ir.amu.ac.inir.amu.ac.in/12063/1/T10629.pdf · STUDIES ON HUMAN IgG MODIFIED BY METHYLGLYOXAL UNDER...

STUDIES ON HUMAN IgG MODIFIED BY METHYLGLYOXAL UNDER NORMAL AND HIGH GLUCOSE CONCENTRATIONS:

LIKELY ROLE OF DAMAGED IgG IN THE ONSET OF ARTHRITIS IN TYPE 2 DIABETES MELLITUS PATIENTS

THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

Doctor of Philosophy in

Biochemistry

Submitted By

Mohd Adnan Khan

Dated:……………………..

Approved:…………………

………………………………

Prof. Khursheed Alam (Supervisor)

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF MEDICINE JAWAHARLAL NEHRU MEDICAL COLLEGE

ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)

2018


We certify that the work presented in the

modified by methylglyoxal under normal and high glucose concentrations

role of damaged IgG in the onset of arthritis in type 2 diabetes mellitus patients”

has been carried out by

under our direct supervision and is suitable for the award of Ph.D. degree

Biochemistry of the Ali

Professor Jamal AhmadCo-Supervisor Ex-Director Rajiv Gandhi Centre for DiabetesEndocrinology Faculty of Medicine Jawaharlal Nehru Medical CollegeAligarh Muslim UniversityAligarh-202002, UP (INDIA)E-mail: [email protected]

DEPARTMENT OF BIOCHEMISTRYFaculty of Medicine

Jawaharlal Nehru Medical CollegeAligarh Muslim University

Aligarh-202002, UP (INDIA)

Certificate

work presented in the thesis entitled “Studies on human

modified by methylglyoxal under normal and high glucose concentrations


has been carried out by Mr. Mohd Adnan Khan in the Department of

direct supervision and is suitable for the award of Ph.D. degree

Aligarh Muslim University, Aligarh.

Jamal Ahmad

Rajiv Gandhi Centre for Diabetes &

Medical College Aligarh Muslim University

(INDIA) mail: [email protected]

Professor Khursheed AlamSupervisor Department of Biochemistry Faculty of MedicineJawaharlal Nehru Medical CollegeAligarh Muslim UniversityAligarh-202002, UPE-mail: [email protected]


Jawaharlal Nehru Medical College

Studies on human IgG

modified by methylglyoxal under normal and high glucose concentrations: likely


the Department of Biochemistry

direct supervision and is suitable for the award of Ph.D. degree in

Khursheed Alam

Department of Biochemistry of Medicine

Jawaharlal Nehru Medical College Aligarh Muslim University

202002, UP (INDIA) mail: [email protected]

My for their

encouragement

Dedicated to

My Beloved Family their love, supportencouragement

Beloved Family support &

i

ACKNOWLEDGEMENTS

In the name of Allah, the Most Beneficent and the Most Merciful

Alhamdulillah, all praise is to Almighty Allah, verily to him is owed all thanksgiving and

gratitude I bow before Allah for all the blessings showered upon me till date, and endowing me

with that capacity that made this humble effort possible. My accomplishments would be

incoherent without a formal salutation to the enigmatic force of that Almighty, who guided me in

every difficult moment and gave me the strength and courage to surge ahead in this onerous task.

I would like to take this opportunity to express sincere and profound gratitude to my

supervisor Prof. Khursheed Alam for his genuine concern and constant encouragement throughout

the course of my research work. His share of abstract reasoning, experience and constructive

criticism broadened the horizons of my knowledge. He is a perfect mentor and I would remain

indebted to him for the intensive training that I received under his guidance. His humble

temperament and optimistic attitude had been a driving force for the completion of this work. His

in-depth knowledge on a broad spectrum of free radical biology and autoimmunity enabled me to

learn more than what my Ph.D. work embodies. I sincerely thank him for giving immense freedom

to work during the course of this doctoral programme and training in writing manuscripts. This

thesis would not have been in its present form without his guidance and utmost careful review.

His enthusiasm and passion for science significantly motivated me.

I am indebted to Prof. Jamal Ahmad, Ex-Director, Rajiv Gandhi Centre for Diabetes and

Endocrinology, J.N. Medical College, AMU, Aligarh for his keen interest and valuable

suggestions during my research as co-supervisor.

I am deeply indebted to Dr. Zarina Arif for her timely guidance throughout my Ph.D.

tenure. She was ever-ready to render any kind of help that was needed during my work.

I would also express profound gratitude to Prof. Moinuddin, chairman of the department

for their valuable suggestions and benign co-operation for departmental development and

instrumentation facility. I am grateful to all the teachers of my department including Prof.

Najmul Islam, Prof. Asif Ali, Prof. Shagufta Moin, Prof. Khushtar Anwar Salman, Dr. Abul

Faiz Faizy, Dr. Safiya Habib, Dr. Sufia Naseem and Dr. Akif Ahsan for their goodwill, support

and invaluable suggestions.

I am also thankful to Prof. Akbar Husain and Dr. Shah Mohd Khan, Department of

Psychology, AMU, Aligarh for their help in analyzing my data and interpreting it.

ii

I am also thankful to my seniors Dr. Abdul Rauf, Dr. Badrul Islam, Dr. Farzana Wasi,

Dr. Manzoor Gatoo, Dr. Parvez Ahmad, Dr. Mir Yasir Arfat, Dr. Shafeeque Ahmad, Dr.

Shaziya Allarakha, Dr. Wasil Hasan, Dr. Sidra Islam for their help and coorperation extended to

me. I would also like to express heartiest thanks to my lab mates Dr. Arif Iqubal , Shireen Naaz

Islam, Asim Badar, Akhlas and Moasfar for their unconditional help, constant moral support and

co-operation, making this journey easier and comfortable. I am grateful to all my juniors including

Talha, Shoeb, Shireen, Minhal, Sana, Mustafa, Sharib, Shifa, Dr. Masum, Somaiya, Sumayya and

Shahida for their support, encouragement and motivational ideas.

I was fortunate to pursue my Ph.D. studies with very gifted, wonderful and talented

person, Asif Zaman. It was always amazing to work with him for late nights in the laboratory

during thesis writing. Beautiful moments spend with him while sharing tea cups will always be

cherished in my heart and shall be treasured long in the memory. I would like to express sincere and

profound regards to my batchmates cum friends Irfan and Azhar and for the countless favours

and help they extended during this whole journey. They worked together with me, discussed the

problems, and thus made a wonderful company.

I owe sincere and heartfelt gratitude to the office staff, clinical laboratory staff and all

the other members of the department specially Mr. Nasir, Mr. Faisal, Mrs. Huma and Mr. Faizan

,Mr. Ashfaque for their never-ending assistance and co-operation and for providing all sort of

technical help required for the successful completion of my research work. I would also like to

mention Raju Bhai and Yogender Bhai for maintaining a clean and hygienic environment in the

department which was a prime need for any experimental protocol.

I may fall short of words while expressing thanks to my dearest friends Rashid Jamal

Khan, Sufiyan Khan, Ahtesham Ahmad, Shah Ahmad, Naiyyer, Nabeel, Mustafa, Furqan,

Imran, Rizwan, Saif, Afzal, Sultan, Kamran, Saddam, Hashim, Zaidan, Asfahan, Adil, Atif,

Saad, Khaliqur Rahman, Mujibur Rahman, Abdus Samad who hold a very special place in my

heart. They were my stress busters, who loved and cared for me unconditionally, and always stood

by me in all the ups and downs, thereby easing all my difficulties. List of my friends is very long

and so I am extremly thankful to all my friends whose names I have not mentioned for their

constant moral support and encouragement throughout this journey of uncertainties.

I would like to take this opportunity in expressing a very special thanks to all my family

members. My words fail to express my appreciation for my wife Zeba for her unconditional love,

co-operation and wholehearted moral support. She deserves a special acknowledgment owing to her

sacrifices and relentless efforts. She always stood by me and became a sink of my outbursts

iii

whenever I felt downright depressed. I feel deeply indebted to Ammi, Abbu, late Nani and Nana

for their sacrifices and devotion. I thank Almighty for such caring, selfless and generous parents

whose love and affection is irreplaceable. They had always been a real source of strength and

motivation, and nurtured me in the best possible ways. Their faith in me boosted my confidence

leading to the successful accomplishment of this task.

I would also like to sincerely acknowledge all the loving and affectionate members of my

family including for their timely help, sincere advice and prayers, without which I would not have

been able to accomplish this work.

Council of Scientific and Industrial Research (CSIR), New Delhi is gratefully

acknowledged for providing financial assistance for my research work in the form of Junior

Research fellowship and Senior Research fellowship. Finally, I would like to pay heartiest homage

to Late Sir Syed Ahmad Khan, the founder of this great university, who would be remembered till

the end of time.

Mohd Adnan Khan CSIR Research Fellow

Abstract

1

Abstract

IgG protects the body from infection that may occur due to entry of bacteria, viruses,

fungi etc. In order to perform its functions the molecule must maintain its native form.

It is rich in lysine and arginine residues and has a half life of over three weeks.

Studies have shown that non-enzymatic glycosylation of IgG by reactive sugars or

their metabolites impairs its role as defender. Furthermore, during hyperglycemia the

rate of non-enzymatic glycation of proteins increases manifold. This leads to the

formation of advanced glycation end products (AGEs) which have been implicated in

plethora of diseases. Methylglyoxal is highly reactive dicarbonyl produced from

dihydroxyacetone phosphate and glyceraldehydes-3-phophate and at an elevated

concentration it may modify virtually every protein. It is 20,000 times more reactive

than glucose. Under normal physiology methylglyoxal is readily detoxified by

glyoxalase system. Alterations in IgG structure due to methylglyoxylation may lead to

unmasking and pooling of the cryptic epitopes.

In this study, we have modified the human IgG by methylglyoxal alone as well as in

combination of normoglycemic and hyperglycaemic concentrations of glucose and

carried out detailed biophysical, biochemical and immunological studies. The IgG

was purified from healthy human sera on Protein-A-agarose affinity matrix and then

incubated with different concentrations of methylglyoxal alone and also with normal

(5 mM) and high glucose (10 mM) for 7 days at 37 0C in capped vials. The

biophysical modifications in IgG have been studied by UV-visible, Fourier transform-

infrared (FT-IR), Fluorescence, Circular dichroism (CD), Dynamic light scattering

(DLS), and Dfferential scanning calorimetry (DSC). Furthermore, formation of

Amadori products were studied by NBT reduction assay and estimation of

hydroxymethyl furfural (HMF), and the effective protein hydrophobicity by ANS

fluorescence. The oxidative stress was determined as protein carbonyl by DNPH

assay and the free sulfhydryl by Ellman’s reagent. The aggregates were detected by

Congo red and thioflavin T (ThT) dye. Morphological changes have been analyzed by

scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

The formation of carboxymethyl lysine (CML) was confirmed by LC-MS studies and

formation of fluorogenic AGEs by fluorescence spectroscopy. The thermostability of

IgG was determined by thermal denaturation studies. Immunogenicity of modified-

IgG samples was tested in healthy and diabetic rabbits. Antibody titre and specificity

Abstract

2

of the induced antibodies was determined by enzyme immunoassay. Antigen-antibody

interactions were visualized by gel retardation assay.

Rabbits were injected with alloxan to produce experimental diabetes. The induction of

diabetes was confirmed from glucose and insulin level. Biochemicals markers of

stress, inflammation etc have been determined in the sera of healthy rabbits, diabetic

rabbits, healthy human subjects and T2DM patients. Rheumatoid factor has also been

estimated in the sera of healthy rabbits, diabetic rabbits, healthy human subjects and

T2DM patients.

All modified-IgG preparations exhibited hyperchromicity which may be due to

exposure of buried chromophoric amino acids residues vis-a-vis unfolding. We also

observed increase in absorbance between 300-400 nm which may be attributed to IgG

crosslinking and aggregation during the course of modification. The observed

quenching in tryptophan fluorescence in modified-IgG preparations suggests change

in its microenvironment. Formation of fluorogenic AGEs was subsequently

confirmed from increase in emission intensity specific to fluorescent-AGEs when the

samples was excited at 370 nm. Furthermore, CML (a non-fluorescent AGEs) was

also found in our modified-IgG preparations when tested on LC-MS. The effective

protein hydrophobicity was increased due to exposure of buried hydrophobic regions.

The Amadori generation between methylglyoxal-IgG mixture completed by 72 h. But

co-incubation with glucose accelerated the process and the Amadori formation

completed by 24 h. After that, AGEs formation started. The molecule gained

thermostabilty due to crosslinking and aggregation as shown by increase in melting

temperature. Furthermore, the Fab fragment under identical conditions was found to

be more susceptible to denaturation than Fc fragment. The results of hydrodynamic

studies suggest increase in the size of modified-IgG molecules. The secondary and

tertiary structure were disturbed during modification. FT-IR results suggested

decrease in α-helical content and gain in β-pleated sheet of the modified-IgG

preparations. It is a clear indication of changes in the secondary structure of modified-

IgG preparations. CD results suggested that the tertiary structure of modified-IgG has

also changed which may be correlated with oxidation of thiol. Increase in protein

carbonyl and decrease in free sulfhydryl suggests that the modification introduced

into the IgG by methylglyoxal and/or glucose produces oxidative stress. The results

gathered from Congo red, Thioflavin T, SEM and TEM speaks in favour of crosslinks

and aggregates in modified-IgG preparations. The IgG-MGO-high glucose

Abstract

3

preparations showed maximum structural alterations as compared to other modified-

IgG preparations. It clearly shows that modification of IgG depends upon

concentrations of modifying agents.

Enzyme immunoassay results suggests that structural alterations and aggregates have

mounted immunogenicity on modified-IgG preparations. Furthermore, the

immunogenicity was more aggressive in diabetic rabbits as compared to healthy

rabbits. This may be attributed to exposure of cryptic epitopes during the course of

modification of IgG and pooling of such epitopes due to aggregation. Furthermore,

the increase in biochemicals like C-reactive protein, IL-1, IL-6, TNFα and rheumatoid

factor in diabetic animals (as compared to healthy animals) immunized with

modified-IgG preparations was also observed in the sera of T2DM patients and there

was a similarity in the pattern of the above biochemicals between diabetic animals

and diabetic patients. Analysis carried out by ANOVA and Tukey posthoc test

suggests that the results are statistically significant and there is a positive correlation

between disease duration and age group. The findings indicate how rheumatoid

arthritis like co-morbidity may arise in T2DM patients.

CONTENTS

Page no.

ACKNOWLEDGEMENT i-iii

LIST OF FIGURES iv-xi

LIST OF TABLES xii-xiii

LIST OF ABBREVIATIONS xiv-xvii

ABSTRACT xviii-xx

HYPOTHESIS & OBJECTIVES xxi

Chapter 1

Introduction and review of literature 1-12

Chapter 1(a): Biochemical and biophysical studies on IgG modified with methylglyoxal.

1. Materials & Methods 13-23

2. Results 24-50

Chapter 1(b): Biochemical and biophysical studies on IgG co-modified with methylglyoxal and normal (5 mM)/high glucose (10 mM).

1. Materials & Methods 51

2. Results 52-81

Combined discussion of Chapter 1(a) and Chapter 1(b) 82-84

Combined references of Chapter 1(a) and Chapter 1(b) 85-96

Chapter 2


Chapter 2(a): Induction and characterization of antibodies raised against IgG modified with methylglyoxal, and IgG co-modified with methylglyoxal and normal (5 mM)/high (10 mM) glucose in healthy rabbits.


2. Results 106-128

Chapter 2(b): Induction and characterization of antibodies raised against IgG modified with methylglyoxal, and IgG co-modified with methylglyoxal and normal (5 mM)/high (10 mM) glucose in diabetic rabbits.


2. Results 132-155

Combined discussion of Chapter 2(a) and Chapter 2(b) 156-157

Combined references of Chapter 2(a) and Chapter 2(b) 158-160

Chapter 3


Chapter 3: Binding profile of autoantibodies in T2DM sera of different age group and disease duration with native and modified-IgG preparations and estimation of rheumatoid factor, IL-1, IL-6, C-reactive protein and TNFα in the sera.


2. Results 167-195

3. Discussion 196

4. References 197-199

LIST OF PUBLICATIONS xxii

LIST OF PRESENTATIONS IN CONFERENCES xxiii

iv

LIST OF FIGURES

Fig. no. Figure Legends Page no.

Chapter 1(a)

Fig. 1(a) UV absorption profile of native IgG isolated from a

healthy human serum

25

Fig. 1(b) UV absorption spectra of native IgG and MGO-

modified-IgG

25

Fig. 2 Emission spectra of native IgG and MGO-modified-IgG

excited at 285 nm

27

Fig. 3 Emission spectra of native IgG and MGO-modified-

IgG excited at 370 nm

29

Fig. 4 Estimation of ε-amino groups in native IgG and MGO-

modified-IgG

32

Fig. 5 Hydroxymethylfurfural content in native IgG and MGO-

modified-IgG

32

Fig. 6 Estimation of Amadori adducts in MGO-modified-IgG 33

Fig. 7

Fig. 8

Fig. 9

Fig. 10(a-c)

Fig. 11

Fig. 12

Fig. 13

Fig. 14

Fig. 15

Emission spectra of ANS bound to native IgG and

MGO-modified-IgG

Carbonyl content in native IgG and MGO-modified-IgG

Free sulfhydryl level in native IgG and MGO-modified-

IgG

FT-IR profile of native IgG and MGO-modified-IgG

Far UV CD spectra of native IgG and MGO-modified-

IgG

Near UV CD spectra of native IgG and MGO-modified-

IgG

Absorption spectra of Congo red bound to native IgG

and MGO-modified-IgG Emission profiles of Thioflavin T interaction with native

IgG and MGO-modified-IgG SEM images of native IgG and MGO-modified-IgG

33

35

35

36-37

39

40

41

41

44

v

Fig. 16

Fig. 17

Fig. 18

Fig. 19

Fig. 20

Fig.21(a)

Fig.21(b)

Fig.22

Fig. 23

Fig. 24

Fig. 25

Fig. 26

Fig. 27

Fig. 28

Fig. 29

Fig. 30(a-e)

Fig. 31

Fig. 32

Fig. 33

Fig. 34

Fig. 35

TEM images of native IgG and MGO-modified-IgG Melting profiles of native IgG and MGO-modified-IgG DSC thermograms of native IgG and MGO-modified-

IgG DLS profiles of native IgG and MGO-modified-IgG LC-MS analysis of native IgG and MGO-modified-IgG

Chapter 1(b)

UV absorption spectra of native IgG and IgG co-

modified with methylglyoxal and normal glucose

UV absorption spectra of native IgG and IgG co-

modified with methylglyoxal and high glucose

Emission spectra of native IgG and modified-IgG

excited at 285 nm

Emission spectra of native IgG and modified-IgG

excited at 370 nm

Estimation of ε-amino groups in native IgG and

modified-IgG

Hydroxymethylfurfural content in native IgG and

modified-IgG

Estimation of Amadori adducts in modified-IgG

Emission spectra of ANS interaction with native IgG

and modified-IgG

Carbonyl content in native IgG and modified-IgG

Free sulfhydryl level of native IgG and modified-IgG

FT-IR profile of native IgG and modified-IgG

Far UV CD spectra of native IgG and modified-IgG

Near UV CD spectra of native IgG and modified-IgG

Absorption spectra of Congo red bound to native IgG

and modified-IgG

Emission profiles of Thioflavin T interaction with native

IgG and modified-IgG

SEM images of native IgG and modified-IgG

45

46

47

48-49

50

54

55

55

58

58

60

60

63

64

64

65-67

68

69

72

73

74

vi

Fig. 36

Fig. 37(a-d)

Fig. 38

Fig. 39

Fig. 40

Fig. 41(a)

Fig. 41(b)

Fig. 42(a)

Fig. 42(b)

Fig. 43

Fig. 44(a)

Fig. 44(b)

Fig. 45(a)

Fig. 45(b)

Fig. 46

Fig. 47

Fig. 48

Fig. 49

TEM images of native IgG and modified-IgG

Melting profiles of native IgG and modified-IgG

DSC thermograms of native IgG and modified-IgG

DLS profiles of native IgG and modified-IgG

LC-MS analysis of native IgG and modified-IgG

Chapter 2(a)

Direct ELISA of experimentally induced antibodies

against native IgG in healthy rabbits


against MGO-modified-IgG in healthy rabbits

Inhibition ELISA of serum antibodies against native IgG

in healthy rabbits

Inhibition ELISA of serum antibodies against MGO-

modified-IgG in healthy rabbits

UV absorption profile of IgG isolated from antiserum

against MGO-modified-IgG

Direct ELISA of IgG isolated from antiserum raised



against MGO-modified-IgG in healthy rabbits

Inhibition ELISA of IgG isolated from antiserum of

native IgG raised in healthy rabbits

Inhibition ELISA of IgG isolated from antiserum of

MGO-modified-IgG raised in healthy rabbits

Gel retardation assay of IgG isolated from antiserum

raised against methylglyoxal-modified-IgG

Rheumatoid factor status in control and methylglyoxal-

modified-IgG immunized healthy rabbits

Tumor necrosis factor-α status in control and

methylglyoxal-modified-IgG immunized healthy rabbits

Interleukin-1 status in control and methylglyoxal-


75

76-77

78

79-80

81

107

107

108

108

109

109

110

110

111

111

114

115

116

vii

Fig. 50

Fig. 51

Fig.52(a)

Fig.52(b)

Fig.53(a)

Fig.53(b)

Fig. 54(a)

Fig. 54(b)

Fig. 55(a)

Fig. 55(b)

Fig. 56

Fig. 57

Fig. 58



C-reactive protein status in control and methylglyoxal-



against IgG co-modified with methylglyoxal and normal

glucose in healthy rabbits


against IgG co-modified with methylglyoxal and high


Inhibition ELISA of serum antibodies against IgG co-

modified with methylglyoxal and normal glucose in

healthy rabbits

Inhibition ELISA of serum antibodies against IgG co-

modified with methylglyoxal and high glucose in

healthy rabbits

Inhibition ELISA of IgG isolated from antiserum raised







raised against IgG co-modified with methylglyoxal and

normal glucose



high glucose

Rheumatoid factor status in control and modified-IgG

immunized healthy rabbits

Tumor necrosis factor-α status in control and modified-

IgG immunized healthy rabbits

Interleukin-1 status in control and modified-IgG

116

117

119

119

120

120

121

121

124

124

125

126

127

viii

Fig. 59

Fig. 60

Fig. 61

Fig. 62

Fig.63(a)

Fig.63(b)

Fig.64(a)

Fig.64(b)

Fig. 65(a)

Fig. 65(b)

Fig. 66

Fig. 67

Fig. 68

Fig. 69




C-reactive protein status in control and modified-IgG


Chapter 2(b)

Changes in blood glucose level of rabbits after a single

dose of alloxan as compared to rabbits who did not

receive alloxan for diabetes induction

Insulin level in control rabbits and alloxan treated

rabbits Direct ELISA of experimentally induced antibodies

against native IgG in diabetic rabbits


against MGO-modified-IgG in diabetic rabbits

Inhibition ELISA of serum antibodies against native IgG

in diabetic rabbits

Inhibition ELISA of serum antibodies against MGO-

modified-IgG in diabetic rabbits




against MGO-modified-IgG in diabetic rabbits


raised against methylglyoxal-modified-IgG in diabetic

rabbits

Rheumatoid factor status in control and methylglyoxal-

modified-IgG immunized diabetic rabbits

Tumor necrosis factor-α status in control and

methylglyoxal-modified-IgG immunized diabetic rabbits



127

128

133

133

135

135

136

136

137

137

138

141

142

143

ix

Fig. 70

Fig. 71

Fig. 72(a)

Fig. 72(b)

Fig.73(a)

Fig.73(b)

Fig.74(a)

Fig.74(b)

Fig. 75(a)

Fig. 75(b)

Fig. 76

Fig. 77

Fig. 78



C-reactive protein status in control and methylglyoxal-




glucose in diabetic rabbits




Inhibition ELISA of serum antibodies against IgG

co-modified with methylglyoxal and normal glucose in

diabetic rabbits

Inhibition ELISA of serum antibodies against IgG

co-modified with methylglyoxal and high glucose in

diabetic rabbits









normal glucose in diabetic rabbits



high glucose in diabetic rabbits

Rheumatoid factor status in control and modified-IgG

immunized diabetic rabbits

Tumor necrosis factor-α status in control and modified-

IgG immunized diabetic rabbits


143

144

146

146

147

147

148

148

151

151

152

153

154

x

Fig. 79

Fig. 80

Fig. 81

Fig. 82

Fig. 83

Fig. 84

Fig. 85

Fig. 86

Fig. 87

Fig. 88

Fig. 89




C-reactive protein status in control and modified-IgG


Chapter 3

Direct ELISA of serum autoantibodies in healthy

subjects on wells coated with modified-IgG preparations

Direct ELISA of serum autoantibodies in T2DM patients

with disease duration of <5 years on wells coated with

modified-IgG preparations


with disease duration of 5 to <10 years on wells coated

with modified-IgG preparations


with disease duration of 10 to <15 years on wells coated

with modified-IgG preparations


with disease duration of >15 years on wells coated with

modified-IgG preparations Inhibition ELISA of serum autoantibodies in T2DM

patients with disease duration of <5 years on wells

coated with modified-IgG preparations

Inhibition ELISA of serum autoantibodies in T2DM

patients with disease duration of 5 to <10 years on wells



patients with disease duration of 10 to <15 years on

wells coated with modified-IgG preparations


patients with disease duration of >15 years on wells


154

155

168

169

170

171

172

173

174

175

176

xi

Fig. 90

Fig. 91

Fig. 92

Fig. 93

Fig. 94

Inhibition ELISA of IgG (isolated from respective

serum) in T2DM patients with disease duration of

<5 years on wells coated with modified-IgG preparations


serum) in T2DM patients with disease duration of 5 to

<10 years on wells coated with modified-IgG

preparations


serum) in T2DM patients with disease duration of 10 to

<15 years on wells coated with modified-IgG

preparations


serum) in T2DM patients with disease duration of >15

years on wells coated with modified-IgG preparations Polyacrylamide gel photograph of IgG antibodies

interaction with IgG co-modified with methylglyoxal

and high glucose (antigen) in different group of type 2

diabetes mellitus patients

177

178

179

180

181

xii

LIST OF TABLES

Table no. Description Page no.

Chapter 1(a)

1.

Effect of time on 280 nm absorbance of native and MGO-

modified IgG preparations

26

2.

Effect of time on fluorescence intensity of native and MGO-

modified IgG preparations, excited at 285 nm

28

3.

AGEs specific increase in fluorescence of MGO-modified

IgG preparations with respect to time, excited at 370 nm

30

4.

FTIR bands characteristic of native and MGO-modified IgG

preparations

37

Chapter 1(b)

5.

Effect of time on 280 nm absorbance of native IgG and its

modified counterparts

56

6.

Effect of time on fluorescence intensity of native and

modified-IgG preparations, excited at 285 nm

57

7.

AGEs specific increase in fluorescence of modified-IgG

preparations with respect to time, excited at 370 nm

59

8. FTIR bands characteristic of native and modified-IgG

preparations

67

Chapter 2(a)

9.

Cross-reactions of IgG isolated from antiserum raised


112

10


against methylglyoxal-modified-IgG in healthy rabbits

113

11.



concentration of glucose (5 mM) in healthy rabbits

122

12. Cross-reactions of IgG isolated from antiserum raised


concentration of glucose (10 mM) in healthy rabbits

123

xiii

Chapter 2(b)

13.

Effect of alloxan on some biochemicals in rabbits during

eight weeks

132

14.



139

15.


against methylglyoxal-modified-IgG in diabetic rabbits

140

16.



149

concentrations of glucose (5 mM) in diabetic rabbits

17.



concentrations of glucose (10 mM) in diabetic rabbits

150

Chapter 3

18. Demography and investigation details of the subjects in

different groups of T2DM patients

166

19. Estimation of biochemicals in the sera of healthy subjects 183

20.

