2 Sanjay D. Bhalerao

A CROSS-SECTIONAL STUDY TO DETERMINE THE PREVALENCE OF
AUTONOMIC DYSFUNCTION IN TYPE-II DIABETIC MELLITUS INDIVIDUALS IN

RURAL POPULATION RESIDING AT THREE PRIMARY HEALTH CENTER (PHC)
AREAS ASSOCIATED TO JAWAHARLAL NEHRU MEDICAL COLLEGE,
BELGAUM
Thesis submitted to
THE KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,
BELGAUM
(KLE DEEMED UNIVERSITY)
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide
Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited A Grade by NAAC)
[Placed in Category A by MHRD (GoI)]
For the award of the degree of Doctor of Philosophy

In the Faculty of Medicine
(Discipline: Physiology)
by
SANJAY D. BHALERAO
(Registration No: KLEU/Ph.D./09-10 /DOUN09001)
Under the Guidance of
DR. SHIVAPRASAD S. GOUDARMD, MHPE

Professor, Department of Physiology,
J.N.Medical College, KLE University, Belgaum.
NOVEMBER-2014
UNDERTAKING
I, SANJAY D.BHALERAO hereby declare that the information and the data mentioned
in my thesis entitled A CROSS-SECTIONAL STUDY TO DETERMINE THE
PREVALENCE
OF
AUTONOMIC
DYSFUNCTION
IN
TYPE-II
DIABETIC
MELLITUS INDIVIDUALS IN RURAL POPULATION RESIDING AT THREE

PRIMARY HEALTH CENTER (PHC) AREAS ASSOCIATED TO JAWAHARLAL
NEHRU MEDICAL COLLEGE, BELGAUM belongs to me and is original.
I am aware of definition of plagiarism as detailed below:
An act or instance of using or closely imitating the language and thoughts of another
author without authorization and the representation of that authors work as ones own,
as by not crediting the original author.
A piece of writing or other work reflecting such unauthorized use or imitation.
The deliberate or reckless representation of anothers words, thoughts or ideas as ones

own without attribution in connection with submission of academic work, whether
graded or otherwise.
I hereby declare that the thesis prepared by me is original-one and does not
involve plagiarism anywhere. In case at a later stage it is found that I have indulged in
plagiarism, then I am solely responsible for the same and the Institution is at liberty to
take any disciplinary action against me including cancellation of dissertation or any
other penalties imposed by the University.
Date:
(SANJAY D. BHALERAO)
Place:
Lecturer, Dept of Physiology

J.N.Medical College, Belgaum
II
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

BELGAUM
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]

DECLARATION
I hereby declare that the thesis entitled A CROSS-SECTIONAL STUDY TO DETERMINE
THE PREVALENCE OF AUTONOMIC DYSFUNCTION IN TYPE-II DIABETIC
NEHRU MEDICAL COLLEGE, BELGAUM is a bonafide and original research carried out by me
under the guidance of DR. SHIVAPRASAD. S. GOUDARMD,
Professor, Department of
MHPE
Physiology, J.N. Medical College, Belgaum. The thesis or any part thereof has not formed the basis for the
award of any degree/fellowship or similar title to any candidate of any University.
Place: Belgaum
Signature
Date:
(Sanjay D.Bhalerao)
Lecturer, Dept of Physiology,
III


BELGAUM
Certificate
This is to certify that the thesis entitled A CROSS-SECTIONAL STUDY TO DETERMINE
NEHRU MEDICAL COLLEGE, BELGAUM is a bonafide record of original research carried out
by SANJAY D. BHALERAO for the award of degree of DOCTOR OF PHILOSOPHY IN FACULTY
OF MEDICINE (DISCIPLINE: PHYSIOLOGY) under my supervision and guidance.
Place: Belgaum
Date:
Signature
Guide
Dr. Shivaprasad S. Goudar MD, MHPE
Professor, Dept. of Physiology,
IV


BELGAUM
Certificate
This is to certify that the thesis entitled A CROSS-SECTIONAL STUDY TO DETERMINE
NEHRU MEDICAL COLLEGE, BELGAUM is a bonafide and genuine research carried out by
SANJAY D. BHALERAO under the guidance of DR.SHIVAPRASAD S. GOUDAR
MD MHPE,
Professor, Department of Physiology, Jawaharlal Nehru Medical College, Belgaum.
Place: Belgaum
Signature
Date:
Dr. (Mrs.) N.S. Mahantshetti M.D (Pead)

Dean,

V


BELGAUM
Copyright DECLARATION
We hereby declare that KLE ACADEMY OF HIGHER EDUCATION AND

RESEARCH, BELGAUM, KARNATAKA, shall have the rights to preserve, use and
disseminate this thesis in print or electronic format for academic/research purpose.
Signature
Research Scholar
(Sanjay D.Bhalerao)
Signature
Guide
Dr. Shivaprasad S. Goudar MD, MHPE
Professor, Dept. of Physiology
Place: Belgaum
Date:

BELGAUM
ACKNOWLEDGEMENT
VI
Completion of this doctoral thesis was possible with the support of several people. I would like
to express my sincere gratitude to all of them. I whole heartedly thank all those people who have
encouraged me right from the conception of this work till its present form.
First of all, It is my privilege to express my heartfelt gratitude and sincere thanks to my
respected teacher and guide, Dr.Shivaprasad S. Goudar
MD, M.H.P.E
Professor, Department of
Physiology, J. N. Medical College, Belgaum, for his valuable guidance, scholarly inputs and consistent
encouragement I received through the research work. Sir has made himself available to clarify my
doubts despite of his busy schedules and I consider it as a great opportunity to do my doctoral
programme under his guidance and to learn from his research expertise. Thank you so much Sir, for all
your help and support.
The implementation of what was in my mind could not have been possible if it were not
encouraged and promoted timely by our beloved Hon Chancellor Dr Prabhakar Kore
Vice-Chancellor Prof. Dr. Chandrakant Kokate
Ph.D.,
D.Lit
and Hon
who motivated me to register PhD program and
gave platform to carry out the research work continuously throughout until completion.
I sincerely thank to Dr. (Mrs) N.S. Mahantshetti
M.D (Pead),
Principal, J. N. Medical College,
Belgaum for the academic support and facilities provided to carry out the research work at the institute.
I am grateful to Shri. M. D. Mallapur, Asst. Professor (Dept. of Statistics), J.N.Medical College,
Belgaum for helping me in analyzing the research data. My research would not have been possible
without his untiring help and support all the time.
I am sincerely obliged to faculty members Dr. Parwati Patil, Professor & HOD, Dr. R.N.
Raichur, Professor, Dr. (Mrs) V.J.Watve, Professor, Dr. Padmashree Kudchi, Associate Professor, Dr.
Jaysheela Bagi, Associate Professor, Dr Vijaya Dandannavar, Associate professor, Dr. Neha
Kulkarni, Associate Professor, Dr. Sunil S. Vernekar, Asst Professor, Dr. Jaysudha K, Assistant
professor, Dr. Nirmala Anand, Assistant professor, Dr. Amar Barwade, Asst. Professor, Dr. Shantala
Herlekar, Asst Professor, Dr. Basavraj T, Assistant professor, Dr. Harpreet Kour, Lecturer, Dr.
Ramesh, Lecturer, Department of Physiology, J. N. Medical College, Belgaum for giving me consistent
VII
suggestion and support, who were always there to encourage me, because of whom my thesis work has
been one, that I will appreciate forever.
I sincerely thank to Dr. Sunil S. Vernekar, Assistant professor, Dr. Manjunath Somannavar,
Associate professor, and Dr. Smita Sonoli, Associate professor and HOD, Dept of Biochemistry, I am
benefited immensely by their scholarly advises and vast global experiences. I shall forever remain
overwhelmed by their helping, caring, affectionate nature.
I am thankful to all postgraduates, Dr Savita Hiremath, Dr. Girish, Mr. Prem kumar Yadav,
Mr. Santosh Kumar Jha, Mr. Shankar Yadav, Mr. Binod Yadav, Mr. Aditya. Mr. Harishankar, Mr.
Ramesh for their support and co-operation during my entire study period.
I am thankful to Dept of community medicine for giving me permission to do work at community
level and also for all their help during the study. I also extend my thanks to Dept. of Biochemistry for
helping me in biochemical investigation of my research work.
Technical, clerical and supporting staff of the Department of Physiology, J. N. Medical College,
Belgaum, have been very kind enough to extend their help at various phases of this research work,
whenever I approach them. I am grateful to all of them.
I owe a lot to my parents, who encourage and helped me at every stage of my personal and
academic life and longed to see this achievement come true. I am very much indebted to mother-in-law,
sister-in-law and brother-in-law, who supported me in every possible way to see the completion of this
work.
No words can sum up what I feel for my pillar of strength and my inspiration to go on in life, my
wife, Mrs. Mallika. I truly acknowledge her love, support, encouragement and never ending belief in me.
I apologies to Sameer my son and Sirigauri my daughter, for not being available to them
during the course of my studies and I also express my love to Sarvadnya, Akshya, Dhyan and Digant.
VIII
This acknowledgment would be incomplete if I fail in my duty to thank all the study participants,
ASHA workers and Medical officers of all three primary health center areas, who participated in this
study and were indispensable for the completion of the thesis.
I am grateful to Mr Ananad, Mr Arun, Mr Manoj, Vigneshwara Associates, for helping me in
DTP, printing and binding of my doctoral thesis.
I thank God, the one above all of us, for answering my prayers and enriching me with the
wisdom and resolve that he has been bestowed upon me during this research work and indeed, all the
way through my life. I bow my head in respect before God Almighty.
Date:
SANJAY D. BHALERAO
Place:
IX
CHAPTER
1
CONTENT
INTRODUCTION
Page No.
01
1.1
Background
01
1.2
Need for the study
04
1.3
Research question
05
1.4
Objectives of the study
06
1.5
Conceptual framework
07
REVIEW OF LITRATURE
14
2.1 Diabetes mellitus
14
2.1.1
Historical approach
14
2.1.2
Classification
20
2.1.3
Epidemiology
21
2.1.4
Pathophysiology
26
2.1.5
Diagnosis
35
2.1.6
Treatment
36
2.1.7
Management
39
2.1.8
Diabetic complications
42
2.2 Diabetes and Autonomic dysfunction
47
2.3 Diabetic neuropathy
49
2.3.1 Diabetic autonomic neuropathy
49
2.3.2 Classification of diabetic neuropathy
51
CHAPTER
CONTENT
2.3.3 Epidemiology
55
2.3.4 Pathophysiology
56
2.3.5 Cardiovascular autonomic neuropathy
64
2.3.5.1 Epidemiology
65
2.3.5.2 Clinical manifestation
67
2.3.6 Diabetic peripheral neuropathy
72
2.3.6.2 Symptoms of DPN
74
2.3.6.3 clinical assessment of DPN
76
2.3.6.4 Causes of DPN and Laboratory testing
78
79
2.3.7.1 Diagnosis assessment of CAN-AFT
82
2.3.7.2 Diagnosis of CAN- HRV analysis
89
1. Historical view
89
2. Measurement of HRV
90
3. Clinical component of HRV
95
4. Diagnosis of CAN by HRV analysis
98
2.3.7.3 Diagnosis assessment of DPN

