

mainly by hyperglycemia-induced generation of free radicals,

contributes to the development and progression of diabetes and

related contributions, it became clear that ameliorating oxidative stress

through treatment with antioxidants might be an effective strategy for

reducing diabetic complications70.Cellular metabolism generates

reactive oxygen species (ROS).Molecular ground-state oxygen can be

activated to a ROS by means of energy transfer (e.g., under the

influence of ultraviolet radiation), forming singlet oxygen (1O2), or

by electron transfer, forming “incomplete” reduction products, i.e.,

the superoxide anion radical (•O−2 ). Small amounts of oxygen (between

0.4 and 4% of all oxygen consumed) are reduced to •O−2 by the

mitochondrial electron transport chain during the course of normal

oxidative phosphorylation which is essential for generating ATP

.Subsequently, •O−2can be converted into other ROS and reactive

nitrogen species (RNS) . Under normal conditions, •O−2 molecules

are quickly converted to H2O2 by the key mitochondrial enzyme,

manganese superoxide dismutase (Mn-SOD) within the

mitochondria and by copper and zinc (CuZn-SOD) in the cytosol .

H2O2 is then either detoxified to H2O and O2 by glutathione


39

peroxidase (in the mitochondria) in conjunction with glutathione

reductase or diffuses into the cytosol and is detoxified by catalase in

peroxisomes. H2O2 can also be converted to the highly reactive

hydroxyl radical (HO•) in the presence of reduced transition metals

such as Cu or Fe (Fenton reaction). Further reactive oxygen species

may be derived from H2O2, such as the hypochlorite (OCl− ), peroxyl

radicals (ROO•) and alkoxyl radicals (RO•) or from peroxidation of

polyunsaturated fatty acids (PUFA) such as conjugate dienes, lipid

hydroperoxides and malonyldialdehyde (MDA) .Production of one

ROS may lead to the production of others through radical chain

reactions. H2O2 is produced by one electron reduction of oxygen by

several different oxidases including NADPH oxidase, xanthine

oxidase, cyclooxygenase and even endothelial nitric oxide synthase

(eNOS) under certain conditions .RNS include free radicals like nitric

oxide (•NO ) and nitrogen dioxide (• NO−2 ), as well as nonradicals

such as peroxynitrite (ONOO− ). • NO, also known as endothelium-

derived relaxing factor (EDRF), produced from L-arginine by eNOS in

the vasculature is considered a vasculoprotective molecule

.However • NO easily reacts with •O−2, generating the highly reactive

molecule ONOO− . Thus, variation in the production of • NO and

•O−2 by endothelium might provide one mechanism for the regulation

of vascular tone and hence of blood pressure70.


40

Figure 2.13 Endogenous stimuli leading to ROS generation

Although these ROS and RNS differ with regard to their stability,

reactivity and molecular targets, a common denominator is that their

uncontrolled formation in cells, i.e., the generation of a ROS load

exceeding the antioxidant capacity of the cell results in damage and

oxidation of lipids, proteins, and nucleic acids, as well as of several

other biomolecules71.

2.7 Approaches in the Management of Diabetic Nephropathy

The basis for the treatment of diabetic nephropathy is the treatment of

its known risk factors: hypertension, hyperglycemia, smoking, and

dyslipidemia54.

2.7.1 Blood Glucose Control

Reduction of Hb1Ac levels which indicate decreased risk for clinical and

structural manifestations of diabetic nephropathy in type 1 and type 2

diabetic patients. Intensive treatment of diabetes reduces the incidence

of microalbuminuria. In the Kumamoto et al study, a reduction in the


41

conversion from micro to macroalbuminuria was observed with intensive

treatment. Some oral antihyperglycemic agents seem to be especially

useful. Rosiglitazone, as compared with glyburide, has been shown to

decrease urinary albumin excretion (UAE) in patients with type 2

diabetes. This suggests a beneficial effect in the prevention of renal

complications of diabetes7.

2.7.2 Hypertension Management

The beneficial effects of lowering blood pressure are on both

progression of renal disease and overall cardiovascular mortality has

been widely reported51. It has become apparent that progressive fall in

GFR in IDDM patients correlates closely with both increasing

albuminuria and blood pressure. Further, reduction in BP slows the rate

of decline of GFR and increasing albuminuria. A prospective study of 6

years duration demonstrated that effective blood pressure treatment

decreased albumin excretion rate of 50% and the rate of decline of GFR

from 0.9 ml/min/month to 0.29 ml/min/Month72. Antihypertensive

treatment with ACE inhibition is found to have additional benefits.

Enthusiasm has been generated in the nephrological community by the

recent evidence that ACEI and Angiotensin-II receptor blockers (ARBs)

are superior to other antihypertensive agents in attenuating progressive

loss of renal function with improvement in glomerular membrane size, a

selective property seems to be unique to ACEI and is independent of

systemic BP changes72.


42

2.7.3 Protein Restricted Diet

Restriction of protein diet has been included in the treatment of chronic

renal failure and diabetic nephropathy. Effectiveness of protein

restriction in both diabetic and non diabetic renal disease has long-

established that such protein restriction diet improves the renal

function72.

2.8 Role of Antioxidants in Diabetic Nephropathy

Over the last few years, a number of studies have provided evidence of

an important role of ROS (reactive oxygen species) in mediating the

development of oxidative stress. Excessive ROS accumulation may

induce the oxidative modification of cellular macromolecules (lipid,

proteins and nucleic acids) with deleterious potential. In fact, DNA

damage by ROS has been implicated in mutagenesis, oncogenesis and

aging. Oxidative lesions in DNA include base modifications, sugar

damage and strand breaks. Since gene transcription can be regulated

by oxidants, antioxidants and other determinants of the intracellular

redox state, ROS can also produce protein damage, inducing other

types of mutations63. If decreased antioxidant status has an important

role in the development of diabetic nephropathy, antioxidant treatment

could become a key element in the prevention and reversal of diabetic

nephropathy. Potential beneficial actions of antioxidants on diabetes

may be involved in the improvement of diabetic nephropathy, although


43

many in vitro studies have supported the involvement of oxidative stress

in the development of diabetic nephropathy73.

The reactive oxygen intermediates, produced in mitochondria,

peroxisomes, and the cytosol, are scavenged by cellular defending

systems, including enzymatic (ex. superoxide dismutase, glutathione

peroxidase GPx, glutathione reductase and catalase) and

nonenzymatic antioxidants (ex. glutathione GSH, thioredoxin, lipoic

acid, ubiquinol, albumin, uric acid, flavonoids, vitamins A, C and E,

etc.), some are located in cell membranes, others in the cytosol and in

the blood plasma74. One observation concerning antioxidants is their

extraordinary chemical efficiency; these compounds should act rapidly,

as soon as a source of increased amount of ROS appears.

Effective protection from the formation and action of ROS requires

antioxidant activity in both aqueous and lipid environments and in

various part of the cell structure. When one antioxidants (AO) reacts

with a ROS, another antioxidant should be present to regenerate

the first . In addition, these AO may act by coupling to protect

both the membrane and the cytoplasm. AO in cells and blood possess

high capacity and redundancy to always be available to protect

cellular structures and vital molecules (nucleic acids).The role of

antioxidants seems to be to sacrifice themselves to protect the

essential cellular structures and molecules against ROS74.


44

2.9 Clinical Studies on Use of Antioxidants in Diabetic

Nephropathy

Diabetic nephropathy is a serious complication of diabetes and a major

cause of mortality and morbidity in these patients. Results of studies in

animal models and humans have demonstrated that diabetes is

associated with oxidative stress and reduced levels of antioxidants75,76.

It has been observed that the individuals with diabetes have significant

defects of antioxidant protection which may enhance their susceptibility

to oxidative stress. Clinical studies suggest that the use of antioxidants

indirectly helps in the prevention of diabetic nephropathy. In a study

carried out by Farvid et.al. 2005, effects of vitamins C, vitamin E and

their combinations with magnesium and zinc supplementation on the

improvement of glomerular functions in type 2 diabetics has been

observed77.

In diabetic nephropathy elevated levels of urinary albumin excretion rate

predict high risk for progressing to end-stage renal disease41. A four

weeks, randomized cross-over study on effects of vitamin C (1250 mg)

per day and vitamin E (680 IU) per day on the diabetic nephropathy

conducted by Gande et.al, 2001 concludes, the short term treatment

with vitamin C and E in pharmacological doses lowers UAER in diabetic

patients with micro/macro albuminurea78.

Supplementation of vitamin C alleviates oxidative stress and renal cell

injury. CRF is associated with impaired endothelium dependent


45

vasodilation and accelerated atherogenesis. ROS modify endothelial

function in renal failure and it is found that vitamin C reduces oxidative

stress in CRF and improves NO- mediated resistance vessel dilation

and also prevents fragmentation & inhibits conjugate dienes formation

during LDL oxidation79.