Estimation of biochemicals in the sera of T2DM patients

with disease duration of <5 years

184

21.


with disease duration of 5 to <10 years

185

22.


with disease duration of 10 to <15 years

186

23.


with disease duration of >15 years

187

24. Correlations among different biochemicals and different

disease duration of T2DM patients

188-190

25. Correlations among different biochemicals and healthy

subjects and T2DM patients of different age group within

group I to group V

191-195

xiv

LIST OF ABBREVIATIONS

aa : Amino acids

A278 : Absorbance at 278 nm

A280 : Absorbance at 280 nm

AGEs : Advanced glycation end products

ALDH : Aldehyde dehydrogenase

ANS : 8-anilino-1-naphthalenesulfonic acid

AR : Aldose reductase

ATR : Attenuated total reflection

BMI : Body mass index

BSA : Bovine serum albumin

B-cells : B-lymphocytes

β-cells : Pancreatic beta cells

Cat : Catalase

CCP : Cyclic citrullinated peptide

CD : Circular dichroism

CEL : Nԑ-carboxyethyl lysine

CML : Nԑ-carboxymethyl lysine

CR : Congo red

CRP : C-reactive protein

Cu, Zn-SOD : Copper and zinc containing superoxide dismutase

DHAP : Dihydroxyacetone phosphate

DLS : Dynamic light scattering

DNA : Deoxyribonucleic acid

DNPH : 2,4-dinitrophenylhydrazine

DSC : Differential scanning calorimetry

DTNB : 5,5’-dithio-bis(2-nitrobenzoic acid)

EDTA : Ethylene diamine tetra-acetic acid

ELISA : Enzyme linked immunosorbent assay

ERK : Extracellular signal-regulated kinases

Fab : Antigen-binding fragment

FAOXs : Fructosyl-amine oxidases

FcR : Fc receptor

xv

Fc : Fragment crystallizable region

FC : Fructosamine content

FI : Fluorescence intensity

FN3K : Fructosamine-3-kinase

FPG : Fasting plasma glucose

FTA : Serum fructosamine content

FT-IR : Fourier-transform infrared

Glo : Glyoxalase

glu-lys : Glutamic acid-lysine

GLUT : Glucose transporter

GOLD : Glyoxal-lysine dimer

G3P : Glyceraldehyde-3-phosphate

GPx : Glutathione peroxidase

GSH : Glutathione

HbA1c : Glycated haemoglobin

HDL : High density lipoprotein

HMF : 5-hydroxymethylfurfural

HRP : Horseradish peroxidase

IgG : Immunoglobulin G

IκB : Inhibitor of NFκB

IL-4 : Interleukin-4



LC-MS : Liquid chromatography-mass spectroscopy

LDL : Low-density lipoprotein

λem : Emission wavelength

λex : Excitation wavelength

M : Molar

MALDI-TOF : Matrix-assisted laser desorption/ionization-time of

flight

MAPKs : Mitogen-activated protein kinases

MG-H : Methylglyoxal derived hydroimidazolones

mM : Millimolar

MGO : Methylglyoxal

xvi

MOLD : Methylglyoxal-lysine dimer

N : normal

NaBH4 : Sodium borohydride

NADPH : Nicotinamide adenine dinucleotide phosphate (H)

NBT : Nitroblue tetrazolium

NFκB : Nuclear factor kappa-B

NO : Nitric oxide

NOX : NADPH oxidase

O2•− : Superoxide anion radical

PAGE : Polyacrylamide gel electrophoresis

PBS : Phosphate buffer saline

phe : Phenylalanine

pNPP : p-nitrophenyl phosphate

PP : Postprandial

RA : Rheumatoid arthritis

RAGE : Receptor for advanced glycation end products

RBC : Red blood cells

RF : Rheumatoid factor

ROS : Reactive oxygen species

SD : Standard deviation

SDS : Sodium dodecyl sulphate

SEM : Scanning electron microscope

-SH : Thiol

SOD : Superoxide dismutase

TBA : Thiobarbituric acid

TBARS : Thiobrbituric acid reactive substances

TBS : Tris buffer saline

TBS-T : Tris buffer saline containing 0.05% Tween 20

TCA : Trichloroacetic acid

T-cells : T lymphocytes

T2DM : Type 2 diabetes mellitus

TEM : Transmission electron microscope

TEMED : N,N,N’,N’-tetramethylethylenediamine

TG : Triglyceride

xvii

ThT : Thioflavin T

Tm : Melting temperature

TMB : 3,3’,5,5’-tetramethylbenzidine

TNBS : 2.4.6-trinitrobenzenesulphonic acid

TNFα : Tumor necrosis factor α

tyr : Tyrosine

UV-Vi s : Ultraviolet visible

VLDL : Very low density lipoprotein

ZnSO4 : Zinc sulphate

Chapter 1

Chapter 1

1

Introduction and review of literature

The biomolecules made up of one or more long chain of amino acid residues (known

as polypeptide) are called as proteins. They differ from each other primarily in their

amino acid sequence which is decided by the sequence of the nucleotide present on

their gene which results into the folding of protein into specific three dimensional

structure that imparts each protein a specific function. Proteins perform numerous

functions such as providing protection against ‘non self’ molecules, catalysing

metabolic reactions, DNA replication and repair, serving structural roles, help in

transportation of nutrients and serve as signalling molecule to regulate growth and

development and also respond to external stimuli. Like carbohydrates and nucleic

acids, proteins are essential part of organisms and participate in virtually every

process occurring inside the organism. Many proteins are biological catalyst; known

as enzymes that accelerate the rate of metabolic reactions. Proteins also have

structural role that maintains the shape of cells and help in movement, e.g. actin and

myosin. Proteins are also needed for cell cycle, cell division, cell signalling, cell

adhesion and development of immune response against pathogens. In animals,

proteins are also needed in the diet to provide both essential and non-essential amino

acids.

When proteins bind with the other copy(ies) of the same molecule then they

oligomerize to form fibrils; such as in the structural proteins, globular monomers

oligomerize to form rigid fibrous structures (Powers and Powers, 2007). Protein-

protein interactions mediate cell adhesion, control enzymatic activity and cell cycle

progression and also regulate assembly of large protein complexes that carry out

many closely related reactions with similar biological function (Tarsounas et al.,

1997). Proteins can even be integrated into the cell membranes to provide help in

transport of various molecules, to mediate signal transduction etc (Agnati et al.,

2005). During certain conditions proteins may acquire abnormal structures/shapes and

may even loose normal function(s) and lead to proteopathy (Walker and Levine,

2002). Some well known proteopathies are Alzheimer disease, prion diseases,

Parkinson disease, tauropathies, cystic fibrosis and sickle cell disease (Chaudhuri and

Paul, 2006).

Chapter 1

2

Immunoglobulin G

IgG is a glycoprotein which protects the body from infection by binding many kinds

of pathogens such as bacteria (Nordenfelt and Bjorck, 2013), viruses (Gottardo et al.,

2013) and fungi (Jiang et al., 2015). IgG control infection of body tissues since it is

present in largest concentrations in blood and extracellular fluid. It also provides

immune protection to the developing foetus, since IgG is the only immunoglobulin

capable of crossing the placenta (Simister, 2003). IgG secretion also occurs in mother

milk and once it has been ingested by the new born it can be transported into blood

and confers immunity (Gasparoni et al., 1992).

IgG molecules are synthesized and secreted by plasma B-cells (Mayumi et al., 1983)

and are high molecular weight proteins (approx. 150 kDa). Each IgG molecule is

composed of four peptide chains-two identical heavy and light chain, respectively

which is arranged in Y-shaped structure, typical of antibody monomers (Girardi et al.,

2009) and has two antigen binding sites (Welschof et al., 1997). It is the most

common type of antibody found in circulation and represents approximately 75% of

the serum immunoglobulins.

IgG functions through following mechanisms:

By binding to pathogen itself which causes the immobilization of pathogen

and also by binding together via agglutination (Guimaraes et al., 2011).

Coating of pathogen surface by IgG (known as opsonisation) that allows

pathogen recognition and ingestion by phagocytic immune cells leading to the

elimination of pathogen (Shaffer et al., 1993).

Activation of the classical pathway of the complement system by IgG i.e. a

cascade of immune protein production that results in pathogen elimination

(Kochi and Johnson, 1988).

Binding and neutralization of toxins (Pincus et al., 2014).

Type II and Type III hypersensitivity reactions are also manifested by the IgG

(Juchnowicz et al., 2016).

Chapter 1

3

Antibody-dependent cell mediated cytotoxicity is also regulated (Jochems et

al., 2017).

Intracellular antibody-mediated proteolysis in which IgG binds TRIM21 (the

receptor with greatest affinity to IgG in humans) in order to direct the marked

virions to the cytosolic proteasome (Okada et al., 1999).

IgG subclass

There are four subclasses of IgG (IgG1, IgG2, IgG3 and IgG4) in humans named in

order of their abundance in serum; the most abundant being IgG1 and IgG4 being the

least abundant (Schur, 1987; Oxelius, 1984). Their characteristics are enlisted in the

table on next page (Hashira et al., 2000).

General structure of an immunoglobulin molecule

1-Fab fragment

2- Fc fragment

3- Heavy chain

4- Light chain

5- Antigen binding site

6- Hinge region

http://www.jbc.org/search?author1=Kunihiko+Okada&sortspec=date&submit=Submit

Chapter 1

4

IgG

subclass Percent

Whether crosses

placenta?

Complement

activation

potential

Whether

binds with Fc

receptor on

phagocytic

cells?

Half

life

IgG1 66% yes second-highest high affinity 21 days

IgG2 23% no third-highest extremely low

affinity 21 days

IgG3 7% yes highest high affinity 7 days

IgG4 4% yes no intermediate

affinity 21 days

Glycation (or glycosylation)

Glycation can be defined as the non-enzymatic reactions between reducing sugars

such as glucose (Singh et al., 2014), fructose (McPherson et al., 1988), ribose (Wei et

al., 2012) etc and biomolecules such as proteins (Mota et al., 1994), lipids (Miyazawa

et al., 2012) and nucleic acids (Krantz et al., 1986). The food chemist Louis Camille

Maillard described the above reaction for first time in 1912 (Maillard, 1912).

Although he described that these reactions would be important in biology, but it was

only in the year 1980 that advanced glycation end products (AGEs) were highlighted

for their pathophysiological role. The formation of AGEs involves simple and

complex multistep reactions (Hegab et al., 2012). The Maillard reaction proceeds by

attachment of the electrophilic carbonyl group of glucose or other reducing sugars

with the free amino group of lysine and arginine which leads to formation of a non-

stable Schiff base. Further rearrangement of the Schiff base leads to the generation of

a ketoamine known as Amadori (Neelofar and Ahmad, 2015). Both Amadori and

Schiff base are reversible products (Ansari and Dash, 2013). However, they can react

with the amino group of peptide or protein, nucleic acid reactive nitrogenous bases etc

to form irreversible product known as protein adduct or protein cross links (Baynes et

al., 1989; Nemet et al., 2011). Alternatively, these protein adducts can undergo

further oxidation, cyclization, rearrangement, dehydration, polymerization and

oxidative breakdown and form diverse class of AGEs (Prasad et al., 2013) and in

presence of oxygen, reactive oxygen species (ROS) (Nowotny et al., 2015) and redox

active transition metals (Zhang et al., 2009) formation of AGEs is accelerated.

Furthermore, AGEs can be formed not only from sugars but also from dicabonyl

Chapter 1

5

compounds derived from autoxidation of sugars and metabolic pathways (Sun et al.,

2016).

The most prevalent in vivo AGEs is carboxymethyllysine (CML) (Teerlink et al.,

2004) and it is non-fluorescent. CML is formed by the oxidative degradation of

Amadori product or direct addition of glyoxal to lysine. Pentosidine was the first

fluorescent AGEs that was successfuly isolated and characterized. It is formed by

cross linking of pentose sugar with arginine and lysine residues (Monnier et al., 2015;

Wetzels et al., 2017). Glucosepane (Draghici et al., 2015), carboxyethyllysine (CEL)

(Ou et al., 2017), fructosyl-lysine (Ahmed et al., 2005), methylglyoxal-derived

hydroimidazolones (Chen et al., 2015) and pyrraline (Foerster and Henle, 2003) are

also non fluorescent AGEs. Furthermore, crosslink type AGEs include glyoxal-lysine

dimer (GOLD) (Yamada et al., 2004) and methylglyoxal-lysine dimer (MOLD)

(Chellan and Nagaraj, 1999) which are also non-fluorescent in nature.

Exogenous glycation products

Exogenous AGEs are formed when sugars are cooked with proteins, fats etc at

temperatures greater than 120°C (~248 °F). Moreover, lower temperatures with

prolonged cooking time also accelerate AGEs formation. These compounds are

absorbed by body with 10% efficiency during digestion. Recent work has highlighted

the important of exogenous glycation products in inflammation as well as other

diseases (Frimat et al., 2017). AGEs have been exploited by the food manufacturers

as colorants (Zhang et al., 2015) and flavour enhancers to improve appearance of food

(Hong et al., 2016). Foods with very high exogenous glycation products include

barbecued meats, cake, donuts and dark coloured soda pops.

Endogenous glycation products

Non-enzymatic glycosylation (glycation) of proteins (Snow et al., 2007), nucleic

acids (Wagner et al., 2016) and lipids (Garg et al., 2017) is considered to be one of

the major factors contributing to cellular and organismal aging. Glycation process is

often called as glycooxidation since rective oxygen species are formed during

glycation and also contribute to glycation induced protein modifications (Piwowar et

al., 2008). The complex pathway which leads to the formation of AGEs involves

oxidative stress and that is the reason for accumulation of AGEs during oxidative

Chapter 1

6

stress (Hung et al., 2017) and inflammation (Hudson and Lippman, 2018). In proteins

arginine, lysine and sulfur-containing amino acid residues such as free cysteines are

particularly vulnerable to glycoxidation (Ottum and Mistry, 2015).

Endogenous glycation mainly occurs in the bloodstream (Zurawska-Plaksej et al.,

2018). Fructose and galactose have apparently ten times more glycation activity than

glucose, the primary body fuel (Bousova et al., 2011; Gugliucci, 2017).

Environmental factors, such as smoking (Prasad and Mishra, 2017) and diet (Aragno

and Mastrocola, 2017) influence the rate of formation of AGEs. Moreover, it seems

that the level of circulating AGEs is genetically determined, as shown by a cohort

study of healthy monozygotic and heterozygotic twins (Prakash et al., 2015).

List of glycating agents, their physiological concentrations, and the situations

under which the concentrations are raised

Sl.

No.

Name

Concentration

in blood of

healthy

subjects

Concentration

is elevated in

following condition

References

1 D-glucose 90-100 mg/dl Diabetes mellitus (Janghorbani and

Amini, 2011)

2 D-fructose 8-9 µM Diabetes mellitus/

NAFLD

(Kawasaki et al.,

2012) (Gugliucci,

2017)

3 D-ribose 100 µM Excessive intake of

ribose supplements

(Chen et al., 2017)

4 D-galactose 0.1-5 µM Galactosemia (Ning and Segal,

2000)

5 Glyoxal 80 nM Diabetes mellitus/

uremia

(Thornalley, 2008)

6 Methylglyoxal 340 nM Diabetes mellitus/

hyperglycaemia

(Aikawa et al.,

2017; Thornalley,

2008)

7 3-deoxygluco-

sone

35nM Diabetes mellitus/

hyperglycaemia

(Thornalley, 2008)

Chapter 1

7

Maillard reaction

The AGEs content in the organism does not depend only upon the rate of their

formation but also by the rate of their removal. Intrinsic detoxifying pathways have

been developed by many cells against accumulation of AGEs. The glyoxalase system,

which consists of glyoxalase (Glo) I and II, has an important role in providing defense

against glycation (Wu et al., 2002). This system uses reduced glutathione (GSH) for

reaction that catalyze the conversion of glyoxal, methylglyoxal and other α-

oxoaldehydes to the less toxic D-lactate via formation of S, D-lactoylglutathione.

The fructosyl-amine oxidases (FAOXs) (Lin and Zheng, 2010) and fructosamine

kinases (Deppe et al., 2011) are the other enzymatic system which is relatively new

classes of enzymes which recognize and break Amadori products. However, the

expression of FAOXs or “amadoriases” has been limited only in bacteria, yeast and

fungi but not found in mammals. They break Amadori products oxidatively but act

mostly on low molecular weight compounds (Lin and Zheng, 2010). Although

fructosamine kinases are expressed in various genomes including humans. These

Chapter 1

8

intracellular enzymes causes phosphorylation and destabilization of Amadori products

which leads to their spontaneous breakdown. Fructosamine-3-kinase (FN3K), is one

of the most studied enzymes in this system, is expressed in human tissues

ubiquitously including the skin. Thus, it plays important role in the intracellular

breakdown of Amadori products.

Role of Amadori and advanced glycation end products in human diseases

Enhanced glycation in fructosemia is due to fructose. Some AGEs are inert but some

are more reactive than the reducing sugars from which they are derived. AGEs have

been implicated in many patho-physiological conditions such as cardiovascular

diseases (Andrades et al., 2009; Sveen et al., 2015; Gupta and Uribarri, 2016; Morais

et al., 2012), Alzheimer's disease (Stock et al., 2016), peripheral neuropathy (Roman-

Pintos et al., 2016) and deafness (Sternberg et al., 2011). This range of diseases is the

consequence of the interference of AGEs at molecular and cellular level and release

of oxidants like hydrogen peroxide that causes the oxidative stress within the body

and thus impairs the normal body functioning (Zhou et al., 2017). Endogenous AGE

formation increases in diabetes mellitus (Di Pino et al., 2017). In diabetes, damage by

glycation also results in stiffening of the collagen in the blood vessel walls (Chang et

al., 2009), leading to high blood pressure. The collagen weakening also occurs in the

wall of the blood vessels caused by glycation which may lead to micro- or macro-

aneurisms; thus leading to the stroke (Wendl et al., 2015). Long-lived proteins such as

crystallins of the lens (Chaudhury et al., 2017) and cornea (Ljubimov, 2017) and long

lived cells such as nerves (Man et al., 2015) and different types of brain cell

(Furukawa et al., 2017) may accumulate AGEs during diabetes mellitus and may

cause complications like diabetic retinopathy, neuropathy and nephropathy.

Advanced glycation end products exert their deleterious actions not only due to their

biological properties per se, but also through their interaction with specific receptors

(Wang et al., 2017). Receptor for advanced glycation end product (RAGE) is a

member of the immunoglobulin superfamily of cell surface receptors, encoded by a

gene on chromosome 6 near the major histocompatibility complex III region (Lin et

al., 2016). It seems to be a pattern recognition receptor (Xie et al., 2008) binding, in

addition to AGEs, various other molecules such as S-100A, high mobility group

protein B1 (amphoterin) (Gasparotto et al., 2018), β-amyloid peptides (Fei et al.,

Chapter 1

9

2017) and β-sheet fibrils (Leung et al., 2016). The binding of ligands to RAGE

stimulates various signaling pathways which include the extracellular signal-regulated

kinases (ERK) 1 and 2 (Wu et al., 2016), phosphatidyl-inositol 3 kinase (Chen et al.,

2014), p21 Ras (Azizan et al., 2017), mitogen-activated protein kinases (MAPKs)

(Lei et al., 2017), stress-activated protein kinase/c-Jun-N-terminal kinase and the

janus kinases (Origlia et al., 2008). RAGE stimulation results in activation of the

nuclear factor kappa-B (NFκB) (Zhou et al., 2016) which is a transcription factor and

also subsequent transcription of many proinflammatory genes such as TNF-alpha

(Wen and Yin, 2017), IL-4 etc (Zhou et al., 2017). RAGE-induced activation of NFκB

transcription factor is characterized by a sustained and self-perpetuating action,

through induction of positive feedback loops and overwhelming of the autoregulatory

negative feedback loops. The activation of RAGE leads to the synthesis of new the

transcriptionally active subunit p65, which overwhelms the newly synthesized

inhibitor IκBα. Moreover NFκB further increases expression of RAGE, which itself

further stimulates NFκB, forming a vicious cycle of self-renewing and perpetuating

proinflammatory signals. The activation of RAGE can directly induce oxidative stress

(Hong et al., 2017) by activation of nicotinamide adenine dinucleotide phosphate

(NADPH)-oxidase (NOX) (Kay et al., 2016), and decreasing the activity of

superoxide dismutase (SOD) (Soliman et al., 2017), catalase (Ren et al., 2017) and

indirectly by reducing cellular antioxidant defenses, like GSH (Lin et al., 2012) and

ascorbic acid (May, 2016). Furthermore, the reduction of GSH leads to decreased

activity of Glo I, which provides the major cellular defense system against

detoxifying methylglyoxal, therefore supporting further production of AGEs. RAGE

is expressed ubiquitously in the organism, typically at low levels, and its expression is

upregulated under various pathological conditions. In the skin, RAGE expression was

observed in both dermis and epidermis (Davies et al., 2009) and it was increased in

sun exposure compared with UV irradiation-protected areas. Keratinocytes (Liu et al.,

2017), dendritic cells (Zhang et al., 2017), fibroblast (Chen et al., 2017) and to a

certain lesser extent endothelial cells (Li et al., 2017) and lymphocytes (Oh et al.,

2018) express RAGE. Various skin cells types have been shown to express RAGE in

vivo as well as in vitro conditions (Abe et al., 2004).

AGEs accumulation has been detected in various tissues during diabetes (Kender et

al., 2012) and aging (Hause et al., 2018), including articular collagen (Ozawa et al.,

Chapter 1

10

2017), smooth vascular (Ren et al., 2009) and skeletal muscles (Son et al., 2017) or

basement membrane of the glomeruli (Yagihashi and Kaseda, 1978). The AGEs

deposited in these tissues have been implicated in diabetes- or age-associated

pathologies such as diabetic angiopathy (Capitao and Soares, 2016), age- and

diabetes-associated macular degeneration (Ciulla et al., 2003) and osteoarthritis (Zhao

et al., 2017). Once formed, the AGEs cannot be removed unless the modified protein

is degraded.

Methylglyoxal as glycating agent

The AGEs formation is now thought to mainly result from the actions of various

reactive metabolites of glucose such as methylglyoxal (Banerjee and Chakraborti,

2017), glyoxal (Banerjee, 2017) and 3-deoxyglucosone (Niwa et al., 1998).

Methyglyoxal is a potent glycating agent that causes glycation of collagen (Paul and

Bailey, 1999), bovine serum albumin (Wang et al., 2016), IgG (Khan et al., 2017) etc

and leads to the formation of glycation adducts. The reactivity of methylglyoxal is

about 20,000 times more than that of glucose (Ramasamy et al., 2006). Methylglyoxal

can modify proteins (Mey et al., 2018) and nucleic acids (Vilanova et al., 2017).

Methylglyoxal is derived endogenously from metabolic intermediates of proteins,

carbohydrates and fatty acids (Bellahcene et al., 2017). Most of the methylglyoxal is

formed as a byproduct of glycolysis from the nonenzymatic degradation of

glyceraldehyde-3-phosphate and dihydroxy acetone phosphate. Under

normoglycaemic conditions, the formation of methylglyoxal takes place at the rate of

120 µM/day. Although methylglyoxal production constitutes only of about 0.1%

glucotriose flux (Thornalley, 1988), but its biological effects are relevant.

Furthermore, autooxidation of glucose, degradation of glycated proteins, oxidation of

acetone in the catabolism of ketone bodies during diabetic ketoacidosis, catabolism of

threonine and lipid peroxidation also generate some amount of methylglyoxal

(Bellahcene et al., 2017).

Chapter 1

11

A putative scheme for the aggregation of ribosylated protein into highly

cytotoxic molten globules. Glycation of α-synuclein with D-ribose induces protein

misfolding and results in the formation of aggregates having high cytotoxicity

(Chen et al., 2010)

Methylglyoxal formation, degradation and reaction

MGO is mainly formed as a byproduct of glycolysis and autoxidation of glucose.

Other sources of MGO are catabolism of threonine and acetone, lipid peroxidation

and degradation of glycated proteins. Increased levels of MGO are predominantly

detoxified by the glyoxalase system, which converts MGO into its endproduct D-

lactate, via the formation of the intermediate S-D-lactoylglutathione. In addition,

ALDH and AR comprise minor pathways of MGO detoxification. If MGO production

exceeds the detoxification capacity, MGO can modify arginine residues to form MG-

H1, -H2 and -H3, AP and THP. When MGO reacts with lysine, it forms CEL and

MOLD, whereas it forms MODIC when it forms a dimer cross-link with arginine and

lysine (Maessen et al., 2015).

Chapter 1

12

Methylglyoxal also plays important role in diabetes, obesity, cancer, disorders of the

central nervous system, hypertension, ageing, atherosclerosis etc. MGO is also

involved in epigenetics (Maessen et al., 2015).

Chapter 1(a)

Biochemical and biophysical studies on IgG modified with methylglyoxal

Chapter 1(a)

13

Materials & methods

MATERIALS

Chemicals

Acrylamide, bisacrylamide, N,N,N’,N’-tetramethylethylenediamine (TEMED)

and ammonium persulphate were procured from Bio-Rad Laboratories, USA. 2,4,6-

trinitrobenzenesulphonic acid (TNBS), sodium dodecyl sulphate (SDS), ammonium

bicarbonate, 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB), succinic acid, and

nitroblue tetrazolium (NBT) were obtained from Sisco Research Laboratories, India.

Ammonium sulphate, sodium nitrite, silver nitrate, sodium carbonate and bicarbonate,

sodium hydroxide pellets, copper sulphate, sodium potassium tartarate, sodium

acetate, tris (hydroxymethyl) aminomethane, trichloroacetic acid, thiobarbituric acid

(TBA), hydrochloric acid, glycine, glycerol, bromophenol blue, methanol, sodium

thiosulphate, formic acid, acetonitrile, sodium chloride, isopropanol, formaldehyde,

ethanol, orthophosphoric acid, glacial acetic acid, EDTA, and oxalic acid were

purchased from Qualigens fine Chemicals, India. D-glucose, sodium borohydride

(NaBH4), 2, 4-dinitrophenylhydrazine (DNPH), and ethylacetate were purchased from

Merck, India. Protein-A-agarose (2.5 ml pre-packed column), agarose, 8-anilino-1-

naphthalenesulfonic acid (ANS), sodium azide, dialysis tubing, methylglyoxal, Congo

red, Thioflavin T, Folin & Ciocalteu’s phenol reagent and Coomassie Brilliant Blue

G-250 were purchased from Sigma Chemical Company, USA. All others chemicals

and reagents used were of the highest analytical grade available.

Equipments

Spectrophotometer (model , UV-1700; Shimadzu, Japan) attached with a

temperature-programmer and controller unit, Spectropolarimeter (model, Jasco J-815;

USA), Spectrofluorometer (model, RF-5301; Shimadzu, Japan), pH meter (model,

L1-120; ELICO, India), Polyacrylamide gel electrophoresis assembly (Bio-Rad

Laboratories, USA), Avanti 30 table top high speed refrigerated centrifuge (Beckman,

USA), Gel-doc (Bio-Rad laboratories, USA), an orthogonal time of flight (TOF) mass

spectrometer (Applied Biosystems, Mariner atmospheric pressure ionization TOF

workstation, Framingham, MA, USA), VP-DSC microcalorimeter (MicroCal, MA,

USA), DynaPro-TC-04 DLS (Wyatt Technology, CA, USA), LC-MS; Micromass

Chapter 1(a)

14

Quattro II triple quadrupole mass spectrometer (Beverly, MA, USA), and FT-IR

spectrometer (PerkinElmer, Massachusetts, USA) were the major equipments used in

this study.

METHODS

Determination of protein concentration

Protein estimation was carried out by Bradford (Cheng et. al., 2016) and

Lowry et al (1951) methods.

Lowry method

Protein estimation by Lowry method utilizes use of alkali (for keeping the

high pH), Cu2+

ions (to chelate proteins) and tartarate (to keep the Cu

2+ ions in solution

at high pH).

(a) Folin-Ciocalteau reagent

Before use, commercially available Folin-Ciocalteau reagent was

diluted 1:4 with distilled water.

(b) Alkaline copper reagent

The alkaline copper reagent components were prepared as follows;

(i) Two percent sodium carbonate in 100 mM sodium hydroxide.

(ii) 0.5 percent copper sulphate in 1.0 percent sodium-potassium

tartarate.

Freshly prepared working reagent was used. Working reagent was

prepared by mixing components (i) and (ii) in 50:1 ratio.

(c) Procedure

Varying amounts of bovine serum albumin were taken in 1.0 ml

volume and mixed with 5.0 ml of freshly prepared alkaline copper reagent

and then incubated at room temperature for 10 min. One ml working Folin-

Ciocalteau reagent was added followed by 30 min incubation at room

temperature. The absorbance was recorded at 660 nm. Protein content in

Chapter 1(a)

15

unknown samples was determined from the standard plot of bovine serum

albumin.

Bradford method

This assay is based on the change in absorption maximum of an acidic

solution of Coomassie Brilliant Blue G-250 dye from 465 nm to 595 nm when protein

binds to it. Both hydrophobic and ionic interactions stabilize the anionic form of the

dye, causing a visible colour change.

Dye preparation

One hundred mg of Coomassie Brilliant Blue G-250 was dissolved in 50 ml of

95% ethanol and 100 ml of 85% (v/v) orthophosphoric acid added. The resulting

solution was diluted to 1 litre and filtered through Whatman filter paper (No. 1) to

remove undissolved particles.

Protein assay

To one ml of solutions containing 10-100 mg protein, 5.0 ml of dye solution

was added and the contents were mixed by vortexing. The absorbance was read at 595

nm after 5 min against the blank reagent.

Isolation of IgG from healthy human serum

Blood samples were obtained from healthy human subjects after their verbal

consent and allowed to coagulate at 37 0C for 30 min. Sera were separated by

centrifugation at 3000 rpm. IgG was isolated from the serum by affinity

chromatography using Protein A-agarose column (Goding, 1978). Concentration of

isolated IgG was determined considering 1.4 OD278nm = 1.0 mg IgG/ml and then

subjected to dialysis against PBS, pH 7.4. Protein concentration in dialyzate was

determined by absorbance measurement at 278 nm. Homogeneity of the IgG was

evaluated on 7.5% SDS-PAGE and the material was stored at -20 0C with 0.1%

sodium azide.

Modification of purified IgG by methylglyoxal

IgG (6.67 µM) was mixed with different concentrations of methylglyoxal

(1.67, 3.33, 5.0, 6.67 and 8.33 µM) and incubated. The reaction was carried out in 10

Chapter 1(a)

16

mM PBS, pH 7.4 under sterile conditions for 7 days at 37 0C. IgG (6.67 µM) devoid

of methylglyoxal was kept under identical experimental conditions and served as

control. At the end of the incubation the solutions were extensively dialyzed against

10 mM PBS, pH 7.4 to remove excess methylglyoxal and then stored at -20 0C.

Absorption spectroscopy

Absorption profiles of samples were recorded on Shimadzu UV-1700

spectrophotometer in the wavelength range of 240-400 nm using quartz cuvette of 1

cm path length. Hyperchromicity at 280 nm was calculated from the following

equation:

% hyperchromicity at 280 nm =

Fluorescence studies

The structural changes in MGO-modified IgG preparations were studied by

the fluorescence measurement at room temperature. The tryptophan fluorescence was

monitored after excitation of samples at 285 nm and the emission spectra were

recorded in the wavelength range of 290-400 nm (Shaklai et al., 1984). The decrease

in the fluorescence intensity (F.I.) was calculated from the following equation:

% decrease in F.I. =

AGE specific fluorescence was measured by exciting the samples at 370 nm

and the emission spectra were recorded in the wavelength range of 400-600 nm

(Yanagisawa et al., 1998). The increase in fluorescence intensity (F.I.) was calculated

from the following equation:

% increase in F.I. =

Estimation of ε-amino groups in native and methylglyoxal-modified IgG

preparations by TNBS

Free ε-amino groups were quntitated in native and MGO-modified IgG

preparations with the help of 2,4,6-trinitrobenzenesulphonic acid (TNBS) reagent.