MATERIAL AND METHODS ..
3.1
71
2.3.6.1 Epidemiology
2.3.7 Diagnosis of diabetic neuropathy
3.
Page No
Experimental designs and sampling methods

XI
99
105
105
CHAPTER
CONTENT
Page No.
3.2
Sample size estimation
106
3.3
Sampling methods
107
3.4
Study area and sampling distribution plan
108
3.5
Study protocol
112
3.6
Personal detail and History
113
3.7
Anthropometrical measurements
114
3.8
Biochemical screening methods for Type 2 DM
116
3.9
BP measurement
118
3.10
Assessment of CAN by HRV analysis
118
3.11
Autonomic function tests
121
3.12
CAN scoring methods
136
3.13
Tests for diabetic peripheral neuropathy
137
RESULTS
4.1
Sociodemogrpahic and anthropometric
140
141
characteristics of study participants

4.2
Prevalence of T2DM
141
4.3
Association between various risk factors and
142
prevalence of T2DM
4.4
Risk factors of T2DM
142
4.5
Prevalence of CAN and DPN
157
4.6
Sympathovagal status among T2DM HRV
157
XII
CHAPTER
CONTENT
4.7 Association between potential risk factors and
Page No
158
prevalence of CAN- HRV analysis

4.8 Risk factors of CAN HRV analysis
4.9
Prevalence of CAN Ewings and Bellavare
159
167
criteria
4.10 Sympathovagal status among T2DM participants
167
Ewings criteria
168
prevalence of CAN - Ewings tests and

Bellavare criteria
4.12 Risk factors of CAN
169
4.13 Distribution of T2DM participants by various
169
cardiovascular reflex tests

4.14 Prevalence of DPN
179
179
prevalence of DPN-DNS and DNE methods

4.16 Risk factors of DPN
180
DISCUSSION.
189
SUMMARY
202
CONCLUSION..
205
MISCELLANEOUS..
206
A. Scope for future studies
XIII
206
CHAPTER
CONTENT
B. Limitations of the study
Page No
207
BIBLIOGRAPHY.
208
10
ANNEXURES
247
Annexure I
Annexure - II
: Ethical clearance certificate
256
Annexure III
: Proforma
257
Annexure IV
: DNS questionnaire
258
Annexure V
: DNE questionnaire
259
Annexure VI
: Photographs
260
Annexure VII :
Publications
264
Annexure VIII :
Poster presentation certificate
283
Informed consent
XIV
247
LIST OF ABBREVIATIONS
AFT
Autonomic Function Tests
ADA
American Diabetic Association
ANS
Autonomic Nervous System
AAN
American Association of Neurology
AGEs
Advance Glycation End Products
BMI
Body Mass Index
BMR
Basal Metabolic rate
CAN
Cardiovascular Autonomic Neuropathy
CVD
Cardiovascular Disease
DNS
Diabetic Neuropathy Symptom Score
DNE
Diabetic Neuropathy Examination
DAN
Diabetic Autonomic Neuropathy
DN
Diabetic Neuropathy
DM
Diabetes Mellitus
DPN
Diabetic Peripheral Neuropathy
DDM
Duration of Diabetes Mellitus
DKS
Diabetic Ketoacidosis
ETS
Environmental Tobacco Smoke
EURODIAB
European Diabetes Complication Prospective Study
FHDM
Family History of Diabetes Mellitus
FBS
Fasting Blood Sugar
FFT
Fast Fourier Transform
FPG
Fasting Plasma Glucose
FPI
Fasting Plasma Insulin
FDA
Food and Drug Association
HC
Hip Circumference
XV
HRV
Heart Rate Variability
HHS
Hyperglycemia Hyperosmolar State
IHD
Ischemic Heart Disease
ICMR
Indian Council of Medical Research
IRS
Insulin Substrate Receptor
ID
International Dollars
IGT
Impaired Glucose Tolerance
IDF
International Diabetes Federation
NHP
National Health Policy
NIDDM
Non-Insulin Dependent Diabetes Mellitus
NCD
Non-Communicable Diseases
NCV
Nerve Conduction Velocity
NTT
Number Needed to Treat
OGTT
Oral Glucose Tolerance Test
PHC
Primary Health Center
PDS
Power Density Spectrum
ROS
Reactive Oxygen Stress
SSPP
Steady state Plasma Protein
SPSS
Software Package for Social Sciences
T1DM
Type 1 Diabetes mellitus
T2DM
Type 2 Diabetes mellitus
TNF
Tumor Necrosis Factor
TCA
Tricyclic Antidepressant
WHO
World Health Organization
WC
Waist Circumference
XVI
LIST OF TABLES
SL.No.
1.5.1
Particulars
Page No
Risk factors for type 2 diabetes mellitus
09
2.1.3.1
Prevalence of diabetes in urban cities of India
24
2.1.3.2
Prevalence of diabetes in rural India
25
2.1.5.1
Diagnostic criteria for diabetes
35
2.1.6.1
Oral agents used in the management of T2DM
41
2.3.2.1
Clinical classification of diabetes neuropathies by Bolton & ward
49
2.3.2.2
Clinical classification of diabetic neuropathies by Thomas
49
2.3.2.3
Clinical classification of diabetic neuropathies by Waltans and
50
Edmonds
2.3.2.4
Classification of diabetic neuropathies
54
2.3.4.1
Possible pathological mechanism of diabetic neuropathy
59
Prevalence of CAN reported by various studies
66
2.3.5.1.1
2.3.6.1.1 Prevalence of DPN reported by various studies
73
2.3.6.2.2 Clinical feature of small and large fiber diabetic peripheral neuropathy
76
2.3.6.4.1 Causes of DPN
79
2.3.7.1
Differential diagnosis of diabetic neuropathy
80
2.3.7.2
Diagnostic tools for diabetic autonomic neuropathy
81
2.3.7.1.1 Normal, Borderline and abnormal values of cardiovascular tests
88
XVII
2.3.7.2.1 Selected time domain measures of HRV analysis
93
2.3.7.2.2 Selected frequency domain measures of HRV analysis
94
3.8.3.1
Testing glucometer accuracy
117
3.11.3.1
Flow plan for performing tests of Cardiovascular autonomic function
134
3.11.2.2
Cardiovascular autonomic function tests with reference value of
135
parasympthathetic tests
3.11.3.3
Cardiovascular autonomic function tests with reference value of
135
sympathetic tests
4.1.1
Distribution `of study participants by age and gender
144
4.1.2
Distribution of study participants according to literacy
145
4.1.3
Distribution of study participants by dietary habit, BMI, central and
146
truncal obesity
4.1.4
Distribution of study participants by smoking habits, alcohol
147
consumption and family history of diabetes

4.1.5
Comparison of numerical outcome like age, height, weight, BMI,
148
Waist-Hip ratio, FBS, SBP and DBP in diabetic and non-diabetic

participants.
4.3.1
Association between age, gender and prevalence of T2DM
150
4.3.2
Association between literacy, occupation and prevalence of T2DM
151
4.3.3
Association between diet, BMI, central obesity, truncal obesity and
152
prevalence of T2DM
4.3.4
Association between smoking, alcohol consumption, family history of
153
diabetes and prevalence of T2DM

4.4.1
Socio-demographic risk factors of T2DM
154
4.4.2
Socio-demographic and anthropometric risk factors of T2DM
155
XVIII
4.4.3
Socio-demographic risk factors of T2DM
156
4.6.1
Parameters of HRV analysis reflect autonomic status among T2DM
161
participants
4.7.1.1
Association between age, gender, smoking, alcohol consumption ,
162
family history diabetes and prevalence of CAN-Time domain method

4.7.1.2
Association between diet, BMI, central obesity, truncal obesity,
163
duration diabetes and prevalence of CAN Time domain method

4.7.2.1
Association between various sociodemogrpahic variables and
164
prevalence of CAN Frequency domain method

4.7.2.2
Association between various sociodemogrpahic and anthropometric
165
variables and prevalence of CAN-Frequency domain method

4.8
Sociodemogrpahic and anthropometric risk factors for CAN HRV
166
analysis
4.10.1
Autonomic function test parameters depicting sympathovagal status
172
among T2DM participants

4.11.1.1
173
family history of diabetes and prevalence of CAN- Bellavare criteria

4.11.1.2
Association between diet, BMI, central , truncal obesity, duration of
174
diabetes and prevalence of CAN- Bellavare criteria

4.11.2.1
175
family history of diabetes and prevalence of CAN- Ewings criteria

4.11.2.2
176
diabetes and prevalence of CAN- Ewings criteria

4.12.1
Sociodemogrpahic and anthropometric risk factors for CAN
177
4.13.1
Distribution of T2DM participants by various cardiovascular reflex
178
XIX
tests
4.15.1.1
182
family history of diabetes and prevalence of DPN- DNS method

4.15.1.2
183
diabetes and prevalence of DPN- DNS method

4.15.2.1
185
family history of diabetes and prevalence of DPN- DNE method

4.15.2.2
186
diabetes and prevalence of DPN- DNE method

4.16.1
Sociodemogrpahic and anthropometric risk factors for DPN
XX
188
LIST OF FIGURES
SL.No.
Particulars
Page No
1.5.1
Conceptual framework model for prevalence of T2DM ( present study)
10
1.5.2
Conceptual framework model for estimation of prevalence of autonomic
12
dysfunction (CAN and DPN)