Antihypertensive angiotensin receptor blocker agents have a slowing

effect on the progression of diabetic nephropathy and on the

development of proteinuria in diabetes shows that long-lasting

hyperglycemia in type 1 diabetic patients induces permanent alterations

of endothelial function by increased oxidative stress, even when

glycemia is normalized. This study data shows that neither the

normalization of glycemia nor vitamin C treatment alone was able to

normalize endothelial dysfunction or oxidative stress. However,

combining insulin and vitamin C normalized endothelial dysfunction and

decreased oxidative stress to normal levels80.

Reduced glutathione is considered as the most powerful, versatile and

the most important antioxidant which reduces the oxidative stress 61.

Costagliola et al, 1992 has shown that, the therapy on patients

undergoing treatment with reduced glutathione could represent it to be a

useful drug in the treatment and management of anemia in patients

affected by chronic renal failure81.

Mattia et.al, 1998 found that one hour GSH infusion (1.35 gm/m2/hr)

simultaneously improves the intraerythrocytic GSH/SSH ratio and


46

insulin sensitivity in NIDDM patients and reduces the oxidative stress.

Further investigations are needed explain these findings and clarify the

molecular mechanisms involved in the effect of GSH on glucose

metabolism35.Reduced glutathione is the most affluent low molecular

weight thiol and GSH/ glutathione disulfide is the major redox couple in

animal cells82. Hagen et.al, 1988 has experimentally proved the

glutathione uptake shows protection against oxidative injury in isolated

kidney cells, this study indicates that GSH may be taken up intact from

the diet, providing a means by which GSH may be therapeutically

administered to augment plasma GSH concentrations83.

2.10 Drugs used in present study

2.10.1 Vitamin E

The term vitamin E is used to describe eight lipophilic, naturally

occurring compounds that include four tocopherol and four tocotrienols

designated as α-, β-,γ-and δ-.The most well known function of vitamin E

is that of a chain breaking antioxidant that prevents the cyclic

propagation of lipid peroxidation.

Figure 2.14 Vitamin E


47

Mode of action: Its main function is to prevent the peroxidation of

membrane phospholipids and avoids cell membrane damage through

its antioxidant action. The lipophilic character of tocopherol enables it to

locate in the interior of the cell membrane bilayers. Tocopherol-OH can

transfer a hydrogen atom with a single electron to a free radical, thus

removing the radical before it can interact with cell membrane proteins

or generate lipid peroxidation. When tocopherol-OH combines with the

free radical, it becomes tocopherol-O·, itself a radical. When ascorbic

acid is available, tocopherol-O· plus ascorbate (with its available

hydrogen) yields semidehydroascorbate (a weak radical) plus

tocopherol-OH. By this process, an aggressive ROI is eliminated and a

weak ROI (dehydroascorbate) is formed, and tocopherol-OH is

regenerated. Despite this complex defence system, there are no known

endogenous enzymatic antioxidant systems for the hydroxyl radical82.

Alpha tocopherol + LOO˙ Alpha tocopherol˙+ LOOH

Alpha tocopherol˙+ LOO˙ LOO-alpha tocopherol

2.10.2 Vitamin C

Vitamin C (ascorbic acid) is a powerful antioxidant and a cofactor in

collagen biosynthesis, which affects platelet activation, prostaglandin

biosynthesis and the polyol pathway. Vitamin C acts as an antioxidant

both in vitro and in vivo and protects plasma lipids and lipid membranes.

It has the power to spare and to increase plasma-reduced glutathione.


48

The antioxidative ability of vitamin E can be continuously restored

through its recycling by other antioxidants82.

Figure 2.15 Vitamin C

Mode of action: The action of vitamin C is attributed to two of its

functions. It is a water-soluble chain breaking antioxidant. As an

antioxidant, it scavenges free radicals and reactive oxygen molecules

which are produced during metabolic pathways of detoxification82. One

important property is its ability to act as a reducing agent (electron

donor). Ascorbic acid is a reducing agent with a hydrogen potential of

+O.08V, making it capable of reducing such compounds as a molecular

oxygen, nitrate and cytochromes. Donation of one electron by ascorbate

gives the semi-dehydroascorbate radical (DHA). Ascorbate reacts

rapidly with O2·⁻and even more rapidly with ·OH to give DHA. DHA, itself

can act as a source of vitamin C82.

Ascorbic acid + 2O2·⁻ + 2H⁺ H2O2 + DHA


49

Moreover, ascorbic acid alone can act as a "pro-oxidant" or reducing

agent to react with copper or iron salts. Ferric iron (Fe3+) formed by the

reaction,

Fe2+ + H2O2 HO + ·OH + Fe3+, is converted by ascorbic acid to

ferrous (Fe2+) ion. Ferrous iron is therefore recycled to promote the

conversion of more H2O2 to ·OH.

Mode of action of combination of vitamin E + vitamin C: Its ability to

function in cooperation. Effective protection from the formation and

action of ROS requires antioxidant activity in both aqueous and lipid

environments and in various part of the cell structure. When one AO

reacts with a ROS, another antioxidant should be present to

regenerate the first . In addition, these AO may act by coupling to

protect both the membrane and the cytoplasm. AO in cells and blood

possess high capacity and redundancy to always be available to

protect cellular structures and vital molecules (nucleic acids). The

role of antioxidants seems to be to sacrifice themselves to protect

the essential cellular structures and molecules against ROS71.

Figure 2.16 Interaction between antioxidants in the process of

detoxifying lipid peroxides


50

2.10.3 Reduced glutathione

Reduced L-glutathione is a well-known tripeptide, γ-glutamyl-cysteinyl-

glycine, abbreviated GSH. It is the most abundant low molecular weight

thiol, and GSH/glutathione disulfide is the major redox couple in animal

cells. “Glutathione deficiency contributes to oxidative stress which plays

a key role in aging and the pathogenesis of many diseases including

kwashiorkor, seizure, HIV, AIDS, cancer, heart attack, diabetes and its

complications82.

Figure 2.17 Reduced glutathione

Mode of action: GSH recycling is catalyzed by glutathione disulfide

reductase which uses reducing equivalents from NADPH to reconvert

GSSG to 2GSH. The reducing power of ascorbate helps conserve

systemic GSH. GSH is used as a cofactor by multiple peroxidase

enzymes to detoxify peroxides generated from oxygen radical attack on

biological molecules; transhydrogenases to reduce oxidized centers on

DNA, proteins, and other biomolecules; and glutathione S-transferases

(GST) to conjugate GSH with endogenous substances (e.g., estrogens)

and to exogenous electrophiles (e.g., arene oxides, unsaturated

carbonyls, organic halides) and diverse xenobiotics82.Glutathione


51

peroxidase reduces H2O2 to H2O by oxidizing glutathione (GSH)

(Equation A). Reduction of the oxidized form of glutathione (GSSG) is

then catalysed by glutathione reductase (Equation B). These enzymes

also require trace metal cofactors for maximal efficiency including

selenium for glutathione peroxidase; copper, zinc, or manganese for

SOD; and iron for catalase.

H2O2 GSSG + 2 H2O (equation A)

GSSG + NADPH + H+ 2 GSH + NADP+ (equation B)

Based on above literature it could be understood that, the diabetic

nephropathy is associated with oxidative stress leads to abnormal

generation of free radicals, so to counteract the oxidative stress, there

has been need to use of externally administered antioxidants to

attenuate diabetic nephropathy as well as to control the upcoming

complications, for these reasons, antioxidant especially vitamins

become a key element in prevention and reversal of diabetic

nephropathy especially combination of vitamin E and C plus reduced

glutathione .

CHAPTER 3 NEED FOR THE STUDY

52

3 NEED FOR THE STUDY

India leads the World with largest number of diabetic subjects earning

the dubious distinction of being termed the “diabetes capital of the

world”. According to the Diabetes Atlas 2011 published by the

International Diabetes Federation, the number of people living with

diabetes is expected to rise 552 million by 2030, unless urgent

preventive steps are taken. In diabetes, the worsen condition of

sufferers is mostly due to poor control of complications associated with

diabetes even though huge facilities available to control its growth rate.

Thus, apart from managing diabetes alone, it becomes necessary to

control its associated complications such as nephropathy which is

associated with oxidative stress because chronic hyperglycemia

increases oxidative stress leads to abnormality in structure and function

of proteins and lipids. These modified products could contribute to the

morphological and functional abnormalities seen in the kidney of

patients with diabetes. Management of diabetic nephropathy is

extremely expensive and frustrating. Therefore, prevention is better by

supplementation of antioxidants, especially antioxidant vitamins along

with regular antidiabetic therapy. Treating diabetes complications

require correct diagnosis, exercise and sticking to the strict drug-dose-

food-intake regimen. There has been a dearth of clinical data which

would support the therapy in treating diabetic patients, throw light on its

pros & cons. The current study highlights the effect of antioxidant

CHAPTER 3 NEED FOR THE STUDY

53

vitamins in nephropathy. For these reasons, there has been need in the

use of externally administered antioxidants to attenuate diabetic

nephropathy as well as to control the worsen condition of the patients.

This could support for treating diabetes along with OHA to reduce the

doses of other therapies, it would also excel in assorting confidence in

the diabetic population regarding the therapy because supplementation

of antioxidants when it comes to treat patients with diabetes which is

claimed to be devoid less of side-effects.