Under mild conditions, TNBS specifically reacts with ε-amino groups to form

Chapter 1(a)

17

trinitrophenyl derivatives (Hashimoto et al., 2013). Briefly, 150 µl of 0.5% TNBS

(w/v) was added to 0.75 ml of samples and incubated for 1 h at 37 0C. At the end of

the incubation the samples were solubilised in 0.375 ml of 10% SDS followed by 0.15

ml of 1N HCl. Absorbance was recorded at 420 nm and ε-amino groups were

calculated using molar extinction coefficient of 19,200 cm-1

mol-1

. Furthermore, the

extent of MGO-modification in IgG was evaluated from the following formula:-

% modification of IgG ε-amino groups =

×100

Detection of 5-hydroxymethylfurfural

HMF (5-hydroxymethylfurfural) generated from the Amadori product of

MGO-modified IgG preparations was detected by thiobarbituric acid (TBA) reagent

according to the method described by Ney et al., 1981. Briefly, 1 ml each of native

IgG and MGO-modified IgG samples were mixed with 1 ml of 1 M oxalic acid and

incubated at 100 0C for 2 h. Then the protein from the assay mixture was removed by

precipitation with 40% trichloroacetic acid. 0.25 ml of TBA (0.05 M) was added to

0.75 ml of protein free filtrate and incubated at 40 0C for 40 min. The colour was read

at 443 nm and amount of HMF was calculated using molar extinction coefficient of

40,000 cm-1

mol-1

.

Detection of Amadori adducts by NBT reagent

The Amadori adduct in MGO-modified IgG preparations was determined by

NBT reduction assay as described previously (Baker et. al., 1993). Samples (300 µl

each) were mixed with 3 ml of 100 mM sodium carbonate buffer (pH 10.35)

containing 0.25 mM NBT and thereafter incubated at 37 0C for 2 h. The absorbance

was read at 525 nm against distilled water and Amadori adduct (fructosamine) was

determined using molar extinction coefficient of 12,640 M-1

cm-1

for monoformazan.

Increase in fructosamine content (FC) was calculated from the following equation:

% increase in F.C. =

Chapter 1(a)

18

Effective protein hydrophobicity

Binding of ANS to native and MGO-modified IgG preparations was evaluated

in terms of fluorescence (Cardamone and Puri, 1992). The fluorescence of ANS is

affected by the exposure or masking of hydrophobic patches in proteins. A fresh stock

solution of ANS was prepared in distilled water and the concentration was determined

spectrophotometrically using the molar extinction coefficient of 5000 M-1

cm-1

at 350

nm. The molar ratio of protein to ANS was adjusted to 1:50 and emission spectra

were obtained in the wavelength range of 400-600 nm after exciting the samples at

380 nm. Increase in ANS binding to modified samples was calculated using the

following equation:


Determination of protein bound carbonyl

Carbonyl content in native and MGO-modified IgG preparations was

determined after reaction with DNPH (Levine et al., 1990). The final absorbance was

read at 360 nm against appropriate blank. The carbonyl content was determined using

a molar extinction coefficient of 22,000 M-1

cm-1

and expressed as nmol/mg protein.

Determination of free sulfhydryl

The free sulfhydryl groups in native and MGO-modified IgG preparations

were determined by Ellman’s reagent (Ellman, 1959). Following solutions were

prepared:

DTNB Stock: 50 mM sodium acetate in distilled water containing 2 mM

DTNB

Tris buffer: 1M Tris, pH 8.0

DTNB working reagent was prepared by mixing 100 µl Tris buffer, 840 µl

distilled water and 50 µl of DTNB stock. Ten µl of the sample was mixed with 990 µl

of DTNB working reagent. The solution was thoroughly mixed and incubated for 5

min at 37 0C. Absorbance was recorded at 412 nm. The free sulfhydryl content was

determined using the molar extinction coefficient of 13,600 M-1

cm-1

.

Chapter 1(a)

19

FT-IR spectroscopy

Native and MGO-modified IgG preparations were analyzed by FT-IR spectra.

FT-IR spectra of native and MGO-modified IgG samples were recorded on

PerkinElmer FT-IR spectrometer in the wavenumber range of 1400 to 1800 cm-1

which covers the typical amide I and amide II peaks (Kumosinski and Unruh, 1996).

Ten microliters of native/modified sample was placed on the attenuated total

reflection (ATR) device and IR spectra were acquired.

Circular dichroism studies on native and methylglyoxal-modified IgG

preparations

MGO-induced conformational/structural changes in the samples were studied

using CD machine and far- and near-UV CD spectra were generated. The instrument

was calibrated with D-10-camphorsulfonic acid and spectra were collected using cell

of 1.0 and 10 mm path length. The protein concentration was kept at 6.67 μM. The

far- and near-UV CD spectra were recorded in the wavelength range of 250-190 and

350-250 nm, respectively at a scan speed of 100 nm/min and response time of 1 s

(Arfat et al., 2014). The CD shape constant (S value) was calculated to evaluate the

effect of MGO modification on the secondary structure of IgG.

Aggregate(s) detection by Congo red (CR) and Thioflavin T (ThT) dye

Congo red is the sodium salt of 3,3’-([1,1’-biphenyl]-4,4’-diyl) bis (4-

aminonaphthalene sulfonic acid). It is a secondary diazo nonfluorescent dye. Apple

green birefringence of Congo red stained samples under polarized light is indicative

of amyloid fibrils and protein aggregation. Likely formation of protein aggregates in

MGO-modified IgG samples were carried out by spectral measurements of CR

binding in the wavelength range of 300-700 nm. The molar ratio of sample to Congo

red was kept at 1:2 and the assay mixture was incubated for 30 min at room

temperature (Frid et al., 2007).

Chapter 1(a)

20

Native and MGO-modified IgG preparations were incubated with ThT in a

molar ratio of 1:2 for 60 min at 25 0C and the F.I. was measured (Hudson et al.,

2009). Increase in ThT binding was calculated from the following equation:


Scanning electron microscopy of methylglyoxal-modified IgG preparations

Scanning electron microscope (SEM) is a type of electron microscope that

produces images of samples with a focused beam of electrons which interact with the

atoms in the samples producing various signals that give information about the

samples’ composition and surface topography.

50 µl of MGO-modified IgG preparations were placed on glass slides and

covered with cover slip and dehydrated overnight at 37 °C and 50% humidity.

Samples’ images were recorded using JEOL JSM-6510LV microscope working at an

acceleration voltage of 5 kV (Zaman et al., 2017).

Transmission electron microscopy of methylglyoxal-modified IgG preparations

Transmission electron microscopy (TEM) gives information about the

structure and composition of the specimen. The variations in structural information

create an image which undergoes magnification by a series of electromagnetic lenses

and detected.

MGO-modified IgG preparations were first fixed and then dehydrated with

ethanol. The preparation was then passed through propylene oxide and the grid was

prepared and coated with formvar film for stabilization during passage of electron.

The image was obtained using iTEM and TIA softwares.

Thermal denaturation studies

The thermal denaturation analysis of the preparations was carried out to

ascertain the effect of MGO modification on IgG thermostability (Zaman et al.,

2017). Mid point melting temperature (Tm) of the samples was determined from

absorbance values at different temperatures. All samples were melted from 20 0C to

95 0C at a rate of 1

0C/min. The change in absorbance at 280 nm was recorded with

Chapter 1(a)

21

increase in temperature. Percent denaturation was calculated from the following

equation:

where,

AT = Absorbance at temperature T 0C

Amax = Final absorbance on the completion of denaturation (95 0C)

A20 = Initial absorbance at 20 0C

Differential scanning calorimetry (DSC) of native and methylglyoxal-modified

IgG preparations

Thermal denaturation of native and modified-IgG preparations was monitored

on VP-DSC microcalorimeter (MicroCal, MA, USA). The DSC scans were run from

20 to 90 °C at the rate of 1 °C min−1

. Buffer-buffer baselines were run under identical

conditions and subtracted from each sample scan. Since transitions of IgG are

irreversible, the experimental values of melting temperatures and enthalpies were

considered as “apparent” values (Arfat et al., 2016).

Dynamic light scattering (DLS) of native and methylglyoxal-modified IgG

preparations

DLS properties of native and MGO-modified IgG preparations were studied

using DynaPro-TC-04 DLS equipment (Wyatt Technology, CA, USA). Before

measurement, all solutions were spun at 10,000 rpm for 10 min and passed through a

2 μm pore sized filter directly into a 12 μl quartz cuvette. All data were analyzed

using Dynamics 6.10.0.10 software (Arfat et al., 2014).

LC-MS analysis of native and methylglyoxal-modified IgG preparations

IgG modified with MGO was subjected to LC/MS studies for the

identification of AGEs i.e. specifically CML. Prior to loading, the native and MGO-

modified samples were hydrolyzed in 6N HCl at 95 0C for 24 h (Schleicher et al.,

1981). The hydrolysates were filtered through a medium grade filter paper. Reversed-

phase separation was carried out on an Agilent 1100 series capillary HPLC system

equipped with a synergi C18 analytical column (2×250 mm with 5 µm particle size).

The chromatographic conditions were as follows: 0.4% acetic acid (Solvent A), 0.2%

acetonitrile (Solvent B) each containing 2% formic acid. Gradient elution protocol

was as follows: 0% to 2% solvent B in the first 5 min from 2% to 6% solvent B over

Chapter 1(a)

22

19 min, 6% to 80% solvent B in 11 min and then raised to 80% to wash the residual

material off the column at the constant flow rate of 0.5 ml/min. Mass spectrometric

analysis was then carried out on Micromass Quattro Ultima Triple Quadropole Mass

spectrometer interfaced to an Agilent 1100 capillary HPLC system. The mass

spectrometer was operated in a positive ion mode and full scan mass spectra were

recorded in 0-500 m/z range.

SDS-polyacrylamide gel electrophoresis

SDS-PAGE was performed as described earlier (Laemmli, 1970). The

following stock solutions were prepared:-

Acrylamide-bisacrylamide (30:0.8)

A stock solution was prepared by dissolving 15 gm acrylamide and 0.4 gm

bisacrylamide in distilled water to a final volume of 50 ml. The solution was filtered

and stored at 4 0C in an amber colour bottle.

Resolving gel buffer

A stock solution was prepared by dissolving 9.08 gm Tris base in 40 ml

distilled water. The pH was adjusted to 8.8 by 6N HCl and the final volume brought

to 50 ml with the distilled water.

Electrode buffer

3 gm Tris base, 14.4 gm glycine and 1.0 gm SDS were dissolved in distilled

water, pH adjusted to 8.3 and the final volume was made to one litre.

Procedure of SDS-PAGE

Thoroughly cleaned glass plates separated by 1.0 mm thick spacer were used.

The resolving gel mixture (7.5%) was prepared by mixing the components and poured

between the glass plates and allowed to polymerize at room temperature. Protein

samples mixed with one fourth of sample dye (10% SDS, 50% glycerol, 1M Tris, pH

6.8 and 1% bromophenol blue) were loaded into the wells and electrophoresis was

carried out at 80 volts for 3-4 h in Tris-glycine buffer. Protein bands were visualized

by silver staining and Coomassie brilliant blue staining.

Chapter 1(a)

23

Recipe for 7.5 % resolving gel

Component Volume

Acrylamide-bisacrylamide (30:0.8)

2.7 ml

Resolving gel buffer 2.5 ml

Distilled water 4.6 ml 10% SDS 0.1 ml 1.5% ammonium persulphate

0.1 ml

TEMED

5.0 µl

Silver staining

At the end of electrophoresis, the protein bands were visualized by silver

staining. The procedure involved fixing of protein bands for 10 min in a mixture of

40% methanol and 13.5% formaldehyde followed by two washings with distilled

water, at an interval of 5 min. The gel was then immersed in 0.02% sodium

thiosulphate solution for 1 min followed by two washings with distilled water at an

interval of 20 sec. The gel was immersed in 0.1% silver nitrate solution for 10 min

and finally washed with distilled water. The gel was then immersed in the developer

solution (3% sodium carbonate, 0.02% sodium thiosulphate and 0.05% formaldehyde)

to facilitate staining and on appearance of bands the reaction was stopped by a stopper

solution (25% isopropanol, 10% glacial acetic acid).

Coomassie brilliant blue staining

The protein bands were also visualized with coomassie brilliant blue staining

(Arndt et al., 2012). The procedure involved staining of gel for 60 min in a mixture of

45% methanol and 10% glacial acetic acid and 45% distilled water and 0.1%

coomassie brilliant blue dye followed by overnight destaining in a mixture of 10%

methanol and 10% glacial acetic acid and 80% distilled water.

Statistical analysis

Data are presented as mean ± standard deviation. Statistical significance of the

data was determined by Student’s–t test and a p-value of <0.05 was considered as

statistically significant.

Chapter 1(a)

24

Results

UV characterization of methylglyoxal-modified IgG preparations

Human IgG (6.67 µM) isolated on Protein-A agarose affinity column was incubated

with different concentrations of methylglyoxal (1.67, 3.33, 5.00, 6.67 and 8.33 µM)

under sterile and aseptic conditions for 7 days at 37 0C. The preparations were then

dialyzed and absorption spectra were recorded. The photograph of affinity purified

IgG from normal healthy human serum run on 7.5% SDS-polyacrylamide gel is

shown in inset to Fig. 1a. The λmax of native IgG was found to be at 280 nm. The

MGO-modified preparations showed hyperchromicities at 280 nm (Fig. 1b). There

was 57.44, 58.11, 58.76, 59.18 and 63.13% hyperchromicity in IgG modified by 1.67,

3.33, 5.00, 6.67 and 8.33 µM MGO, respectively. The data have been summarized in

Table 1.

Fluorescence studies on methylglyoxal-modified IgG preparations

Tryptophan’s intrinsic fluorescence was exploited to obtain information about

changes in the fluorescence properties of MGO-modified IgG (Vivian and Callis,

2001). Native and MGO-modified IgG preparations were excited at 285 nm and the

emission intensities were recorded and the result are shown in Fig. 2. The attachment

of methylglyoxal with IgG caused reduction in the emission intensity of tryptophan

intrinsic fluorescence. We observed 17.06, 53.05, 57.61, 62.16 and 69.84% decrease

in emission intensity at 1.67, 3.33, 5.00, 6.67 and 8.33 µM MGO, respectively. The

data have been summarized in Table 2.

Fresh samples of MGO-modified IgG were excited at 370 nm to detect fluorogenic

AGEs. Native IgG excited at 370 nm showed negligible AGE specific fluorescence

(Fig. 3 & Table 3). Under similar experimental conditions the emission intensities of

the MGO-modified IgG preparations increased; which provides clear indication of

formation of fluorogenic AGEs during modification (Olar et al., 2015). The

attachment of methylglyoxal with IgG caused increase in the emission intensity due to

presence of fluorescent AGEs. We observed 86.06, 88.81, 89.50, 90.11 and 90.65%

increase in emission intensity at 1.67, 3.33, 5.00, 6.67 and 8.33 µM MGO,

respectively. The data have been summarized in Table 3.

Chapter 1(a)

25

Fig. 1(a) UV absorption spectra of native IgG isolated from a healthy human serum

on protein A-agarose affinity matrix. Inset: SDS-gel photograph of purified

IgG on 7.5% polyacrylamide gel.

Fig. 1(b) UV absorption spectra of native IgG (filled triangle) and IgG modified with

1.67 μM (open triangle), 3.33 μM (filled circle), 5.00 μM (open circle), 6.67

μM (filled square) and 8.33 μM MGO (open square) after 7 days of

incubation.

Chapter 1(a)

26

Table 1

Effect of time on 280 nm absorbance of MGO-modified IgG preparations

Sample Absorbance at 280 nm

0h 24h 48h 72h 96h 120h 144h 168h

Native

IgG 0.76 0.77 0.76 0.78 0.79 0.80 0.80 0.80

IgG

treated

with

1.67

µM

MGO

1.51

(49.66)

1.55

(50.32)

1.62

(53.08)

1.67

(53.29)

1.72

(54.06)

1.75

(54.28)

1.83

(56.28)

1.88

(57.44)

IgG

treated

with

3.33

µM

MGO

1.6

(52.5)

1.64

(53.04)

1.65

(53.93)

1.71

(54.38)

1.76

(55.11)

1.82

(56.04)

1.86

(56.98)

1.91

(58.11)

IgG

treated

with

5.00

µM

MGO

1.72

(55.81)

1.76

(56.25)

1.77

(57.06)

1.82

(57.14)

1.85

(57.29)

1.88

(57.44)

1.89

(57.67)

1.94

(58.76)

IgG

treated

with

6.67

µM

MGO

1.78

(57.30) 1.81

(57.45)

1.84

(58.69)

1.87

(58.28) 1.89

(58.20) 1.92

(58.33)

1.94

(58.76)

1.96

(59.18)

IgG

treated

with

8.33

µM

MGO

1.89

(59.78) 1.92

(59.89) 1.93

(60.62)

1.99

(60.80) 2.02

(60.89) 2.05

(60.97) 2.08

(61.53) 2.17

(63.13)

Note: The data within parentheses indicate percent change in absorbance at 280 nm

compared to native IgG.

Chapter 1(a)

27

Fig. 2 Fluorescence emission profiles of native IgG (open square) and IgG modified

with 1.67 μM (open triangle), 3.33 μM (open circle), 5.00 μM (filled square),

6.67 μM (filled triangle) and 8.33 μM MGO (filled circle). The samples were

excited at 285 nm.

Chapter 1(a)

28

Table 2

Effect of time on fluorescence intensity of native and MGO-modified IgG preparation

Sample

Fluorescence intensity

0 h 24 h 48 h 72 h 96 h 120 h 144 h 168 h

Native

IgG

950

900

850

818

750

743

726

703

IgG

treated

with

1.67 µM

MGO

900

(5.26)

835

(7.22)

780

(8.23)

733

(10.39)

670

(10.66)

626

(15.74)

603

(16.94)

583

(17.06)

IgG

treated

with

3.33 µM

MGO

850

(10.52)

794

(11.77)

740

(12.94)

696

(14.91)

500

(33.33)

400

(46.16)

350

(51.79)

330

(53.05)

IgG

treated

with

5.00 µM

MGO

800

(15.78)

754

(16.22)

700

(17.64)

657

(19.68)

400

(46.66)

350

(52.89)

312

(57.02)

298

(57.61)

IgG

treated

with

6.67 µM

MGO

700

(26.31)

646

(28.22)

606

(28.70)

576

(29.58)

350

(53.33)

300

(59.62)

276

(61.98)

266

(62.16)

IgG

treated

with

8.33 µM

MGO

600

(36.84)

564

(37.33)

514

(39.52)

478

(41.56)

300

(60.00)

250

(66.35)

224

(69.14)

212

(69.84)

Note: (i) The data within parentheses indicate percent decrease in F.I. compared to

native IgG. (ii) All samples were excited at 285 nm.

Chapter 1(a)

29

Fig. 3 Detection of fluorogenic AGEs in IgG modified with 1.67 μM (filled triangle),

3.33 μM (filled square), 5.00 μM (open circle), 6.67 μM (open triangle) and

8.33 μM MGO (open square) in comparison to native IgG (filled circle). The

samples were excited at 370 nm for AGE specific fluorescence.

Chapter 1(a)

30

Table 3

AGEs specific increase in fluorescence of MGO-modified IgG preparations with

respect to time

Sample

AGEs specific fluorescence intensity 0 h 24 h 48 h 72 h 96 h 120 h 144 h 168 h

Native

IgG 55 65 75 85 85 85 85 85

IgG

treated

with

1.67

µM

MGO

100

(45.00)

300

(78.33)

360

(79.16)

420

(79.76)

440

(80.68)

475

(82.10)

510

(83.33)

610

(86.06)

IgG

treated

with

3.33

µM

MGO

150

(63.33)

460

(85.86)

540

(86.11)

620

(86.29)

640

(86.71)

675

(87.40)

710

(88.02)

760

(88.81)

IgG

treated

with

5.00

µM

MGO

200

(72.50)

500

(87.00)

585

(87.17)

680

(87.50)

695

(87.76)

710

(88.02)

750

(88.66)

810

(89.50)

IgG

treated

with

6.67

µM

MGO

250

(78.00)

550

(88.18)

640

(88.28)

730

(88.35)

745

(88.59)

760

(88.81)

810

(89.50)

860

(90.11)

IgG

treated

with

8.33

µM

MGO

325

(83.07)

600

(89.16)

695

(89.20)

790

(89.24)

800

(89.375)

810

(89.50)

860

(90.11)

p910

(90.65)

Note: (i) The data within parentheses indicate percent increase in F.I. compared to

native IgG. (ii) All samples were excited at AGEs specific wavelength of

370 nm.

Chapter 1(a)

31

Estimation of ε-amino groups in native and methylglyoxal-modified IgG

preparations

As shown in Fig. 4, addition of different concentration of MGO to IgG has consumed

some ε-amino moieties and it came down from 230.0±1.72 nmol/mg in native IgG to

83.33±0.72 nmol/mg in IgG treated with 8.33 µM MGO (Sashidhar et al., 1994).

Estimation of hydroxymethyl furfural (HMF) in native and methylglyoxal-


Treatment of Amadori product with weak acid (oxalic acid or acetic acid) yields

HMF, which reacts with thiobarbituric acid (TBA) and forms a derivative having

λmax at 443 nm (Sun et al., 2001). The TBA assay measures the amount of ketoamine

bound to the protein. It is based on the release, by hydrolysis, of the adducted MGO

as 5-hydroxymethyl furfural (HMF). The amount of HMF formed in IgG modified

with 1.67, 3.33, 5.00, 6.67 and 8.33 µM MGO was found to be 11.332±0.13,

13.2433±0.14, 14.4234±0.17, 15.5234±0.18 and 16.9846±0.25 nmol/mg, respectively

(Fig. 5).

Nitroblue tetrazolium reduction assay for determination of early glycation

products (Amadori products) in native and methylglyoxal-modified IgG

preparations

Methylglyoxal-induced glycation of IgG was assessed from appearance of Amadori

products (ketoamine moieties) by NBT dye (Arif et al., 2012). Native IgG lacked

ketoamine. Under our experimental conditions the Amadori formation completed at

36 h (Fig. 6). After that the formation of AGEs started. The ketoamine content of IgG

modified with 3.33 µM and 6.67 µM MGO was found to be 135±2.01 nmoles/mg and

160±3.07 nmoles/mg, respectively.

Probing effective protein hydrophobicity by ANS

ANS (8-anilinonaphthalene-1-sulfonic acid) is an organic fluorescent probe and binds

preferentially with hydrophobic regions/patches in proteins. This property of ANS

was used to study gain or loss in hydrophobicity of IgG during MGO-induced

modification (Pasdar and Li-Chan, 2000). The IgG modified by methylglyoxal and

mixed with ANS showed 64.91, 69.23, 73.33, 76.47 and 78.94% jump in fluorescence

intensity as compared to native IgG incubated with same amount of ANS (Fig.7). The

results suggest that MGO-induced modification of IgG has increased its

hydrophobicity.

Chapter 1(a)

32

Fig. 4 Estimation of ε-amino groups in native IgG (black bar) and IgG modified with

1.67 (red bar), 3.33 (green bar), 5.00 (blue bar), 6.67 (yellow bar) and 8.33 µM

(purple bar) MGO. Each bar represents the mean ± S.D. of three independent

assays in similar experimental conditions. *The p value was <0.05 and

considered statistically significant as compared to native IgG.

Fig. 5 Hydroxymethylfurfural content in native IgG (black); IgG modified with 1.67

μM MGO (red); IgG modified with 3.33 μM MGO (green); IgG modified with

5.00 μM MGO (blue); IgG modified with 6.67 μM MGO (orange); and IgG

modified with 8.33 μM MGO (purple). Each bar represents the mean ± S.D. of

three independent assays in similar experimental conditions. *The p value was

<0.05 and considered statistically significant as compared to native IgG.

Chapter 1(a)

33

Fig. 6 Level of Amadori adducts in IgG modified by 3.33 µM (open square) and 6.67

µM (open triangle) MGO, respectively as determined by NBT dye.

Fig. 7 Emission spectra of ANS binding with native IgG (filled circle) and IgG

modified with 1.67 μM (filled triangle), 3.33 μM (filled square), 5.00 μM

(open circle), 6.67 μM (open triangle) and 8.33 μM MGO (open square),

respectively. The samples were excited at 380 nm.

Chapter 1(a)

34

Estimation of protein carbonyl in native and methylglyoxal-modified IgG

preparations

The oxidative stress generated during the course of MGO-induced modification of

IgG was assessed in terms of carbonyl; a known bio-marker of oxidative stress

(Pradeep et al., 2013). Carbonyls, biomarker of the oxidative stress, are outcome of

the oxidation of lysine, arginine, threonine and proline residues. The side chains of

lysine, arginine, threonine and proline residues can be oxidized and carbonyl may be

formed. Upon reaction with dinitrophenylhydrazine, carbonyls form 2,4-

dinitrophenylhydrazone derivatives which can be measured spectrophotometrically at

360 nm. The carbonyl content of IgG increased to 87.12±4.83 and 130.68±6.73

nmol/mg protein, respectively when treated with 3.33 and 6.67 µM MGO (Fig. 8).

The average carbonyl content in native IgG was found to be 43.56±0.97 nmol/mg of

IgG.

Estimation of free sulfhydryl in native and methylglyoxal-modified IgG

preparations

Besides carbonyl, decrease/loss in free sulfhydryl groups is an authentic parameter to

demonstrate oxidation during the course of a reaction (Ellman, 1959). Using Ellman’s

reagent the free sulfhydryl content in native IgG was found to be 222±1.9 nmoles/mg

of protein (Fig. 9). We observed a significant decrease in IgG sulfhydryl when

modified with 3.33 µM MGO (148±0.7 nmoles/mg protein) and 6.67 µM MGO

(111.195±1.52 nmoles/mg protein). The results suggest that the MGO modification of

IgG has affected protein’s redox equilibrium in favour of oxidation.

FT-IR spectroscopy

FT-IR spectroscopy is a reliable technique for recording changes in the secondary

structure of the proteins based on the stretching and bending of bonds in peptide

backbone. In case of human IgG the bands located between the wavenumbers 1624

and 1642 cm-1

and 1510 and 1550 cm-1

have been assigned to amide I and II,

respectively which corresponds to β-sheets (Manning, 2005). The FT-IR spectra of

native and MGO-modified IgG samples are shown in Fig. 10 (a-c). The peak position

of amide I band in native IgG (1642 cm-1

) shifted to 1636 cm-1

and 1634.2 cm-1

,

respectively for IgG modified with 3.33 µM and 6.67 µM MGO, respectively.

Furthermore, the peak position of the amide II bands in native IgG observed at 1542

cm-1

shifted to 1536 cm-1

for IgG modified with 3.33 µM MGO and 1532.4 cm-1

for

Chapter 1(a)

35

IgG modified with 6.67 µM MGO. It suggests consumption of amino groups during

MGO modification vis-a-vis stretching and bending of bonds involved in peptide

backbone. The data has been compiled in Table 4.

Fig. 8 Carbonyl content in native IgG (black), treated with 3.33 µM MGO (red) and

6.67 µM MGO (blue). Each bar represents the mean ± S.D. of three

independent assays in similar experimental conditions. *The p value was


Fig. 9 Free sulfhydryl content in native IgG (black), treated with 3.33 µM MGO (red)

and 6.67 µM MGO (blue). Each bar represents the mean ± S.D. of three

independent assays in similar experimental conditions. *The p value was


Chapter 1(a)

36

Fig. 10(a) FTIR profile of IgG in PBS, pH 7.4

Fig. 10(b) FTIR profile of IgG treated with 3.33 µM MGO.

Chapter 1(a)

37

Fig. 10(c) FTIR profile of IgG treated with 6.67 µM MGO.

Table 4

Bands characteristic in FTIR of native and MGO-modified IgG preparations

Sample

Band type and wavenumber

Amide I

Amide II

Extra band

Native IgG

IgG treated with 3.33 µM MGO

IgG treated with 6.67 µM MGO

1642

1636

1634.2

1542

1536

1532.4

1585

1581

1579.60

CD spectroscopy

To study the secondary structure of MGO-modified IgG samples, the preparations

were subjected to ellipticity measurements in far-UV region (190-250 nm). The CD

profile of IgG in its native conformation in far-UV region (190-250 nm) exhibited

characteristic feature of a β-sheeted protein with negative minima at 217 nm, positive

maxima at 200 nm and zero ellipticity at 209 nm (Fig. 11). The far-UV CD of MGO-

Chapter 1(a)

38

modified IgG at different concentrations of MGO showed enhanced ellipticity at

217 nm and decreased ellipticity at 200 nm as compared to native IgG. The findings

indicate that MGO modification of IgG has caused decrease in α-helical conformation

and increase in β-sheet and random coil structure (Arfat et al., 2014). Furthermore,

information on β-sheet packaging of IgG was obtained from S value. An S value of 1

indicates weakly packed β-sheets, while values greater than 1 and less than 1 indicate

strongly packed β-sheets and loosely packed β-sheets, respectively. In our study,

the S values of native and modified IgG were calculated to be 1.383 and 0.892

respectively. It suggests that β-sheets in MGO-modified IgG are loosely packed. In

near-UV region (250-350 nm) the CD profile of modified IgG showed increase in

negative ellipticity at 262 and 267 nm and decrease in positive ellipticity at 290 nm

compared to native IgG (Fig. 12) which may be due to modification of lysine and

arginine residues.

The findings indicate that MGO modification of IgG has caused decrease in α-helical

conformation, and increase in β-sheet and random coil structure. CD studies in near-

UV region (250-350 nm) shows that for modified-IgG samples the negative ellipticity

at 262 and 267 nm, and positive ellipticity at 290 nm has increased compared to

native IgG (Fig. 12). It indicates MGO modification induced environment change in

aromatic residues, and oxidation of thiol groups-all associated with the tertiary

structure.