1.5.3
Conceptual framework model for estimation of related risk factors for
13
CAN and DPN (present study)

2.1.1.1
Frederick Banting and Charles Best, 1924
17
2.1.3.1
Number of people with diabetes by IDF region, 2013
22
2.1.3.2
Top 10 countries showing number of people with diabetes (20-79 years),
22
2013
2.1.4.1
Normal glucose homeostasis
27
2.1.4.2
Physiology of insulin secretion and action
28
2.1.4.3
Insulin signal transduction
29
2.1.4.4
Etiology and pathophysiology of type 2 diabetes
30
2.1.4.5
Pathophysiological progression T2DM as seen from pancreatic beta cell
32
function
2.1.4.6
Pathophysiological factors responsible for impaired insulin secretions
32
2.1.4.7
Insulin transduction system
34
2.1.6.1
The management of paradigm for T2DM presentation of onset of
36
proactive management of early stage diabetes

2.1.6.2
Flow chart presenting the treatment for diabetes
38
2.1.7.1
Diabetic complications
46
XXI
2.2.1
Physiology of autonomic nervous system
48
2.3.4.1
Pathogenesis of diabetic neuropathy
58
2.3.4.2
Pathophysiological factors in diabetic neuropathy
60
2.3.4.3
Oxidative stress generated in diabetic neuropathy
61
2.3.4.4
The sorbitol pathway
63
2.3.4.5
Schematic depiction of glucose metabolism in normal & diabetic nerve
63
2.3.5.2.1 The autonomic innervations of the heart and the effect of diabetes
2.3.6.1
67
Most commonly affected areas of diabetic peripheral neuropathy
72
2.3.6.2.1 Stocking and glove distribution of diabetic peripheral neuropathy
75
2.3.6.3.1 Semmes-Weinstein monofilament examination
77
2.3.7.1.1 Baroreflex mechanism
86
2.3.7.2.3 Power spectral density of HRV analysis ( frequency domain method)
95
3.3.1
Population details
107
3.10.1
Flow chart summarizing individuals steps used where recording and
120
processing the ECG signal in order to obtain the HRV data

3.11.3.1
ECG recording during OTT
124
3.11.3.2
ECG recording Valsalva Maneuver
129
4.2.1
Prevalence of T2DM in the study area
149
4.2.2
Prevalence of T2DM in the different sub centers and villages in the study
149
area
4.5.1
Prevalence of CAN in the study area - HRV analysis
161
4.9.1
Prevalence of CAN - Ewing test criteria
170
XXII
4.9.2
Prevalence of CAN - Bellavere criteria
170
4.11.1
Association between age groups and prevalence of CAN
171
4.11.2
Association between duration of diabetes and prevalence of CAN
171
Prevalence of DPN estimated by DNS and DNE scoring
181
4.15.1.1
Association between age groups and prevalence of DPN-DNS method
184
4.15.1.2
Association between duration of diabetes and prevalence of DPN DNS
185
4.14
method
4.15.2.1
Association between age groups and prevalence of DPN - DNE method
187
4.15.2.2
Association between duration of diabetes and prevalence of DPN DNE
197
method
XXIII
ABSTRACT
Introduction: Diabetes Mellitus (DM) is emerging as a major health problem owing to its
serious complications. Within India, inter-regional disparities in burden of type 2 diabetes
mellitus (T2DM) are expected because of varying lifestyles and demographic patterns. DM is
also well known for its chronic complication such as tripathy which include neuropathy,
retinopathy and nephropathy. Diabetic neuropathy [particularly, Cardiac autonomic neuropathy
(CAN) and Diabetic peripheral neuropathy (DPN)] is most common clinical complication of
DM. Its presence is associated with worsening prognosis and poorer quality of life. The risk of
developing autonomic dysfunction in DM depends on several factors. It is important to assess
these factors for developing interventions that can decrease and or arrest the progression of the
disease.
Objectives:
Primary: To estimate the prevalence and autonomic dysfunction, assessed by heart rate
variability analysis and autonomic function tests, among T2DM individuals of the rural
population residing in areas of three primary health centers associated with Jawaharlal Nehru
Medical College, Belgaum.
Secondary:
1. To estimate the prevalence of DPN among T2DM individuals.
2. To determine the association of various risk factors such as age, gender, literacy,
occupation, diet, body mass index, central and truncal obesity, smoking, alcohol
consumption and family history of diabetes with prevalence of T2DM, CAN and DPN.
XXIV
Material and Methods: A cross sectional study was conducted to find out the prevalence and
autonomic dysfunction in T2DM individuals of age above 30 years residing at three primary
health center areas associated with Jawaharlal Nehru Medical College, Belgaum. 2684 selected
participants (using multistage cluster sampling method) were screened for type 2 diabetes mellitus
as per WHO criteria. Participants identified as T2DM were screened for autonomic dysfunction by
using various cardiovascular reflex tests (Ewing tests criteria). Diabetic peripheral neuropathy was
assessed by using diabetic neuropathic examination score (DNE) and diabetic symptom scoring
system (DNS).
Results: - Overall prevalence of T2DM was found to be 17.7%. Age, diet, BMI, truncal obesity,
smoking, alcohol consumption and family history of diabetes appear to increase the risk for
developing T2DM. Furthermore, advance age and duration of diabetes appear to be risk factors for
developing CAN and DPN in the T2DM participants.
Conclusion: - High prevalence of T2DM, CAN and DPN, even in rural community, suggests
the impact of socioeconomic transition on the occurrence of the disease. HRV analysis and
autonomic function tests can be used as screening tools for assessment of CAN in community
settings. There is need to institute screening and awareness programs for early detection of
diabetic complication so as to prevent long term consequences. The findings of this study
suggest the need for the promotion of preventive measures to prevent or delay the development
of chronic complications of diabetes through good glycaemic control, regular monitoring,
lifestyle modification, practice of exercise and yoga to maintain the normal balance of
sympathetic and parasympathetic tone.
Key words: autonomic function tests; type 2 diabetes mellitus; cardiovascular autonomic
neuropathy; diabetic peripheral neuropathy; heart rate variability; diabetic symptom score; diabetic
neuropathy examination score.
XXV
Introduction
1.1 Background:
Diabetes mellitus is a group of metabolic disorders due to either the inability of
the pancreas to produce enough insulin or diminished responsiveness of the cells to
the insulin that is produced known as insulin resistance.1 Type 2 diabetes mellitus
[T2DM] ( also known as non-insulin dependent diabetes mellitus {NIDDM} or adultonset diabetes) is non-autoimmune, complex, heterogeneous and polygenic metabolic
disease condition in which the body fails to produce sufficient beta cell insulin,
impaired insulin effectiveness and characterized by abnormal glucose homeostasis.2
The development of T2DM is caused by a combination of lifestyle and genetic
factors.3 The impact of T2DM is significant as a lifelong disease. It increases
morbidity, mortality and worsens the quality of life.4 Furthermore, these disease and
its complications also cause a heavy economic burden for patients, their families and
society. International Diabetes Federation (IDF) has estimated that the global burden
of T2DM for 2013 was 382 million people. India is estimated to have 65.1 million
people with diabetes in 2013, which is projected to increase above 72.1 million by
2035 5, 6, 50 and due to which India is referred as the diabetic capital of the world.
Prevalence of Diabetes in India Study (PODIS-2002), a nationally represented
multi-centric (49 urban and 59 rural centers) study conducted on 41,000 volunteers,
estimated the prevalence of DM ranging from 5.6 to 12.4% in urban area and 2.4 to
2.7% in rural area.
7, 8, 9
This urban rural difference in the prevalence of DM within
the same ethnic group (Indians) is attributed primarily to the modern lifestyle
followed by urban Indians compared to the traditional lifestyle of rural Indians. The
differences in lifestyle factors in urban-rural India include dietary pattern, physical
activity and mental stress. The prevalence of DM differs not only transversely across
KLE University, Belgaum
Introduction
the rural-urban divide but also in the different Indian states which may be due to the
different stages of demographic transition.10
The World Health Organization (WHO) has stressed the importance of
research on epidemiology of diabetes to enable the development of appropriate
interventions. National Health Policy-2002 recognizes the need to establish baseline
estimates for non-communicable diseases like diabetes in a longer timeframe.
According to national health policy, prevalence of diabetes in rural population is an
important public health issue. 10, 11, 12
There is widespread agreement that specific tests are necessary to monitor for
early signs of diabetic complications. DM is expected to emerge as a major public
health problem due to serious complications.13 The major long-term complications
relate to damage of the blood vessels and peripheral nerves (motor and sensory
nerves). Complications of DM is characterized by macrovascular and microvascular,
which will double the risk of cardiovascular disease.14 The main macrovascular
diseases are linked to atherosclerosis of larger arteries like IHD,
stroke
and peripheral vascular disease.15