CHAPTER 4 OBJECTIVES AND HYPOTHESIS

54

4 OBJECTIVES AND HYPOTHESIS

To study the effect of antioxidants supplementation on

microalbuminuria level in patients with diabetic nephropathy.

To measure the effectiveness of antioxidants for diabetic

nephropathy by analyzing various renal parameters such as

(a) Microalbuminuria

(b) Serum albumin

(c) Urine albumin

(d) Urine creatinine

(e) Serum creatinine

(f) Blood urea nitrogen

To achieve good glycemic control by monitoring fasting plasma

glucose (FPG), postprandial plasma glucose (PPG) & Glycosylated

hemoglobin(HbA1c).

CHAPTER 5 METHODOLOGY

55

5 METHODOLOGY

This study was undertaken to further substantiate the effects of

externally administered antioxidants on the renal function of the patients

who have been diagnosed to have macroalbuminuria.

A total of 216 Indian diabetic patients were enrolled in the study on the

basis of inclusion & exclusion criteria. Both men and women patients

were enrolled after signing the informed consent form from inpatient as

well as outpatient departments of the hospital. The present study was a

randomized, open controlled clinical trial conducted on type 2 diabetic

patients aged 35–60 years who have had diabetes for at least 5 years

with renal function of the patients who have been diagnosed to have

microalbuminuria (Urine albumin excretion >20 mg/dL). Each patient

was interviewed for their past medical as well as medication history for

diabetes before participation in the study.

5.1 Permission from Institutional Human Ethical Committee &

Hospital Authority: This research study protocol were obtains an

approval from Institutional Human Ethics Committee of R. C. Patel

Institute of Pharmaceutical Education & Research previously known as

R. C. Patel College of Pharmacy, Shirpur, Dhule, Maharashtra, India.

(Reference number Annexure-I RCPCOP/IHEC/2008-09/11).

It is customary that every research work carried out in the hospital has

to be approved by the hospital authority and should be informed to

concern physician and other healthcare professionals of the hospital. So


56

a protocol of the study which included the objectives, methodology, and

detailed procedure was submitted to the medical superintendent of the

hospital and the authorization from the medical superintendent was

procured.

5.2 Patient Data Collection: The patient data entry form called

proforma was prepared in order to collect all the essential information of

the patients such as patients name, age, sex, weight, height, body mass

index (BMI), address, blood pressure, social history, family history,

associated diseases, present complaints etc. (at every visit to hospital).

It also includes all the records of biochemical as well as hematological

parameters such as renal parameters, fasting plasma glucose,

postprandial plasma glucose, glycosylated hemoglobin (HbA1c) etc.,

follow up medication details was also included in proforma. The patients

when came to the diabetic nephropathy were observed for weight,

height and blood pressure measurement and their initial sign and

symptoms, which is helpful for the determination of body mass index.

The clinical examination of patients was done and it was confirmed by

urine and serum tests (Annexure- II).After confirmation of diagnosis with

diabetic nephropathy; patients were prescribed with antioxidants

therapy.

5.3 Informed Consent Process: Informed consent is documented by

means of a written, signed and dated. All the patients were enrolled in

the study after explanation of research procedure and at the last by


57

getting their written informed consent. Process by which a subject

voluntarily confirms his/her willingness to participate in a particular

clinical study after having been informed of all aspects of the study like

purpose of the study, its benefits, procedures and hazards of the trial.

The Subjects were free to leave the trial at any time.

5.4 Study Design: After the run-in period, patients entered a

randomized open controlled trial and were randomly assigned in to four

groups, first group considered as positive control group (n=54), second

group considered as vitamins groups (n=54) in which patients received

combination of antioxidant vitamin E plus vitamin C. Third group

considered as reduced glutathione group (n=47) and fourth group was

combination of antioxidant vitamin E and vitamin C plus reduced

glutathione (n=61). All four groups received treatment for diabetes and

ACE, ARB therapies. Each subjects of second group was received one

tablet (vitamin E-400 mg and vitamin C-500 mg) per day for a period of

4 months. Each subject of third group received one tablet of reduced

glutathione 50 mg (Incyto WANBURY) per day whereas subjects of

fourth group received reduced glutathione along with vitamin E plus

vitamin C. Patients were instructed to take the tablets as per physician

direction. During study, patient’s urine and serum samples were

collected for evaluation of various biochemical as well as hematological

parameters were done at the beginning of the run-in phase at

randomization and at the end of each treatment period.


58

Figure 5.1 Screening of diabetic nephropathy patients

Figure 5.2 Design of study

CONTROL GROUP

VITAMIN GROUP

GLUTATHIONE GROUP

GLUTATHIONE+ VITAMINS

4 MONTHS

DURATION OF THERAPY

B

A

S

E

L

I

N

E

F

I

N

A

L

CONTROL

GROUP

54 patients

VITAMINS

+ GLUTATHIONE

61 patients

GLUTATHIONE

GROUP

47 patients

VITAMIN

GROUP

54 patients

Urine Albumin Excretion

>20 mg/dL

Base line readings of renal and serum parameters

4 Months follow-up period

Final readings of renal and serum parameters


59

5.5 Study Site: Patients were recruited from the outpatient and

inpatient department of Indira Gandhi Memorial Hospital, Shirpur

Maharashtra (INDIA).

5.6 Inclusion Criteria: Type 2 diabetic patients according to WHO

definition and albumin excretion rate in the range >20 mg/dL with ACE-

inhibitors or ARBs is continued for all four groups and hypertensive

patients84-85.

5.7 Exclusion Criteria: Systolic blood pressure higher than 200 mmHg

or diastolic blood pressure higher than 110 mmHg, prior myocardial

infarction or congestive heart failure, prior dialysis or kidney

transplantation, known cause of albuminuria other than diabetes and

history of allergic or adverse response to any vitamin, requiring renal

replacement therapy within 1 year, ESRD (End stage renal disease)

and less life expectancy of the patients84-85.

5.8 Biochemical and Haematological Estimations: The urine and

serum samples were analyzed for various haematological and renal

biochemical parameters64,86.

5.8.1 Specimen Collection and Storage: Appropriate sample

acquisition, storage, and handling are essential. However, it is crucial

that all specimens to be used for experimental investigation have been

collected from the appropriate patients using standardized protocols

and placed in the appropriately labeled specimen collection tubes and

stored in a manner that maintained specimen integrity. In this study,


60

blood samples were obtained from selected diabetic subjects by a

research nurse in Indira Gandhi Memorial Hospital, Shirpur. Blood

samples were collected in clean polypropylene tubes, labeled and

biochemical investigations were done. Although in most cases urine is

an easier specimen to obtain than blood, collection requirements must

also be strictly adhered. Furthermore, all urine specimens appropriately

stored to minimize the risk of growth. In this study, urine specimens

were collected in clean polypropylene tubes from selected diabetes

subjects in the hospital. Urine specimens were labeled and biochemical

investigations were done. All specimens (blood and urine) were

alequoted into 2 ml and storage at -20°C. All frozen specimens were

thawed completely and mixed well before the appropriate analysis was

performed.

Various laws and regulations apply to the shipment and or

transportation of biological specimens. It is very important factor that

appropriate temperature must be maintained during the transportation

of the clinical specimens. Commercially available containers with dry ice

were used for the transportation of all specimens (blood and urine) for

analysis.

5.8.2 Determination of Blood Glucose Level (fasting &

postprandial): Glucose is a major carbohydrate present in the blood

and serves as primary energy source. It is usually obtained from


61

ingested starch and sugar. The glucose concentration is normally

maintained at constant level.

Principle: The substrate β D- glucose is oxidized by glucose oxidase to

form gluconic acid and hydrogen peroxide. The hydrogen peroxide so

generated oxidizes the chromogen system consisting of 4-Amino

antipyrine and phenolic compound to a red quinoeimine dye. The

intensity of the color produced is proportional to the glucose

concentration.

GOD

Glucose + O2 + H2O Gluconic acid + H2O2

POD 2 H2O2 + 4 Aminophenazone + Phenol Quinonimine+ 4 H2O

Method: GOD-PAP Method was used to estimate the blood glucose

level. It was estimated by using commercially available standard kit

manufactured by AGAPPE Diagnostic Ltd, Ernakulum, Kerala, India.

Determination of blood glucose level was done by using Micro plate

Reader biochemistry analyzer (Bio Tek).


62

Table 5.1 Assay parameters for blood glucose determination

Parameters Inference

Mode of reaction End Point

Slope of reaction Increasing

wavelength (nm) 505 nm (490-550)

Temperature 37° C

Standard Concentration 100 mg/dL

Linearity 600 mg/dL

Blank Reagent

Incubation time (min) 10 min

Sample volume (µl) 10 µl

Reagent volume (µl) 1000 µl

Cuvette 1 cm light path

Table 5.2 Procedure for Blood glucose test

Reagents Blank Standard Test

Working Reagent 1000 µl 1000 µl 1000 µl

Standard _ 10 µl _

Test _ 10 µl

Mix and incubate for 10 minutes at 37°C. Measure the absorbance of

test and standard against the reagent blank.