Aggregate(s) detection in methylglyoxal-modified IgG preparations by dye

The absorbance of Congo red is significantly enhanced when bound by protein

aggregates (Bose et al., 2010). Upon binding with aggregates in MGO-modified IgG a

shift in the dye λmax and a characteristic apple-green birefringence due to expansion

of the conjugated π-electron system of the dye occurred (Fig. 13). No significant

change was observed or no red shift was observed in spectrum of the dye upon its

incubation with native IgG. Thus, the result of Congo red binding to modified

samples support formation of aggregates upon modification of protein by the MGO.

Aggregate formation was further probed with another dye Thioflavin T (ThT), a

fluorescent dye (Biancalana and Koide, 2010). ThT showed more binding with MGO-

modified IgG samples compared to native IgG (Fig.14). We observed 83.33%,

Chapter 1(a)

39

88.02%, 88.81% and 89.50% increase in emission intensities of IgG samples modified

by MGO at 1.67, 3.33, 5.00 and 6.67 µM, respectively.

Fig. 11 Far UV CD spectra of native IgG kept in PBS (bold line) and IgG treated with 3.33

µM MGO (dotted line) (a) and 6.67 µM MGO (dotted line) (b).

-40

-30

-20

-10

0

10

20

30

40

50

190 200 210 220 230 240 250

Ell

ipti

city

(m

deg

)

Wavelength (nm)

a

b

Chapter 1(a)

40

Fig. 12 Near UV CD spectra of native IgG kept in PBS (bold line) and IgG treated

with 3.33 µM MGO (dotted line) (a) and 6.67 µM MGO (dotted line) (b).

-25

-20

-15

-10

-5

0

5

10

15

20

25

250 270 290 310 330 350

Ell

ipti

city

(m

deg

)

Wavelength (nm)

a

b

Chapter 1(a)

41

Fig. 13 Absorption spectra of Congo red (open circle) bound to native IgG (filled

circle), treated with 3.33 µM MGO (open square) and 6.67 µM MGO (filled

square).

Fig. 14 Emission profiles of ThT bound to native IgG (filled circle) modified with

MGO at 1.67 μM (filled triangle), 3.33 μM (filled square), 5.00 μM (open

circle) and 6.67 μM (open triangle). The samples were excited at 435 nm.

Chapter 1(a)

42

SEM and TEM analysis of native and methylglyoxal-modified IgG preparations

MGO-induced generation of IgG aggregates was viewed under SEM and the images

are shown in Fig. 15 a-c. Native IgG was visualized as rod (Fig. 15a) while MGO-

modified samples appeared as small and large aggregates at 3.33 µM (Fig. 15b) and

6.67 µM MGO-modified IgG (Fig. 15c), respectively.

TEM images of native and MGO-modified IgG samples are depicted in Fig. 16 a-c.

Native human IgG (Fig. 16a) appeared as stretch of globules while both preparations

of MGO-modified IgG at 3.33 and 6.67 µM of MGO (Fig. 16b and c) appeared as

extended branched protein networks with large surface area and amorphous and

irregular shape.

Thermal denaturation profile of native and methylglyoxal-modified IgG

preparations

Modifications-induced effect on the stability of protein was assessed by controlled

heating at 1 0C/min. Heat-induced structural transitions in native and modified-IgG

samples was monitored at 280 nm by heating at 1 0C/min using Peltier device (Zaman

et al., 2017). The native and modified-IgG samples were heated from 20-95 0C and

absorbance was recorded at 280 nm. The increase in absorbance at 280 nm was taken

as a measure of denaturation. Melting temperatures of protein samples were

determined from fraction of proteins denatured (fD) at different temperatures.

The melting temperature (Tm) of native IgG was found to be 72.5 0C. The IgG

samples modified with 3.33 µM and 6.67 µM MGO showed Tm of 75.8 0C and 76.4

0C, respectively (Fig. 17a & b). In case of native IgG the unfolding started at 40

0C

which was delayed by 10 0C in case of MGO-modified IgG. The results suggest that

MGO-modified IgG is thermostable as compared to native IgG.

Differential Scanning Calorimetry (DSC) studies of native and methylglyoxal-


Thermal stability of native and modified-IgG was also examined by DSC. The

thermal denaturation of these samples between 20 and 90 °C resulted in two

independent endothermic transitions (Fig. 18). The second transition was larger in

amplitude. Since IgG is a multi-domain protein, the two peaks observed in the study

may represent the denaturation of Fab (first transition) and Fc (second transition)

domains (Arfat et al., 2014). In case of native IgG, the first transition showed a Tm of

Chapter 1(a)

43

65 °C and the denaturation enthalpy value was found to be 160.1 kcal/mol, while the

second transition Tm was seen at 77.5 °C with an enthalpy of 860.8 kcal/mol. In

comparison to the above, the Tm value of two transitions of MGO-modified IgG was

found at 70 °C and 80.1 °C, and the enthalpies were 172.7 kcal/mol and

866.5 kcal/mol, respectively. The findings suggest that MGO modification of IgG has

conferred stability on IgG domains evident from increase in Tm and enthalpy.

Dynamic light scattering studies on native and methylglyoxal-modified IgG

preparations

DLS is an important technique for determination of the size of the protein particles

(Arfat et al., 2016). For native IgG, the hydrodynamic diameter was found to be

9.8 nm and an apparent molecular weight of 154 kDa with 10.8% polydispersity (Fig.

19a). In case of modified-IgG preparations (Fig. 19 b-e), there is increase in

hydrodynamic diameter i.e. 11.2, 11.6, 11.8 and 12.6 nm respectively for 1.67, 3.33,

5.00 and 6.67 µM MGO-modified IgG and polydispersity i.e. 12.2%, 12.7%, 13.1%

and 13.6% was observed. Our results suggest appreciable increase in IgG size due to

attachment of methylglyoxal and subsequent formation of aggregates.

LC-MS studies on native and methylglyoxal-modified IgG preparations

CML (N ε-carboxymethyllysine), a known standard of non-fluorescent AGEs was

evaluated in the acid hydrolysates of modified-IgG preparations (Schleicher et al.,

1981). Fig. 20 a-c shows mass spectroscopic profiles of standard CML, acid

hydrolyzed native IgG, and acid hydrolyzed MGO-modified preparations (i.e.

modified with 6.67 µM MGO). As shown in Fig. 20c, the acid hydrolysate of MGO-

modified IgG preparations showed peaks at m/z value of 339.1154, 358.2132,

372.2285, 373.2316 matching with m/z value of standard CML peak. No such species

was observed in the hydrolysate of native IgG. Peak identity was assigned by the

retention time, chromatographic pattern and spike of authentic standard.

Chapter 1(a)

44

Fig. 15 SEM images of native IgG (a), 3.33 µM MGO-modified IgG (b), 6.67 µM

MGO-modified IgG (c). All magnification at 2000 x.

(a)

(b)

(c)

Chapter 1(a)

45

Fig. 16 TEM images of native IgG (a), 3.33 µM MGO-modified IgG (b), 6.67 µM

MGO-modified IgG (c). All magnification at 60,000 x.

(b)

(c)

(a)

Chapter 1(a)

46

Fig. 17(a) Melting profiles of native IgG (—) treated with 3.33 µM MGO (- - -).

Fig. 17(b) Melting profiles of native IgG (—) treated with 6.67 µM MGO (- - -).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperatur (0C)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperatue (0C)

a

b

Chapter 1(a)

47

Fig. 18 DSC thermograms of native IgG kept in PBS (bold line) and IgG treated with

3.33 µM MGO (dotted line) (a) and 6.67 µM MGO (dotted line) (b) at

heating rate of 1 0C min

-1.

0

20

40

60

80

100

120

140

160

180

20 30 40 50 60 70 80 90

Cp

(kca

l/m

ol/

0C

)

Temperature (0C)

a

b

Chapter 1(a)

48

a

b

c

d

Chapter 1(a)

49

Fig. 19 DLS profile of native IgG kept in PBS (a), and IgG treated with 1.67 µM

MGO (b), 3.33 µM MGO (c), 5.00 µM MGO (d) and 6.67 µM MGO (e) for

determining hydrodynamic diameter and polydispersity.

e

a

b

Chapter 1(a)

50

Fig. 20 LC-MS analysis of acid hydrolysates of native IgG (a), standard CML (b),

6.67 µM MGO-modified IgG (c).

c

Chapter 1(b)

Biochemical and biophysical studies on IgG co-modified with methylglyoxal and normal

(5 mM)/high glucose (10 mM)

Chapter 1(b)

51

METHODS

Estimation of protein concentration by bicinchoninic acid

Colorimetric estimation of the protein was carried out by bicinchoninic acid

(BCA) reagent (Walker, 1994) using BSA as a standard. The estimation is based on

the reduction of Cu2+

to Cu+

in presence of proteins under alkaline conditions. The

Cu+

is then detected by reaction with BCA reagent which develop intense purple

colour with an absorbance maximum at 562 nm. Briefly, to one ml of protein sample

2.0 ml of BCA working reagent was added. The BCA working reagent was prepared

fresh by mixing 100 ml reagent A (0.1 g sodium bicinchoninate, 2.0 g Na2CO3.H2O,

0.16 g sodium-potassium tartarate, 0.4 g sodium hydroxide, 0.95 g sodium

bicarbonate in 100 ml distilled water) with 2 ml of reagent B (0.4 g CuSO4.5H2O in

10 ml of distilled water). The mixture was incubated at 60 0C for 30 min followed by

cooling at room temperature. The colour intensity was read at 562 nm against blank.

Protein concentration in unknown sample was determined from standard plot

constructed with BSA.

Modification of human IgG by co-treatment with methylglyoxal and normal (5

mM)/high glucose (10 mM)

IgG (6.67 µM) mixed with methylglyoxal (6.67 µM) and 5 mM and 10 mM

glucose, respectively; and IgG treated with only 5 mM and 10 mM glucose were

taken in an assay tube. The reaction was carried out in 10 mM PBS, pH 7.4 under

sterile conditions for 7 days at 37 0C. Appropriate controls were prepared and kept

under identical experimental conditions. At the end of the incubation the solutions

were extensively dialyzed against 10 mM PBS, pH 7.4 to remove excess

methylglyoxal and glucose and then stored at -20 0C.

Note: The other methods used in this Chapter (other than as described above) are

same as described under Chapter 1a.

Chapter 1(b)

52

Results

UV characterization of modified-IgG preparations

IgG co-treated with 6.67 µM MGO and 5/10 mM glucose, respectively; as well as the

IgG treated with 5/10 mM glucose only showed progressive increase in 280 nm

absorbance (Fig. 21a and b). The hyperchromicity was found to be associated with

sugar concentration and incubation time (Table 5). There was 60.0, 68.0, 53.92, and

56.21% hyperchromicity in IgG co-treated with 6.67 µM MGO and 5/10 mM glucose,

respectively; and IgG treated with 5/10 mM glucose only, respectively.

Fluorescence studies on modified-IgG preparations

The tryptophan’s intrinsic fluorescence in IgG was monitored to collect information

about change in the microenvironment of tryptophan residues as a consequence of the

co-treatment by 6.67 µM MGO and/or 5 mM and 10 mM glucose (Hunt, et al., 1988).

Native and modified-IgG preparations were excited at 285 nm and the emission

intensity was recorded over the range of wavelength from 290-400 nm (Fig. 22 &

Table 6). There was a marked decrease in the emission intensity of IgG samples co-

treated with 6.67 µM MGO and 5 mM and 10 mM glucose, respectively; and IgG

treated with 5 mM and 10 mM glucose. The decrease in the fluorescence intensity

(FI) was 57.89, 65.78, 7.89 and 35.52% with increasing concentrations of glucose

and/or MGO (6.67 µM) respectively; in IgG sample co-treated with 6.67 µM MGO

and 5 mM and 10 mM glucose, respectively; and IgG treated with 5 mM and 10 mM

glucose as compared to native IgG.

During the modification of IgG by 5 mM and 10 mM glucose co-treated with and/or

6.67 µM MGO the likely formation of fluorogenic AGEs was assessed by exciting the

samples at 370 nm and recording the emission intensity of the samples over

wavelength range of 400-600 nm. Native IgG after excitation at 370 nm showed

negligible AGE specific fluorescence (Fig. 23 & Table 7). Under similar experimental

conditions the fluorescence intensity of the modified IgG samples gradually increased

in a time dependent and concentration dependent manner which suggests the

formation of fluorogenic AGEs during modification (de la Maza et al., 2012). The

increase in the fluorescence intensity was 89.37, 90.11, 83.33 and 87.85% with

increasing concentrations of glucose and/or MGO (6.67 µM), respectively; in IgG

Chapter 1(b)

53

samples co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose, respectively;

and IgG treated with 5 mM and 10 mM glucose as compared to native IgG. No further

change in fluorescence intensity was observed after 7 days of incubation.

Estimation of ε-amino groups in native and modified-IgG preparations

As shown in Fig. 24, addition of 5 mM (low) glucose to IgG has consumed some ε-

amino moieties and it came down from 230.01±1.72 nmol/mg in native IgG to

200.22±1.54 nmol/mg in IgG containing 5 mM glucose. Addition of methylglyoxal

further decreased the ε-amino moieties to 111.78±0.82 nmol/mg (Sashidhar et al.,

1994). Furthermore, at 10 mM (high) glucose, the consumption of ε-amino group

increased and methylglyoxal addition further engaged the ε-amino group.

Estimation of hydroxymethyl furfural (HMF) in native and modified-IgG

preparations

Treatment of Amadori product with weak acid (oxalic acid or acetic acid) yields

HMF, which reacts with thiobarbituric acid (TBA) and forms a derivative having

λmax at 443 nm (Sun et al., 2001). The TBA assay measures the amount of ketoamine

bound to the protein. It is based on the release, by hydrolysis, of the adducted MGO

and/or glucose co-modified IgG as 5-hydroxymethyl furfural (HMF). The amount of

HMF formed in IgG co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose,

respectively; was 17.2332±0.21and 20.7024±0.27 nmol/mg, respectively (Fig. 25).

The amount of HMF formed in IgG modified with 5 mM glucose and 10 mM glucose

was 9.0752±0.07and 13.344±0.13 nmol/mg, respectively (Fig. 25). Native IgG

incubated under identical conditions without MGO/glucose served as control, had

almost negligible content of HMF. Thus, the highest amount of HMF was released

from IgG co-treated with methylglyoxal and high glucose (10 mM) (Fig. 25).

Nitroblue tetrazolium reduction assay for determination of early glycation

products (Amadori products) of native and modified-IgG preparations

Modification of IgG was assessed from appearance of Amadori products (ketoamine

moieties) by NBT dye (Baker et al., 1994). Native IgG lacked ketoamine. The

ketoamine content of IgG co-treated with 6.67 µM MGO and 5 mM glucose was

(160±2.01 nmoles/mg) and the ketoamine content of IgG co-treated with 6.67 µM

MGO and 10 mM glucose was (180±3.07 nmoles/mg), respectively; and IgG

modified with 5 mM of glucose was (100±1.07 nmoles/mg) and IgG modified with 10

Chapter 1(b)

54

mM glucose was (110±1.17 nmoles/mg). IgG modified with 5 mM and 10 mM

glucose had maximum ketoamine content after 72 hours of incubation (Fig. 26) and

IgG co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose, respectively; had

maximum ketoamine content after 24 hours of incubation (Fig. 26). It showed that

early glycation was maximum after 72 hours of incubation in IgG modified with 5

mM and 10 mM glucose and after 24 hours of incubation in IgG co-treated with 6.67

µM MGO and 5 mM and 10 mM glucose, respectively; and after that AGEs

production would start which lead to the decrease in the ketoamine content.

Fig. 21(a) UV absorption spectra of native IgG (open square) and IgG treated with 5

mM glucose (open rhombus) and co-treated with 3.33 µM MGO and 5 mM

glucose (open triangle) and co-treated with 6.67 µM MGO and 5 mM

glucose (open circle) for 7 days.

Chapter 1(b)

55

Fig. 21(b) UV absorption spectra of native IgG (open square) and IgG treated with

10 mM glucose (open rhombus) and co-treated with 3.33 µM MGO and

10 mM glucose (open triangle) and co-treated with 6.67 µM MGO and 10

mM glucose (open circle) for 7 days.

Fig. 22 Emission profile of native IgG (open square); IgG + 5.0 mM glucose (open

triangle); IgG + 10.0 mM glucose (open circle); IgG + 5.0 mM glucose +

6.67 μM MGO (filled triangle); IgG + 10.0 mM glucose + 6.67 μM MGO

(filled circle) after 7 days of incubation. All samples were excited at 285 nm

for tryptophan fluorescence.

Chapter 1(b)

56

Table 5

Effect of time on 280 nm absorbance of native IgG and its modified counterparts

Sample Absorbance at 280 nm

(0 h) (24 h) (48 h) (72 h) (96 h) (120 h) (144 h) (168 h)

Native

IgG

0.80

(0.0)

0.82

(0.0)

0.84

(0.0)

0.85

(0.0)

0.86

(0.0)

0.87

(0.0)

0.88

(0.0)

0.88

(0.0)

IgG

+ 5 mM

glucose

1.4

(42.85)

1.46

(43.83)

1.54

(45.45)

1.61

(47.20)

1.72

(50.00)

1.8

(51.66)

1.84

(52.17)

1.91

(53.92)

IgG

+ 10 mM

glucose

1.5

(46.66)

1.55

(47.09)

1.62

(48.14)

1.7

(50.00)

1.8

(52.22)

1.9

(54.21)

1.96

(55.10)

2.01

(56.21)

IgG

+ 5 mM

glucose

+ 3.33

µM MGO

1.54

(48.05)

1.62

(49.38)

1.7

(50.58)

1.78

(52.24)

1.86

(53.76)

1.95

(55.38)

2.03

(56.65)

2.1

(58.09)

IgG

+ 5 mM

glucose

+ 6.67

µM MGO

1.64

(51.21)

1.74

(52.87)

1.81

(53.59)

1.91

(55.49)

1.99

(56.78)

2.06

(57.76)

2.13

(58.68)

2.2

(60.00)

IgG

+ 10 mM

glucose

+ 3.33

µM MGO

1.72

(53.48)

1.82

(54.94)

1.89

(55.55)

2.02

(57.92)

2.15

(60.00)

2.27

(61.67)

2.39

(63.17)

2.54

(65.35)

IgG

+ 10 mM

glucose

+ 6.67

µM MGO

1.82

(56.04)

1.94

(57.73)

2.1

(60.00)

2.17

(60.82)

2.29

(62.44)

2.49

(65.06)

2.64

(66.66)

2.75

(68.00)

Note: The data within parentheses indicate percent change in absorbance at 280 nm

compared to native IgG.

Chapter 1(b)

57

Table 6

Effect of time on tryptophan specific fluorescence of various samples

Sample Fluorescence intensity

(0 h) (24 h) (48 h) (72 h) (96 h) (120 h) (144 h) (168 h)

Native

IgG 980 950 917 880 848 800 776 760

IgG

+ 5 mM

glucose

934

(4.69)

900

(5.26)

866

(5.56)

824

(6.36)

792

(6.60)

745

(6.87)

721

(7.08)

700

(7.89)

IgG

+ 10 mM

glucose

912

(7.45)

872

(8.21)

816

(11.01)

743

(15.56)

706

(16.74)

657

(17.87)

540

(30.41)

490

(35.52)

IgG

+ 5 mM

glucose

+ 6.67

µM MGO

700

(28.57)

646

(32.00)

606

(33.91)

576

(34.54)

516

(39.15)

442

(44.75)

396

(48.96)

320

(57.89)

IgG

+ 10 mM

glucose +

6.67 µM

MGO

600

(38.77)

564

(40.63)

514

(43.94)

478

(45.68)

422

(50.23)

376

(53.00)

300

(61.34)

260

(65.78)

Note: (i) The data within parentheses indicate percent decrease in F.I. compared to

native IgG.

(ii) All samples were excited at 285 nm.

Chapter 1(b)

58

Fig. 23 Emission profile of native IgG (filled circle); IgG + 5.0 mM glucose (filled

triangle); IgG + 10.0 mM glucose (filled square); IgG + 5.0 mM glucose +

6.67 μM MGO (open triangle); IgG + 10.0 mM glucose + 6.67 μM MGO

(open square) after 7 days of incubation. All samples were excited at 370 nm

for AGEs.

Fig. 24 Estimation of ε-amino groups in native IgG (black bar) and IgG + 5.0 mM

glucose (red bar); IgG + 10.0 mM glucose (green bar); IgG + 5.0 mM glucose

+ 6.67 μM MGO (blue bar); IgG + 10.0 mM glucose + 6.67 μM MGO (purple

bar). Each bar represents the mean ± S.D. of three independent assays in

similar experimental conditions. *The p value was <0.05 and considered

statistically significant as compared to native IgG.

Chapter 1(b)

59

Table 7

Effect of time on appearance of AGEs specific fluorescence in modified-IgG

preparations

Sample Fluorescence intensity

(0 h) (24 h) (48 h) (72 h) (96 h) (120 h) (144 h) (168 h)

Native

IgG 55 65 75 85 85 85 85 85

IgG

+ 5 mM

glucose

100

(45.00)

130

(50.00)

250

(70.00)

320

(73.43)

380

(77.63)

440

(80.68)

480

(82.29)

510

(83.33)

IgG

+ 10 mM

glucose

125

(56.00)

200

(67.50)

340

(77.94)

420

(79.76)

480

(82.29)

575

(85.21)

640

(86.71)

700

(87.85)

IgG

+ 5 mM

glucose

+ 6.67

µM MGO

250

(78.00)

350

(81.42)

440

(82.95)

530

(83.96)

615

(86.17)

680

(87.50)

740

(88.51)

800

(89.37)

IgG

+ 10 mM

glucose +

6.67 µM

MGO

325

(83.07)

450

(85.55)

595

(87.39)

690

(87.68)

720

(88.19)

760

(88.81)

810

(89.50)

860

(90.11)

Note: (i) The data within parentheses indicate percent increase in F.I. compared to

native IgG.

(ii) All samples were excited at AGEs specific wavelength of 370 nm.

Chapter 1(b)

60

Fig. 25 Hydroxymethylfurfural content in native IgG (black bar); IgG + 5 mM

glucose (red bar); IgG + 10 mM glucose (green bar); IgG + 5 mM glucose +

6.67 μM MGO (blue bar); and IgG + 10 mM glucose + 6.67 μM MGO (purple

bar). Each bar represents the mean ± S.D. of three independent assays in



Fig. 26 Level of Amadori adducts in IgG + 5 mM glucose (open square); IgG + 10

mM glucose (open triangle); IgG + 5 mM glucose + 6.67 µM MGO (filled

square); and IgG + 10 mM glucose + 6.67 µM MGO (filled triangle).

Chapter 1(b)

61

Probing effective protein hydrophobicity by ANS

ANS (8-anilinonaphthalene-1-sulfonic acid) is an organic fluorescent probe and binds

preferentially with hydrophobic regions/patches in proteins. This property of ANS

was used to study gain or loss in hydrophobicity of IgG during modification

(Csizmadia et al., 1999). Profiles in Fig. 27 suggests that methylglyoxal has led to

exposure of more hydrophobic moieties/patches in IgG co-treated with 6.67 µM

MGO and high glucose (10 mM) compared to low glucose (5 mM).

Estimation of protein carbonyl in native and modified-IgG preparations

The oxidative stress generated during the course of modification of IgG was assessed

in terms of carbonyl; a known bio-marker of oxidative stress (Levine et al., 1990).

Carbonyls, biomarker of the oxidative stress, are outcome of the oxidation of lysine,

arginine, threonine and proline residues. The side chains of lysine, arginine, threonine

and proline residues can be oxidized and carbonyl may be formed. Upon reaction with

dinitrophenylhydrazine, carbonyls form 2,4-dinitrophenylhydrazone derivatives

which can be measured spectrophotometrically at 360 nm. The average carbonyl

content of native IgG was found to be 43.56±0.97 nmol/mg of IgG (Fig. 28). Both

glucose and methylglyoxal produced stress on protein and lead to 2-5 fold increase in

carbonyl (Fig. 28).

Estimation of free sulfhydryl in native and modified-IgG preparations

Modifications induced by glucose and/or MGO on protein is generally accompanied

by oxidative stress which leads to many biochemical changes. A change in the redox

state of protein is also the consequence of oxidative stress, and sulfhydryl estimation

by Ellman’s reagent is an authentic parameter of such an event (Ellman, 1959).

Besides carbonyl, decrease/loss in free sulfhydryl groups is an authentic parameter to

demonstrate oxidation during the course of a reaction. Using Ellman’s reagent the free

sulfhydryl content in native IgG was found to be 222±1.9 nmoles/mg of protein

(Fig. 29). As shown in Fig. 29 there is a reduction in free sulfhydryls when IgG was

treated with glucose. When MGO was added the free sulfhydryls decreased in high

amount compared to what was observed with glucose alone. It simply suggests that

the redox imbalance created in IgG by glucose got augmented in presence of

methylglyoxal during co-treatment.

Chapter 1(b)

62

FT-IR spectroscopy

FT-IR spectroscopy is a reliable technique for recording changes in the secondary

structure of the proteins based on the stretching and bending of bonds involved in

peptide backbone (Pelton and McLean, 2000). In case of human IgG the bands located

between the wavenumbers 1624 and 1642 cm-1

and 1510 and 1550 cm-1

have been

assigned to amide I and II, respectively which corresponds to β-sheets (Pelton and

McLean, 2000). The FT-IR spectra of native and modified-IgG samples co-treated

with 6.67 µM MGO and 5 mM and 10 mM glucose, respectively; and IgG treated

with 5 mM and 10 mM glucose are shown in Fig. 30a-e. However, the peak positions

of the amide I bands shifted from 1642 (native IgG) to 1633 cm-1

(IgG co-treated with

6.67 µM MGO and 5 mM glucose), 1631.6 cm-1

(IgG co-treated with 6.67 µM MGO

and 10 mM glucose), 1639 cm-1

(IgG treated with 5 mM glucose) and 1637 cm-1

(IgG

treated with 10 mM glucose). Furthermore, in the same samples the peak positions of

the amide II bands shifted from 1542 (native IgG) to 1529 cm-1

(IgG co-treated with

6.67 µM MGO and 5 mM glucose), 1527.1 cm-1

(IgG co-treated with 6.67 µM MGO

and 10 mM glucose), 1539 cm-1

(IgG treated with 5 mM glucose) and 1537 cm-1

(IgG

treated with 10 mM glucose). The 1585 cm-1

band in native IgG shifted to 1579 cm-1

(IgG co-treated with 6.67 µM MGO and 5 mM glucose), 1576.4 cm-1

(IgG co-treated

with 6.67 µM MGO and 10 mM glucose), 1583 cm-1

(IgG treated with 5 mM glucose)

and 1582 cm-1

(IgG treated with 10 mM glucose) and that suggest consumption of

protein amino group during modification of IgG. Thus, FT-IR finding strongly

suggests changes in secondary structure of IgG associated with the stretching and

bending of bond involved in peptide backbone and also generation of AGEs. The data

has been compiled in Table 8.

CD spectroscopy

To study the secondary structure of modified-IgG samples, the samples were

subjected to ellipticity measurements in far-UV region (190-250 nm). The CD profile

of IgG in its native conformation in far-UV region (190-250 nm) exhibited

characteristic feature of a β-sheeted protein with negative minima at 217 nm, positive

maxima at 200 nm and zero ellipticity at 209 nm (Fig. 31). The far-UV CD of

modified IgG samples co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose,

respectively; and IgG treated with 5 mM and 10 mM glucose showed enhanced

Chapter 1(b)

63

ellipticity at 217 nm and decreased ellipticity at 200 nm as compared to native IgG.

The findings indicate that modification of IgG has caused decrease in α-helical

conformation, and increase in β-sheet and random coil structure. Furthermore,

information on β-sheet packaging of IgG was obtained from S value. An S value of 1

indicates weakly packed β-sheets, while values greater than 1 and less than 1 indicate

strongly packed β-sheets and loosely packed β-sheets, respectively (Arfat et al.,

2014). In our study, the S values of native and modified-IgG were calculated to be

1.383 and 0.864 respectively. It suggests that β-sheets in modified-IgG are loosely

packed. In near-UV region (250-350 nm) the CD profile of modified IgG showed

increase in negative ellipticity at 262 and 267 nm and decrease in positive ellipticity at

290 nm compared to native IgG (Fig. 32) which may be due to modification of lysine

and arginine residues.

The findings indicate that modification of IgG has caused decrease in α-helical

conformation, and increase in β-sheet and random coil structure. CD studies in near-

UV region (250-350 nm) shows that for modified-IgG samples the negative ellipticity

at 262 and 267 nm, and positive ellipticity at 290 nm has increased compared to

native IgG (Fig. 32). It indicates modification induced environment change in

aromatic residues, and oxidation of thiol groups-all associated with the tertiary

structure.

Fig. 27 Emission profile of ANS binding with native IgG (open triangle); IgG + 5.0

mM glucose (open circle); IgG + 10.0 mM glucose (open square); IgG + 5.0

mM glucose + 6.67 μM MGO (filled triangle); IgG + 10.0 mM glucose +

6.67 μM MGO (filled circle). All samples were excited at 380 nm.

Chapter 1(b)

64

Fig. 28 Carbonyl content of native IgG (black bar) and various samples of modified-

IgG. Each bar represents the mean ± S.D. of three independent assays in



Fig. 29 Free sulfhydryl content of native IgG (black bar) and various samples of

modified- IgG. Each bar represents the mean ± S.D. of three independent

assays in similar experimental conditions. *The p value was <0.05 and

considered statistically significant as compared to native IgG.

Chapter 1(b)

65

Fig. 30(a) FTIR profile of IgG in PBS, pH 7.4

Fig. 30(b) FTIR profile of IgG treated with 5 mM glucose

b

Amide I

Amide II

Chapter 1(b)

66

Fig. 30(c) FTIR profile of IgG treated with 10 mM glucose.