DM is also well known for the microvascular complications as tripathy
includes diabetic neuropathy, retinopathy and nephropathy. Diabetic retinopathy can
lead to visual symptoms including reduced vision and potential blindness. Diabetic
nephropathy occurs due to impact of diabetes on the kidneys which can lead to loss
of small or progressively larger amounts of protein in the urine. It causes chronic
kidney disease which may require dialysis. Another important and common clinical
complication of diabetes is diabetic neuropathy (DN). DN is a heterogeneous disorder
that encompasses a wide range of abnormalities affecting both proximal and distal
Introduction
peripheral sensory and motor nerves as well as the autonomic nervous system.
Primary symptoms of DN include numbness, tingling and pain in the feet and it may
also increase the risk of skin damage due to altered sensation. DN can also affect all
the organ systems in the body including gastrointestinal tract, urogenital tract &
cardiovascular system. Further, together with vascular disease in the legs, neuropathy
contributes to the risk of diabetes related foot problems such as diabetic foot ulcers
which is difficult to treat and occasionally it may require amputation. Factors
involved in the pathogenesis of DN are altered metabolism, vascular insufficiency,
loss of growth factor tropism and autoimmune destruction of nerves in the visceral
and cutaneous distribution. DN is among the least recognized and understood
complications of diabetes despite its significant negative impact on survival and
quality of life. Most common clinical form of DN is diabetic autonomic neuropathy
(DAN) and diabetic peripheral neuropathy (DPN). DAN affects the nerves that
control the heart rate, blood pressure, blood glucose response and other internal
organs. This will cause the problems with digestion, respiratory function, urination,
sexual response and vision. It also affects the system which restores blood glucose
levels to normal after a hypoglycemic episode resulting in loss of the warning signs of
hypoglycemia, such as sweating and palpitations.16, 17
Ziegler et al estimated the prevalence of DAN as 16.7% in type 1and type 2
diabetes patients based on one or more abnormal heart rate variability (HRV) test.18,
19, 20
The prevalence of DPN varies from 5-100% which is depending upon type of
modality used for assessment.21 According to studies, 50% of patients with DPN
experience some degree of painful symptoms and 10% to 20% will have symptoms
related to advance age, longer diabetes duration, higher levels of glycosylated
Introduction
hemoglobin, lower HDL cholesterol, smoking, peripheral vascular disease and insulin
use.22,23
1.2 Need for the study
There are inconclusive data regarding prevalence of diabetes, CAN and DPN
and its reporting. Further, factors that account for the marked variability in reported
prevalence rates include the lack of a standard accepted definition, different
diagnostic methods used, variability in study selection criteria, population and referral
bias.24 In addition, some confounding variables which may affect the prevalence
include age, sex, duration of disease, glycemic control, type of diabetes and other
factors. India is predominantly an agricultural nation with approximately 70-80% of
the population residing in rural areas and there are relatively few studies related to
prevalence of diabetes and its complications among the rural populations. Moreover,
periodical studies are essential for understanding its epidemiology. The baseline data
regarding the prevalence and its predictors is essential before implementation of
National Program for Control of Diabetes, Cardiovascular Diseases and Stroke
(NPDCS).24 Before commencing any kind of management measures, it is important to
assess the factors contributing to the occurrence of the disease so that by limiting
these factors the progression of the disease in diabetic patients can be controlled.
Further, occurrence of T2DM or DN in susceptible individuals can be prevented by
reducing the exposure to the specific predisposing factor.
Although neuropathy has long been recognized as a complication of diabetes
but the impact of this condition has not been adequately established. The prevalence
of DN is virtually unknown because the published studies differ considerably with
regard to definition, method of assessment and patient selection, despite being
Introduction
considered one of the most common long-term complications of diabetes. DN has a
slow and insidious onset which may be responsible for the problem of awareness of
this condition in the patient and hence patients may suffer from the condition
unknowingly for many years. Early diagnosis and intervention are of prime
importance
in
preventing
potentially
serious
consequences
of
diabetic
complications.25, 26, 27 Also, there are not many studies reporting the prevalence of
diabetes and autonomic dysfunction among diabetes individuals in the rural
population. Hence, the present study was carried out to estimate the prevalence and
autonomic dysfunction among T2DM individuals in a rural population of North
Karnataka. Additionally, various predisposing factors for T2DM and its complications
were also assessed.
1.3 Research question

1. What is the prevalence and autonomic dysfunction among T2DM individuals
in rural population of North Karnataka, Belgaum?
2. What is the prevalence of diabetic peripheral neuropathy among T2DM
individuals in the rural population of North Karnataka, Belgaum?
3. What are the risk factors related to T2DM, cardiovascular autonomic
neuropathy and diabetic peripheral neuropathy?
Objectives of the study

1. PRIMARY OBJECTIVE:
To estimate the prevalence and autonomic dysfunction, assessed by heart rate

variability analysis and autonomic function tests, among T2DM individuals
of the rural population residing at three primary health centers associated with
Jawaharlal Nehru Medical College, Belgaum.
2. SECONDARY OBJECTIVES:
To estimate the prevalence of diabetic peripheral neuropathy among T2DM

individuals.
To determine the association of various risk factors such as age, gender,

literacy, occupation, diet, BMI, central and truncal obesity, smoking, alcohol
consumption, family history of diabetes with :
1.
Prevalence of Type 2 Diabetes Mellitus (T2DM)
2.
Prevalence of Cardiovascular Autonomic Neuropathy (CAN) in patients

of Type 2 Diabetes Mellitus (T2DM)
3.
Prevalence of Diabetic Peripheral Neuropathy (DPN)
Based on literature review and guidelines of American diabetes association
(ADA), World Health Organization (WHO), Principles of Internal Medicine, Task Force
European Society of Cardiology and American Academy of Neurology, present study has
used two conceptual framework models for addressing the following research questions:
A. Prevalence and risk factors of Type 2 diabetes mellitus in rural population of
North Karnataka
B. Prevalence and risk factors of autonomic dysfunction among Type 2 diabetic
individuals in rural population of North Karnataka.
A. Prevalence and risk factors of Type 2 diabetes mellitus in rural population of

North Karnataka
According to International Diabetes Federation (IDF) and Prevalence of
Diabetes in India Study (PODIS), prevalence of diabetes estimated by various
type of studies include:i. Global prevalence of diabetes:
According to International Diabetes Federation (IDF) Diabetes Atlas
report, nearly 382 million people worldwide or average of 8.3% of adults are
projected to have diabetes. Approximately 80% live in low and middle-income
countries. If these trends continue, around 592 million people, or in every 10
adults, will have diabetes by 2035.
ii. Global urban-rural distributions of diabetes:
There are more people with diabetes living in urban (246 million) than in
rural (136 million) areas although the figures for rural areas are on the increase. In
low and middle income countries, the number of people with diabetes in urban
areas is 181 million, whereas 122 million live in rural areas. By 2035, the
difference is expected to widen to 347 million people in urban areas and 145
million in rural areas.50
iii. India - Diabetes capital of the world
Diabetes is growing alarmingly in India, being home to more than 65.1
million people with the disease in 2013, compared to 50.8 million in 2010.
According to the International Diabetes Federation (IDF) Atlas report of 2013,
India with 65.1 million diabetic patients is just next to China (98.4 million) in the
race to become the diabetes capital of the world. 50,51
iv. Urban-Rural distribution of diabetes in India
The Prevalence Of Diabetes in India Study (PODIS) was carried out in
108 centres (49 urban and 59 rural) to look at the urban-rural differences in the
prevalence of T2DM and glucose intolerance which showed 4.7 per cent in the
urban and 2.0 percent in the rural population, according to ADA criteria while
according to the WHO criteria prevalence was 5.6 and 2.7 percent among urban
and rural areas respectively.51, 52, 53
v.
Risk factors for Type 2 Diabetes Mellitus

According to Principles of Internal Medicine, American Diabetes
Association and World Health Organization, risk factors for Type 2 diabetes
include the following:1.5.1 Table: Risk factors for Type 2 Diabetes Mellitus
Major risk factors

Family history of diabetes ( i.e. parent or sibling with Type 2 diabetes)
Obesity ( i.e. BMI > 25Kg/m2; 20% over desired body weight)
Age 45 years
Race/ethnicity ( e.g. African, Asian)
History of gestational diabetes or having given birth to a baby weighing >9
pounds or > 4.5 kg
Hypertension(blood pressure 140/90 mm of Hg)
HDL cholesterol level 0.90mmol/L ( 35mg/dL) and /or a triglyceride level
2.82mmol/L (250mg/dL)
Polycystic ovary syndrome
Impaired glucose tolerance (previously identified plasma fasting glucose of 110125mg/dl)
Impaired fasting glucose (previously identified plasma fasting glucose of <
140mg/dl and 2-hr plasma glucose of 140-199mg/dl
Secondary risk factors
Sex ( female)
Education(< High school education)
Income ( Limited income or poverty)
Sedentary lifestyle ( inadequate exercise or physical activity)
In the present study the prevalence and related risk factors for type 2 diabetes
mellitus in rural population have been estimated. The conceptual framework for the
present study is given below:
Independent variables
Dependent variable
1. Personal characteristics
Age
Sex
Education (literacy status)
Occupation
Location (Rural, North
Karnataka, Belgaum)
2. Anthropometry variables
BMI
Overweight
Obese
Truncal obesity
Central obesity
Type 2 Diabetes Mellitus

(T2DM)
3. History of diabetes in first degree relative

(Family history of diabetes)
4.Lifestyle factors
Diet
Smoking
Alcohol consumption
Prevalence and risk

factors for T2DM
Figure 1.5.1: Conceptual framework model for T2DM (Present study)
10
C. Prevalence and risk factors of autonomic dysfunction among Type 2 diabetic
individuals in rural population of North Karnataka.
i.
Prevalence of Diabetic Neuropathy

Estimates of prevalence of diabetic neuropathy vary widely from 5% to
nearly 60% and every so often 100% if patients with asymptomatic abnormalities
of nerve conduction are included
140
. Booya et al has established neuropathy in
10% of diabetic patients at the time of diagnosis and overall in 50% of patients
with a 25-year history of the disease.141 Prevalence diabetic neuropathy depend on
the criteria used for its estimation and a type of population studied. Dyck et al
conducted population-based study on visceral autonomic neuropathy, based upon
symptoms had prevalence of diabetic neuropathy was 5.5%.142 In contrast, Ziegler
et al reported prevalence of DAN was 16.7% based on Heart rate variability
(HRV) tests. 184
ii.
Role of risk factors for diabetic neuropathy

The European Diabetes Complications Prospective Study (EURODIAB),
involving 1,101 patients with type 1 diabetes and type 2 diabetes followed for a
mean of 7.3 years has concluded smoking, glycosylated haemoglobin (HbA1C),
diabetes duration and components of the metabolic syndrome (including
hypertension, obesity, triglycerides and cholesterol) to be associated with an
increased risk of polyneuropathy.145, 182 Duration of diabetes, age, long term poor
glycemic control, high blood pressure, high triglycerides levels, low HDL, high
HbA1C, peripheral arterial disease, high alcohol intake, increased height,
smoking, obesity, retinopathy, renal failure, low socioeconomic status, co-morbid
11
diseases, missed doses of hypoglycaemic agents > 5 times/month, treatment with
insulin are the various factors studied and proved that raises the likelihood of
neuropathy.146-151 However, the pathogenesis of diabetic neuropathy is not fully
understood and factors affecting it are still remains obscure.
In the present study, estimation of the prevalence and related risk factors
for CAN and DPN among T2DM participants will be estimated based on various
literature and as per standard guideline of American Academy of Neurology,
American Diabetes Association, World Health Organization and recommendation
of Task Force European Society of Cardiology.
Based on above criteria, following conceptual framework model was
prepared for the present study:-
T2DM
Diabetic
complication
Microvascular
Diabetic Neuropathy
Acute
sensory
neuropathy
Nephropathy
Autonomic
neuropathy
Diabetic peripheral
neuropathy
CAN
DNS and
DNE scoring
Autonomic
functions tests
Prevalence of
T2DM
Macrovascular
Retinopathy
HRV
Analysis
Figure 1.5.2: - Conceptual framework model for estimation of prevalence of