Calculation for blood glucose level:

Absorbance of test Blood glucose level (mg/dL) = * 100 Absorbance of standard


63

Normal Values: Fasting blood glucose- Up to 100 mg/dL

Postprandial blood glucose-100-140 mg /dL

Clinical Significance: Elevated blood glucose level is found in diabetes

mellitus, hyperthyroidism, hyperadrenalism, and certain liver diseases.

Decreased levels are found in insulinoma, hypothyroidism etc.

5.8.3 Micral (Urine Albumin and Serum Albumin)

Urine Albumin: This test is most often done to detect diabetic

nephropathy in a person who has had diabetes for several years.

Detectable levels of the protein albumin in the urine signal the

beginning of a condition called microalbuminuria and are typical in

disorders such as diabetic nephropathy. It is one of the best markers to

show an early indication of deteriorating renal function and increased

vascular permeability. It may also have an interesting potential for

monitoring blood pressure treatment. It is known that patients with high

blood pressure levels have increased levels of albumin, or

microalbuminuria, and this in itself can be toxic to the kidneys.

Normal Values: Albumin>20 mg/dL or 30-300 mg of albumin in two

different 24-hour urine samples is considered microalbuminuria.

Serum Albumin: The serum albumin test measures the amount of

albumin in serum, the clear liquid portion of blood. This test helps in

determining if a patient has liver disease or kidney disease or if not

enough protein is being absorbed by the body. Albumin is the protein

having highest concentration in plasma and it transports many small

http://www.nlm.nih.gov/medlineplus/ency/article/000494.htm




64

molecules in the blood (for example bilirubin, calcium, progesterone,

and drugs). It is also of prime importance keeping the fluid from the

blood from leaking out into the tissues. This is because, unlike small

molecules such as sodium and chloride, the concentration of albumin in

the blood is much greater than it is in the fluid outside of it. Because

albumin is made by the liver, decreased serum albumin may result from

liver disease.

Principle: Determination of albumin in serum or urine is based on the

binding behaviour of albumin with 33’ 55’ tetrabromo M-cresol

sulfopthalein (BCG) in acidic medium at pH 4.2. The blue green

coloured complex is formed, the conc. of which is proportional to the

albumin present in the sample.

Acidic medium

Albumin + BCG → Albumin-BCG Complex

Method: End point method was used to estimate the blood glucose

level by using commercially available standard kit manufactured by

AGAPPE Diagnostic Ltd, Ernakulum, Kerala, India. Determination of

albumin level was done by using Micro plate Reader biochemistry

analyzer (Bio Tek).


65

Table 5.3 Assay parameters for blood albumin determination


Mode of reaction End Point


wavelength (nm) 630 nm

Temperature Room Temperature

Standard Concentration 3 g/dL

Linearity 6 g/dL

Blank Reagent

Incubation time (min) 01 min




Table 5.4 Procedure for blood albumin test

Reagents Blank Standard Test

Working Reagent 1000 µl 1000 µl 1000 µl

Standard _ 10 µl _

Sample _ 10 µl

Mix and incubate for 1 minute at 37°C. Measure the absorbance of

sample and standard against the reagent blank.

Calculation for albumin level:

Absorbance of sample Albumin concentration (g/dL) = * 3 Absorbance of standard


66

Calculation for globulin level: Serum globulins=Total protein-Albumin

Normal Values: The normal range is 3.4 - 5.4 g/dL

Significance: Lower-than-normal levels of albumin may indicate:

Glomerulonephritis, malnutrition, diabetic nephropathy syndrome

5.8.4 Serum Creatinine: Creatinine is a breakdown product of creatine

which is an important part of muscle. This test measures the amount of

creatinine in the blood which is used to evaluate kidney function.

Creatinine can be converted to the ATP molecule, which is a high-

energy source. The daily production of creatine and subsequently

creatinine depends on muscle mass which fluctuates very little. It is

excreted from the body entirely by the kidneys. With normal renal

excretory function, the serum creatinine level should remain constant

and normal.

Principle: Creatinine reacts with picric acid in alkaline medium to form

an orange coloured complex and the rate of change of this absorbance

is measured at 505 nm at predetermined interval of time. The

concentration of picric acid and sodium hydroxide as well as reaction

time has been optimised in this method to avoid Interference by CLIF

(creatinine like interfering factor) in sample.

NaOH Creatinine + Picric acid → Orange Coloured Complex

Method: Modified Jaffes method was used to estimate the urine

creatinine level by using commercially available standard kit

manufactured by AGAPPE Diagnostic Ltd, Ernakulum, Kerala, India.


67

Determination of serum creatinine level was done by using Micro plate

Reader biochemistry analyzer (Bio Tek).

Table 5.5 Assay parameters for serum creatinine determination


Method Modified Jaffes


wavelength (nm) 492 nm/505 nm

Temperature 37° C

Standard Concentration 2mg/dL

Linearity Up to 24 mg/dL

Blank D I water

Delay time 60 seconds

Interval 60 seconds




Table 2.6 Procedure for serum creatinine test

Reagents Standard Sample

Working Reagent 1000 µl 1000 µl

Standard 100 µl _

Sample - 100 µl


68

Mix and read the optical density (T1) 60 seconds after the sample or

standard addition, exactly 60 second after the first reading take second

reading (T2).

Calculation for serum creatinine level:

(T2-T1) of sample Creatinine concentration (mg/dL) = * 2 (T2-T1) of standard Normal Values: A normal (usual) value is 0.8 to 1.4 mg/dL. Normal

value ranges may vary slightly among different laboratories.

Significance: Higher than normal levels may indicate: Acute tubular

necrosis, diabetic nephropathy, glomerulonephritis, pyelonephritis,

reduced renal blood flow (shock, congestive heart failure), renal failure

and urinary tract obstruction.

5.8.5 Urine Creatinine: Principle for urine creatinine estimation is same

as that of estimation of serum creatinine.

Method: Method for urine creatinine estimation is same as that of

estimation of serum creatinine.

Preparation of Working Reagent: Mix 1 volume of (R1) with 1 volume

reagent 2 (R2). Urine sample should be diluted 100 times with distilled

water prior to use and observed results should be multiplied by 100.

Urine (diluted 1/100 with distilled water).


69

Table 5.7 Assay parameters for urine creatinine determination


Method Modified Jaffes


wavelength (nm) 492 nm/505 nm

Temperature 37° C

Standard Concentration 2mg/dL


Blank D I water


Interval 60 seconds




Table 5.8 Procedure for urine creatinine test

Reagents Standard Sample

Working Reagent 1000 µl 1000 µl

Standard 100 µl _

Sample - 100 µl

Mix and read the optical density (T1) 60 seconds after the sample or

standard addition, exactly 60 second after the first reading take second

reading (T2).


70

Calculation for urine creatinine level:

(T2-T1) of sample Creatinine concentration (mg/dL) = * 2 (T2-T1) of standard Normal Value: A normal (usual) value is 0.6 to 1.2 mg/dL. Normal

value ranges may vary slightly among different laboratories.

5.8.6 Blood Urea Nitrogen: Urea nitrogen forms when protein breaks

down. A test can be done to measure the amount of urea nitrogen in

the blood.

Principle: Methods are based on preliminary hydrolysis of urea by

urease followed by an analytical process that quantitates the

ammonium ion

Urease

Urea + 2H2O 2NH4++ CO3

- -

Method: Ultraviolet method was used to estimate the BUN level by

using commercially available standard kit manufactured by AUTOPAK,

Siemens healthcare diagnostic Ltd, Baroda, Gujarat, India.

Determination of BUN level was done by using Micro plate Reader

biochemistry analyzer (Bio Tek).

Preparation of Working Reagent: Dissolve the content of one bottle of

reagent 1 with one bottle of reagent 1A. Mix by gentle swirling.


71

Table 5.9 Assay parameters for BUN determination


Mode of reaction UV

Reaction type Fixed time

Slope of reaction Decreasing

wavelength (nm) 340 nm

Flow cell Temperature 30° C

No. of reading 2


Interval 60 seconds



Reagent volume 1 ML

Standard conc. 20 mg/dL

Zero setting with Distilled water

Table 5.10 Procedure for BUN test

Reagents Test

Reconstituted Reagent 1 mL

Sample 10 µl

Mix and read immediately

Normal Values: 7-20 mg/dL. Note that normal values may vary among

different laboratories.


72

Significance: Higher-than-normal levels may be due to: Excessive

protein levels, Heart attack, and Kidney disease, including

glomerulonephritis, pyelonephritis acute tubular necrosis and urinary

tract obstruction where as lower levels may be due to malnutrition,

over-hydration.

5.8.7 Total Protein: The total protein test is a rough measure of all the

proteins found in the fluid portion of blood. Proteins are important parts

of all cells and tissues. This test is often done to diagnose nutritional

problems, kidney disease or liver disease.

Principle: Protein, in an alkaline medium, binds with the cupric ion

present in the biuret reagent to form a blue- violet coloured complex.

The intensity of coloured from is directly proportional to amount of

proteins present in the sample.

Proteins + Cu++ → Blue violet coloured complex

Method: End point method was used to estimate the total protein level

by using commercially available standard kit manufactured by Span

Diagnostic Ltd, Surat, Gujarat, India. Determination of total protein level

was done by using Micro plate Reader biochemistry analyzer (Bio Tek).