Fig. 30(d) FTIR profile of IgG co-treated with 5 mM glucose and 6.67 µM MGO.

c

Amide I

Amide II

d

Amide II Amide I

Chapter 1(b)

67

Fig. 30(e) FTIR profile of IgG co-treated with 10 mM glucose and 6.67 µM MGO.

Table 8

Bands characteristic in FTIR of native and modified-IgG preparations

Sample

Band type and wavenumber

Amide I

Amide II

Extra band

Native IgG

IgG treated with 5 mM glucose

IgG treated with 10 mM glucose

IgG co-treated with 5 mM glucose and

6.67 µM MGO

IgG co-treated with 10 mM glucose and

6.67 µM MGO

1642

1639

1637

1633

1631.6

1542

1539

1537

1529

1527.1

1585

1583

1582

1579

1576.4

e

Amide I

Amide II

Chapter 1(b)

68

Fig. 31 Far UV CD spectra of native IgG kept in PBS (bold line) and IgG treated with

5 mM glucose (dotted line) (a), co-treated with 5 mM glucose and 6.67 µM

MGO (dotted line) (b) and co-treated with 10 mM glucose and 6.67 µM

MGO (dotted line) (c).

-40

-30

-20

-10

0

10

20

30

40

50

190 200 210 220 230 240 250

Ell

ipti

city

(m

deg

)

Wavelength (nm)

-40

-30

-20

-10

0

10

20

30

40

50

190 200 210 220 230 240 250

Ell

ipti

city

(m

deg

)

Wavelength (nm)

-40

-30

-20

-10

0

10

20

30

40

50

190 200 210 220 230 240 250

Ell

ipti

city

(m

deg

)

Wavelength (nm)

a

b

c

Chapter 1(b)

69

Fig. 32 Near UV CD spectra of native IgG kept in PBS (bold line) and IgG treated

with 5 mM glucose (dotted line) (a), co-treated with 5 mM glucose and 6.67

µM MGO (dotted line) (b) and co-treated with 10 mM glucose and 6.67 µM

MGO (dotted line) (c).

-25

-20

-15

-10

-5

0

5

10

15

20

25

250 270 290 310 330 350

Ell

ipti

city

(m

deg

)

Wavelength (nm)

-25

-20

-15

-10

-5

0

5

10

15

20

25

250 270 290 310 330 350

Ell

ipti

city

(m

deg

)

Wavelength (nm)

-25

-20

-15

-10

-5

0

5

10

15

20

25

250 270 290 310 330 350

Ell

ipti

city

(m

deg

)

Wavelength (nm)

a

b

c

Chapter 1(b)

70

Aggregate(s) detection in modified-IgG samples by dye

The absorbance of Congo red is significantly enhanced when bound by protein

aggregates (Khurana et al., 2001). Upon binding with aggregates in modified IgG

samples a shift in the dye λmax and a characteristic apple-green birefringence due to

expansion of the conjugated π-electron system of the dye occurred (Fig. 33). No

significant change was observed or no red shift was observed in spectrum of the dye

upon its incubation with native IgG. Thus, the result of Congo red binding to

modified-IgG samples support formation of aggregates upon modification of protein

by the MGO and/or glucose. Similarly under identical conditions the dye showed

enhancement and shift in its λmax when bound by glucose and/or MGO-modified

counterparts of IgG (Fig. 33). The findings indicate presence of aggregates.

Aggregate formation was further probed with another dye Thioflavin T (ThT), a

fluorescent dye (Groenning, 2010). Further evidence of aggregates formation came

from enhancement in the emission intensities of modified counterparts of IgG mixed

with ThT (Fig. 34).

SEM and TEM analysis of native and modified-IgG preparations

Modification-induced generation of IgG aggregates was viewed under SEM and the

images are shown in Fig. 35a-e. Native IgG was visualized as rod (Fig. 35a) while

modified-IgG preparations appeared as small and large aggregates, respectively

(Fig. 35b-e).

TEM images of native and modified-IgG preparations are depicted in Fig. 36a-e.

Native human IgG (Fig. 36a) appeared as stretch of globules while low and high

glucose versions of MGO-modified IgG appeared as extended branched protein

networks with large surface area and amorphous and irregular in shape (Fig. 36b-e).

Thermal denaturation profile of native and modified-IgG preparations

Modifications-induced effect on the stability of protein was assessed by controlled

heating at 1 0C/min. Heat-induced structural transitions in native and modified-IgG

samples co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose, respectively;

and IgG treated with 5 mM and 10 mM glucose was monitored at 280 nm by heating

at 1 0C/min using Peltier device. The native and modified-IgG samples were heated

http://www.jbc.org/search?author1=Ritu+Khurana&sortspec=date&submit=Submit

Chapter 1(b)

71

from 20-95 0C and absorbance was recorded at 280 nm. The increase in absorbance at

280 nm was taken as a measure of denaturation. Melting temperatures of protein

samples were determined from fraction of proteins denatured (fD) at different

temperatures (Zaman et al., 2017).

The melting temperature (Tm) of native IgG was found to be 72.5 0C. The IgG

samples treated with 5 mM and 10 mM glucose showed Tm of 73.4 0C and 74.8

0C,

respectively (Fig. 37a and b) whereas, the IgG samples co-treated with 6.67 µM MGO

and 5 mM and 10 mM glucose, respectively; showed Tm of 77.2 0C and 78.9

0C,

respectively (Fig. 37c and d). In case of native IgG the unfolding started at 40 0C

which was delayed by around 10 0C in case of modified-IgG samples. The results

suggest that modified-IgG samples co-treated with 6.67 µM MGO and 5 mM and 10

mM glucose, respectively; and IgG treated with 5 mM and 10 mM glucose is

thermostable as compared to native IgG. The data in (Fig. 37a-d) suggests that the

modification introduced by glucose and/or methylglyoxal has lead to structural

reorganization and the molecule (IgG) has gained stability as observed in the case of

modified-IgG samples.

Differential Scanning Calorimetry (DSC) studies of native and modified-IgG

preparations

Thermal stability of native and modified-IgG was examined by DSC. The thermal

denaturation of these samples between 20 and 90 °C resulted in two independent

endothermic transitions (Fig. 38). The second transition was larger in amplitude.

Since IgG is a multi-domain protein, the two peaks observed in the study may

represent the denaturation of Fab (first transition) and Fc (second transition) domains

(Arfat et al., 2016). In case of native IgG, the first transition showed a Tm of 65 °C

and the denaturation enthalpy value was found to be 160.1 kcal/mol, while the second

transition Tm was seen at 77.5 °C with an enthalpy of 860.8 kcal/mol. In comparison

to the above, the Tm value of two transitions of modified IgG samples co-treated with

10 mM glucose and 6.67 µM MGO was found at 77 °C and 82.1 °C, and the

enthalpies were 192.4 kcal/mol and 888.2 kcal/mol, respectively. The findings suggest

that modification of IgG has conferred stability on IgG domains evident from increase

in Tm and enthalpy.

Chapter 1(b)

72

Dynamic light scattering studies on native and modified-IgG preparations

DLS is an important technique for determination of the size of the protein particles

(Arfat et al., 2016). For native IgG, the hydrodynamic diameter was found to be

9.8 nm and an apparent molecular weight of 154 kDa with 10.8% polydispersity

(Fig. 39a). In case of modified-IgG samples (Fig. 39b-e), there is increase in

hydrodynamic diameter i.e. 10.3, 10.9, and 13.2, 13.9 nm respectively for 5 mM and

10 mM glucose and IgG co-treated with 6.67 µM MGO and 5 mM and 10 mM

glucose, respectively; and polydispersity i.e. 11.2%, 11.5 %, 14.8% and 15.9% was

observed. Our result suggests appreciable increase in IgG size due to attachment of

methylglyoxal and glucose residues on IgG in modified-IgG samples, and subsequent

formation of aggregates.

Fig. 33 Absorption profile of Congo red (open square) bound to: native IgG (open

triangle); IgG + 5 mM glucose (open circle); IgG + 10 mM glucose (filled

square); IgG + 5 mM glucose + 6.67 μM MGO (filled triangle); and IgG + 10

mM glucose + 6.67 μM MGO (filled circle).

Chapter 1(b)

73

Fig. 34 Emission profile of Thioflavin T bound to native IgG (filled circle); IgG + 5

mM glucose (filled triangle); IgG + 10 mM glucose (filled square); IgG + 5

mM glucose + 6.67 μM MGO (open triangle); and IgG + 10 mM glucose +

6.67 μM MGO (open square). All samples were excited at 435 nm.

Chapter 1(b)

74

Fig. 35 SEM images of native IgG (a), IgG + 5 mM glucose (b), IgG + 5 mM glucose

+ 6.67 µM MGO (c), IgG + 10 mM glucose (d) and IgG + 10 mM glucose +

6.67 µM MGO (e). All magnification at 1500 x and at an acceleration voltage

of 15 kV.

Chapter 1(b)

75

Fig. 36 TEM images of native IgG (a), IgG + 5 mM glucose (b), IgG + 5 mM glucose

+ 6.67 µM MGO (c), IgG + 10 mM glucose (d) and IgG + 10 mM glucose +

6.67 µM MGO (e). All magnification at 60000 x and at an acceleration

voltage of 200 kV.

Chapter 1(b)

76

Fig. 37(a) Melting profiles of native IgG (—) treated with 5 mM glucose (- - -).

Fig. 37(b) Melting profiles of native IgG (—) treated with 10 mM glucose (- - -).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperatur (0C)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperatur (0C)

a

b

Chapter 1(b)

77

Fig. 37(c) Melting profiles of native IgG (—) co-treated with 6.67 µM MGO and 5

mM glucose (- - -).

Fig. 37(d) Melting profiles of native IgG (—) co-treated with 6.67 µM MGO and 10

mM glucose (- - -).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperature (0C)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

20 30 40 50 60 70 80 90

fD

Temperature (0C)

c

d

Chapter 1(b)

78

Fig. 38 DSC thermograms of native IgG kept in PBS (bold line) and IgG treated with

5 mM glucose (dotted line) (a), co-treated with 6.67 µM MGO and 5 mM

glucose (dotted line) (b) and IgG co-treated with 6.67 µM MGO and 10 mM

glucose (dotted line) (c) at heating rate of 1 0C min

-1.

0

20

40

60

80

100

120

140

160

180

20 30 40 50 60 70 80 90

Cp

(kca

l/m

ol/

0C

)

Temperature (0C)

0

20

40

60

80

100

120

140

160

180

20 30 40 50 60 70 80 90

Cp

(kca

l/m

ol/

0C

)

Temperature (0C)

0

20

40

60

80

100

120

140

160

180

20 30 40 50 60 70 80 90

Cp

(kca

l/m

ol/

0C

)

Temperature (0C)

a

b

c

Chapter 1(b)

79

LC-MS studies on native and modified-IgG preparations

CML (N ε-carboxymethyl lysine) a known standard of non-fluorescent AGEs, was

evaluated in the acid hydrolysates of modified-IgG preparations (Schleicher et al.,

1981). Fig. 40a-d shows mass spectroscopic profile of standard CML, acid hydrolyzed

native IgG, and IgG co-treated with 6.67 µM MGO and 5 mM and 10 mM glucose,

respectively. As shown in Fig. 40 c and d, the acid hydrolysate of modified-IgG

preparations showed peaks at m/z value of 339.1148, 358.2127, 372.2280, 373.2312

matching with m/z value of standard CML peak. No such species was observed in the

hydrolysate of native IgG. Peak identity was assigned by the retention time,

chromatographic pattern and spike of authentic standard.

a

b

Chapter 1(b)

80

Fig. 39 DLS profile of native IgG kept in PBS (a), treated with 5 mM glucose (b), 10

mM glucose (c), co-treated with 6.67 µM MGO and 5 mM glucose (d) and

IgG co-treated with 6.67 µM MGO and 10 mM glucose (e) for determining

hydrodynamic diameter and polydispersity.

c

d

e

Chapter 1(b)

81

Fig. 40 LC-MS analysis of acid hydrolysates of native IgG (a), standard CML (b),

IgG co-treated with 6.67 µM MGO and 5 mM glucose (c) and IgG co-treated

with 6.67 µM MGO and 10 mM glucose (d).

a

b

c

d

82

Combined discussion of Chapter 1(a) and Chapter 1(b)

AGEs formation and accumulation are related to various pathological conditions such

as diabetes and its complications (Brownlee, 1995; Peppa et al., 2003), cardiovascular

disease (Hegab et al., 2012), rheumatoid arthritis (Ahmed et al., 2014) and various

age-related diseases (Gkogkolou and Bohm, 2012). Methylglyoxal is a highly reactive

dicarbonyl and rapidly forms AGEs upon reaction with biomolecules (Ramasamy et

al., 2006) at an accelerated rate during hyperglycaemia in diabetes and its associated

complications (Price and Knight, 2009). Under physiological conditions, the MGO is

readily detoxified into D-lactate by glyoxylase system (Silva et al., 2013; Jack and

Wright, 2012). But under hyperglycaemia, due to increased formation of ROS the

glyoxylase system is unable to perform its functions since it requires reduced

glutathione for its activity (Suh et al., 2015).

IgG is primarily a defence protein and constitutes about 75-80% of the total serum

immunoglobulins. Furthermore, IgG has quite long half-life and possesses 80 lysine

residues and thus it is a better target for methylglyoxal and glucose modification

(Mankarious et al., 1988; Arfat et al., 2014). Critical modifications in IgG structure

may affect its defence role (Bliesener and Gerbitz, 1990) and accumulation of this

aberrant protein may cause or exacerbate diseases. Under hyperglycemic conditions

excessively generated methylglyoxal and glyoxal may covalently bind with IgG and

in due course of time AGEs may form. Participation of modified-IgG in autoimmune

disorders (especially rheumatoid arthritis) has been reported by several authors

(Sokolove et al., 2014). It has been demonstrated that AGE-IgG is immunogenic and

autoantibodies against AGE-IgG have been reported in diseases (Kaneshige, 1987;

Arfat et al., 2014; Ercan et al., 2010).

The hyperchromicities shown by modified-IgG preparations in presence of normal

glucose, high glucose and/or MGO may be due to enhanced exposure of

chromophoric aromatic amino acid residues which were otherwise buried in native

IgG (Traverso et al., 1997). Furthermore, the increased absorbance that we observed

between 300-400 nm wavelength in modified-IgG preparations indicate aggregate

formation as reported earlier (Arfat et al., 2014). IgG incubated with glucose and/or

MGO showed decrease in ε-amino groups. This may be attributed to blocking of

ε-amino groups by glucose and/or MGO.

83

The apparent loss in tryptophan content and change(s) in its microenvironment may

be the apparent reason for observed quenching in tryptophan fluorescence (Davies et

al., 1987; Vivian & Callis, 2001). A similar observation has been made earlier when

haemoglobin was modified by glyoxal (Iram, et.al., 2013). Formation of fluorogenic

AGEs in modified-IgG preparations was evident from increase in fluorescence

intensity at the excitation wavelength of 370 nm (Mustafa and Bano, 2016).

IgG modified by glucose and/or MGO generated Amadori products and was

confirmed by NBT dye and HMF. This further underwent rearrangement reactions

such as cyclization, condensation etc and formed AGEs (Faisal et al., 2017; Pamplona

et al., 1995). Furthermore, increase in protein carbonyl and decrease in sulfhydryl

suggests that the reaction has produced oxidative stress in the system (Yilmaz et al.,

2017; Elosta et al., 2017).

The shifts in position of amide I and amide II bands due to C=O stretching and N-H

bending is a quasi evidence of changes in IgG secondary structure during

modification by glucose and/or MGO (Ashraf et al., 2015). Amide I band is mainly

associated with the C=O stretching vibration of the peptide bond of the protein and

corresponds mainly to α helix of the protein (Kyriakidou et al., 2017). The decrease

observed in amide I band corresponds to the decrease in α helical content of the

protein and thus increase in random coil conformation as well as β pleated sheet

conformation. Furthermore, amide II band derives mainly from N-H in-plane bending

and C-N stretching vibrations (Kyriakidou et al., 2017). Appearance of extra band

around 1585 cm-1

may be related to attachment of carbonyl (C=O) of glucose and/or

methylglyoxal.

The far-UV CD results of modified-IgG preparations showed enhanced ellipticity at

217 nm and decreased ellipticity at 200 nm as compared to native IgG. It suggests that

modification of IgG has caused decrease in α-helical conformation, and increase in β-

sheet and random coil. The above observations were further supported by FT-IR

results which showed loss in the secondary structure of modified-IgG preparations.

Near-UV CD results suggests exposure of some residues and oxidation of thiol groups.

This has possibly resulted in increase in the fraction of surface hydrophobic patches

and cross-linking (Arfat et al., 2014) as also shown by increase in the fluorescence

intensity of ANS dye when bound by modified-IgG preparations.

84

The aggregates and cross-links in modified-IgG preparations were authenticated from

increase in ThT fluorescence, red shift in the Congo red spectrum and the SEM and

TEM images. It has been shown that protein aggregates may lead to various

pathological conditions, collectively known as proteopathies (Moreau & King, 2012).

Enlargement in hydrodynamic size of modified-IgG preparations was shown by DLS

results and it may be linked to attachment of glucose and/or methylglyoxal to IgG.

The denaturation of Fab and Fc domains of native and modified-IgG preparations

represents set of structural transitions and the results generated from DSC

experiments justify those transitions. The DSC results showed increase in

thermostability and enthalpy of modified-IgG preparations. The thermostability

conferred on modified-IgG preparations as shown by DSC and Tm result may be due

to formation of new disulphide bridges from the oxidation of free sulfhydryl groups

and cross-links on modified-IgG preparations which contributed to the resistance in

thermal induced melting. Thus, modification of IgG has caused an irreversible change

that has resulted in crosslinking and aggregation. It implies that increased

concentration of MGO and glucose during hyperglycemia may cause aggregation of

IgG. Critical numbers of the aggregated IgG may invoke immune response and

rheumatoid arthritis like features may develop as a co-morbidity in type 2 diabetes

mellitus patients.

85

Combined references of Chapter 1(a) and 1(b)

Abe, R., Shimizu, T., Sugawara, H., Watanabe, H., Nakamura, H., Choei, H., Sasaki,

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Chapter 2

97


An antigen is a molecule that binds specifically to its specific receptors but may not

induce immune response by itself in the body of an organism (Gras et al., 2018).

Antigens are usually lipids (Mori and Libero, 2012), polypeptides/proteins (Maurer

and Callahan, 1980) and polysaccharides (Weintraub, 2003). The molecules other

than polypeptides, such as lipids and polysaccharides, qualify as antigens but not as

immunogen since they cannot elicit immune response on their own. For peptide to

induce immune response i.e. activation of T- cells by antigen presenting cells it

should be large enough in size, since too small peptides will also not elicit immune

response (Geginat et al., 2015). The antigen may originate from within the body of an

organism called self antigens (Anderson and Kuchroo, 2003) or from the external

environment known as non-self antigens (Cohn, 2009). Usually the immune system

does not react to self antigens under normal homeostatic conditions due to T-cell

negative selection (Guidos et al., 1990) in the thymus and is assumed to identify and

attack only non-self invaders from the external environment or modified/harmful

substances present in the body under distressed conditions. Antigen is presented by

antigen presenting cells (Unanue, 1984) in the form of peptides on histocompatibility

molecules (Pierce, 1994). The T-cells of the adaptive immune system recognize the

antigens. Depending on the type of histocompatibility and antigen different types of

T-cell are activated (Kondo et al., 2017). For the recognition of the peptides by T-cell

receptor it must be processed into the small fragment inside the cell and must be

presented by a major histocompatibility complex.

Immunogenicity is the ability of a particular substance to provoke an immune

response in the body of an animal or human being. In other words, immunogenicity is

the ability to induce a cell-mediated and/or humoral immune responses. Distinction

has to be made between unwanted and wanted immunogenicity. Unwanted

immunogenicity is the immune response shown by an organism against a therapeutic

antigen (such as monoclonal antibody or recombinant protein). This reaction leads to

production of anti-drug-antibodies inactivating the therapeutic effects of the drug

treatment and, in some cases, inducing adverse effects (Dhanda et al., 2018; Jaki et

al., 2016). The predictions of the immunogenic potential of novel protein therapeutics

thus pose a challenge in biotherapy. Wanted immunogenicity is related with vaccines,

https://en.wikipedia.org/wiki/Immune_response

https://en.wikipedia.org/wiki/Immune_response

https://en.wikipedia.org/wiki/Cell-mediated

https://en.wikipedia.org/wiki/Monoclonal_antibody

https://en.wikipedia.org/wiki/Adverse_effects

https://en.wikipedia.org/wiki/Vaccines

Chapter 2

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where the injection of an antigen (the vaccine) provokes an immune response against

the pathogen (such as bacteria, virus etc.) aiming to protect the organism (Yu et al.,

2009; Heininger, 2008).

Proteins are significantly more immunogenic than oligosaccharides and

polysaccharides. Since nucleic acids and lipids are generally non-immunogenic

haptens, so they require conjugation with an epitope such as a polysaccharide or

protein before they can evoke an immunologic response. Immunogenicity is

influenced by various characteristics of an antigen such as phylogenetic distance,

epitope density, protein structure, aa-polymers, glu-lys, tyr, phe, molecular size,

chemical composition and heterogeneity, degradability (ability to be processed &

presented to T cells) and D-amino acids.

The structural basis of human IgG antigenicity (Hunneyball and Stanworth, 1976)

delineation is of particular interest due to the involvement of anti-IgG antibodies in

the immunopathology of various disorders such as rheumatoid arthritis (Abruzzo and

Heimer, 1974). The nature of the 'autoantigenic' determinants which interact with

'general' rheumatoid factors is of concern (Parekh et al., 1985). These autoantigenic

determinants are said to found within the Fc fragment of both human and rabbit IgG

(McDuffie et al., 1965). Human IgG and various other proteins show immunological

behaviour in its native conformation, but glycation of proteins by glucose, ribose,

fructose etc and methylglyoxal and glyoxal leads to structural perturbations and

enhanced immunogenic behaviour. This may further lead to the generation of

autoantibodies and contribute in the the development of rheumatoid arthritis, diabetic

complications etc (Neelofar et al., 2017; Akhter et al., 2015; Islam et al., 2017).

Human IgG is a lysine and arginine rich major serum glycoprotein and thus prone to

glycation. The dominant factor in glycation process appears to be half-life of protein;

proteins with longer half-life showed enhanced glycation (Austin et al., 1987). Since

IgG has longer biological half-life (approximately 25.8 days) (Mankarious et al.,

1988) so it can undergo high degree of glycation in vivo.

Islets of Langerhans of pancreas contain relatively small amounts of various

antioxidant enzymes such as CuZn-SOD, Mn-SOD, catalase, and glutathione

peroxidase (GPx) (Kim et al., 2017). It has been demonstrated that β cells were

sensitive to peroxide in rats (Graciano et al., 2016). These and various other

https://en.wikipedia.org/wiki/Protein

https://en.wikipedia.org/wiki/Polysaccharide

https://en.wikipedia.org/wiki/Hapten

https://en.wikipedia.org/wiki/Conjugate_vaccine

https://en.wikipedia.org/wiki/Epitope

https://en.wikipedia.org/wiki/Genetic_distance

https://en.wikipedia.org/wiki/Epitope

https://en.wikipedia.org/w/index.php?title=Aa-polymer&action=edit&redlink=1

https://en.wikipedia.org/wiki/Glutamic_acid

https://en.wikipedia.org/wiki/Lysine

https://en.wikipedia.org/wiki/Tyrosine

https://en.wikipedia.org/wiki/Phenylalanine

https://en.wikipedia.org/wiki/Molecular_size

https://en.wikipedia.org/wiki/Amino_acid

Chapter 2

99

observations have amply clarified that the intrinsically low level of antioxidant

activity of islets renders them at risk for ROS-induced damage (Emre et al., 2007). To

provide protection against highly toxic hydroxyl radical and various other ROS, the β

cells must be able to metabolize hydrogen peroxide via activation of catalase and GPx

(Cumaoglu et al., 2011; Newsholme et al., 2012; Robertson et al., 2007). This

untoward situation makes the β cells easy target for ROS. Nitrosative and oxidative

stress play a major role in the onset of diabetic complications (Thakur et al., 2018). In

presence of biopterin, NADPH and oxygen, arginine is oxidized to citrulline by the

enzyme nitric oxide synthase (Adak et al., 2000). In general, nitric oxide produces

many beneficial effects to the vascular system at physiological level. However,

increased oxidative stress and activation of the transcription factor NF-kappa B has

been linked to the development of diabetic complications (Rashid et al., 2017). The

NF-kappa B increases nitric oxide production which thus cause β cell damage (Burke

et al., 2013).

Mechanism of action of alloxan on pancreas

Alloxan (2, 4, 5, 6-tetraoxypyrimidine; 5, 6-dioxyuracil) was first described by

Brugnatelli in the year 1818 and its diabetogenic property was first reported by Dunn

et al. in 1944 in rabbits and they observed specific necrosis of pancreatic islet. Since

then, alloxan has been utilized for producing insulin dependent diabetes mellitus

(IDDM) in animal model (Gupta et al., 1996). Alloxan is a toxic analogue of glucose

which destroys selectively insulin producing β cells in the pancreas when

administered to rodents and many other animal species (Chaudhry et al., 2007),

because it accumulates preferentially in β cells through uptake via the GLUT2

transporter (Elsner et al., 2006). In the presence of intracellular thiols, alloxan

generates ROS in a cyclic reaction and dialuric acid is formed. Further, it is then

reoxidized back to alloxan thus establishing a redox cycle which generates superoxide

radicals. Superoxide radicals undergo dismutation to hydrogen peroxide. Highly

reactive hydroxyl radicals are then formed by Fenton reaction in presence of Fe2+

and

hydrogen peroxide. The hydroxyl radical action has been reported following alloxan

treatment both in vitro as well as in vivo. Alloxan has two well known pathological

effects: glucose-induced insulin secretion is selectively inhibited by it through specific

inhibition of glucokinase, the glucose sensor of the β cell and it causes a state of

insulin-dependent diabetes through its ability to induce ROS formation, thus resulting

Chapter 2

100

in the selective necrosis of β cells. These two effects can be explained on the basis of

the specific chemical properties of alloxan, i.e. its selective cellular uptake and

accumulation of alloxan by the β cells (Elsner et al., 2006).

Note: In the following two chapters (i.e. Chapter 2a and 2b) we have evaluated the

immunogenicity of human IgG modified with methylglyoxal in low and high glucose

in healthy and diabetic rabbits.

Chapter 2(a)

Induction and characterization of antibodies raised against IgG modified with methylglyoxal

and IgG co-modified with methylglyoxal and normal (5 mM)/ high (10 mM) glucose in

healthy rabbits

Chapter 2(a)

101

Materials & Methods

MATERIALS

Chemicals

Freund’s complete and incomplete adjuvants, p-nitrophenyl phosphate

(PNPP), anti-rabbit IgG alkaline phosphatase conjugate, and alloxan monohydrate

were purchased from Sigma Chemical Company, USA. All others chemicals and

reagents used were of the highest analytical grade available.

Equipments

ELISA reader (Bio-Rad, USA) and glucometer (Accu-chek).

Experimental animals

Ten female New Zealand rabbits (1-1.8 kg) were obtained from the Central Animal

House facility of the Jawaharlal Nehru Medical College, Aligarh Muslim University,

India. Animals were housed in cages (with wide square mesh at the bottom to avoid

coprophagy) and placed in a well ventilated animal room with 12 h light/ 12 h dark

cycle, 50% humidity and 28±2 0C temperature. The animals were acclimatized to

laboratory conditions for about a week. All animals received a standard diet and water

ad libitum. The protocol and procedure was duly approved by the Institutional Animal

Ethics Committee.

Ethical statement

All experiments on animals were performed according to the instructions of the

Council of International Organizations of Medical Sciences for the use and care of

laboratory animals. The immunization protocol was approved by the Institutional

Animal Ethics Committee (IAEC) vide letter no. 401/GO/Re/S/2001/CPCSEA.

All animals were healthy and divided into five groups and each group had two

animals.

Group I: - Injected with 0.9% saline only.

Group II: - Injected with native IgG (6.67 µM).

Group III:- Injected with methylglyoxal-modified-IgG. The concentration of IgG as

well as methylglyoxal was 6.67 µM.

Group IV:- Injected with IgG co-modified with methylglyoxal and normal glucose.

The concentration of IgG as well as methylglyoxal was 6.67 µM and

glucose was 5 mM.

Chapter 2(a)

102

Group V:- Injected with IgG co-modified with methylglyoxal and high glucose. The

concentration of IgG as well as methylglyoxal was 6.67 µM and glucose

was 10 mM.

All animals were maintained for eight weeks.

Immunization schedule

All rabbits of group II to group V were immunized with 500 µg of native IgG or

modified-IgG preparations per week. The antigens were emulsified with an equal

volume of Freund’s complete adjuvant and injected intramuscularly at multiple sites.

Subsequent injections were given in Freund’s incomplete adjuvant.

Withdrawl of blood

One week after the last dose the marginal ear veins of animals were punctured and

blood was carefully collected to prevent hemolysis. Sera were separated and

complement proteins were inactivated by heating at 56 0C for 30 min. All sera were

stored in small aliquots at -20 0C with 0.1 % sodium azide as preservative.

Enzyme linked immunosorbent assay (ELISA): Following reagents were prepared

Antigen coating buffer: 15 mM sodium carbonate, 35 mM sodium bicarbonate, pH

9.6

Tris buffered saline (TBS): 10 mM Tris, 150 mM NaCl, pH 7.4

Tris buffered saline containing Tween 20 (TBS-T): 20 mM Tris, 144 mM NaCl,

2.68 mM KCl, pH 7.4 containing 500 µL Tween 20 per litre.

Carbonate-bicarbonate buffer: 15 mM sodium carbonate, 35 mM sodium

bicarbonate, pH 9.6 containing 2 mM magnesium chloride.

Substrate: 500 µg p-nitrophenyl phosphate (PNPP) in one ml of carbonate-

bicarbonate buffer, pH 9.6.