Autonomic dysfunction among T2DM participants
12
Independent variables
Dependant variable
2. Personal characteristics
Age (Years)
Sex
Education (literacy status)
Occupation
Location (North
Karnataka, Belgaum)
Cardiovascular Autonomic
Neuropathy (CAN) and
Diabetic Peripheral
Neuropathy (DPN) among
T2DM
3. Anthropometry variables
BMI
Overweight
Obese
Truncal obesity
Central obesity
4. History of diabetes in first degree relative

(Family history of diabetes)
5. Duration of Diabetes (Years)
6. Lifestyle factors
Diet
Smoking
Alcohol consumption
Prevalence and risk

factors for CAN and
DPN
Figure1.5.3: Conceptual framework model for estimation of related

risk factors for CAN and DPN (Present study)
13
Review of literature
2.1 DIABETES MELLITUS
2.1.1 Historical aspects
i. Origin of the term Diabetes mellitus
Diabetes mellitus is derived from the Greek word diabetes meaning siphon
through and the Latin word mellitus meaning honeyed or sweet. It was also
acknowledged in the 17th century as the Pissing evil This sweet taste had been
noticed in urine by the ancient Greeks, Chinese, Egyptians, Indians and Persians as is
evident from their literature. The term "diabetes" or "to pass through" was first used in
230 BC by the Greek Appollonius of Memphis. Thomas Willis further added the
term mellitus or from honey in the late 16th century to distinguish DM from Diabetes
Insipidus which is also associated with frequent urination.28-31
ii. Discovery of Diabetes

The history of diabetes is delineated into different historical periods that
reflect the understanding and management of the disease.29,30 Ancient period was
eyewitness to the first clinical description of diabetes mellitus given by Egyptian
physicians 3,500 years ago who had been ruthless to diagnose and treat diabetes.29, 30
Around 6-7th the century, Sushruta and Charaka, two outstanding Indian physicians
first reported a sweet tasting substance called as madhumeha present in the urine of
patients with polyuria, being sticky to touch and also they observed that urine was
attracted strongly by ants.28 Indian physician given justification of this time appear to
distinguish two forms of diabetes one affecting older, fatter people and the other thin
14
people who did not survive long that put up the resemblance to the current subdivision
of type I and type II diabetes. 31, 32
The first inclusive clinical explanation of diabetes was provided by
the Ancient Greek physician Aretaeus of Cappadocia (1st century CE), who also
noted that excess of urine excretion.33, 34 Diabetes mellitus appears to have been a
deathlike judgment in the ancient era. Hippocrates does not point out anything about
the disease as he felt the disease was untreatable. Aretaeus attempted to treat diabetes
but found poor prognosis and, hence, commented that life with diabetes is short,
disgusting and painful.35 The disease must have been atypical during the time of
the Roman Empire with Galen commenting that he had observed only two cases during
his vocation.
In Medieval Persia, Avicenna (9801037) has given a complete report on
diabetes mellitus in the Canon of Medicine, in which he described about abnormal
appetite and the collapse of sexual functions" and also documented the sweet taste of
diabetic urine. Analogous to Aretaeus and prior to him, Avicenna recognized primary
and secondary diabetes. He also described about diabetic gangrene, and treated
diabetes by using a mixture of lupin, Trigonella (fenugreek) and Zedoary seed, which
produces a considerable reduction in the excretion of sugar, a kind of treatment which
is still prescribed in modern times.29, 35
Avicenna also described about diabetes insipidus very accurately for the first
time, though it was much later that Thomas Willis differentiated it from diabetes
mellitus in a section of his book Pharmaceutice rationalis (1674).29,30 The sweet urine
symptom of diabetes is apparent in the Chinese name for diabetes, Tng nio
bng means "sugar-urine disease".33
15
During 1776, Mathew Dobson established that the sweet taste comes from an
excess of a kind of sugar present in the urine and blood.33Although diabetes has been
recognized since ancient times and treatments of various efficacies have been known
in various regions for much longer, pathogenesis of diabetes has only been understood
experimentally since 1900. The discovery of a role for the pancreas in diabetes is
generally accredited to Joseph Von Mering and Oskar Minkowski. In 1889, they
found that dogs whose pancreas was removed, developed all the signs and symptoms
of diabetes and then died shortly later.35 In the 1910, Sir Edward Albert Shapey
commented that people with diabetes were deficient in a single chemical that was
normally produced by the pancreas. He then proposed calling this substance insulin
which was taken from the Latin word insula which connotes island in reference to the
insulin producing islets of Langerhans in the pancreas.36
The endocrine role of the pancreas in metabolism and indeed the existence of
insulin were further clarified in 1921 when Sir Frederick Grant Banting and Charles
Herbert Best repeated the work of Von Mering and Minkowski and went on to
demonstrate that they could reverse induced diabetes in dogs by giving them an
extract from the pancreatic islets of Langerhans of healthy dogs.36
The islets of Langerhans were discovered in 1869 by an anatomist
named Paul Langerhans. He identified the key cells in the pancreas which produce the
main substance that controls glucose levels in the body.37
Banting, Best and colleagues (especially the chemist Collip) went on to purify
the hormone insulin from bovine pancreases at the University of Toronto. This led to
the ease of use of an effective treatment of insulin injections and the first patients
were treated in 1922. For this work, Banting and laboratory director John MacLeod
16
received the Nobel Prize in Physiology or Medicine in 1923. They both shared their
prize money with others in the team who were not recognized.
Banting and Best made the patent accessible with no charge and did not
endeavour to control commercial production. Mainly, as a result of this decision,
insulin production and therapy quickly spread around the world and Banting was
honored with celebration of World Diabetes Day on his birthday on November 14.
The peculiarity between what is now known as T1DM and T2DM was first clearly
made by Sir Harold Percival Hemsworth and then published in January 1936.36,38
Figure 2.1.1.1: Frederick Banting & Charles Best, 192436
17
Landmark discoveries 34
Development of the long acting insulin NPH in the 1940s by Novo-Nordisk.
Identification of the first of the Sulfonylureas in 1942
Reintroduction of the use of Biguanides for T2DM in the late 1950s. The
initial Phenformin was withdrawn worldwide (in the U.S. in 1977) due to its
potential for sometimes fatal lactic acidosis and Metformin was first marketed in
France in 1979, but not until 1994 in the US.
The determination of the amino acid sequence of insulin (by Sir Frederick Sanger
, for which he received a Nobel Prize in the 1958)
The radioimmunoassay for insulin, as discovered by Rosalyn Yalow and Solomon

Berson (gaining Yalow the 1977 Nobel Prize in Physiology or Medicine)39
The three-dimensional structure of insulin [Dorothy Hodgkin's laboratory in 1969

(PDB file 1ins) ]
Dr Gerald Reavens identification of the constellation of symptoms now

called metabolic syndrome in 1988.
Diabetes Control and Complications Trial Research Group had demonstrated in

1993 that intensive glycemic control in T1DM reduces chronic side effects more
as glucose levels approach 'normal' in a large longitudinal study and also in
T2DM in other large studies.40
Identification of the first Thazolidineone as an effective insulin sensitizer during

the 1990s.
18
iii.
Development of Insulin
In 1980, U.S. biotech company Genentech developed biosynthetic human
insulin. The insulin was isolated from genetically altered bacteria (the bacteria contain
the human gene for synthesizing synthetic human insulin), which produce large
quantities of insulin. The purified insulin was distributed to pharmacies for use by
diabetes patients. Initially, this development was not regarded by the medical
profession as a clinically meaningful development. However, in 1996 the advent of
insulin analogues which had vastly improved absorption, distribution, metabolism,
and excretion characteristics which were clinically meaningful based on this early
biotechnology development.
The U.S. Food & Drug Administration (FDA) approved the first inhaled
insulin Exubera or insulin human (rDNA origin) inhalation in January 2006 for either
T1DM or T2DM. Inhaled insulin is one of the greatest breakthroughs for people who
must take short-acting insulin.41,
42
Oral-Lyn is an oral spray formulation of human
insulin indicated for the treatment of T1DM and T2DM. Generex Biotechnology
started it clinical use of Oral-Lyn among T2DM in Ecuador in 2005.43
19
2.1.2
Classification of Diabetes mellitus

According to World Health Organization, Diabetes mellitus is a heterogeneous
metabolic disorder characterised by common feature of chronic hyperglycemia with