73

Table 5.11 Assay parameters for total protein determination


Mode of reaction End point

wavelength (nm) 578 nm (550-580 nm)

Flow cell Temperature 37° C

Optical path length 1 cm

Blanking Reagent blank

Linearity Up to 20 g/dL

Incubation time 5 min.


Reagent volume 1000 µl

Standard conc. 6.5 g/dL

Stability of final colour 2 hrs

Permissible reagent blank

absorbance

<0.2 AU

Units g/dL

Table 5.12 Procedure for total protein test

Pipette into tube Blank Standard Test

Serum/plasma - - 10 µl

Reagent 2 - 10 µl -

Reagent 1 1000 µl 1000 µl 1000 µl


74

Mix well and incubate at 37°C for 5 min. Programme the analyser as per

assay parameters such as blank the analyser with reagent blank and

measure absorbance of standard followed by test.

Calculation for total protein:

Absorbance of test Total protein concentration (g/dL) = * 6.5 Absorbance of standard

Normal Values: The normal range is 6.0 to 8.3 gm/dL

Significance: Higher-than-normal levels may be due to: Excessive

protein levels, Hypovolemia, and heart attack, kidney disease including

glomerulonephritis, pyelonephritis and acute tubular necrosis, kidney

failure, urinary tract obstruction whereas lower-than-normal levels may

be due to low protein diet leads to malnutrition.

5.8.8 HbA1c: The measurement of HbA1c is recommended for

monitoring the long term care of people with diabetes mellitus. The

HbA1c reflects the average level of blood sugar concentration within the

red blood cell over the previous 2-3 months, so it is recommended to

measure the HbA1c concentration after every 3 months. The level of

HbA1c rises proportionately in patients with higher level of blood sugar

such as those with uncontrolled or undiagnosed diabetes.

Principle: The HbA1c test (hemoglobin A1c, glycosylated hemoglobin

A1c, glycohemoglobin A1c, or A1c test) is a lab test, which reveals

average blood glucose over a period of two to three

months. Specifically, it measures the number of glucose molecules


75

attached to hemoglobin, a substance in red blood cells. People who do

not have diabetes generally have an HbA1c level of less than 6 %. This

means that less than 6 % of their hemoglobin molecules have glucose

permanently attached (normal reference value 4-6 %).

Based on the results of studies such as the Diabetes Control and

Complications Trial (DCCT), which showed that tight blood glucose

control could reduce the risk of diabetic eye, kidney and nerve disease,

the American Diabetes Association (ADA) recommends that people with

diabetes try to keep their HbA1c level below 7 %. Patient’s daily blood

glucose tests provide only a snapshot of glycemic control at the moment

of test. The HbA1c test, on the other hand, gives the big picture by

showing how patient blood glucose control has been over the previous

couple of months. It is helpful to physician because they give an

immediate indication of patient blood glucose control. Over a longer

period of time, consecutive HbA1c tests may provide an overall trend in

patient diabetes control. If HbA1c is progressively rising each time, there

is a need to modify treatment plan of patient.


76

Figure 5.3 Diabetes control card

HbA1c (%) Materials and Method: It was estimated by using commercially

available standard kit supplied by Biorad Laboratories Ltd, Gurgaon,

Haryana, India.

The Micromat II HbA1c test uses boronate affinity chromatography to

separate the glycated hemoglobin fraction from the non-glycated

fraction. After a test cartridge has been placed into the instrument, a

small sample of blood is added to the first sample tube. The blood is

instantly lysed to release the hemoglobin and the boronate affinity resin

binds the glycated hemoglobin. After a short incubation step, the liquid

is poured into the central funnel of the test cartridge and non-glycated

fraction is collected in an optical chamber where the hemoglobin

concentration is photometrically measured. The glycated hemoglobin

remains bound to the boronate affinity resin, which sits at the bottom of

test cartridge funnel. The boronate affinity resin is then washed with


77

content of second tube. The final step is elution of the glycated

hemoglobin off the boronate affinity resin using the third tube. The

glycated hemoglobin concentration is measured and the HbA1c

concentration in the sample is calculated by the instrument.

Reagents in the Cartridge

Each cartridge contains 3 tubes of buffer solution.

1. The first tube (Red capped tube) contains boronate affinity resin,

surfactant, 20 mM Herpes buffer and sodium azide (≤ 0.1 %).

2. The second wash buffer tube (Blue capped tube) contains 20 mM

Herpes buffer and sodium azide (≤ 0.1 %).

3. The third Elution buffer tube (Clear capped tube) contains

surfactants, 50 mM ammonium acetate buffer and phenoxyethanol.

Procedure for HbA1c determination:

Step 1: The cartridges were placed into the instrument and

immediately push it down until it clicks into place. The instrument was

then check that the test cartridge was valid. When this check was

completed, in several seconds, an audible beep and flashing red light

was appear at the position 1.

Step 2: The white rim of the test cartridge was hold and rotate it

clockwise through 900 to position 1. The test cartridge was click into its

new position and first sample tube was rise from the cartridge. The

sample tube was removed from the test cartridge and unscrewed the

cap. The blood was taken from the patient by pricking the finger of the


78

patients with sterile needle. The Microsafe pipette was touch the small

drop of blood on patient’s finger, leaving the pipette in contact with the

blood until the blood covers the air hole. The tip of the Microsafe

pipette was placed into the liquid of the sample tube and squeezes the

bulb to release the blood. The cap was replaced and mixes the

contents by gently inverting the tube 5 times. The incubation period

was started immediately pressing the enter button. A 60 second

countdown was appears on display.

Step 3: The end of the incubation period was indicated by an

intermittent beep and the appearance of the “insert/mix” and “pour

reagent” icons on display. Remix the content of the tube by gently

inverting it 3 times. Remove the cap and pour the entire content into the

central funnel of the test cartridge. Replace the cap on the tube and

place the tube back in the cartridge.

Step 4: The contents of tube was poured into the funnel, a 50 second

countdown was appear while the instrument takes a measurement.

After this intermittent beep was sound, the light was flash at position 2

and the “rotated cartridge” was appear on the display.

Step 5: The cartridge was rotated clockwise through 900 to position 2,

the second tube was rise from the cartridge, the cap was unscrew and

the entire content was poured into the central funnel of test cartridge.

The enter button was pressed; a 40 second countdown was appear on


79

the display, during which the liquid was gradually disappear into the test

cartridge.

Step 6: At the end of countdown, the light was flash at position 3; the

cartridge was rotated to the position 3. The 3rd tube was rise from the

cartridge. When prompted by the “pour reagent” icon, the tube was

removed from the cartridge and the entire content was poured into the

central funnel. Again the instrument automatically senses the liquid and

the intermittent beeps continue for up to 20 seconds while the

instrument takes reading. The final 80 second countdown was then

appears on display.

Step 7: At the end of countdown, the rotated icon appears on the

display. The test cartridge was rotated to its starting position and

removed from the instrument. Upon removal of the test cartridge, the

instrument displays the percentage HbA1c value for the sample. The

enter button was pushed to allowed a new test to be carry out.

Normal Values: 6–7%. Note that normal values may vary among

different laboratories.


80

Figure 5.4 Bio-rad micromat II HbA1c monitoring instrument

5.8.9 Blood pressure estimation: Blood pressure is a measurement of

the force applied to the walls of the arteries as the heart pumps blood

through the body. The pressure is determined by the force and amount

of blood pumped and the size and flexibility of the arteries. Blood

pressure is continually changing depending on activity, temperature,

diet, emotional state, posture, physical state, and medication use.

Significance: High blood pressure increases the risk of heart failure,

heart attack, stroke, and kidney failure.

Normal Values: In adults, the systolic pressure should be less than 120

mmHg and the diastolic pressure should be less than 80 mmHg.

5.9 Statistical Analysis

All the biochemical as well as hematological parameters means and

differences were determined by Paired t- Test. The measurements of all

parameters were done at the start of the run-in phase at randomization

and at the end of each treatment during the study period.





CHAPTER 6 RESULTS AND DISCUSSION

81

6 RESULTS AND DISCUSSION

This study was undertaken to demonstrate the possessions of orally

administered antioxidants on the renal function of the patients who have

been diagnosed to have microalbuminuria. This study demonstrate

therapeutic value of orally administered vitamin ‘E’, vitamin ‘C’ and

reduced glutathione in patients with diabetic nephropathy.

It is important to note that diabetic nephropathy is a multistage condition

that takes several years to become clinically overt. At the onset of

diabetes, there are usually changes in renal function such as glomerular

hyperfiltration, increased renal blood flow and hypertrophy of the

kidney91.Most of these changes can be reversed at an early stage by

good glycemic control but they persist in many patients and may be

important in the later development of clinical nephropathy6.Traditionally,

nephropathy is divided into two types based on microalbuminuria,

incipient and overt nephropathy usually occurs after 6-15 years of

diabetes7.