Procedure

ELISA was carried out on flat bottom polysorp plates as described earlier (Ali and

Alam, 2002). Briefly, microtitre wells were coated with one hundred microlitre of

native and modified-IgG samples (10 µg/ml dissolved in antigen coating buffer) and

incubated for 2 h at 37 0C and overnight at 4

0C. Each sample was coated in duplicate

and half of the plate, devoid of antigen, served as control. The test wells were emptied

and washed thrice with TBS-T to remove the unbound antigen. Unoccupied sites were

blocked with 150 µl of 2% non-fat dry milk (in TBS, pH 7.4) for 4-5 h followed by

single wash with TBS-T. In direct binding ELISA, test serum (serially diluted in TBS-

T) or purified IgG were directly added into antigen-coated wells and incubated for 2 h

Chapter 2(a)

103

at 37 0C and overnight at 4

0C. The wells were emptied and thoroughly washed with

TBS-T. Anti-immunoglobulin G alkaline phosphatase conjugate wad added to each

well and incubated at 37 0C for 2 h and then the plates were washed thrice with TBS-

T followed by a single wash with distilled water. Para-nitrophenyl phosphate was

added and the developed colour was read at 405 nm on a microplate reader. The result

was expressed as mean of difference of absorbance values in test and control wells

(Atest – Acontrol).

Inhibition ELISA

The antigenic specificity of the antibodies was determined by the inhibition ELISA

(Khan et al., 2007). Varying amounts of inhibitors (0-20 µg/ml) were mixed with

constant amount of antiserum or purified IgG. The mixture was incubated at room

temperature for 2 h and overnight at 4 0C. Immune complexes were coated in the

wells instead of the antiserum. The remaining steps were the same as in direct binding

ELISA. Percent inhibition was calculated using the equation as follows:

Percent inhibition =

Gel retardation assay

For the visual detection of antigen-antibody complex formation, gel retardation assay

was performed (Khan et al., 2007). Immune complexes were prepared by incubating

constant amount of native and modified-IgG samples (25 µg/ml) with varying

amounts of affinity purified IgG from respective antisera (25, 50 and 100 µg/ml ) and

incubated for 2 h at 37 0C and overnight at 4

0C. At the end of incubation, one-fourth

volume of sample buffer was added and the samples were electrophoresed on 7.5%

SDS-polyacrylamide gel for 4 h at 80 V. The gels were stained with Coomassie

brilliant blue and photographed.

Purification of IgG from healthy human sera and rabbit sera

Blood samples were withdrawn from healthy human subjects and rabbits and allowed

to coagulate at 37 0C for 1 h. Sera were separated by centrifugation and IgG was

isolated using Protein-A-agarose affinity column (Goding, 1978). The concentration

of IgG was determined and subjected to dialysis against 10 mM PBS, pH 7.4. The

homogeneity of the isolated IgG was checked on 7.5% SDS-polyacrylamide gel and

the material was stored at -20 0C with 0.1% sodium azide.

Blood samples were withdrawn from the marginal ear vein of rabbits and sera were

separated. Following parameter was determined in all rabbit sera.

Chapter 2(a)

104

(i) Rheumatoid factor: The rheumatoid factor (RF) was determined as per the

instructions of kit manufacturer (Thermo Fisher Scientific). The assay employs the

qualitative enzyme immunoassay technique. The microtiter plate was pre-coated with

antigen provided with the kit. Sera (60-120 µl) were carefully transferred into the

wells alongwith anti-rabbit IgG conjugated to horseradish peroxidase (HRP). Then the

plates were washed to remove any unbound reagent, a substrate solution was added to

the wells. Colour developed in proportion to the amount of rheumatoid factor in the

serum. The reaction was stopped and the intensity was measured. Detection

wavelength: 450 nm versus 620 nm within 30 min after adding the stop solution.

Result was calculated from standard curve plot drawn from standard solution of

rheumatoid factor and then the amount of RF was calculated in test samples (Ashraf et

al., 2015).

(ii) Tumor necrosis factor-α: Estimation of TNF-α was carried as per the

instructions of Thermo Fisher Scientific, Invitrogen (Massachusetts, USA). Briefly,

empty wells of the ELISA plates were filled and rinsed six times by diluted assay

buffer. After the last wash, the plate was gently tapped and placed up side down on

absorbent to remove residual assay buffer. Now, 100 µl of CRP TMB substrate

solution was added to each well. Then the plate was covered with plastic film and

incubated in dark for 25 min at room temperature. At the end of incubation 100 µl of

CRP HRP stop solution was added to each well. The absorbance was recorded at 450

nm. The TNFα concentration in test samples and control was determined from the

standard plot (Ashraf et al., 2015).

(iii) Interleukin-6: The IL-6 was estimated as per the instructions of Thermo Fisher

Scientific, Invitrogen (Massachusetts, USA). Briefly, 100 μL of the standard diluent

buffer was added to zero wells. Well(s) reserved for chromogen blank was left empty.

Then 100 μL each of control, standard and sample were added into the microtiter

wells. Fifty μL of biotinylated anti-IL-6 (Biotin conjugate) was pipetted into each

well, except the chromogen blank(s) and incubated for 2 h at room temperature. The

wells were then washed six times and 100 μL of Streptavidin-HRP (horseradish

peroxidase) working solution was added to each well, except the chromogen blank(s)

and re-incubated for 30 min at room temperature. The wells were again washed for

six times. Hundred μL of stabilized chromogen was added to each well. The liquid in

the wells turned blue. Hundred μL of stop solution was added to each well and the

Chapter 2(a)

105

absorbance was measured at 450 nm. The IL-6 concentration in test samples and

control was determined from the standard plot (Ashraf et al., 2015).

(iv) Interleukin-1: The method for estimation of IL-6 and IL-1 was similar except

that for IL-1 estimation biotinylated-anti-IL-1 was used.

(v) C-reactive protein: The CRP estimation was carried out as per the instructions of

the kit manufacturer (Cayman Chemical). Briefly, 50 µL of sample diluent

(containing 0.15% sodium azide) was added into 96 well plate. This was followed by

addition of 75 µL of standard or sample in duplicate. The plate was covered and

incubated at room temperature for 1 h. After five washes 100 µL of biotinylated

antibody reagent (containing 0.15% sodium azide) was added to the wells and re-

incubated at room temperature for 1 h. The plate was again washed five times to

remove the unbound antigen. Hundred µL of Streptavidin HRP (horseradish

peroxidase) reagent was added to each well and incubated at room temperature for 30

min. After washing five times 100 µL TMB (3,3′,5,5′-tetramethylbenzidine) substrate

was added to each well and incubated in the dark for 30 min. Hundred µL of stop

solution was added to each well and the absorbance was read at 450 and 550 nm. The

average absorbance (450 nm minus 550 nm) against each standard concentration was

placed on the Y-axis and the corresponding CRP concentration on the X-axis and the

standard plot was constructed (Ashraf et al., 2015).


Data are presented as mean ± standard deviation. Statistical significance of the

data was determined by Student’s–t test and a p-value of <0.05 was considered as

significant.

Chapter 2(a)

106

Results

Immunogenicity of native and methylglyoxal-modified-IgG in healthy rabbits Native IgG injected into healthy rabbits induced moderate antibody response with a

titre of <1:3200 as revealed by direct ELISA (Fig. 41a). In contrast, the

methylglyoxal-modified-IgG seems to be a potent immuogen as it induced high titre

antibodies in experimental animals (>1:6400) (Fig. 41b). Preimmune serum included

as control showed negligible binding with respective immunogens under identical

conditions.

Inhibition ELISA results showed that the induced antibodies were quite specific

towards respective immunogens (Fig. 42a and b). A maximum of 59.78% inhibition

was observed in serum antibodies against native IgG with the native IgG as inhibitor

(Fig. 42a), whereas serum antibodies against methylglyoxal-modified-IgG showed

68.72% inhibition when methylglyoxal-modified-IgG was used as inhibitor

(Fig. 42b).

Characterization of IgG isolated from antisera

IgG was isolated from rabbits’ antisera as well as preimmune sera on Protein-A

agarose affinity column (Fig. 43). The isolated IgG was found to be homogeneous

when analyzed on SDS-polyacrylamide gel and moved as a single compact band

(Fig. 43 inset). The 278/251 absorbance ratio of the isolated IgG was found to be >2,

which is an index of purity. Direct ELISA of the IgG isolated from rabbit antisera

showed strong binding towards respective immunogens (Fig. 44a and b) and were

specific (Fig. 45a and b).


Antigen-antibody interaction was visualized in gel after staining with coomassie dye.

Constant amount of methylglyoxal-modified-IgG (antigen) was incubated with

increasing amount of IgG isolated from antiserum against methylglyoxal-modified-

IgG and the results are shown in Fig. 46. The pattern of bands in lane 2, 3 and 4

suggest antibody binding and formation of high molecular weight immune complex.

Cross-reaction studies on IgG isolated from rabbit antisera raised against native

IgG and methylglyoxal-modified-IgG

The results of cross-reaction studies are shown in Table 9 and 10. The IgG isolated

from antisera was found to be polyspecific in nature because it also showed binding

with an array of other biomolecules.

Chapter 2(a)

107

Fig. 41(a) Level of antibodies induced against native IgG in healthy rabbits as

determined by direct ELISA on plate coated with native IgG. Preimmune

serum (open square) and immune serum (open triangle).

Fig. 41(b) Level of antibodies induced against MGO-modified-IgG in healthy rabbits

as determined by direct ELISA on plate coated with MGO-modified-IgG.

Preimmune serum (open square) and immune serum (open triangle).

Chapter 2(a)

108

Fig. 42(a) Inhibition ELISA of serum antibodies against native IgG.

Fig. 42(b) Inhibition ELISA of serum antibodies against MGO-modified-IgG.

Chapter 2(a)

109

Fig. 43 UV absorption profile of the IgG isolated from antiserum against MGO-

modified-IgG on protein A-agarose affinity matrix. Inset: SDS-gel photograph

of the purified IgG on 7.5% polyacrylamide gel.

Fig. 44(a) Direct ELISA of IgG isolated from antiserum raised against native IgG

(open square). Binding of IgG isolated from preimmune serum of the

same animal is shown by open triangle.

Chapter 2(a)

110

Fig. 44(b) Direct ELISA of IgG isolated from antiserum raised against MGO-

modified-IgG (open square). Binding of IgG isolated from preimmune

serum of the same animal is shown by open triangle.

Fig. 45(a) Inhibition ELISA of IgG isolated from antiserum raised against native IgG.

Chapter 2(a)

111

Fig. 45(b) Inhibition ELISA of IgG isolated from antiserum raised against MGO-

modified-IgG.

1 2 3 4

Fig. 46 Polyacrylamide gel photograph of methylglyoxal-modified-IgG (antigen) and

antibody interaction. Electrophoresis was performed on 7.5% SDS-

polyacrylamide gel at 80 V for 4 h. Lane 1 contains methylglyoxal-modified-

IgG (25 µg). Lane 2, 3 & 4 contains methylglyoxal-modified-IgG plus 25, 50

and 100 µg of IgG isolated from antiserum raised against methylglyoxal-

modified-IgG.

Chapter 2(a)

112

Table 9

Cross-reactions of IgG isolated from antiserum raised against native IgG in

healthy rabbits

Inhibitor

Maximum % inhibition

at 20 µg/ml

Concentration for 50 %

inhibition (µg/ml)

Percent

relative

affinity

Native IgG

IgG modified with

6.67 µM MGO

IgG co-modified with

6.67 µM MGO and

5 mM glucose


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO

H2A histone modified

with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

66.72

29.62

31.67

29.82

48.72

46.62

43.82

24.62

26.67

28.83

25.62

23.83

21.73

15

-

-

-

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(a)

113

Table 10

Cross-reactions of IgG isolated from antiserum raised against

methylglyoxal-modified-IgG in healthy rabbits

Inhibitor

Maximum %

inhibition

at 20 µg/ml


inhibition (µg/ml)

Percent

relative

affinity

IgG modified with

6.67 µM MGO

Native IgG


6.67 µM MGO and

5 mM glucose


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

76.83

32.62

54.67

52.62

28.62

26.82

23.52

49.32

46.67

47.73

41.12

39.13

38.21

12.50

-

17.50

18.75

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(a)

114

Estimation of biochemicals in rabbit sera immunized with methylglyoxal-

modified-IgG preparations

Rheumatoid factor

Serum samples from control animals and those immunized with native IgG and

methylglyoxal-modified-IgG were processed for rheumatoid factor (RF) by

immunoassay and the results are shown in Fig. 47. High value of rheumatoid factor

(14.73±1.30 IU/ml) was observed in sera of animals injected with methylglyoxal-

modified-IgG as compared to control or native IgG group.

Fig. 47 Rheumatoid factor status in control and immunized rabbits. * The p value was

<0.05 and considered statistically significant as compared to control.

Tumour necrosis factor-α


methylglyoxal-modified-IgG were processed for tumour necrosis factor-α by

immunoassay and the results are shown in Fig. 48. High value of tumour necrosis

factor-α (44.12±1.89 pg/ml) was observed in sera of animals injected with

methylglyoxal-modified-IgG as compared to control or native IgG group.

Chapter 2(a)

115

Fig. 48 Tumour necrosis factor-α status in control and immunized rabbits. * The p

value was <0.05 and considered statistically significant as compared to

control.

Interleukin-1


methylglyoxal-modified-IgG were processed for interleukin-1 by immunoassay and

the results are shown in Fig. 49. High value of interleukin-1 (343.72±4.84 pg/ml) was

observed in sera of animals injected with methylglyoxal-modified-IgG as compared to

control or native IgG group.

Interleukin-6




observed in sera of animals injected with methylglyoxal-modified-IgG as compared to

control or native IgG group.

Chapter 2(a)

116

Fig. 49 Interleukin-1 status in control and immunized rabbits. * The p value was




Chapter 2(a)

117

C-reactive protein


methylglyoxal-modified-IgG were processed for C-reactive protein by immunoassay

and the results are shown in Fig. 51. High value of C-reactive protein (17.20±1.14

mg/L) was observed in sera of animals injected with methylglyoxal-modified-IgG as

compared to control or native IgG group.

Fig. 51 C-reactive protein status in control and immunized rabbits. * The p value was


Chapter 2(a)

118

Immunogenicity of IgG co-modified with methylglyoxal and glucose (normal and

high) in healthy rabbits

The IgG co-modified with methylglyoxal under normal glucose (5 mM) induced

antibodies having titre >1:6400 (Fig. 52a), whereas the IgG co-modified with

methylglyoxal under high glucose (10 mM) was found to be more potent immuogen

(titre >1:12800) than its low glucose counterpart (Fig. 52b). Preimmune serum

included as control showed negligible binding with respective immunogens under

identical conditions.


towards respective immunogens (Fig. 53a and b). A maximum of 80.20% and 82.38%

inhibition was observed in serum antibodies against normal and high glucose version

of methylglyoxal-modified-IgG with their respective immunogens (Fig. 53a and b).

Cross-reaction studies on IgG isolated from rabbit antisera raised against IgG

co-modified with methylglyoxal and glucose






Constant amount of the IgG co-modified with methylglyoxal and glucose (antigen)

was incubated with increasing amount of the IgG isolated from respective antisera and

the results are shown in Fig. 55a and b. The pattern of bands in lane 2, 3 and 4 suggest

antibody binding and formation of high molecular weight immune complex.

Chapter 2(a)

119

Fig. 52(a) Level of antibodies induced against IgG co-modified with methylglyoxal

and normal glucose in healthy rabbits as determined by direct ELISA on

plate coated with IgG co-modified with methylglyoxal and normal

glucose. Preimmune serum (open square) and immune serum (open

triangle).

Fig. 52(b) Level of antibodies induced against IgG co-modified with methylglyoxal

and high glucose in healthy rabbits as determined by direct ELISA on plate

coated with IgG co-modified with methylglyoxal and high glucose.

Preimmune serum (open square) and immune serum (open triangle).

Chapter 2(a)

120

Fig. 53(a) Inhibition ELISA of serum antibodies against IgG co-modified with

methylglyoxal and normal glucose.

Fig. 53(b) Inhibition ELISA of serum antibodies against IgG co-modified with

methylglyoxal and high glucose.

Chapter 2(a)

121

Fig. 54(a) Inhibition ELISA of IgG isolated from antiserum raised against IgG co-

modified with methylglyoxal and normal glucose.

Fig. 54(b) Inhibition ELISA of IgG isolated from antiserum raised against IgG co-

modified with methylglyoxal and high glucose.

Chapter 2(a)

122

Table 11

Cross-reactions of IgG isolated from antiserum raised against IgG co-modified

with methylglyoxal and normal concentration of glucose (5 mM)

Inhibitors


at 20 µg/ml


inhibition (µg/ml)

Percent relative

affinity


6.67 µM MGO and

5 mM glucose

Native IgG

IgG modified with

6.67 µM MGO


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

82.78

36.12

59.17

62.82

31.12

36.62

33.22

39.12

49.67

49.13

43.42

41.63

42.97

6.00

-

15.00

15.00

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(a)

123

Table 12


with methylglyoxal and high concentration of glucose (10 mM)

Inhibitors

Maximum %

inhibition

at 20 µg/ml


inhibition (µg/ml)

Percent relative

affinity


6.67 µM MGO and

10 mM glucose

Native IgG

IgG modified with

6.67 µM MGO


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

88.90

39.12

62.17

66.32

35.62

39.82

38.02

42.62

44.67

46.13

46.32

42.33

40.87

5.00

-

15.00

15.00

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(a)

124

1 2 3 4

Fig. 55(a) Polyacrylamide gel photograph of IgG co-modified with MGO and 5 mM

glucose (antigen) and antibody interaction. Electrophoresis was performed

on 7.5% SDS- polyacrylamide gel at 80 V for 4 h. Lane 1 contains IgG co-

modified with MGO and 5 mM glucose (25 µg). Lane 2, 3 & 4 contains

IgG co-modified with MGO and 5 mM glucose plus 25, 50 and 100 µg of

IgG isolated from antiserum raised against IgG co-modified with MGO and

5 mM glucose.

1 2 3 4

Fig. 55(b) Polyacrylamide gel photograph of IgG co-modified with MGO and 10 mM






10 mM glucose.

Chapter 2(a)

125

Estimation of biochemicals in rabbit sera immunized with methylglyoxal-

glucose-modified IgG preparations

Rheumatoid factor

Serum samples from control animals and those immunized with native IgG or its

modified counterparts were processed for rheumatoid factor (RF) by immunoassay

and the results are shown in Fig. 56. Highest value of rheumatoid factor (17.86 ± 2.07

IU/ml) was observed in sera of animals injected with IgG co-modified with

methylglyoxal and 10 mM glucose as compared to other groups.

Fig. 56 Rheumatoid factor status in control and immunized rabbits. * The p value was




modified counterparts were processed for tumour necrosis factor-α by immunoassay

and the results are shown in Fig. 57. Highest value of tumour necrosis factor-α (59.67

± 2.17 pg/ml) was observed in sera of animals injected with IgG co-modified with


Chapter 2(a)

126

Fig. 57 Tumour necrosis factor-α status in control and immunized rabbits. * The p

value was <0.05 and considered statistically significant as compared to

control.

Interleukin-1


modified counterparts were processed for interleukin-1 by immunoassay and the

results are shown in Fig. 58. Highest value of interleukin-1 (424.32 ± 4.17 pg/ml) was

observed in sera of animals injected with IgG co-modified with methylglyoxal and 10

mM glucose as compared to other groups.

Interleukin-6




observed in sera of animals injected with IgG co-modified with methylglyoxal and 10

mM glucose as compared to other groups.

Chapter 2(a)

127





Chapter 2(a)

128

C-reactive protein


modified counterparts were processed for C-reactive protein by immunoassay and the

results are shown in Fig. 60. Highest value of C-reactive protein (35.54 ± 1.07 mg/L)

was observed in sera of animals injected with IgG co-modified with methylglyoxal

and 10 mM glucose as compared to other groups.

Fig. 60 C-reactive protein status in control and immunized rabbits. * The p value was


Chapter 2(b)

Induction and characterization of antibodies raised against IgG modified with methylglyoxal

and IgG co-modified with methylglyoxal and normal (5 mM)/high (10 mM) glucose in

diabetic rabbits

Chapter 2(b)

129

Materials & Methods

Experimental animals

Ten female New Zealand rabbits (1-1.8 kg) were obtained from the Central Animal

House facility of the Jawaharlal Nehru Medical College, Aligarh Muslim University,

India. Animals were housed in cages (with wide square mesh at the bottom to avoid

coprophagy) and placed in a well ventilated animal room with 12 h light/ 12 h dark

cycle, 50% humidity and 28±2 0C temperature. The animals were acclimatized to

laboratory conditions for about a week. All animals received a standard diet and water

ad libitum. The protocol and procedure was duly approved by the Institutional Animal

Ethics Committee.

All animals were made diabetic and divided into five groups. The experimental model

of diabetes was produced by alloxan.

Group I: - Injected with 0.9% saline only.

Group II: - Injected with native IgG (6.67 µM).

Group III:- Injected with methylglyoxal-modified-IgG. The concentration of IgG as

well as methylglyoxal was 6.67 µM.

Group IV:- Injected with IgG co-modified with methylglyoxal and normal glucose.

The concentration of IgG as well as methylglyoxal was 6.67 µM and of

glucose was 5 mM.

Group V:- Injected with IgG co-modified with methylglyoxal and high glucose. The

concentration of IgG as well as methylglyoxal was 6.67 µM and of

glucose was 10 mM.

All animals were maintained for sixteen weeks.

Experimental induction of diabetes mellitus in rabbits

Rabbits were fasted for 12 h and given a single intravenous injection of alloxan (100

mg/kg body weight) dissolved in 0.9% (w/v) sterile saline in maginal ear vein.

Weekly blood glucose level was measured with glucometer (Accu-chek) and insulin

level by radioimmunoassay method using commercially available kit (Insulin IRMA

Kit, Inc., Immunotech). Induction of diabetes mellitus was ascertained by sustained

hyperglycaemia and hypoinsulinemia.

Chapter 2(b)

130

Blood samples were withdrawn from the marginal ear vein of rabbits and sera were

separated. Following parameter was determined in all rabbit sera.

(i) Fructosamine: The fructosamine was determined by NBT reduction assay as

described previously (Baker et al., 1993).

(ii) Superoxide dismutase: The SOD activity was determined by the method of

Marklund and Marklund 1974. Briefly, fifty microlitre serum was mixed with 0.1 ml

of 8 mM pyrogallol, dissolved in 50 mM Tris succinate buffer, pH 8.2. The blank

contained distilled water instead of serum. The change in absorbance at 420 nm was

recorded every 30 sec for 3 min. SOD activity was expressed as U/mg protein.

(iii) Catalase activity: The catalase activity was measured as described earlier (Aebi

et al., 1974). The assay tube contained 50 µl serum, 1.0 ml of 30 mM H2O2 and 1.95

ml of 50 mM phosphate buffer, pH 7.0. The contents were mixed and change in

absorbance at 240 nm was recorded every 30 sec for 2 min. Catalase activity was

expressed as U/mg of protein.

(iv) Glutathione peroxidase activity: The GPx activity was measured as described

earlier (Flohe and Gunzler, 1984). Briefly, 100 µl serum was mixed with 1 mM

EDTA, 2.4 U/mL glutathione reductase and 10 mM glutathione and the volume was

made to 3.0 ml with 20 mM potassium phosphate buffer, pH 7.4. The reaction mixture

was incubated for 10 min at 37 0C followed by addition of 0.19 mM of NADPH and

12mM tertiary-butyl hydroperoxide. The decrease in absorbance was recorded at 340

nm for 3 min. GPx activity was expressed as U/mg of protein.

(v) Reduced glutathione: The reduced glutathione was determined by DTNB

reagent. (Ellman, 1959). One ml serum was precipitated with 1 ml of 4%

sulphosalicylic acid and left for 1 h at 4 0C. The samples were then centrifuged at

1,200 g for 15 min at 4 0C. To 400 µl of the supernatants, 2.2 ml of phosphate buffer

(0.1 mM, pH 8.1) and 400 µl of 3 mM 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB)

was added. The yellow colour was measured at 412 nm and the amount of the reduced

glutathione was calculated using the molar extinction coefficient value of 13,640 cm-

1mol

-1.

(vi) Malondialdehyde: The serum malondialdehyde was measured as thiobarbituric

acid reactive substances (TBARS) as described (Gallou et. al., 1993). Briefly, 10 µl of

1 mM EDTA was added to 0.5 ml of serum followed by 1 ml of 15% (w/v)

Chapter 2(b)

131

trichloroacetic acid. The contents were mixed and centrifuged at 3000 g for 10 min to

facilitate protein precipitation. The supernatant was then treated with 200 µl of 1%

(w/v) thiobarbituric acid and immersed in a boiling water bath for 60 min. After

cooling, 600 µl of the chromogen was mixed with 600 µl of n-butanol and extracted.

This was followed by centrifugation at 900 g for 10 min. Finally, 250 µl was removed

from the butanolic phase and absorbance was read at 535 nm in a microplate reader.

The concentration of TBARS was calculated using the molar extinction coefficient

value of 1,56,000 cm-1

mol-1

(Stocks and Dormandy, 1971).

(vii) Protein carbonyl: The protein carbonyl was measured by the method of Levine

et. al., (1990). Briefly, 100 µl of serum was precipitated with 10% (w/v) ice-cold

TCA and incubated for 10 min at 4 0C. The samples were centrifuged at 11,000 g for

3 min. The pellet was re-suspended in 500 µl of 10 mM 2, 4-dinitrophenylhydrazine

(DNPH) prepared in 2M HCl. The samples were then vortexed at room temperature

for 1 h and precipitated with 0.5 ml of 20% (w/v) TCA followed by 3 min

centrifugation at 11,000 g. After centrifugation, the pellet was washed thrice with 1.0

ml of ethanol/ethyl acetate (1:1) to remove extra DNPH reagent. The protein pellet

was finally suspended in 1 ml of 6M guanidinium hydrochloride dissolved in 20 mM

potassium phosphate buffer (adjusted to pH 2.3 with trifluoroacetic acid). The

absorbance was recorded at 360 nm and the concentration of protein carbonyls was

calculated using molar absorption coefficient of 22,000 cm-1

mol-1

and expressed as

nmol/mg of protein.

(viii) Nitrite: The serum nitrite was quantitated with Griess reagent (1 g/L

sulfanilamide, 25 g/L phosphoric acid and 0.1 g/L N-1-naphthylethylenediamine

dihydrochloride) according to the method of Green et. al., (1982). Briefly, 200 µl of

sera were deproteinized with 20% (w/v) ZnSO4. The samples were vortexed and

incubated at room temperature for 15 min. After incubation, the samples were

centrifuged at 3000 g for 5 min and 100 µl of the supernatant was added to 96-well

microtiter plates in duplicate. Hundred microlitre of Griess reagent was added to each

well and incubated at 37 0C for 10 min. The purple colour was read at 540 nm on a

microplate reader. The concentration of serum nitrite was determined using sodium

nitrite as the standard.

The method used in this Chapter (other than as described above) is same as been

described in Chapter 2a.

Chapter 2(b)

132

Results

Experimental induction of diabetes mellitus in rabbit by alloxan

The effect of alloxan on blood glucose and insulin is shown in Fig. 61 and 62,

respectively. The data clearly indicate that the diabetes has been successfully induced

in rabbits. Henceforth the animals will be referred to as diabetic animals/rabbits.

Furthermore, the effect of alloxan on some important biochemicals (related to in vivo

stress) during eight weeks time is summarized in Table 13.

Table 13

Effect of alloxan on some biochemicals in rabbits during eight weeks

Week Control rabbits Diabetic rabbits

00

01

02

03

04

05

06

07

08

FTA SOD Cat GPx GSH CL NO FTA SOD Cat GPx GSH CL NO

120

122

130

133

139

144

139

148

155

53

52

53

50

49

47

46

48

47

51

48

47

46

44

43

44

42

44

23

22

22

22

21

20

20

19

18

253

248

247

246

244

243

244

242

241

20

21

22

21

23

27

29

28

30

16

15

18

17

16

18

17

16

15

140

300

360

420

480

530

590

640

670

54

46

41

36

33

30

27

25

25

52

36

31

26

21

19

16

14

12

24

18

16

14

13

10

11

9

6

257

236

221

206

183

160

137

107

93

22

44

66

99

122

154

199

210

223

33

42

72

111

133

167

203

233

254

where,

FTA stands for serum fructosamine content (nmol/mg)

SOD stands for superoxide dismutase activity (U/mg)

Cat stands for catalase activity (U/mg)

GPx stands for glutathione peroxidase activity (U/mg)

GSH stands for glutathione content (nmol/mg)

CL stands for level of protein carbonyl (nmol/mg)

NO stands for nitric oxide concentration (nM)

Chapter 2(b)

133

Fig. 61 Changes in blood glucose level in rabbits given a single dose of alloxan (filled

square) as compared to rabbits who did not receive alloxan (open circle).

Fig. 62 Insulin level in control rabbits (blue bar) and alloxan treated rabbits (red bar).

Chapter 2(b)

134

Immunogenicity of native and methylglyoxal-modified-IgG in diabetic rabbits

Native IgG injected into diabetic rabbits induced antibodies with a titre of <1:6400 as

determined by direct ELISA (Fig. 63a). In contrast, the methylglyoxal-modified-IgG

was more immunogenic in diabetic animals (>1:12800) as compared to native IgG

(Fig. 63b). Preimmune serum included as control showed negligible binding with

respective immunogens under identical conditions.


towards respective immunogens (Fig. 64a and b). A maximum of 64.86% inhibition

was observed in serum antibodies against native IgG with the native IgG as inhibitor

(Fig. 64a) whereas serum antibodies against methylglyoxal-modified-IgG showed

74.60% inhibition when methylglyoxal-modified-IgG was used as inhibitor

(Fig. 64b).