disturbances in carbohydrate, fat and protein metabolism.44
Diabetes mellitus is mainly classified into four broad categories such as Type
1, Type 2, Gestational diabetes and other specific types. The "other specific types" are
a compilation of a few dozen individual causes.45
i. Type 1 diabetes (T1DM)
T1DM is characterized by destruction of the insulin-producing beta cells of the islets
of Langerhans in the pancreas which leads to insulin deficiency. This type can be
further classified as immune-mediated or idiopathic. The majority of T1DM is of the
immune-mediated nature, in which the beta cell loss is due to T-cellmediated autoimmune attack.45 T1DM mainly affects the children or adults so
traditionally termed as "juvenile diabetes" because a majority of these diabetes cases
were in children. The term "T1DM has replaced several former terms such as
childhood-onset diabetes, juvenile diabetes and insulin-dependent diabetes mellitus
(IDDM).
ii. Type 2 diabetes (T2DM)
T2DM is characterized by insulin resistance, which may be collective with
relatively reduced insulin secretion. The defective responsiveness of body tissues to
insulin is believed to involve the insulin receptor. However, specific defects are not
known. T2DM is the most common type. In the early stage of T2DM, the
predominant abnormality is reduced insulin sensitivity and causes hyperglycemia. The
term T2DM has replaced several former terms which include adult-onset diabetes,
obesity related diabetes and noninsulin-dependent diabetes mellitus (NIDDM). 44, 45
20
iii. Gestational diabetes
Gestational diabetes mellitus (GDM) resembles T2DM in several respects and
involves a combination of moderately inadequate insulin secretion and sensitivity. It
occurs in about 25% of all pregnancies and may progress or disappear after delivery.
Gestational diabetes is completely treatable but requires careful medical supervision
throughout the pregnancy. About 2050% of affected women develop T2DM later on
in life.46, 47, 48, 49
2.1.3
Epidemiology
i. Global prevalence
Diabetes is among one of the most common non-communicable diseases
(NCDs). It is the fourth leading cause of death in most developing countries and
according to extensive evidence; it is widespread in many economically developing
and newly industrialized countries.
Diabetes is undoubtedly one of the most challenging health problems of the
21st century. Diabetes mellitus has emerged as a major health care problem
worldwide. According to latest report of International Diabetes Federation report the
low and middle income countries face the greatest burden of diabetes.50, 51
According to International Diabetes Federation (IDF) Diabetes Atlas report,
nearly 382 million people worldwide or average of 8.3% of adults are projected to
have diabetes. Approximately 80% live in low and middle-income countries. If these
trends continue, then by 2035, around 592 million people, or one in every 10 adults,
will have diabetes. This equates to roughly three new cases every 10 seconds or
almost 10 million per year. The major increases will take place in the regions where
increasing economies are predominant.50
21
Figure 2.1.3.1: Number of people with diabetes by IDF Region, 201350
Figure 2.1.3.2:- Top 10 countries showing number of people with diabetes (age,
20-79 years), 201350
22
ii. Global urban-rural distributions of diabetes
There are numerous people with diabetes living in urban (246 million) than in
rural (136 million) areas although the figures for rural areas are on the amplify. In low
and middle income countries, the number of people with diabetes in urban areas is
181 million, whereas 122 million live in rural areas. By 2035, the difference is
expected to widen as 347 million people living in urban areas and 145 million in rural
areas.50
iv. India - Diabetes capital of the world
Diabetes is growing alarmingly in India, being home to more than 65.1 million
people with the disease in 2013, compared to 50.8 million in 2010. Ominously,
obesity is reaching epidemic proportions among India's middle-class children and
adolescents, as young people choose Western fast food over traditional cuisine.
Doctors in India are fitting gastric bands on children as young as 13 years. India with
65.1 million diabetic patients is just next to China (98.4 million) in the race to become
the diabetes capital of the world, according to the International Diabetes Federation
(IDF) Atlas, 2013.50,51
v. Urban-Rural distribution of diabetes in India
An urban rural variation in the prevalence of diabetes has been consistently
reported from India. Earlier study conducted by ICMR reported that the prevalence
was 2.1 per cent in urban and 1.5 per cent in rural areas. The prevalence of diabetes in
India study (PODIS) was carried out in 108 centres (49 urban and 59 rural) to look at
the urban-rural differences in the prevalence of T2DM and glucose intolerance
showed 4.7 per cent in the urban and 2.0 percent in the rural population, according to
ADA criteria while according to the WHO criteria prevalence was 5.6 and 2.7 percent
among urban and rural areas respectively.51, 52, 53
23
Table 2.1.3.1:- Prevalence of diabetes in urban cities of India 54
Place
Year
Authors
Region
Prevalence
(%)
Thriuvananthapuram
1999
Raman et al. 1999
South
16.3
Hyderabad
2001
Ramachandran et al. 2001
South
16.6
Bengaluru
2001
South
12.4
Chennai
2006
Mohan et al. 2006
South
14.3
Ernakulam
2006
Menon et al. 2006
South
19.5
Vellore
2007
Raghupathy et al. 2007
South
3.7
Tamil Nadu
2008
South
18.6
Multi-centric
2008
Mohan et al. 2008 (WHO-
Multi-
7.1
ICMR)
centric
vi. Undiagnosed diabetes cases worldwide:

IDF estimates that as many as 175 million people worldwide or close to half of
all people with diabetes are unaware of their disease. Most of these cases are T2DM.
Early diagnosis of the diabetes will lead to better chances of preventing harmful and
costly complications. Hence there is need to provide appropriate tools for early
diagnosis.53, 54,56,57
24
Table 2.1.3.2:-Prevalence of diabetes in rural India 54
Place
Year
Prevalence (%)
References
Delhi
1991
1.5
Ahuja 1991
Punjab
1994
4.6
Wander et al. 1994
Srinagar
2000
4.0
Zargar et al. 2000
India
2001
2.7
Sadikot et al. 2004
Rajasthan
2004
1.8
Aggarwal et al. 2004
Mysore
2005
3.8
Basavanagowdappa et al. 2005
Maharashtra
2006
9.3
Deo et al. 2006
Nagpur
2007
3.7
Kokiwar et al.2007
Vellore
2007
2.1
Raghupathy et al. 2007
TamilNadu
2008
9.1
Multi-centric
2008
3.1
Mohan et al. 2008
25
2.1.4
Pathophysiology
T2DM is the most widespread type of diabetes. It typically occurs in adults,
but is also increasingly seen in children and adolescents. In T2DM, the body is able to
produce insulin but either this is not sufficient or the body is unable to respond to its
effects (also known as insulin resistance), which then leads to an upsurge of glucose
in the blood. Many people with T2DM remain unaware of their poor health for a long
time since symptoms may take years to appear, during which time the body is being
damaged by excess blood glucose. They are often diagnosed only when complications
of diabetes have already developed.58,
59
Although the risk factors responsible for
developing T2DM are not so clear, various studies have identified several important
risk factors which include the following:
Genetics
Obesity
Poor diet
Physical inactivity
Advancing age
Family history of diabetes
Ethnicity
High blood glucose during pregnancy
26
i. Normal glucose homeostasis:
Glucose metabolism is critical to normal physiological functioning. Glucose
acts both as a source of energy and as a source of starting material for almost all types
of biosynthetic reactions which is necessary for regular and normal functioning of
body.
Figure 2.1.4.1: Normal glucose homeostasis 58
ii.
Physiology of Insulin Secretion

Insulin is a hormone that is produced in pancreas. Insulin allows glucose to
enter the bodys cells, where it is converted into energy. In Canada in 1921, scientist
27
Frederick Banting and medical student Charles Best isolated a substance from the
pancreas of dogs, which they named isletin and which is now recognized as insulin.58
Figure 2.1.4.2:- Physiology of insulin secretion and
action58
iii. Insulin transduction (signaling) pathway

The insulin transduction pathway is an important biochemical pathway
beginning at the cellular level affecting homeostasis. This pathway is also influenced
by fed versus fasting states, stress levels and a variety of other hormones. When
carbohydrates are consumed, digested and absorbed, the pancreas senses the
subsequent rise in blood glucose concentration and releases insulin to promote an
uptake of glucose from the blood stream. When insulin binds on the cellular insulin
receptor, it leads to a cascade of cellular processes that promote the usage or, in some
cases, the storage of glucose in the cell. The effects of insulin vary depending on the
28
tissue involved, e.g., insulin is most important in the uptake of glucose by muscle and
adipose tissue.
This insulin signal transduction pathway is composed of trigger mechanisms
(autophosphorylation mechanisms) that serve as signals throughout the cell.
The insulin receptor is a tyrosine kinase that undergoes autophosphorylation and
catalyses the phosphorylation of cellular protein such as member of the IRS family,
Shc and CbI. Upon tyrosine phosphorylation, these proteins interact with signaling
molecules through their SH2 domains, resulting in diverse series of signaling
pathways including activation of PI(3)K and downstream PtdIns (3,4,5) P3-dependant
protein kinase, ras and MAP kinase cascade and CbI/CAP and the activation of
TC10. These pathways act in the concerted fashion to coordinate the regulation of
vesicle trafficking, protein synthesis, enzyme activation and inactivation and gene
expression, which results in the regulation of glucose, lipid and protein metabolism.75
Figure 2.1.4.3:- Insulin signal transduction75
29
iv. Pathogenesis58
The pathophysiology of T2DM is characterized by peripheral insulin
resistance, impaired regulation of hepatic glucose production and declining -cell
function, eventually leading to - cell failure. The primary events are believed to be
an initial deficit in insulin secretion and subsequently relative insulin deficiency in
association with peripheral insulin resistance.58
v. Etiology:T2DM is caused by a combination of genetic factors related to impaired
insulin secretion and insulin resistance and environmental factors such as obesity,
overeating, lack of exercise, and stress, as well as aging. It is typically a multifactorial
disease involving multiple genes and environmental factors to unstable extents.
Figure 2.1.4.4: Etiology and pathophysiology of type 2 diabetes58
30
vi. Genetic factors involved in the pathogenesis of diabetes
The development of T2DM is undoubtedly associated with a family history of
diabetes. The significantly higher concordance rate between monozygotic twins than
between dizygotic twins suggests the substantial involvement of genetic factors. The
pathogenesis has been assumed to involve genetic abnormality in the molecules
related to the regulatory system of glucose metabolism. The analyses of candidate
genes targeted at glucose-stimulated insulin secretion of pancreatic cells and the
molecules comprising the molecular mechanism for insulin action have identified
genetic abnormalities that can be independent causes of pathogenesis which includes
those in glucokinase genes, mitochondrial genes and insulin receptor genes. Recently,
a genome wide association study (GWAS) has identified the mutation in the KCNQ1
gene linked to insulin secretion abnormality as an important disease susceptible gene
associated with the pathogenesis of diabetes in Asian ethnic groups.58, 59, 60
vii. Role of environmental factors
Several studies reported that aging, obesity, insufficient energy consumption,
alcohol consumption and smoking, etc. are independent risk factors of pathogenesis.
Obesity include predominantly visceral fat obesity due to a lack of exercise is
accompanied by a decrease in muscle mass, induces insulin resistance, and is closely
associated with the rapid increase in the number of middle and towering aged patients.
55, 56, 61
viii. Impaired insulin secretion