Persistent microalbuminuria is universally considered as being the first

clinical sign of diabetic nephropathy92,93. Although microalbuminuria

may progress to overt nephropathy, it has been shown that at these

state glomerular changes can be modified and restoration of impaired

glomerular filtration can be achieved. Several mechanisms have been

proposed as being involved in the onset and progression of diabetic-

related nephropathy94.Among them, increased oxidative stress


82

associated with diabetic-related chronic hyperglycemia seems to play

the most important role.

Microalbuminuria is the first sign of deteriorating kidney function. As

kidney function declines, the amount of albumin in the urine increases

and microalbuminuria becomes proteinuria. The level and type of

proteinuria strongly determine the extent of damage and a person in at

risk for developing progressive kidney failure93.

Figure 6.1 Origin of microalbuminuria

Decreased antioxidant status has an important role in development of

diabetic nephropathy, so antioxidant treatment could become a key

element in prevention and reversal of diabetic nephropathy. Vitamin E

ameliorated increase in GFR after 2 weeks of diabetes and reduced

albumin in urea at 10 weeks. In addition to well known effect of

antioxidant effect, direct increase in (DAG) diacylglycerol kinase in

inhibition of PKC may contribute to protective effect of vitamin E on

diabetic nephropathy60.


83

Priya et.al, 2009 have confirmed that the role of free radical-mediated

damage on lipids (measured as MDA levels) and effect on antioxidants

(vitamin C and vitamin E) defense mechanisms was studied in chronic

renal failure (CRF) patients before dialysis to show the role of

antioxidant in preventing the progression of CRF and for monitoring and

optimization of antioxidant therapy. CRF is associated with impaired

endothelium dependent vasodilation and accelerated atherogenesis79.

ROS modify endothelial function in renal failure and it was found that

vitamin C reduces oxidative stress in CRF and improves NO- mediated

resistance vessel dilation; similarly glutathione reduces the oxidative

stress in diabetes patients71. Costagliola et.al, 1992 has demonstrate

that, the therapy on patients undergoing treatment with reduced

glutathione (1200 mg/day) could represent it to be a useful drug in the

treatment and management of anemia in patients with chronic renal

failure71 but in our study we have used low dose of vitamin E-400 mg

and vitamin C-500 mg, glutathione 50 mg per day for a period of 4

months.

It is well known that free radical production is increased in diabetes

patients and might play a role in the genesis of late diabetic patients.

Mattia et.al, 1998 found that one hour GSH infusion (1.35 gm/m2/hr)

simultaneously improves the intraerythrocytic GSH/SSH ratio and

insulin sensitivity in NIDDM patients and reduces the oxidative stress81.


84

Glutathione deficit contributes to the oxidative stress and plays a key

role in the pathogenesis of many diseases including diabetes82. Hagen

et al, 1988 has proved the glutathione uptake and protection against

oxidative injury in isolated kidney cells. This study indicates that GSH

may be taken up intact from the diet, providing a means by which GSH

may be therapeutically administered to augment plasma GSH

concentrations. This may be significant in those pathological conditions

where hepatic GSH efflux is impaired. Under the conditions of oxidative

stress the declined GSH is thought to lead to deleterious oxidative

processes83. So as discuss earlier, antioxidants play key role to counter

act diabetic nephropathy.


85

Table 6.1 Effect of antioxidants on renal and hematological parameters in DN

Paired t- Test: *p < 0.05 (Significant); **p < 0.01 (Very significant); ***p < 0.001 (extremely significant)

Parameters Control group Vitamin group Glutathione group Vitamin plus glutathione

Base Final Base Final Base Final Base Final

Microalbuminuria 33.8±2 34.1±1 33.2±2 30.5±2** 31.4±2 30.1±1** 33.3±2 27.2±2***

FPG 162 ± 31.11 161 ± 1.11 150 ± 31.30 148 ± 35.11 157 ± 31.10 150 ± 30.10 161 ± 30.11 149 ± 20.10

PPG 246 ± 34.27 239 ± 26.65 240 ± 33.27 235 ± 30.10 241 ± 30.20 235 ± 30.11 240 ± 27.10 221 ± 27.10

Urine creatinine 0.933±3 0.937±3 0.949±4 0.86±3.9 0.916±2 0.89±2 0.939±3 0.742±3*

Serum creatinine 1.1±0.06 1.2±0.06* 1.4±0.1 1.1±0.1* 1.1±0.1 1.2±0.08 1.2±0.05 0.8±0.04*

BUN 30.9±2.2 29±2.1 30.6±3 27±3 29.2±2.9 27.4±2.9 29±2 21.3±2*

Serum albumin 4.4±0.2 4.1±0.2* 4.8±0.1 4.5±0.2** 3.9±0.2 3.9±0.1 4.5±0.2 4.1±0.2*

Serum globulin 2.7±0.2 2.5±0.1 2.8±0.2 2.7±0.1 2.4±0.2 2.6±0.2 2.8±0.3 2.6±0.2*

HbA1c 8.7±0.4 8.7±0.4 7.2±0.5 7.1±0.5** 7.5±0.2 6.8±0.2* 6.9±0.4 6.5±0.3*

Total protein 7.5±0.3 7.5±0.3 8±0.4 7.6±0.3** 7.1±0.2 7.1±0.2 7.8±0.3 7.5±0.3*

Systolic BP 157.7±7 148±6* 129.2±5 131.2±4 155.7±6 153.6±5 145.7±7 147.1±4

Diastolic BP 88.8±2 85.8±2 83.8±2 85±2 88.6±3 86.4±2 85±3 83.9±3


86

6.1 Effect of Antioxidants on Microalbuminuria in DN

Detectable levels of the protein albumin in the urine signal the beginning

of a condition called microalbuminuria diabetic nephropathy. It is one of

the best markers to show an early indication of deteriorating renal

function and increased vascular permeability. Protective effects of

treatment with vitamin C and E on renal injury in diabetic nephropathy

patients have been reported; it also reduced albumin excretion rate.

GSH is an extremely important cell protectant; it directly quenches

reactive hydroxyl free radicals, oxygen-centered free radical, radical

centers on DNA and other biomolecules78.

Although previous study demonstrates pharmacological dose of

reduced glutathione, vitamin E, vitamin C supplementation improves

renal function by reducing the albumin excretion rate and oxidative

stress82,85 but the, supplementation of vitamin E alone in patients with

diabetes and vascular disease had no significant effect on micro

vascular outcomes including nephropathy57. In a previous study it was

reported that the high doses of vitamin E (dose >1000 mg/day) and

vitamin C (dose >1000 mg/day) reduce albumin excretion rate68 but in

our study we have used low dose of vitamin E (400 mg) and C (500 mg)

plus reduced glutathione as compare to earlier studies because our

intention was to reduce the doses of other agents as well as resistance

of the patients and it was observed that four months vitamin and

reduced glutathione treated groups shows significant (p<0.009 and


87

p<0.002) decreased microalbuminuria levels from 33.2±2 to 30.5±2 and

31.4±2 to 30.1±1 respectively as compared to control group.

Interestingly, treatment of glutathione plus vitamins exhibited significant

decreased (p<0.0001) values from 33.3±2 to 27.2±2. (Figure 6.2).

Because GSH is an extremely important cell protectant, it directly

quenches reactive hydroxyl free radicals, oxygen-centered free radical,

radical centers on DNA and other biomolecules which shows protective

effects in DN.

Figure 6.2 Effect of antioxidants on microalbuminuria in DN

Values are expressed as mean ± SEM. Significance of difference in the means was determined by Paired t- Test. (*p< 0.05; **p< 0.01;***p< 0.001)

33.8±2 33.2±2 31.4±2

33.3±2 34.1±1

30.5±2**

30.1±1** 27.2±2***

0

5

10

15

20

25

30

35

40

Control group Vitamin group Glutathione group Gluta+ Vit group

Mic

roal

bu

min

uri

a m

g/d

l

Microalbuminurea

Base line Final


88

6.2 Effects of Antioxidants on Fasting and Postprandial Blood

Glucose in DN

The tight glycemic control and reduction of elevated lipid levels are

primary goals in the prevention of cardiovascular complications in type 2

diabetics. Poor glycemic controls in type 2 diabetes associated with

hyperlipidemia are independent risk factors for cardiovascular events.

Thus, an ideal therapy required suitable antidiabetic agents along with

antioxidants; it would improve both glycemic control and dyslipidemias.

In the present study a slight significant positive correlation was noted in

mean fasting and postprandial plasma glucose level between baseline

and at the end of study (Table 6.1).