Cross-reaction studies on IgG isolated from antisera raised against native IgG

and methylglyoxal-modified-IgG in diabetic rabbit


from respective antisera was polyspecific because it also showed binding with an

array of other biomolecules.



Constant amount of methylglyoxal-modified-IgG (antigen) was incubated with

increasing amount of IgG isolated from antiserum raised against methylglyoxal-

modified-IgG in diabetic rabbits and the results are shown in Fig. 66. The pattern of

bands in lane 2, 3 and 4 suggest antibody binding and formation of high molecular

weight immune complex.

Chapter 2(b)

135

Fig. 63(a) Level of antibodies induced against native IgG in diabetic rabbits on plate

coated with native IgG. Preimmune serum (open square) and immune

serum (open triangle).

Fig. 63(b) Level of antibodies induced against MGO-modified-IgG in diabetic rabbits

on plate coated with MGO-modified-IgG. Preimmune serum (open

square) and immune serum (open triangle).

Chapter 2(b)

136

Fig. 64(a) Inhibition ELISA of serum antibodies against native IgG in diabetic

rabbits.

Fig. 64(b) Inhibition ELISA of serum antibodies against MGO-modified-IgG in

diabetic rabbits.

Chapter 2(b)

137

Fig. 65(a) Direct ELISA of IgG isolated from antiserum raised against native IgG

(open square) in diabetic rabbits. Binding of IgG isolated from preimmune

serum of the same animal is shown by open triangle.

Fig. 65(b) Direct ELISA of IgG isolated from antiserum raised against MGO-

modified-IgG (open square) in diabetic rabbits. Binding of IgG isolated

from preimmune serum of the same animal is shown by open triangle.

Chapter 2(b)

138

1 2 3 4

Fig. 66 Polyacrylamide gel photograph of methylglyoxal-modified-IgG (antigen) and

antibody interaction. Electrophoresis was performed on 7.5% SDS-

polyacrylamide gel at 80 V for 4 h. Lane 1 contains methylglyoxal-

modified-IgG (25 µg). Lane 2, 3 & 4 contains methylglyoxal-modified-IgG

plus 25, 50 and 100 µg of IgG isolated from antiserum raised against

methylglyoxal-modified-IgG.

Chapter 2(b)

139

Table 14

Cross-reactions of IgG isolated from antiserum raised against native IgG in

diabetic rabbits

Inhibitors


at 20 µg/ml


inhibition (µg/ml)

Percent relative

affinity

Native IgG

IgG modified with

6.67 µM MGO

IgG co-modified

with

6.67 µM MGO and

5 mM glucose

IgG co-modified

with

6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO

H2A histone

modified

with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose

H2A histone

modified

with 5 mM glucose

64.86

31.57

32.99

30.82

31.72

33.82

34.45

23.73

27.61

28.42

24.33

22.54

21.73

10.00

-

-

-

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(b)

140

Table 15

Cross-reactions of IgG isolated from antiserum raised against

methylglyoxal-modified-IgG in diabetic rabbits

Inhibitors


at 20 µg/ml

Concentration for

50 %

inhibition (µg/ml)

Percent

relative

affinity

IgG modified with

6.67 µM MGO

Native IgG


6.67 µM MGO and

5 mM glucose


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

75.64

30.88

57.61

55.43

26.44

27.34

24.93

49.66

49.82

48.84

40.62

40.93

39.97

10.00

-

15.00

17.50

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(b)

141

Estimation of biochemicals in diabetic rabbit sera immunized with

methylglyoxal-modified-IgG preparations

Rheumatoid factor


methylglyoxal-modified-IgG were processed for rheumatoid factor (RF) by

immunoassay and the results are shown in Fig. 67. High value of rheumatoid factor

(15.98±1.42 IU/ml) was observed in sera of diabetic animals injected with


Fig. 67 Rheumatoid factor status in non-immunized diabetic rabbits (control) and

modified-IgG immunized diabetic rabbits. * The p value was <0.05 and

considered statistically significant as compared to control.



methylglyoxal-modified-IgG were processed for tumour necrosis factor-α by

immunoassay and the results are shown in Fig. 68. High value of tumour necrosis

factor-α (63.76±2.27 pg/ml) was observed in sera of diabetic animals injected with


Chapter 2(b)

142

Fig. 68 Tumour necrosis factor-α status in non-immunized diabetic rabbits (control)

and modified-IgG immunized diabetic rabbits. * The p value was <0.05 and


Interleukin-1




observed in sera of diabetic animals injected with methylglyoxal-modified-IgG as


Interleukin-6




observed in sera of diabetic animals injected with methylglyoxal-modified-IgG as


Chapter 2(b)

143

Fig. 69 Interleukin-1 status in non-immunized diabetic rabbits (control) and modified-

IgG immunized diabetic rabbits. * The p value was <0.05 and considered

statistically significant as compared to control.




Chapter 2(b)

144

C-reactive protein


methylglyoxal-modified-IgG were processed for C-reactive protein by immunoassay

and the results are shown in Fig. 71. High value of C-reactive protein (31.36±1.54

mg/L) was observed in sera of diabetic animals injected with methylglyoxal-

modified-IgG as compared to control or native IgG group.

Fig. 71 C-reactive protein status in non-immunized diabetic rabbits (control) and



Chapter 2(b)

145

Immunogenicity of IgG co-modified with methylglyoxal and glucose (normal and

high) in diabetic rabbits

The IgG co-modified with methylglyoxal under normal glucose (5 mM) induced

antibodies in diabetic rabbits having the titre of >1:12800 (Fig. 72a), whereas the IgG

co-modified with methylglyoxal under high glucose (10 mM) showed more

immunogenicity (titre >1:25600) (Fig. 72b). Preimmune serum included as control

showed negligible binding with respective immunogens under identical conditions.


towards respective immunogens (Fig. 73a and b). A maximum of 81.40% and 88.38%

inhibition was observed in serum antibodies against normal and high glucose version

of methylglyoxal-modified-IgG with their respective immunogens (Fig. 73a and b).

Cross-reaction studies on IgG isolated from antisera raised against IgG

co-modified with methylglyoxal and glucose in diabetic rabbits






Constant amount of the IgG co-modified with methylglyoxal and glucose, normal or

high (antigen) was incubated with increasing amount of the IgG isolated from the

respective antisera and the results are shown in Fig. 75a and b. The pattern of bands in

lane 2, 3 and 4 suggest antibody binding and formation of high molecular weight

immune complex.

Chapter 2(b)

146

Fig. 72(a) Level of antibodies induced against IgG co-modified with methylglyoxal

and normal glucose in diabetic rabbits as determined by direct ELISA on

plate coated with the immunogen. Preimmune serum (open square) and

immune serum (open triangle).

Fig. 72(b) Level of antibodies induced against IgG co-modified with methylglyoxal

and high glucose in diabetic rabbits as determined by direct ELISA on

plate coated with the immunogen. Preimmune serum (open square) and

immune serum (open triangle).

Chapter 2(b)

147

Fig. 73(a) Inhibition ELISA of serum antibodies against IgG co-modified with

methylglyoxal and normal glucose in diabetic rabbits.

Fig. 73(b) Inhibition ELISA of serum antibodies against IgG co-modified with

methylglyoxal and high glucose in diabetic rabbits.

Chapter 2(b)

148

Fig. 74(a) Inhibition ELISA of IgG isolated from antiserum raised against IgG co-

modified with methylglyoxal and normal glucose in diabetic rabbits.

Fig. 74(b) Inhibition ELISA of IgG isolated from antiserum raised against IgG co-

modified with methylglyoxal and high glucose in diabetic rabbits.

Chapter 2(b)

149

Table 16


with methylglyoxal and normal concentration of glucose (5 mM) in

diabetic rabbit

Inhibitors


at 20 µg/ml


inhibition (µg/ml)

Percent

relative

affinity


6.67 µM MGO and

5 mM glucose

Native IgG

IgG modified with

6.67 µM MGO


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

80.20

37.63

58.67

60.73

30.83

37.73

35.54

38.87

49.62

47.14

42.64

40.87

40.45

6.00

-

15.00

15.00

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(b)

150

Table 17


with methylglyoxal and high concentration of glucose (10 mM) in

diabetic rabbits

Inhibitors

Maximum %

inhibition

at 20 µg/ml


inhibition (µg/ml)

Percent

relative

affinity


6.67 µM MGO and

10 mM glucose

Native IgG

IgG modified with

6.67 µM MGO


6.67 µM MGO and

10 mM glucose

Native HSA

Native BSA

Native H2A histone

HSA modified with

6.67 µM MGO

BSA modified with

6.67 µM MGO


with 6.67 µM MGO

HSA modified with

5 mM glucose

BSA modified with

5 mM glucose


with 5 mM glucose

88.38

37.77

60.59

64.39

37.44

38.73

36.27

41.64

41.47

45.42

45.44

40.87

38.63

5.00

-

15.00

15.00

-

-

-

-

-

-

-

-

-

-

100

-

-

-

-

-

-

-

-

-

-

-

Chapter 2(b)

151

1 2 3 4

Fig. 75(a) Polyacrylamide gel photograph of IgG co-modified with MGO and 5 mM






5 mM glucose.

1 2 3 4

Fig. 75(b) Polyacrylamide gel photograph of IgG co-modified with MGO and 10 mM






10 mM glucose.

Chapter 2(b)

152

Estimation of biochemicals in diabetic rabbit sera immunized with

methylglyoxal-glucose-modified IgG preparations

Rheumatoid factor


modified counterparts were processed for rheumatoid factor (RF) by immunoassay

and the results are shown in Fig. 76. Highest value of rheumatoid factor (19.27 ± 2.19

IU/ml) was observed in sera of diabetic animals injected with IgG co-modified with


Fig. 76 Rheumatoid factor status in non-immunized diabetic rabbits (control) and





modified counterparts were processed for tumour necrosis factor-α by immunoassay

and the results are shown in Fig. 77. Highest value of tumour necrosis factor-α (64.42

± 2.47 pg/ml) was observed in sera of diabetic animals injected with IgG co-modified

with methylglyoxal and 10 mM glucose as compared to other groups.

Chapter 2(b)

153

Fig. 77 Tumour necrosis factor-α status in non-immunized diabetic rabbits (control)

and modified-IgG immunized diabetic rabbits. * The p value was <0.05 and


Interleukin-1




observed in sera of diabetic animals injected with IgG co-modified with


Interleukin-6




observed in sera of diabetic animals injected with IgG co-modified with


Chapter 2(b)

154







Chapter 2(b)

155

C-reactive protein


modified counterparts were processed for C-reactive protein by immunoassay and the

results are shown in Fig. 80. Highest value of C-reactive protein (38.96 ± 1.09 mg/L)

was observed in sera of diabetic animals injected with IgG co-modified with


Fig. 80 Level of C-reactive protein in non-immunized diabetic rabbits (control) and



156

Combined discussion of Chapter 2(a) and Chapter 2(b)

The non-enzymatic glycation involves the attachment of reducing sugars (McPherson

et al., 1988), dicarbonyls (Banerjee and Chakraborti, 2017) etc with free amino

groups in biomolecules (Mota et al., 1994). Before forming AGEs the non-enzymatic

glycation passes through Schiff and Amadori stage. In this part of study, the human

IgG modified with methylglyoxal alone as well as along with 5 mM and 10 mM

glucose has been tested for immunogenicity in healthy (Chapter 2a) and diabetic

rabbits (Chapter 2b). The modified-IgG samples were found to be more immunogenic

than native IgG, both in healthy and diabetic rabbits. The immunogenicity exhibited

by the modified-IgG samples may be due to alterations in IgG structure and the

resulting aggregates which was recognized as non-self by the rabbits. Although

induced antibodies were specific towards their respective immunogens but also

showed cross-reactivity with modified BSA, HSA, histones etc due to phenomenon of

epitope sharing.

Furthermore, the IgG co-modified with 10 mM glucose and methylglyoxal having

highest level of aggregate was found to be the most effective immunogen under our

experimental conditions. The immunogenicity of aggregated proteins may be

potentiated due to pooling of potentially immunogenic epitopes which were otherwise

scattered on the molecule. Previous studies have also reported similar observations

but under different conditions (Ahmad et al., 2012; Arif et al., 2012; Mir et al., 2016;

Ansari and Dash, 2013; Islam et al., 2017).

The corresponding enhancement in the immunogenicity of respective immunogens in

diabetic animals may be due to further in vivo modification of the immunogens under

increased oxidative stress and sustained hyperglycaemia. Such observation has been

made earlier also by Austin et al., 1987.

In this study, we have also carried out biochemical investigations on serum samples of

healthy and diabetic rabbits immunized with native and modified-IgG for features of

rheumatoid arthritis as a co-morbidity. We observed elevated levels of rheumatoid

factor, CRP, TNF alpha, IL-6 and IL-1 in healthy as well as diabetic rabbits.

However, the level of biochemicals was more prominent in diabetic animals. The

findings/observations hints at the likely role of modified-IgG, especially the IgG

modified with hyperglycaemic concentrations of methylglyoxal and glucose, in the

157

induction of some features of rheumatoid arthritis in T2DM patients. In this context it

is highlighted that role of proteins in rheumatoid arthritis has been suggested by

others also (Khan et al., 2012; Newkirk et al., 1998).

158

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Chapter 3 Binding profile of autoantibodies in T2DM sera of different age group and disease duration with

native and modified-IgG preparations and estimation of rheumatoid factor, IL-1, IL-6,

C-reactive protein and TNFα in the sera

Chapter 3

161


Diabetes mellitus and non-enzymatic glycation

Diabetes mellitus, commonly known as diabetes is characterized by hyperglycemia,

polyuria, polyphagia and polydipsia. If the sugar level is not tightly controlled it may

lead to macrovascular and microvascular complications. Diabetic complications have

been mainly attributed to non-enzymatic glycation of proteins. Importance of

methylglyoxal, which increases manifold during diabetes has been clearly appreciated

in the disease complications (Morioka et al., 2017; Groener et al., 2013).

Furthermore, diabetic patients have shown higher plasma levels of methylglyoxal and

methylglyoxal-derived AGEs (Fosmark, et al. 2006). Methylglyoxal may cause

oxidative stress (Frandsen and Narayanasamy, 2017), apoptosis (Do et al., 2017) and

genotoxicity (Ahmad et al., 2011) in cells. Advanced glycation end products (AGEs)

seem to play a pivotal role in diabetes related complications (Forbes et al., 2004).

Rheumatoid arthritis and advanced glycation end products

Rheumatoid arthritis (RA) is an autoimmune disorder that primarily affects joints. The

underlying mechanism of RA involves the body's immune system attacking the

joints. The presentations are; warm, swollen and painful joints. The underlying

mechanism of RA involves the body’s immune system attacking the joints. It also

affects the underlying cartilage and bone. Being autoimmune in nature its diagnosis

relies on the presence of autoantibodies known as rheumatoid factor (Hauser and

Harre, 2017). Level of AGEs like pentosidine and Nε-carboxymethyl lysine in

rheumatoid arthritis has emphasized the importance of glycation in the disease

(Ahmed et al., 2014).

Besides arthropathy, RA has several extra-articular manifestations like lung fibrosis

and inflammation (Borthwick, 2016), anaemia (Corrado et al., 2017), osteoporosis

(Guler-Yuksel et al., 2018), premature atherosclerosis (Castaneda et al., 2018) and

profound fatigue (Wolfe and Michaud, 2004) and heart disease (Castaneda et al.,

2018). The laboratory investigations of RA include the clinical assessments of various

general inflammatory markers such as C-reactive protein and erythrocyte

sedimentation rate (Terato et al., 2018). Assessment of white blood cell count

(Ingawale and Patel, 2018) and determination of concentration of uric acid can assist

https://en.wikipedia.org/wiki/Autoimmune_disorder

https://en.wikipedia.org/wiki/Synovial_joint

https://en.wikipedia.org/wiki/Immune_system

Chapter 3

162

in the exclusion, or inclusion, of infection or gout as a differential diagnosis (Liu et

al., 2018; Khondker and Khan, 2014). The rheumatoid arthritis mainstay diagnostic

testing is for autoantibodies i.e. RF (Falkenburg et al., 2018). Rheumatoid factor

seems to be a primary diagnostic test; however, its sensitivity is approximately 75%,

but its specificity is limited due to the fact that RF is also found in patients of Sjogren

syndrome (Sandhya et al., 2017), various infectious diseases (such as hepatitis and

tuberculosis) (Rosdahl et al., 2017; Pyo et al., 2017), and also to a certain extent in

the healthy population (3–5%) and healthy elderly individuals (10–30%). In healthy

subjects the upper limit for RF is 8 IU/ml; above 8 IU/ml inflammatory disorder other

than rheumatoid arthritis is suspected. But level above 14 IU/ml indicate rheumatoid

arthritis. Furthermore, antibodies against CCP (cyclic citrullinated peptide) have been

identified as another screening marker and also as a marker of disease progression

(Grover et al., 2016). The sensitivity of anti-CCP positivity for detection of early

stage RA is 70–78% and it has specificity of 88–96%, although 30–40% of RA

patients are negative to the anti-CCP antibody test also.

RA often shows symptoms such as fatigue (Wolfe and Michaud, 2004) and general

malaise (Yanagawa et al., 2012), weight loss (Baker et al., 2018), fever and myalgia

(Chan and Leung, 2017), with some difficulty in performing day to day activities such

as dressing, walking and use of hands. As the rheumatoid arthritis conditions progress

there is symmetrical inflammatory process in the joints, usually in the joint of hand

and feet but any joint can be affected (Astorri et al., 2015). Firstly, joint become

swollen, tender and decreased degree of motion of joints (Michelsen et al., 2017) and

then due to ligament damage, tendon tightening and articular destruction due to

inflammation the joint become deformed and are destroyed (Macovei and Rezus,

2016). Then classic deformities of the upper limb develop which include

Boutonnieres and Swan-neck deformities at the distal and proximal interphalangeal

joints of the fingers, radial deviation at the wrist and ulnar deviation of the metacarpo-

phalangeal joints (Sharif et al., 2017). There is subsequent muscle atrophy and loss of

power (Krajewska-Wlodarczyk, 2016) and pincer grip (Eberhardt and Fex, 1995).

Autoantibodies against glycated proteins and their clinical relevance

Protein glycation by glucose, ribose, fructose, glyoxal and methylglyoxal can lead to

structural perturbations, aggregations etc. This may confer immunogenicity on

Chapter 3

163

molecule. Furthermore, autoantibodies against glycated-IgG has been reported in

T2DM and rheumatoid arthritis (Newkirk et al., 1998). Autoantibodies against

glycated-H2A histone has been reported in SLE patients (Alam et al., 2015) while

autoantibodies against glycated HSA has been found in diabetic nephropathy,

retinopathy etc (Khan et al., 2010). Autoantibodies against glycated-LDL has been

reported in patients of diabetes mellitus and dyslipidemia (Gonzalez et al., 1997;

Akhter et al., 2016) and autoantibodies against glycated-collagen in diabetic patients

(Wu, 1993).

Note: In this Chapter, we have assayed autoantibodies against modified-IgG

preparations in the sera of T2DM patients having different disease duration. Level of

C-reactive protein, TNFα, IL-1, IL-6 and rheumatoid factor have been also evaluated

in the above sera of T2DM patients.

Chapter 3

164

Materials

Blood samples

Blood samples were collected from type 2 diabetes mellitus patients who

were enrolled at the OPD of Rajiv Gandhi Centre for Diabetes and Endocrinolgy,

Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh after

obtaining their verbal consent. It was ensured that none of the diabetic patients were

suffering from other auto-immune diseases. Blood from healthy subjects was also

collected. All blood samples were collected in plain vacutainers and left for clot

formation. Sera were separated by centrifugation at 3000 rpm for 10 min and then

decomplemented by heating it at 56 0C for 45 min to inactivate complement proteins.

They were stored in small aliquots at -20 0C with 0.1% sodium azide as preservative.

The protocol and procedure for the experiment was duly approved by the Institutional

Ethics Committee of the Jawaharlal Nehru Medical College, Faculty of Medicine,

Aligarh Muslim University, India vide letter no. 374/FM.

The collected sera were divided into five groups and each group was further

divided into three sub-groups and the details are given below.

Group I: Normal healthy human subjects

No. of samples: 30 divided into three subgroups

Subgroup Ia: Age < 25 years

Subgroup Ib: Age 25 to < 45 years

Subgroup Ic: Age 45 to < 65 years

Group II: Type 2 diabetes mellitus patients with disease duration of < 5 years


Subgroup IIa: Age < 25 years

Subgroup IIb: Age 25 to < 45 years

Subgroup IIc: Age 45 to < 65 years

Chapter 3

165

Group III: Type 2 diabetes mellitus patients with disease duration of 5 to < 10

years


Subgroup IIIa: Age < 25 years

Subgroup IIIb: Age 25 to < 45 years

Subgroup IIIc: Age 45 to < 65 years

Group IV: Type 2 diabetes mellitus patients with disease duration of 10 to < 15

years


Subgroup IVa: Age < 25 years

Subgroup IVb: Age 25 to < 45 years

Subgroup IVc: Age 45 to < 65 years

Group V: Type 2 diabetes mellitus patients with disease duration of >15 years


Subgroup Va: Age < 25 years

Subgroup Vb: Age 25 to < 45 years

Subgroup Vc: Age 45 to < 65 years

Note: The procedure of direct binding ELISA, inhibition ELISA and gel retardation

assay as well as estimation of C-reactive protein, TNFα, IL-1, IL-6 and rheumatoid

factor is essentially the same as given under Chapter 2a.


For the comparison of RF, TNFα, IL-1, IL-6 and C-reactive protein status in different

groups and subgroups of healthy human subjects and T2DM patients one-way

analysis of variance (ANOVA) and Tukey posthoc test was performed using R 3.3.2

statistical software. Statistical significance was considered if Sig. (2-tailed) value is

.000.

Chapter 3

166

The demography and investigation details of the subjects in different groups is

mentioned in Table 18.

Table 18

Group

Subgroup

Average

H/W/BMI

Average

Plasma glucose

(mg/dl)

Average

HbA1c

(%)

Average

cholesterol

(mg/dl)

Average

TG

(mg/dl)

Average

HDL LDL/ VLDL

(mg/dl) FPG PP

I Ia; M= 5, F= 5

Ib; M= 5, F= 5

Ic; M= 5, F= 5

152/56/

24.23

154/57/

24.03

159/58/

22.94

91

99

107

101

119

132

4.62

5.56

5.84

183.00

188.00

179.00

162.00

169.00

183.00

41 /120/22

43 /124/21

38 /119/22

II IIa; M= 5, F= 5

IIb; M= 5, F= 5

IIc; M= 5, F= 5

157/54/

21.90

161/66/

25.46

158/58/

23.23

133

156

163

199

234

256

6.63

6.84

7.03

202.00

213.00

222.00

188.00

197.00

213.00

33/132/37

39/139/35

42/130/50

III IIIa; M= 5, F= 5

IIIb; M= 5, F= 5

IIIc; M= 5, F= 5

154/56/

23.61

159/63/

24.91

162/64/

24.38

177

193

212

188

233

288

7.46

7.38

8.00

242.00

233.00

255.00

231.00

221.00

239.00

53/145/44

48/134/41

70/145/40

IV IVa; M= 7, F= 3

IVb; M= 5, F= 5

IVc; M= 5, F= 5

159/73/

28.87

162/68/

25.91

166/69/

25.03

224

202

236

272

268

293

8.10

8.04

8.20

267.00

288.00

293.00

283.00

271.00

296.00

52/167/48

60/180/48

69/183/41

V Va; M= 5, F= 5

Vb; M= 5, F= 5

Vc; M= 5, F= 5

165/71/

26.07

159/62/

24.52

157/73/

29.61

249

273

257

298

313

302

8.73

8.93

8.67

301.00

320.00

316.00

299.00

313.00

319.00

50/ 195/56

85/ 185/50

81/ 165/70

where,

H = height in cm

W = weight in kilogram

BMI = Body mass index in kg/m2

FPG = Fasting plasma glucose (mg/dl)

PP = Postprandial glucose (mg/dl)

Chapter 3

167

Results

Detection of autoantibodies against native and modified-IgG preparations in sera

of T2DM patients and healthy subjects by enzyme immunoassay

All sera were diluted to 1:100 with TBS and subjected to direct binding ELISA on

microtitre plates coated with equal amount of native IgG and three preparations of

modified-IgG namely-methylglyoxal-modified-IgG, IgG co-modified with

methylglyoxal and normal glucose (5 mM) and IgG co-modified with methylglyoxal

and high glucose (10 mM), respectively and the results are shown in Fig. 81-85. It

indicates gradual appearance of autoantibodies in T2DM patients of all four groups

against modified-IgG preparations. Furthermore, IgG co-modified with methylglyoxal

under high glucose (10 mM) was found to be the preferred antigen for autoantibodies

in T2DM sera of all age group.

The antigen binding specificity of some selected sera from each T2DM group was

evaluated against native IgG and three modified-IgG preparations by inhibition

ELISA and the results are shown in Fig. 86-89. The IgG isolated from the respective

sera maintained the specificity (Fig. 90-93).

Gel retardation of IgG autoantibodies isolated from one serum each of four

groups of T2DM patients

A constant amount of IgG co-modified with methylglyoxal and 10 mM glucose

(antigen) was incubated with increasing amount of IgG isolated from one serum each

of four groups of T2DM patients. The bands were stained with coomassie dye and the

results are shown in Fig. 94 a-d. The pattern of bands in lane 2, 3 and 4 of all

photographs suggests that autoantibodies are increasing with duration of the disease

and aggressively interacting with epitopes present on IgG co-modified with

methylglyoxal and high glucose.

Chapter 3

168

Fig. 81 Direct ELISA of serum autoantibodies in healthy subjects on wells coated

with native IgG (red bar), MGO-modified-IgG (orange bar), IgG co-modified

with MGO under normal glucose (blue bar), and high glucose (purple bar).

Group Ia

Group Ib

Group Ic

Chapter 3

169

Fig. 82 Direct ELISA of serum autoantibodies in T2DM patients of disease duration

<5 years on wells coated with native IgG (red bar), MGO-modified-IgG

(orange bar), IgG co-modified with MGO under normal glucose (blue bar),

and high glucose (purple bar).

Group IIa

Group IIb

Group IIc

Chapter 3

170

Fig. 83 Direct ELISA of serum autoantibodies in T2DM patients of disease duration 5

to <10 years on wells coated with native IgG (red bar), MGO-modified-IgG



Group IIIa

Group IIIb

Group IIIc

Chapter 3

171


10 to <15 years on wells coated with native IgG (red bar), MGO-modified-IgG



Group IVa

Group IVb

Group IVc

Chapter 3

172


>15 years (Group V) on wells coated with native IgG (red bar), MGO-

modified-IgG (orange bar), IgG co-modified with MGO under normal glucose

(blue bar), and high glucose (purple bar).

Group Va

Group Vb

Group Vc

Chapter 3

173

Fig. 86 Inhibition ELISA of serum autoantibodies in T2DM patients of disease

duration <5 years with native IgG (open circle), MGO-modified-IgG (filled

circle), IgG co-modified with MGO and normal glucose (open square) and

high glucose (filled square).

Group IIa

Serum no. 07

Group IIb

Serum no. 08

Group IIc

Serum no. 01

Chapter 3

174


duration 5 to <10 years with native IgG (open circle), MGO-modified-IgG

(filled circle), IgG co-modified with MGO and normal glucose (open square)

and high glucose (filled square).

Group IIIa

Serum no. 08

Group IIIb

Serum no. 07

Group IIIc

Serum no. 07

Chapter 3

175


duration 10 to <15 years with native IgG (open circle), MGO-modified-IgG

(filled circle), IgG co-modified with MGO and normal glucose (open square)

and high glucose (filled square).

Group IVa

Serum no. 02

Group IVb

Serum no. 07

Group IVc

Serum no. 09

Chapter 3

176


duration >15 years with native IgG (open circle), MGO-modified-IgG (filled

circle), IgG co-modified with MGO and normal glucose (open square) and

high glucose (filled square).

Group Va

Serum no. 10

Group Vb

Serum no. 07

Group Vc

Serum no. 04

Chapter 3

177

Fig. 90 Inhibition ELISA of IgG (isolated from respective serum) in T2DM patients

of disease duration <5 years with native IgG (open circle), MGO-modified-

IgG (closed circle), IgG co-modified with MGO and normal glucose (open

square), and high glucose (closed square).

Group IIa

IgG of serum no.

07

Group IIb

IgG of serum no.

08

Group IIc

IgG of serum no.

01

Chapter 3

178


of disease duration 5 to <10 years with native IgG (open circle), MGO-

modified-IgG (closed circle), IgG co-modified with MGO and normal glucose

(open square), and high glucose (closed square).

Group IIIa

IgG of serum no.

08

Group IIIb

IgG of serum no.

07

Group IIIc

IgG of serum no.

07

Chapter 3

179


of disease duration 10 to <15 years with native IgG (open circle), MGO-

modified-IgG (closed circle), IgG co-modified with MGO and normal

glucose (open square), and high glucose (closed square).

Group IVa

IgG of serum no.

02

Group IVb

IgG of serum no.

07

Group IVc

IgG of serum no.

09

Chapter 3

180


of disease duration >15 years with native IgG (open circle), MGO-modified-

IgG (closed circle), IgG co-modified with MGO and normal glucose (open

square), and high glucose (closed square).

Group Va

IgG of serum no.

10

Group Vb

IgG of serum no.

07

Group Vc

IgG of serum no.

04

Chapter 3

181

1 2 3 4 1 2 3 4

1 2 3 4 1 2 3 4

Fig. 94 Polyacrylamide gel photograph of IgG autoantibodies interaction with

antigen. Electrophoresis was performed on 7.5% SDS-polyacrylamide gel

at 80 V for 4 h.

(a) Lane 1 contains IgG co-modified with methylglyoxal and 10 mM

glucose (25 µg). Lane 2, 3 & 4 contains IgG co-modified with

methylglyoxal and 10 mM glucose plus 25, 50 and 100 µg of IgG

autoantibodies of a group II T2DM patient.