Several researches reported that impaired insulin secretion is generally
progressive, and its progression involves glucose toxicity and lipotoxicity and if it is
untreated it causes a decrease in pancreatic cell mass. The progression of the
impairment of pancreatic cell function greatly affects the long term control of blood
31
glucose whereas in early stages after disease onset mostly show an increase in
postprandial blood glucose as a result of increased insulin resistance and decreased
early phase secretion. The sequence of the deterioration of pancreatic cell function
subsequently causes permanent elevation of blood glucose.61
Figure 2.1.4.5: Pathophysiological progression of type 2 diabetes as seen from

pancreatic cell function 58
Figure 2.1.4.6:-Pathophysiological factors responsible for

cell failure and impaired insulin secretion 75
32
ix. Insulin resistance:Insulin resistance is a condition in which insulin in the body does not exert
sufficient action proportional to its blood concentration. The impairment of insulin
action in major target organs such as liver and muscles is a common
pathophysiological feature of T2DM. Insulin resistance develops and expands prior to
disease onset. The investigation into the molecular mechanism for insulin action has
clarified how insulin resistance is related to genetic factors and environmental factors
(hyperglycemia, free fatty acids, inflammatory mechanism, etc. Known genetic
factors, include not only insulin receptor and insulin receptor substrate (IRS)-1 gene
polymorphisms that directly affect insulin signals but also polymorphisms of thrifty
genes such as the 3 adrenergic receptor gene and the uncoupling protein (UCP) gene,
associated with visceral obesity and promote insulin resistance. Glucolipotoxicity and
inflammatory mediators are also important as the mechanisms for impaired insulin
secretion and insulin signaling impairment.
Recent attention has focused on the involvement of adipocyte-derived
bioactive substances (adipokines) in insulin resistance. While TNF- , leptin, resistin,
and free fatty acids act to increase resistance, adiponectin improves resistance.
Clinical tests to assess the extent of insulin resistance include homeostasis model
assessment for insulin resistance (HOMA-IR), insulin sensitivity test (loading test),
steady state plasma glucose (SSPG), minimal model analysis and insulin clamp
technique.58, 59, 60, 61
33
Figure 2.1.4.7: Insulin transduction system75
The Matsuda index is now gaining appreciation as a relatively simple

procedure that can concurrently evaluate insulin resistance in the liver and muscles.
After performing OGTT, this index is calculated by the formula: Matsuda Index
=10,000/ (FPG x FPI x (mean PG x mean PI), where FPG is fasting plasma glucose
and FPI is fasting plasma insulin. A more convenient way to estimate the degree of
resistance is to check for the presence of high fasting blood insulin, visceral obesity,
and hypertriglyceridemia etc.58
x. Burden of mortality worldwide
Approximately 5.1 million people aged between 20 and 79 years died from
diabetes in 2013, accounting for 8.4% of worldwide all-cause mortality among people
in this age group. This estimated number of deaths is similar in magnitude to the
combined deaths from several infectious diseases that are major public health
priorities and is correspondent to one death every six seconds. Close to half (48%) of
34
deaths due to diabetes are in people under the age of 60. The highest number of deaths
due to diabetes occurred in countries with the largest numbers of people with the
disease: China, India, USA and the Russian Federation.50
xi. Global financial burden of diabetes
Global health spending to treat diabetes and manage complications totaled at
least USD 548 billion in 2013. By 2035, this number is projected to exceed USD 627
billion. Expressed in International Dollars (ID), which correct for differences in
purchasing power, global health spending on diabetes was estimated to be at least ID
581 billion in 2013 and ID 678 billion in 2035. An estimated average of USD 1,437
(ID 1,522) per person with diabetes was spent globally on treating and managing the
disease in 2013.50, 57, 58
2.1.5 Diagnosis of diabetes:The clinical diagnosis of diabetes is often prompted by symptoms such as increased
thirst and urine volume, recurrent infections, unexplained weight loss and in severe
cases, drowsiness and coma; high levels of glycosuria are usually present. Single
blood glucose can establish the diagnosis in diabetes.45
The World Health Organization classified the diagnostic criteria for both type 1 and
type 2 as given below:
Table 2.1.5.1: Diagnostic criteria for diabetes 45
Condition
Normal
Impaired fasting glucose
(IFG)
Impaired glucose
tolerance (IGT)
Diabetes mellitus
Fasting Blood
glucose
<6.1mmol/l (<110
mg/dl)
6.1to 6.9 mmol/l
(110 to 126mg/dl)
<7.0
mmol/l(<126mg/dl)
7.0mmol/l
(126 mg/dl)
35
Postprandial
blood glucose
<7.8mmol/l
(<140mg/dl)
<7.8
(<140mg/dl)
7.8
(140mg/dl)
11.1
(200mg/dl)
HbA1c (%)
<6.0
6.06.4
6.06.4
6.5
2.1.6 Treatment for diabetes
Early initiation of intervention is also important for curbing the progression of
pathophysiological conditions. Early efforts to remove the effect of glucose toxicity as
much as possible and to preserve pancreatic cell function are vital prerequisites for
long term management of diabetes. Microvascular disease is more closely associated
with long term blood glucose control. The treatment paradigm desires to be
considered from the viewpoints of not only controlling vascular complications but
also preventing the progression of pathophysiological conditions. Ideally, the primary
aim should be to prevent the onset of diabetes among individuals with IGT (primary
prevention). In addition to proactive intervention for lifestyle improvement, we need
to accelerate the deliberate about whether to use pharmacological intervention.58, 76
Figure 2.1.6.1: The management paradigm for type 2 diabetes: prevention

of onset and proactive management of early-stage diabetes58
36
i. Treatment policy
The goal of diabetes treatment is to secure a quality of life (QOL) and lifespan
and a prerequisite for attaining this goal is the prevention of onset and progression of
vascular complications. The risk of disease such as microvascular and macrovascular
complication is increased in diabetic individuals with marginal blood glucose levels
which in turns underscoring the need for early intervention.
Reports on the interventions to prevent the onset of diabetes, control its
complications and to improve the prognosis have been demonstrated in the following
facts:
1. Lifestyle improvement and anti-diabetic drugs (-Glucosidase inhibitor,
Metformin, Thiazolidine) to treat IGT and to suppress the risk of developing
T2DM.62-65
2. Sulfonylurea drugs, Metformin and insulin are effective in controlling both
microvascular disease and macrovascular disease and doing earlier
intervention is essential to the control of macrovascular disease.66-69
3. Comprehensive intervention including blood pressure and lipid management is
extremely effective in controlling vascular complications and reducing
mortality rate.70, 71
4. Pioglitazone suppresses the recurrence of cardiovascular disorders.72 Earlier
and more comprehensive (including blood glucose, blood pressure and lipid)
intervention is more effective in controlling vascular complications and
improving prognosis.
37
Figure 2.1.6.2: Flow-chart presenting the treatment of diabetes58
38
2.1.7 Management
Management of T2DM mainly focuses on lifestyle interventions, lowering
other cardiovascular risk factors and maintaining blood glucose levels in the
normal range Self-monitoring of blood glucose for people with newly diagnosed
T2DM was recommended by the British National Health Service in 2008 and it is
considered as one of the important tool for management of diabetes, however the
benefit of self-monitoring in those not using multi dose insulin is
questionable.76,78 Several studies reported that by managing other cardiovascular
risk factors such as hypertension, high cholesterol and microalbuminuria
improves a person's life expectancy.76 - 81
i. Lifestyle
A proper diet and exercise are the fundamentals of diabetic care with a greater
amount of exercise acquiescent better results. Aerobic exercise leads to a decrease
in HbA1c and improved insulin sensitivity. Resistance training is also useful and
the combination of both types of exercise may be most effective.81 several study
reported that having diabetic diet that promotes weight loss which may be useful
to reduce the insulin resistance.82 Carey et al commented that having low
glycemic index diet has been found to improve blood sugar control.82
ii. Pharmacological therapy
Current therapeutic agents available for T2DM include sulfonylureas and
related compounds, biguanides, thiazolidenediones, -glucosidase inhibitors and
insulin. A rational approach would be to begin with the agents particularly suited
to the stage and nature of the disease with its progression and if necessary to
combination therapy. These Pharmacological agents act through different
39
mechanisms of action and should be chosen to improve glucose values to
minimize the adverse effects.
iii. Sulfonylureas and related agents
Sulfonylureas have been used to treat T2DM since 1942.83,
84
Initially
Sulfonylureas such as biguanides, phenformin were withdrawals from the U.S.

market in 1975 and then second generation biguanides, Metformin was introduced
and widely distributed throughout Western Europe, Canada and Mexico.
By using these drugs lowering of glucose occurs primarily by decreasing
hepatic glucose production and to the lesser extent by decreasing peripheral
insulin resistance. The drug acts by causing the translocation of glucose
transporters from the microsomal fraction to the plasma membrane of hepatic and
muscle cells. It does not stimulate insulin release and does not cause
hypoglycemia.85 Furthermore, it does not cause any weight gain and it also
improves the lipid profile by causing a decline in total and very low density
lipoprotein triglyceride, total cholesterol and very low density cholesterol levels
and an increase in high density lipoprotein cholesterol levels. 86-91
iv. Thiazolidenediones
Thiazolidenediones appears to act by binding to the peroxisome proliferators
activator receptor-.92, 93, 94 This nuclear receptor influences the differentiation of
fibroblasts into adipocyte and lowers free fatty acid levels.93 Clinically, its major
effect is to decrease peripheral insulin resistance, although at higher doses it may
also decrease hepatic glucose production.92, 93 Although it act at a different site
than metformin both Troglitazone and Metformin appear to function as insulin
sensitizers and require the presence of insulin for their effects.94-97
40
v. -Glucosidase inhibitors
Members of this class act by slowing the absorption of carbohydrates from
the intestines and thereby minimize the postprandial rise in blood glucose.98
Gastrointestinal side effects require gradual dosage increments over weeks to
months after therapy is initiated. Serious adverse reactions are rare and weight
gain may be minimized with this therapy.98
vi. Insulin Treatment
Insulin therapy is indicated in the treatment of T2DM for initial therapy of
severe hyperglycemia after failure of oral agents.
multiple combinations in T2DM.99,
100
Insulin has been used in
The first available insulin analogue is
Lispro insulin, representing a two-amino acid modification of regular human

insulin. Lispro insulin does not form aggregates when injected, allowing it to have
a more rapid onset and a shorter duration of action than regular insulin.101
Table 2.1.7.1:- Oral agents used in the management of type 2 diabetes mellitus58
Class
Mechanism of action
Indication(n)
Sulfonylureas and
Repaglinide
Biguanides
Increase insulin secretion
Insulinopenia
Thiazolidenediones
a-Glucosidase
inhibitors
Decrease hepatic
gluconeogenesis
Decrease peripheral insulin
resistance
Decrease peripheral insulin
resistance
Reduce fatty acids
Slow absorption of
carbohydrates
41
Obesity 1 insulin
resistance
Insulin resistance
Postprandial
hyperglycemia
2.1.8 Diabetic complication 102-119
Wider health problems accelerate the lethal effects of diabetes with
combined effect of smoking, elevated cholesterol levels, obesity, high blood
pressure and lack of regular exercise.102 Various types diabetic complication is
given below:
i. Diabetic ketoacidosis
Diabetic Ketoacidosis (DKA) is an acute and treacherous complication. A
low insulin level causes liver to turn fatty acid to ketone for fuel (i.e., ketosis)
however; ketone bodies are intermediate substrates in that metabolic sequence.
This is normal when periodic, but can become a serious problem if sustained.
Elevated levels of ketone bodies in the blood in turn decrease the blood's pH,
leading to DKA. Ketoacidosis is much more common in T1DM and T2DM.103
ii.
Hyperglycemia hyperosmolar state (Hyperosmolar coma)