6.3 Effects of Antioxidants on Urine Creatinine & Serum Creatinine

in DN

Creatine is synthesized in the liver, pancreas, and kidneys from the

amino acids and it is transported through the circulatory system to

muscle, brain and other organs, where it is converted to

phosphocreatine and acts as an energy reservoir much like ATP. The

creatinine enters the blood supply, where it is removed through the

kidneys92. If kidney function is abnormal, creatinine levels will increase

in the blood due to decreased excretion of creatinine in the urine. Its

levels also vary according to a person's size and muscle mass. In

previous study It was found that chronic blockade of the oxidative stress

pathway in renal artery stenosis using oral antioxidant vitamin


89

supplementation improves renal hemodynamics and decreases

oxidative stress, intrarenal inflammation and tubulointerstitial fibrosis in

the kidney32.These underscore the role of increased oxidative stress in

the pathogenesis of ischemic nephropathy and suggest a role for

antioxidant vitamins in preserving the function and structure of the

stenotic kidney33. Short duration of vitamin C and E treatment with

pharmacological doses in type 2 diabetic patients with

microalbuminuria/macroalbuminuria significantly lowers AER33. In

contrast to our study we have used combination of vitamin E and C plus

reduced glutathione to get the synergistic effect and it was observed

that, the effect of antioxidants on urine creatinine in patients with control

group, vitamins group, and reduced glutathione group shows no

significant difference between baseline and final values, whereas, four

months treatment of vitamin plus reduced glutathione treated groups

shows significant (p<0.05) differences in urine creatinine from 0.939±3

to 0.742±3 (Figure 6.3). In contrast, the effect of antioxidants on serum

creatinine shows no significant results in patients with control group and

glutathione group but significant observation was found in vitamins

group and glutathione plus vitamins group because of synergistic action

of combination therapy from (p<0.05 and p<0.01) decline from1.4±01 to

1.1±01 and 1.2±0.05 to 0.8±0.04 respectively (Figure 6.3 and 6.4).


90

Figure 6.3 Effects of antioxidants on urine creatinine in DN


0.933±3 0.949±4 0.916±2 0.939±3 0.937±3

0.86.±3.9 0.89±2

0.742±3*

0.1

0.3

0.5

0.7

0.9

1.1


Uri

ne

Cre

atin

in m

g/d

l Urine Creatinin

Base line Final


91

Figure 6.4 Effects of antioxidants on serum creatinine in DN


6.4 Effect of Antioxidants on Blood Urea Nitrogen (BUN) in DN

Increase in the concentration of urea nitrogen in the blood may indicate

kidney failure86. One of the major functions of the kidney is the

elimination of nitrogenous products of protein catabolism94.Urea is

nitrogenous end products of metabolism which is the primary metabolite

derived from dietary protein and tissue protein turnover. Urea is having

relatively small molecular size (60 Daltons) that distribute throughout

total body water. A slight rise of plasma urea and creatinine

concentrations is a common incidental finding, especially in elderly

1.1±0.06

1.4±0.1

1.1±0.1 1.2±0.05 1.2±0.06*

1.1±0.1*

1.2±0.08

0.8±0.04*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


Seru

m C

reat

inin

mg/

dl

Serum Creatinin

Base line Final


92

subjects95. The urea nitrogen test is an indicator of renal disorders and

may be used to determine the source of disorder like prerenal, renal or

postrenal. In present study, the effects of antioxidants on level of BUN

in patients with glutathione plus vitamin group shows significant

difference (p<0.01) in BUN level from 29±2 to 21.3±2. as compared to

control group, vitamin group and glutathione group (Figure 6.5).

Figure 6.5 Effects of antioxidants on blood urea nitrogen in DN


6.5 Effect of Antioxidants on Serum Albumin and Globulin in DN

Determination of albumin helps if a patient has liver disease or kidney

disease76 which allows albumin to escape into the urine .Albumin is the

protein of the highest concentration in plasma and transports many

30.9±2.2 30.6±3 29.2±2.9 29±2 29±2.1

27±3 27.4±2.9

21.3±2*

0

5

10

15

20

25

30

35

40


BU

N m

g/d

l

Blood Urea Nirtogen

Base line Final


93

small molecules in the blood (for example, bilirubin, calcium,

progesterone, and drugs).Decreased lysosomal activity can lead to

decreased albumin fragmentation and increased immunoreactive

albumin excretion. This apparent albuminuria can occur without any

changes in glomerular permeability96.

Figure 6.6 Working model of albuminuria, particularly for albumin

excretion in humans

Previous study shows, an elevated serum albumin level is indicative of

dehydration, in which an insufficient amount of fluid in the blood leads to

increase its level86. The blood protein albumin is found at slightly higher

concentrations in the urine. Because only a small amount of albumin is

excreted, this stage is known as ‘microalbuminuria’. Therefore increase

in serum albumin causes excess excretion of albumin through urine.

The present study shows that antioxidant therapy significantly reduces

the serum albumin in patients treated with control group, vitamin group

and glutathione plus vitamin group shows significant (p<0.05,p<0.001

and p<0.1) decline in serum albumin level from 4.4±0.2 to 4.1±0.2.,

4.8±0.1 to 4.5±0.2 and 4.5±0.2 to 4.1±0.2 respectively (Figure 6.7). In


94

contrast to globulin concentration there was no significant results in

patients with control group, vitamin group, glutathione group as

compared to vitamins plus glutathione combination shows significant

(p<0.05) result from 2.8±0.3 to 2.6±0.2 (Figure 6.8).

Figure 6.7 Effects of antioxidants on serum albumin in DN


4.4±0.2

4.8±0.1

3.9±0.2

4.5±0.2

4.1±0.2*

4.5±0.2**

3.9±0.1

4.1±0.2*

0

1

2

3

4

5

6


Seru

m A

lbu

min

g/d

l

Serum Albumin

Base line Final


95

Figure 6.8 Effects of antioxidants on serum globulin in DN


6.6 Effect of Antioxidants on (Glycosylated Hemoglobin) HbA1c in

DN

The HbA1c test (hemoglobin A1c, glycosylated hemoglobin A1c,

glycohemoglobin A1c, or A1c test) which reveals average blood glucose

over a period of two to three months. Specifically, it measures the

number of glucose molecules attached to hemoglobin. People who do

not have diabetes generally have an HbA1c level of less than 6 %. This

means that less than 6 % of their hemoglobin molecules have glucose

permanently attached97-99. Base on the result of studies such as the

2.7±0.2 2.8±0.2

2.4±0.2

2.8±0.3

2.5±0.1

2.7±0.1 2.6±0.2 2.6±0.2*

0

0.5

1

1.5

2

2.5

3

3.5


seru

m G

lob

ulin

g/d

l Serum Globulin

Base line Final


96

Diabetes Control and Complications Trial (DCCT), which showed that

tight blood glucose control could reduce the risk of diabetic eye, kidney

and nerve disease. The American Diabetes Association (ADA)

recommends that people with diabetes try to keep their HbA1c level

below 7%100. Aggressive glycemic control has been demonstrated to

decrease micro vascular and macro vascular complications, although

the latter claim remains controversial101. The Canadian Diabetes

Association 2003 clinical practice guideline for the prevention and

management of diabetes recommends a target hemoglobin A1c

concentration of 7.0 % or less for all patients with diabetes102. The

UKPDS (United Kingdom Prospective Diabetes Study) confirmed what

was already evident to most physicians in type 2 diabetes i.e. eventually

most patients will not be able to maintain glycemic control only with a

single antidiabetic agent. The UKPDS indicated that by 6 years after the

diagnosis of diabetes more than half of the patients needed

supplementary therapy such as antioxidants along with pharmacological

agent to maintain glycemic control103. Therefore, HbA1c is a useful

indicator of how well the blood glucose level has been controlled in the

recent past and may be used to monitor the effects of diet, exercise,

and drug therapy etc. on blood glucose in diabetic patients104-107. In the

present study a significant positive correlation was noted in mean HbA1c

level between baseline and at the end of study in patients with vitamin

group, glutathione group and glutathione plus vitamin group shows


97

significant difference (p<0.002, p<0.01 and p<0.02) decline in HbA1c

level from 7.2±0.5 to 7.1±0.5, 7.5±0.2 to 6.8±0.2, and 6.9±0.4 to 6.5±0.3

respectively as compare to control group (8.7 ± 0.4 to 8.7 ± 0.4) (Figure

6.9).

It has been suggested that initiating therapy with combination of

antioxidants vitamins have complementary effects can increase the

overall efficacy. The early use of antioxidants with OHA either alone or

in combination is expected to improve both acute and long-term

outcomes in patients with type 2 diabetes108. A once daily dose of

antioxidants has been shown to effectively reduce blood glucose levels

with a more rapid onset of action coupled with a longer duration of

action compared with other OHA alone109-112.


98

Figure 6.9 Effects of antioxidants on HbA1c in DN


6.7 Effect of Antioxidants on Total Protein in DN

The total protein test is a rough measure of all the proteins found in the

fluid portion of blood. It is commonly used for diagnosing and monitoring

various primary and secondary nephropathies. Increased protein level

associated with systemic and metabolic disorders such as hypertension,

diabetes, the kidney may undergo slow but progressive deterioration,

leading eventually to renal failure113. Renal involvement in these

disorders is often first manifested by gradual increase in proteinuria, for

which sensitive assays are therefore desirable86,114. The current study

shows significant difference (p<0.001 and p<0.05) decline in level from

8.7 ± 0.4

7.2±0.5 7.5±0.2

6.9±0.4

8.7±0.4

7.1±0.5** 6.8±0.2*

6.5±0.3*

0

1

2

3

4

5

6

7

8

9

10


Hb

A1

c %

Glycosylated Haemoglobin

Base line Final


99

8±0.4 to 7.6±0.3; 7.8±0.3 to 7.5±0.3 in patients treated with vitamin and

glutathione plus vitamin combination as compared to control and

glutathione group alone (Figure 6.10).