(b) Lane 1 contains IgG co-modified with methylglyoxal and 10 mM



autoantibodies of a group III T2DM patient.

(c) Lane 1 contains IgG co-modified with methylglyoxal and 10 mM



autoantibodies of a group IV T2DM patient.

(d) Lane 1 contains IgG co-modified with methylglyoxal and 10 mM



autoantibodies of a group V T2DM patient.

(a) (b)

(c) (d)

1 2 3 4 1 2 3 4

Chapter 3

182

Estimation of biochemicals in sera of T2DM patients and healthy subjects

All serum samples of healthy subjects and T2DM patients included in this study were

processed for rheumatoid factor (RF), C-reactive protein, tumor necrosis factor-α, IL-

1 and IL-6 by immunoassay and the results are shown in Table 19-23. In general, we

observed raised level of all biochemicals in T2DM sera but the level was highest

amongst patients having longest history of the disease. Out of the five biochemicals

that we have analyzed in the sera four are related to inflammation (C-reactive protein,

tumor necrosis factor-α, IL-1 and IL-6) but presence of rheumatoid factor beyond

cutoff value alongwith other four biochemicals of inflammation in T2DM patients is

noteworthy, and it hints at rheumatoid arthritis like disease as co-morbidity in T2DM

patients.

Results of statistical analysis

There was a positive correlation between status of each biochemicals (such as RF,

TNFα, IL-1, IL-6 and C-reactive protein) and disease duration, and also between

status of RF, TNFα, IL-1, IL-6 and C-reactive protein and age group. The results

finally suggests that the level of biochemicals in different age group and different

group of T2DM patients has statistical significance based on their Pearson correlation

(R) and Sig. (2-tailed) as shown in Table 24a-c and Table 25a-e.

Chapter 3

183

Table 19

Estimation of biochemicals in sera of healthy subjects

Group Serum number Biochemicals

RF TNFα IL-1 IL-6 CRP

Ia

01

02

03

04

05

06

07

08

09

10

2.12

2.34

2.03

2.67

2.89

3.13

3.67

2.89

3.03

3.54

6.03

5.44

6.19

7.21

8.42

6.33

7.19

8.83

9.09

8.67

60.30

50.00

61.28

70.96

80.12

61.28

70.00

80.48

90.12

80.88

63.12

54.84

67.00

64.43

73.00

54.48

64.88

72.38

88.80

94.00

2.00

1.67

2.00

2.33

2.67

1.67

2.33

1.67

3.00

2.67

Ib

01

02

03

04

05

06

07

08

09

10

2.73

3.69

4.04

2.73

3.13

3.63

2.89

2.93

3.55

3.93

8.18

10.21

8.67

10.11

8.97

9.63

5.56

9.19

8.77

12.09

90.90

100.00

121.12

100.00

80.88

110.24

101.24

90.76

80.67

120.44

97.33

106.24

123.12

112.00

93.84

119.42

117.84

98.88

88.84

128.00

3.00

3.67

4.00

3.33

2.67

5.33

3.33

3.00

1.67

4.00

Ic

01

02

03

04

05

06

07

08

09

10

3.76

4.00

4.14

2.31

3.93

4.16

3.87

3.93

4.07

4.62

13.12

11.03

15.54

12.83

11.67

10.71

14.83

13.31

12.88

10.11

130.32

110.67

115.54

121.76

119.67

100.76

151.14

133.67

120.24

100.00

138.84

117.24

154.00

128.82

119.44

108.00

156.42

134.48

128.84

109.18

5.33

3.67

5.00

3.33

3.67

3.00

5.67

4.33

4.00

1.67

where,

RF stands for rheumatoid factor content; cut off value 14 IU/ml

TNFα stands for tumor necrosis factor α level: cut off value 10 pg/ml

IL-1stands for interleukin-1 level: cut off value 209 pg/ml

IL-6 stands for interleukin-6 level: cut off value 200 pg/ml

CRP stands for C-reactive protein content: cut off value 5 mg/L

Note: All cut off values were provided by the kit manufacturer.

Chapter 3

184

Table 20

Estimation of biochemicals in sera of T2DM patients with disease duration of

<5 years



IIa

01

02

03

04

05

06

07

08

09

10

6.78

11.66

7.34

8.33

6.56

5.54

4.33

5.56

3.56

6.93

11.72

33.00

13.39

16.76

17.82

19.33

38.00

17.23

23.72

21.00

111.76

330.00

131.24

162.84

177.67

190.84

380.00

171.00

234.00

210.00

112.24

348.00

134.44

167.84

172.96

194.44

398.00

178.84

256.62

219.40

3.67

11.00

4.33

5.33

5.67

6.33

12.67

5.67

7.67

7.00

IIb

01

02

03

04

05

06

07

08

09

10

7.67

5.66

12.84

6.78

8.87

6.76

5.54

12.96

8.83

7.66

16.33

19.82

39.00

19.33

20.87

21.06

20.98

43.00

16.82

17.33

160.62

190.87

390.00

190.00

200.00

210.84

211.76

434.00

167.83

173.88

167.67

194.45

404.00

198.80

204.00

219.96

218.84

448.00

177.62

178.00

5.33

6.33

13.00

6.33

6.67

7.00

7.66

14.33

5.33

5.67

IIc

01

02

03

04

05

06

07

08

09

10

14.20

8.76

9.34

14.40

5.87

8.98

13.93

8.83

6.67

5.56

44.00

24.72

27.83

46.00

24.88

27.00

43.00

29.62

33.00

27.83

440.00

240.48

270.12

463.00

241.48

279.88

436.00

294.12

332.00

273.88

468.00

244.42

278.88

494.12

248.62

288.84

454.00

302.27

337.00

280.84

14.67

8.33

9.00

15.33

7.67

9.33

14.33

9.67

11.33

9.67

where,







Chapter 3

185

Table 21


5 to <10 years



IIIa

01

02

03

04

05

06

07

08

09

10

15.65

11.66

8.99

8.33

16.62

6.73

8.45

14.93

7.83

9.67

47.00

30.12

23.72

27.00

45.00

31.33

27.82

48.00

29.76

24.44

479.00

301.84

232.00

278.00

453.00

313.79

279.88

484.00

296.32

243.84

494.80

308.00

239.96

284.50

484.12

319.96

288.80

498.00

298.00

248.40

16.33

10.00

7.67

9.00

15.00

10.33

9.33

16.00

9.67

8.00

IIIb

01

02

03

04

05

06

07

08

09

10

7.67

16.67

12.84

6.78

8.87

6.76

14.83

10.79

8.83

7.66

26.33

48.00

31.67

33.33

27.84

29.09

51.00

39.00

27.84

29.62

262.88

489.00

313.33

337.74

274.48

291.00

517.00

397.88

272.84

294.56

269.00

504.00

318.84

348.86

282.00

298.30

534.20

411.67

278.80

300.00

9.33

16.00

10.33

11.00

9.67

9.00

17.00

13.00

9.00

10.67

IIIc

01

02

03

04

05

06

07

08

09

10

10.60

16.67

9.34

14.40

5.87

8.98

13.93

8.83

17.30

15.54

31.33

51.00

29.82

54.00

34.62

36.67

54.12

31.48

49.00

32.67

313.67

517.00

298.84

544.00

343.12

362.88

549.00

311.11

494.84

487.00

319.96

534.42

306.48

554.00

349.96

378.00

562.00

319.67

506.00

503.40

10.33

17.00

9.67

18.00

11.33

12.00

18.00

10.33

16.33

10.67

where,







Chapter 3

186

Table 22


10 to <15 years



IVa

01

02

03

04

05

06

07

08

09

10

11.64

17.84

8.99

10.67

16.62

17.84

8.45

14.93

7.88

17.83

35.54

59.12

37.84

31.44

61.46

64.00

33.33

59.00

39.00

41.42

354.48

599.50

373.18

319.92

614.00

644.00

332.88

598.00

391.87

484.00

367.33

614.40

378.80

324.00

622.80

654.00

338.84

617.09

399.84

506.42

11.67

19.67

12.33

10.33

20.33

21.33

11.00

19.67

13.00

13.67

IVb

01

02

03

04

05

06

07

08

09

10

17.67

15.43

12.84

16.78

8.87

10.97

14.83

10.79

8.83

16.88

67.00

69.00

37.12

63.00

44.44

42.00

69.00

43.00

39.82

67.44

672.00

698.00

373.84

639.98

441.42

427.84

698.88

434.00

397.00

678.88

698.80

714.40

378.67

648.00

448.44

434.00

705.50

448.62

404.12

696.80

21.33

23.00

12.33

21.00

14.67

14.00

23.00

14.33

13.00

21.33

IVc

01

02

03

04

05

06

07

08

09

10

16.93

12.10

9.34

17.80

5.87

17.33

13.93

8.83

17.30

15.54

69.00

39.42

37.68

71.12

39.88

71.00

39.62

43.44

73.00

42.48

698.88

394.44

373.00

711.00

399.84

717.00

392.00

438.12

739.00

424.44

714.00

400.20

378.62

718.90

404.32

725.00

400.33

446.80

748.00

430.42

23.00

13.00

12.33

23.67

13.00

21.67

13.33

14.33

24.33

14.00

where,







Chapter 3

187

Table 23

Estimation of biochemicals in sera of T2DM patients with disease duration

of >15 years



Va

01

02

03

04

05

06

07

08

09

10

11.64

17.84

16.76

10.67

16.62

12.10

15.93

14.83

7.83

17.63

35.54

79.00

76.00

44.48

83.84

44.12

84.48

88.00

42.88

87.00

354.98

797.00

765.00

442.00

838.88

447.00

849.00

887.00

445.20

878.00

359.48

808.33

784.12

448.00

850.48

454.33

868.76

900.00

454.67

894.33

11.67

26.33

25.33

14.67

27.67

14.33

28.00

29.33

14.67

29.00

Vb

01

02

03

04

05

06

07

08

09

10

17.67

10.23

16.67

12.63

17.93

10.97

14.83

15.66

8.83

16.88

87.12

69.00

89.00

63.00

83.00

42.00

84.12

88.00

39.98

93.00

879.98

693.44

892.00

634.00

838.84

456.00

843.00

887.00

393.72

931.31

904.00

714.40

908.80

638.48

848.20

460.30

854.00

900.62

400.80

948.00

29.00

23.00

29.67

21.00

27.67

14.33

28.00

29.33

13.67

31.00

Vc

01

02

03

04

05

06

07

08

09

10

16.93

15.92

9.34

17.80

5.87

17.33

13.93

8.83

17.30

15.54

89.00

87.24

37.76

88.00

39.44

85.54

44.12

43.44

85.00

90.12

898.84

872.00

373.88

886.00

399.00

857.00

394.44

431.00

852.00

907.80

908.00

899.67

375.54

896.32

404.48

864.48

400.32

444.67

860.32

916.84

29.67

29.00

12.33

29.33

13.00

28.33

13.67

14.33

28.33

30.00

where,







Chapter 3

188

Table 24a

Correlations among different biochemicals and different disease duration of T2DM

patients and healthy subjects

Patient age: <25 years

Subgroup: Ia + IIa + IIIa + IVa + Va, collectively represented as subgroup A

Subgroup Biochemicals Correlation and

significance


A

RF

Pearson

Correlation 1 .876

** .884

** .887

** .878

**

Sig. (2-tailed) .000 .000 .000 .000

N 50 50 50 50 50

TNFα

Pearson

Correlation .876

** 1 .999

** .998

** 1.000

**

Sig. (2-tailed) .000 .000 .000 .000

N 50 50 50 50 50

IL-1

Pearson

Correlation .884

** .999

** 1 1.000

** .999

**

Sig. (2-tailed) .000 .000 .000 .000

N 50 50 50 50 50

IL-6

Pearson

Correlation .887

** .998

** 1.000

** 1 .999

**

Sig. (2-tailed) .000 .000 .000 .000

N 50 50 50 50 50

CRP

Pearson

Correlation .878

** 1.000

** .999

** .999

** 1

Sig. (2-tailed) .000 .000 .000 .000

N 50 50 50 50 50

Note: **Correlation is significant at the 0.01 level (2-tailed).

Chapter 3

189

Table 24b



Patient age: 25 to <45 years

Subgroup: Ib + IIb + IIIb + IVb + Vb, collectively represented as subgroup B

Subgroup Biochemicals Correlation

and

significance


B

RF

Pearson

Correlation 1 .905

** .904

** .904

** .897

**

Sig. (2-

tailed)

.000 .000 .000 .000

N 50 50 50 50 50

TNFα

Pearson

Correlation .905

** 1 .999

** .999

** .998

**

Sig. (2-

tailed) .000

.000 .000 .000

N 50 50 50 50 50

IL-1

Pearson

Correlation .904

** .999

** 1 1.000

** .999

**

Sig. (2-

tailed) .000 .000

.000 .000

N 50 50 50 50 50

IL-6

Pearson

Correlation .904

** .999

** 1.000

** 1 .998

**

Sig. (2-

tailed) .000 .000 .000

.000

N 50 50 50 50 50

CRP

Pearson

Correlation .897

** .998

** .999

** .998

** 1

Sig. (2-

tailed) .000 .000 .000 .000

N 50 50 50 50 50


Chapter 3

190

Table 24c



Patient age: 45 to <65 years

Subgroup: Ic + IIc + IIIc + IVc + Vc, collectively represented as subgroup C

Subgroup Biochemicals Correlation

and

significance


C

RF

Pearson

Correlation 1 .861

** .875

** .878

** .859

**

Sig. (2-

tailed)

.000 .000 .000 .000

N 50 50 50 50 50

TNFα

Pearson

Correlation .861

** 1 .995

** .994

** .998

**

Sig. (2-

tailed) .000

.000 .000 .000

N 50 50 50 50 50

IL-1

Pearson

Correlation .875 .995

** 1

** 1.000

** .993

**

Sig. (2-

tailed) .000 .000

.000 .000

N 50 50 50 50 50

IL-6

Pearson

Correlation .878

** .994 1.000

** 1

** .993

**

Sig. (2-

tailed) .000 .000 .000

.000

N 50 50 50 50 50

CRP

Pearson

Correlation .859

** .998

** .993 .993

** 1

**

Sig. (2-

tailed) .000 .000 .000 .000

N 50 50 50 50 50


Chapter 3

191

Table 25a

Correlations among different biochemicals and healthy subjects of different age group

within group I

Group Biochemicals Correlation and

significance


I

RF

Pearson

Correlation 1 .609

** .618

** .608

** .471

Sig. (2-tailed) .000 .000 .000 .009

N 30 30 30 30 30

TNFα

Pearson

Correlation .609

** 1 .860

** .866

** .749

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-1

Pearson

Correlation .618

** .860

** 1 .956

** .858

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-6

Pearson

Correlation .608

** .866

** .956

** 1 .865

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

CRP

Pearson

Correlation .471

** .749

** .858

** .865

** 1

**

Sig. (2-tailed) .009 .000 .000 .000

N 30 30 30 30 30


Chapter 3

192

Table 25b

Correlations among different biochemicals and T2DM patients of different age group

within group II


significance


II

RF

Pearson

Correlation 1 .690

** .693

** .693

** .678

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

TNFα

Pearson

Correlation .690

** 1 .999

** .998

** .997

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-1

Pearson

Correlation .693

** .999

** 1 .999

** .998

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-6

Pearson

Correlation .693

** .998

** .999

** 1 .996

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

CRP

Pearson

Correlation .678

** .997

** .998

** .996

** 1

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30


Chapter 3

193

Table 25c


within group III


significance


III

RF

Pearson

Correlation 1 .789

** .839

** .844

** .789

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

TNFα

Pearson

Correlation .789

** 1 .958

** .957

** .996

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-1

Pearson


** 1

** .999

** .952

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-6

Pearson

Correlation .844

** .957 .999

** 1

** .951

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

CRP

Pearson

Correlation .789

** .996

** .952 .951

** 1

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30


Chapter 3

194

Table 25d


within group IV


significance


IV

RF

Pearson

Correlation 1

** .780

** .804

** .810 .779

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

TNFα

Pearson

Correlation .780

** 1

** .996

** .994

** .996

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-1

Pearson


** 1

** .999

** .992

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-6

Pearson

Correlation .810

** .994 .999

** 1

** .990

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

CRP

Pearson

Correlation .779

** .996

** .992 .990

** 1

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30


Chapter 3

195

Table 25e


within group V


significance


V

RF

Pearson

Correlation 1

** .873

** .865

** .862 .865

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

TNFα

Pearson

Correlation .873

** 1

** .998

** .998

** .999

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-1

Pearson


** 1

** 1.000

** .999

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

IL-6

Pearson

Correlation .862

** .998 1.000

** 1

** .999

**

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30

CRP

Pearson


** .999

** .999

** 1

Sig. (2-tailed) .000 .000 .000 .000

N 30 30 30 30 30


Chapter 3

196

Discussion

Methylglyoxal is a highly efficient glycating agent (Zhao et al., 2017; Wetzels et al.,

2017) and has been implicated in aging (Tsutsui et al., 2017), diabetes mellitus

(Fosmark et al., 2006) and other disorders (Ahmed et al., 2014; Rabbani et al., 2016).

Many complications of the diabetes mellitus have been repeatedly linked to

methylglyoxal promoted non-enzymatic glycosylation of proteins and other

biomacromolecules (Rabbani and Thornalley, 2018). The major complications are

retinopathy, neuropathy and nephropathy (Fosmark et al., 2006; Hanssen et al., 2017;

Ahmed et al., 2014).

In this chapter, we have determined the profile of autoantibodies against three

preparations of modified-IgG by enzyme immunoassay. Prevalence of autoantibodies

was found to be increasing with age and disease duration. The autoantibodies

preferred IgG co-modified with methylglyoxal under high glucose as antigen. It may

be recalled that the said antigen has been found to be most effective immunogen in

diabetic rabbits (Chapter 2b). It suggests that autoantibodies in T2DM patients might

have formed as a result of immune system reaction against AGEs that has formed

from methylglyoxal assisted non-enzymatic glycation of IgG in hyperglycemia and

accumulated.

Another important result that we want to discuss here is presence of rheumatoid factor

above cut off level in good number of T2DM sera and as well in diabetic sera

immunized with various preparations of modified-IgG. This correlation alongwith

observed increase in level of CRP, TNF-α, IL-6 and IL-1 in diabetic animals as well

as diabetic humans partially supports our thought on rheumatoid arthritis like co-

morbidity in the patients of T2DM.

Chapter 3

197

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Bakker-Jonges, L.E., Haagen, I.A., Lems, W.F., Hamann, D., van

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Forbes, J.M., Yee, L.T., Thallas, V., Lassila, M., Candido, R., Jandeleit- Dahm, K.A.,

Thomas, M.C., Burns, W.C., Deemer, E.K., Thorpe, S.R., Cooper, M.E. and

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Fosmark, D.S., Torjesen, P.A., Kilhovd, B.K., Berg, T.J., Sandvik, L., Hanssen, K.F.,

Agardh, C.D. and Agardh, E. (2006) Metabolism 55, 232-236.

Frandsen, J.R. and Narayanasamy, P. (2017) Redox Biol. 14, 465-473.

Gonzalez, M., Rojas, N., Duran, D., Schade, A., Campos, R. and Milos, C. (1997)

Rev. Med. Chil. 125, 879-885.

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Groener, J.B., Reismann, P., Fleming, T., Kalscheuer, H., Lehnhoff, D., Hamann, A.,

Roser, P., Bierhaus, A., Nawroth, P.P. and Rudofsky, G. (2013) Exp. Clin.

Endocrinol Diabetes 121, 436-439.

Grover, C., Kashyap, B., Daulatabad, D., Dhawan, A. and Kaur, I.R. (2016) Indian J.

Dermatol. 61, 510-514.

Guler-Yuksel, M., Hoes, J.N., Bultink, I.E.M. and Lems, W.F. (2018) Calcif. Tissue

Int. doi: 10.1007/s00223-017-0335-7.

Hanssen, N.M.J., Scheijen, J.L.J.M., Jorsal, A., Parving, H.H., Tarnow, L., Rossing,

P., Stehouwer, C.D.A., Schalkwijk, C.G. (2017) Diabetes 66, 2278-2283.

Hauser, B. and Harre, U. (2017) Calcif. Tissue Int. doi: 10.1007/s00223-017-0370-4.

Ingawale, D.K. and Patel, S.S. (2018) Immunopharmacol. Immunotoxicol. 40, 59-71.

Khan, M.W., Qadrie, Z.L. and Khan, W.A. (2010) Int. Arch. Allergy. Immunol. 153,

207-214.

Khondker, L. and Khan, S.I. (2014) Mymensingh. Med. J. 23, 609-613.

Krajewska-Wlodarczyk, M. (2016) Wiad. Lek. 69, 542-547.

Liu, X.Z., Gao, Y., Fan, J., Xu, X., Zhang, J., Gao, J., Wan, W. and Zhao, D.B. (2018)

Clin. Rheumatol. 37, 219-226.

Macovei, L.A. and Rezus, E. (2016) Rev. Med. Chir. Soc. Med. Nat. Iasi. 120, 70-76.

Michelsen, B., Kristianslund, E.K., Hammer, H.B., Fagerli, K.M., Lie, E., Wierod, A.,

Kalstad, S., Rodevand, E., Kroll, F., Haugeberg, G. and Kvien, T.K. (2017)

Ann. Rheum. Dis. 76, 708-711.

Morioka, Y., Teshiqawara, K., Tomono, Y., Wang, D., Izushi, Y., Wake, H., Liu, K.,

Takahashi, H.K., Mori, S. and Nishibori, M. (2017) J. Pharmacol. Sci. 134, 218-

224.


le-grand) 44, 1129-1138.

Pyo, J., Cho, S.K., Kim, D. and Sung, Y.K. (2017) Korean J. Intern. Med. doi:

10.3904/kjim.2016.222.

Rabbani, N. and Thornalley, P.J. (2018) Antioxid. Redox. Signal. doi:

10.1089/ars.2017.7424.


Rosdahl, A., Herzog, C., Frosner, G., Noren, T., Rombo, L. and Askling, H.H. (2017)

Travel Med. Infect. Dis. doi: 10.1016/j.tmaid.2017.12.004.

Chapter 3

199

Sandhya, P., Mahasampath, G., Mashru, P., Bondu, J.D., Job, V. and Danda, D.

(2017) J. Clin. Diagn. Res. 11, OC33-OC36.

Sharif, K., Sharif, A., Jumah, F., Oskouian, R. and Tubbs, R.S. (2017) Clin. Anat. doi:

10.1002/ca.22980.

Terato, K., Waritani, T., Fukai, R., Shionoya, H., Itoh, H. and Katayama, K. (2018)


Tsutsui, A., Pradipta, A.R., Kitazume, S., Taniguchi, N. and Tanaka, K. (2017) Org.

Biomol. Chem. 15, 6720-6724.


Int. J. Mol. Sci. 18, 1-15.

Wolfe, F. and Michaud, K. (2004) J. Rheumatol. 31, 2115-2120.

Wu, J.T. (1993) J. Clin. Lab. Anal. 7, 293-300.

Yanagawa, Y., Hirano, Y., Kato, H. and Iba, T. (2012) BMJ Case Rep. 2012, doi:

10.1136/bcr.02.2012.5835.


1193-1198.

Chapter 3

197

References

Ahmad, S., Moinuddin, Dixit, K., Shahab, U., Alam, K. and Ali, A. (2011) Biochem.

Biophys. Res. Commun. 407, 568-574.


542.

Akhter, F., Khan, M.S., Alatar, A.A., Faisal, M. and Ahmad, S. (2016) Life Sci. 151,

139-146.

Alam, S., Arif, Z. and Alam, K. (2015) Autoimmunity 48, 19-28.

Astorri, E., Nerviani, A., Bombardieri, M. and Pitzalis, C. (2015) Curr. Pharm. Des.

21, 2216-2224.

Baker, J.F., Kerr, G. and Mikuls, T.R. (2018) Arthritis. Rheumatol. doi:

10.1002/art.40412.

Borthwick, L.A. (2016) Semin. Immunopathol. 38, 517-534.

Castaneda, S., Gonzalez-Juanatey, C. and Gonzalez-Gay, M.A. (2018) Curr. Pharm.

Des. doi: 10.2174/1381612824666180123102632.

Chan, P.S. and Leung, M.H. (2017) Case. Rep. Med. 2017, doi:

10.1155/2017/2592964.

Corrado, A., Di Bello, V., d’Onofrio, F., Maruotti, N. and Cantatore, F.P. (2017) Int.

J. Immunopathol. Pharmacol. 30, 302-307.

Do, M.H., Lee, J.H., Wahedi, H.M., Pak, C., Lee, C.H., Yeo, E.J., Lim, Y., Ha, S.K.,

Choi, I. and Kim, S.Y. (2017) Phytomedicine 36, 26-36.

Eberhardt, K.B. and Fex, E. (1995) J. Rheumatol. 22, 1037-1042.

Falkenburg, W.J.J., von Richthofen, H.J., Koers, J., Weykamp, C., Schreurs, M.W.J.,

Bakker-Jonges, L.E., Haagen, I.A., Lems, W.F., Hamann, D., van

Schaardenburg, D., Rispens, T. (2018) Clin. Chem. Lab. Med. doi:

10.1515/cclm-2017-0988.

Forbes, J.M., Yee, L.T., Thallas, V., Lassila, M., Candido, R., Jandeleit- Dahm, K.A.,

Thomas, M.C., Burns, W.C., Deemer, E.K., Thorpe, S.R., Cooper, M.E. and

Allen, T.J. (2004) Diabetes 53, 1813-1823.

Fosmark, D.S., Torjesen, P.A., Kilhovd, B.K., Berg, T.J., Sandvik, L., Hanssen, K.F.,

Agardh, C.D. and Agardh, E. (2006) Metabolism 55, 232-236.

Frandsen, J.R. and Narayanasamy, P. (2017) Redox Biol. 14, 465-473.

Gonzalez, M., Rojas, N., Duran, D., Schade, A., Campos, R. and Milos, C. (1997)

Rev. Med. Chil. 125, 879-885.

Chapter 3

198

Groener, J.B., Reismann, P., Fleming, T., Kalscheuer, H., Lehnhoff, D., Hamann, A.,

Roser, P., Bierhaus, A., Nawroth, P.P. and Rudofsky, G. (2013) Exp. Clin.

Endocrinol Diabetes 121, 436-439.

Grover, C., Kashyap, B., Daulatabad, D., Dhawan, A. and Kaur, I.R. (2016) Indian J.

Dermatol. 61, 510-514.

Guler-Yuksel, M., Hoes, J.N., Bultink, I.E.M. and Lems, W.F. (2018) Calcif. Tissue

Int. doi: 10.1007/s00223-017-0335-7.

Hanssen, N.M.J., Scheijen, J.L.J.M., Jorsal, A., Parving, H.H., Tarnow, L., Rossing,

P., Stehouwer, C.D.A., Schalkwijk, C.G. (2017) Diabetes 66, 2278-2283.

Hauser, B. and Harre, U. (2017) Calcif. Tissue Int. doi: 10.1007/s00223-017-0370-4.

Ingawale, D.K. and Patel, S.S. (2018) Immunopharmacol. Immunotoxicol. 40, 59-71.

Khan, M.W., Qadrie, Z.L. and Khan, W.A. (2010) Int. Arch. Allergy. Immunol. 153,

207-214.

Khondker, L. and Khan, S.I. (2014) Mymensingh. Med. J. 23, 609-613.

Krajewska-Wlodarczyk, M. (2016) Wiad. Lek. 69, 542-547.

Liu, X.Z., Gao, Y., Fan, J., Xu, X., Zhang, J., Gao, J., Wan, W. and Zhao, D.B. (2018)

Clin. Rheumatol. 37, 219-226.

Macovei, L.A. and Rezus, E. (2016) Rev. Med. Chir. Soc. Med. Nat. Iasi. 120, 70-76.

Michelsen, B., Kristianslund, E.K., Hammer, H.B., Fagerli, K.M., Lie, E., Wierod, A.,

Kalstad, S., Rodevand, E., Kroll, F., Haugeberg, G. and Kvien, T.K. (2017)

Ann. Rheum. Dis. 76, 708-711.

Morioka, Y., Teshiqawara, K., Tomono, Y., Wang, D., Izushi, Y., Wake, H., Liu, K.,

Takahashi, H.K., Mori, S. and Nishibori, M. (2017) J. Pharmacol. Sci. 134, 218-

224.


le-grand) 44, 1129-1138.

Pyo, J., Cho, S.K., Kim, D. and Sung, Y.K. (2017) Korean J. Intern. Med. doi:

10.3904/kjim.2016.222.

Rabbani, N. and Thornalley, P.J. (2018) Antioxid. Redox. Signal. doi:

10.1089/ars.2017.7424.


Rosdahl, A., Herzog, C., Frosner, G., Noren, T., Rombo, L. and Askling, H.H. (2017)

Travel Med. Infect. Dis. doi: 10.1016/j.tmaid.2017.12.004.

Chapter 3

199

Sandhya, P., Mahasampath, G., Mashru, P., Bondu, J.D., Job, V. and Danda, D.

(2017) J. Clin. Diagn. Res. 11, OC33-OC36.

Sharif, K., Sharif, A., Jumah, F., Oskouian, R. and Tubbs, R.S. (2017) Clin. Anat. doi:

10.1002/ca.22980.

Terato, K., Waritani, T., Fukai, R., Shionoya, H., Itoh, H. and Katayama, K. (2018)


Tsutsui, A., Pradipta, A.R., Kitazume, S., Taniguchi, N. and Tanaka, K. (2017) Org.

Biomol. Chem. 15, 6720-6724.


Int. J. Mol. Sci. 18, 1-15.

Wolfe, F. and Michaud, K. (2004) J. Rheumatol. 31, 2115-2120.

Wu, J.T. (1993) J. Clin. Lab. Anal. 7, 293-300.

Yanagawa, Y., Hirano, Y., Kato, H. and Iba, T. (2012) BMJ Case Rep. 2012, doi:

10.1136/bcr.02.2012.5835.


1193-1198.

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