Figure 6.10 Effect of antioxidants on total protein in DN

Values are expressed as mean ± SEM. Significance of difference in the means was determined by Paired t- Test. (*p< 0.05; **p< 0.01;***p< 0.001) 6.8 Effect of Antioxidants on Diastolic and Systolic BP in DN

The relationship between hypertension and poor vascular outcomes

including progression of renal diseases is unequivocal and independent

of other confounding factors. The impact of hypertension on outcomes

is exponential rather than linear. A sustained reduction in blood

pressure seems to be currently the most important single intervention to

7.5±0.3

8±0.4

7.1±0.2

7.8±0.3 7.5±0.3 7.6±0.3**

7.1±0.2 7.5±0.3*

0

1

2

3

4

5

6

7

8

9


Tota

l Pro

tein

g/d

l

Total Protein

Base line Final


100

slow progressive nephropathy in type 1 and type 2 diabetes. Initially

normotensive diabetic subjects without renal disease demonstrate a

blood pressure dependent decline in GFR with blood pressure levels

within the reference range. Patients with a blood pressure

corresponding to 130/80 mmHg rarely develop microalbuminuria and

show an annual decline in GFR close to the age-matched normal

population. Diabetic patients with a blood pressure between 130/80

mmHg and 140/90 mmHg have a greater decline in GFR, with 30% of

patients developing associated microalbuminuria or proteinuria over the

subsequent 12 to 15 years64. The current study shows no significant

difference on systolic as well as diastolic blood pressure with control

group, vitamin group, glutathione and glutathione plus vitamins group

respectively, the reason behind was aggressive glycemic controls

require self care, adherence to the medications, continues monitoring of

BP, glucose level and ideal OHAs along with antihypertensive (Figure

6.11 and 6.12).


101

Figure 6.11 Effect of antioxidants on systolic BP in DN


157.7±7

129.2±5

155.7±6 145.7±7 148±6*

131.2±4

153.6±5 147.1±4

0

20

40

60

80

100

120

140

160

180


Sist

olic

BP

mm

Hg

Systolic Blood Pressure

Base line Final


102

Figure 6.12 Effect of antioxidants on diastolic BP in DN


88.8±2

83.8±2

88.6 ±3

85±3

85.8±2 85±2

86.4±2

83.9±3

74

76

78

80

82

84

86

88

90

92


Dys

tolic

BP

mm

Hg

Dystolic Blood Pressure

Base line Final

CHAPTER 7 CONCLUSION

103

7 CONCLUSION

The most important predictor of diabetic nephropathy is

microalbuminuria which predicts the onset of renal disease in diabetic

patients. Albuminuria reflects glomerular dysfunction in patients with

diabetic nephropathy. The main marker of evaluation of diabetic

patients is long term glycemic control and prediction of risk for

development and progression of diabetic complication.

The present study shows 4 months supplementation of antioxidants

in diabetic nephropathy patients shows significant results on urinary

albumin excretion in patients treated with combination of vitamin E

and C. Interestingly, glutathione plus vitamin E and C exhibited

significant difference as compare to control group.

A Significant correlation was noted in urine creatinine in patients

treated with glutathione plus vitamin E and C. In contrast to serum

creatinine shows positive significant in glutathione plus vitamin E and

C and vitamins group.

Similarly significant results were found in blood urea nitrogen in

patients treated with combination of glutathione plus vitamin E and

C.

Total protein which serves as a marker for glomerular renal function

and blood glucose level. It was observed that, combination therapy

of reduced glutathione plus vitamin E plus vitamin C gives synergistic

CHAPTER 7 CONCLUSION

104

antioxidant activity effect compare to alone administration of either

vitamin E or vitamin C or reduced glutathione .

In contrast to, serum globulin, systolic and diastolic BP shows no

significant change in any treatment groups.

So it has been suggested that initiating therapy with combination of

antioxidants vitamins have complementary effects can increase the

overall efficacy.

The early use of antioxidants with OHAs either alone or in

combination is expected to improve both acute and long-term

outcomes in patients with type 2 diabetes.

A once daily dose of antioxidants has been shown to effectively

reduce blood glucose levels with a more rapid onset of action

coupled with a longer duration of action compared with other OHAs

alone.

These findings also concluded that receiving 400 mg vitamin C, 500

mg vitamin E and 50 mg reduced glutathione decreased the urinary

albumin excretion ratio in diabetic patients.

So it is important to highlight here that such antioxidant therapy, if

started at the earlier stage of disease may help in reduce the harm to

the development of diabetic nephropathy as well other complications. In

this study, patients did not report any side-effects during the course

because supplementation of antioxidants when it comes to treat

patients with diabetes which is claimed to be devoid of side-effects.

CHAPTER 8 REFERENCES

105

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CHAPTER 9 ANNEXURE

I

CHAPTER 9

ANNEXURE-I

CHAPTER 9 ANNEXURE

II

ANNEXURE-II

PROFORMA

Patient Name:

Consultant Name: Dr.

Name of Hospital:

Date of examination:

SEX: Male Female

Age: Yrs Weight: Kg

Height: cm

BMI: Obese / Overweight / Nonobese

Address:

Contact Number:

HOME:

OFFICE:

MOB. :

E-mail:

CHAPTER 9 ANNEXURE

III

Occupation:

Social History: Nonalcoholic / Alcoholic @ the age

Nonsmoker / Smoker @ the age Vegetarian / Mix-vegetarian On Examination:

DATE :

TIME :

G/C :

P/A :

TEMP. :

CNS :

Past Medical History:

Past Medication History:

Present Complaints:

CHAPTER 9 ANNEXURE

IV

Blood Pressure, Pulse & Glycemic Control:

DATE: TIME

Pulse/min

BP mmHg

Blood sugar level

DATE: TIME

Pulse/min

BP mmHg

Blood sugar level

DATE: TIME

Pulse/min

BP mmHg

Blood sugar level

CHAPTER 9 ANNEXURE

V

Diagnostic Test Performed:

Sr.No DATE NAME OF TEST NORMAL RANGE

OBSERVED VALUE

1 Microalbuminuria <30 mg albumin/24h

2 Urine Creatinine 0.6 to 1.2 mg/dL

3 Blood Urea Nitrogen 7 - 20 mg/dL

4 Serum Creatinine 0.8 to 1.4 mg/dL

5 Serum Albumin 3.4 - 5.4 g/dL

6 Serum Globulin 2.6-4.6 g/dL

7 HbA1c 4 - 6 %

8 Total Protein 6.0 to 8.3 gm/dL

9 Urine Albumin 0 to 8 mg/dL

10 Glomerular Filtration Rate

100-130 ml/min.

11 Systolic Blood Pressure

Less than 120 mmHg

12 Diastolic Blood Pressure

Less than 80

mmHg

Other Test Performed:

Impression:

CHAPTER 9 ANNEXURE

VI

Medication Details:

Sr. No

Medication Ingredient / Composition

Dose & Frequency

Route Date

Comments:

CHAPTER 9 ANNEXURE

VII

ANNEXURE-III

Informed Consent Form

Study Number:……………………. Subject Initials:……………………...

Subject’s Name:………………………………………………………………

Date of birth / age:……………………………………………………………

1. I confirm that I have read and understood the information sheet date for the above study and have had the opportunity to ask questions. [ ]

2. I understand that my participation in the study is voluntary and that I am free to withdraw at any time, without giving any reason, without my medical care or legal rights being affected. [ ]

3. I understand that the Sponsor of the above study, others working on the Sponsor’s behalf, the Ethics Committee and the regulatory authorities will not need any permission to look at my health records both in respects of the current study and any further research that may be conducted in relation to it, even if I withdraw from the trial. I agree to this access. However, I understand that my identity will not be revealed in any information released to third parties or published

[ ]

4. I agree not to restrict the use of any data or results that arise from this study. Provided such a use is only for scientific purpose(s).

[ ]

5. I agree to take part in the above study.

[ ]

Signature of the Subject or Representative:……………… Date:…………

CHAPTER 9 ANNEXURE

VIII

Signatory’s Name:……………………………………………………………..

Signature of Clinical Investigator:………………….. Date:…………………

Signature of the Chief Investigator:…………………Date:…………………

Study Investigator’s Name:…………………………………………………...

Signature of the witness: ………………………….. Date:………………….

CHAPTER 9 ANNEXURE

IX

CHAPTER 9 ANNEXURE

X

ANNEXURE –IV

List of Publications

1) V. G. Kuchake, C. D. Upasani. Evaluation of protective effect of

antioxidant vitamins in patients with diabetic nephropathy: Asian

Journal of Pharmaceutical and Clinical Research 2011 ;( 4)52-54.

2) V. G. Kuchake, C. D. Upasani. Effect of Vitamin E and C plus

Reduced Glutathione in Treatment of Diabetic Nephropathy:

International Journal of Pharma Research and Review, 2013.

CHAPTER 9 ANNEXURE

XI

CHAPTER 9 ANNEXURE

XII

CHAPTER 9 ANNEXURE

XIII

CHAPTER 9 ANNEXURE

XIV

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