Hyperglycemia Hyperosmolar State (HHS) is an acute complication
giving out many symptoms with DKA, but having an entirely diverse origin and
having different treatment. In a person with very high (usually considered to be
above 300 mg/dl (16 mmol/L)) blood glucose levels, water is osmotically drawn
out of cells into the blood and the kidneys ultimately begin to unload glucose into
the urine. These will results in loss of water and an increase in blood osmolarity.
If fluid is not replaced by mouth or intravenously the osmotic effect of high
glucose levels, combined with the loss of water, will eventually lead
to dehydration. The body's cells become progressively dehydrated as water is
taken from them and excreted. Lethargy may ultimately progress to a coma,
though this is more common in T2DM than T1DM.102, 103
42
iii. Hypoglycemia
Hypoglycemia or abnormally low blood glucose is an acute complication
of diabetes and its several treatments.
Hypoglycemia patient may become
agitated, sweaty, weak, and have many symptoms of sympathetic activation of

the autonomic nervous system which results in feelings akin to dread and
immobilized panic. Consciousness can be altered or even lost in extreme cases,
leading to coma, seizures, or even brain damage and death.
iv. Diabetic coma
Diabetic coma is a medical emergency in which a person with diabetes
mellitus is comatose (unconscious) because of one of the acute complications of
diabetes such as severe diabetic hypoglycemia, DKA and Hyperosmolar
nonketotic coma102. In patients with diabetes, this may be caused due to several
factors, such as taking too much or incorrectly timed insulin, doing too much or
incorrectly timed exercise (exercise decreases insulin requirements) or not having
enough food (specifically glucose containing carbohydrates)
v. Respiratory infections
The immune response is impaired in individuals with diabetes mellitus.
Cellular studies have revealed that hyperglycemia both reduces the function of
immune cells and increases inflammation. The vascular effects of diabetes also
tend to alter lung function, all of which leads to an increase in susceptibility to
respiratory infections such as pneumonia and influenza among individuals with
diabetes.104
43
vi. Periodontal disease
Diabetes is also associated with periodontal disease (gum disease)105
and it may make diabetes more difficult to treat.106 Gum disease is frequently
related to bacterial infection by organisms such as Porphyromonas gingivitis
and actinobacillus.107 A number of trials have found improved blood sugar
levels in type 2 diabetics who have undergone periodontal treatment.107
vii. Chronic complication108
Chronic elevation of blood glucose level leads to damage of blood
vessels (angiopathy). The endothelial cells lining the blood vessels take in
more glucose than normal, since they do not depend on insulin. They then
form more surface glycoproteins than normal and cause the basement
membrane to grow thicker and weaker. In diabetes, the resulting problems are
grouped under "microvascular disease" (due to damage to small blood vessels)
and "macrovascular disease" (due to damage to the arteries).108
Microvascular: - The damage to small blood vessels leads to
a microangiopathy, which can cause one or more of the following:
Diabetic cardiomyopathy: - damage to the heart, leading to diastolic

dysfunction and eventually heart failure119.
Diabetic nephropathy:- damage to the kidney which can lead to

chronic renal failure, eventually requiring dialysis.116
Diabetic neuropathy:- abnormal and decreased sensation, usually in

a 'glove and stocking' distribution starting with the feet but potentially
in other nerves, later often fingers and hands. When combined with
damaged blood vessels this can lead to diabetic foot. Other forms of
44
diabetic neuropathy may present as mononeuritis or autonomic
neuropathy. Diabetic amyotrophy
is muscle weakness due to
neuropathy,108, 109, 111, 116
Diabetic retinopathy: - abnormal growth of friable and poor-quality

new blood vessels in the retina as well as macular edema (swelling of
the macula), which can lead to brutal vision loss or blindness. Retinal
damage (from microangiopathy) makes it the most widespread cause
of blindness among non-elderly adults in the US.113, 115
Macrovascular disease leads to cardiovascular disease to which accelerated

atherosclerosis is a contributor of:
Coronary artery diseases (CAD)- myocardial infarctions
Diabetic myonecrosis ('muscle wasting')
Peripheral vascular disease which contributes to intermittent

claudication (exertion-related leg and foot pain) Stroke (mainly the
ischemic type)
45
Figure 2.1.8.1: Diabetic complications108
46
2.2 Diabetes and Autonomic Dysfunction
The Autonomic Nervous System (ANS) is an extensive neural network whose

main role is to regulate the milieu intrieur by controlling homeostasis and visceral
functions. ANS has two main divisions (parasympathetic and sympathetic) due to
which it work in balanced form to control the heart rate, force of cardiac contraction,
dilatation and constriction of blood vessels, contraction and relaxation of smooth
muscle in the digestive and urogenital systems, secretions of glands and pupillary
size.120, 128 The ANS is mostly efferent, mainly transmitting impulses from the central
nervous system to peripheral organs. However, it also has an afferent component.
The ANS plays a key role in the modulation of the CVS dynamics by means
of an interaction between the sympathetic and vagal tone which in turn act in negative
feedback manner in physiologic conditions. The metabolic disorders of diabetes lead
to diffuse and widespread damage of peripheral nerves and small vessels. Diabetes
can cause dysfunction of any or every part of the autonomic nervous system which
may leads to wide range of disorders. The most worrying and dangerous conditions
associated to diabetic neuropathy are cardiovascular autonomic neuropathy, diabetic
peripheral neuropathy, silent myocardial infarction (MI), cardiac arrhythmias,
ulceration, gangrene, retinopathy and nephropathy.
47
Figure 2.2.1: Physiology of autonomic nervous system125, 126, 127, 128
48
2.3 : Diabetic Neuropathy
Diabetes mellitus is most commonly associated with autonomic dysfunction
among the people with diabetes. Prolonged chronic hyperglycaemia can leads to
degradation of the microvasculature which intern leads to a specific form of
autonomic dysfunction termed as Tripathy which includes Diabetic neuropathy,
retinopathy and nephropathy. Several studies showed that sympathetic and
parasympathetic innervations in the heart play a major role in the regulation of cardiac
function. Barkley first suggested the existence of sensory nerve endings in the heart in
1894.121 Wollard122 conducted study in the 1926 concluded that a large portion of
cardiac sensory endings were of vagal origin.
Diabetic Neuropathies are a heterogeneous disorder by their symptoms,
pattern of neurologic involvement, course, risk factors, pathologic alterations and
underlying mechanisms that classifies diabetic neuropathies into the mononeuropathy,
polyneuropathies, plexopathies and radiculopathies. It also ranges from subclinical
functional impairment of cardiovascular reflexes and sudomotor functions to severe
cardiovascular, gastrointestinal, or genitourinary dysfunction. Diabetic Autonomic
Neuropathy (DAN) and Diabetic Peripheral Neuropathy (DPN) are the most common
clinical complications of diabetes. Orthostatic hypotension, resting tachycardia and
heart rate unresponsiveness to respiration are the hallmarks of diabetic autonomic
neuropathy. Diabetic peripheral neuropathy greatly affects all areas of a patients life,
including mood, sleep, self-worth, independence, ability to work and interpersonal
relationships.128,129
2.3.1: Diabetic Autonomic Neuropathy
Diabetic Autonomic Neuropathy (DAN) is a complication of diabetes which
will be developing slowly over the years and indistinctly robbing diabetic patients of
49
their ability to sense when they are getting hypoglycemic or having a heart attack.
DAN is among the least recognized and understood complication of diabetes, despite
of its significant negative impact on survival and quality of life in people with
diabetes.130,
131
It impairs the capability to conduct activities of daily living and
increases the jeopardy of death. It also accounts for a large portion of the cost of
care.132
DAN can engross the entire ANS, which involve vasomotor, visceromotor and
sensory fibres that innervates every organ. It may be either clinically apparent or
subclinical. It is manifested by dysfunction of one or more organ systems (e.g.,
cardiovascular, gastrointestinal [GI], genitourinary, sudomotor and ocular).130 Many
organs are dually innervated and receiving fibres from the parasympathetic and
sympathetic division of the ANS. It manifest firstly the longer nerve and certainly,
vagus nerve is the longest of nerves of the ANS which accounts for more than 75% of
all parasympathetic activity due to this early effect of DAN are widespread.131
Clinical symptoms of DAN usually do not occur until extensive after the onset
of diabetes. Whereas symptoms suggestive of autonomic dysfunction may be
common they may recurrently be due to other causes rather than to true autonomic
neuropathy. Several studies showed that subclinical autonomic dysfunction can occur
within a year of diagnosis in T2DM patients and within two years in T1DM
patients.133
In clinical practice, this modulation of diabetic neuropathy is usually assessed
by the well-recognized methods such as heart rate variability (HRV) and
cardiovascular reflex test, which means an analysis of spontaneous and induced
fluctuations that occur in HR (electrocardiographic RR interval) as a result of ANS
sympathetic and parasympathetic activities on sinus node automaticity.129, 134
50
Advances in technology built on decades of research and clinical testing due to
this, now it is possible that identify early stages of various diabetic neuropathies with
the use of careful measurement of autonomic function and then to provide therapeutic
choices that are based on symptom control which might abrogate the underlying
disorder.
2.3.2 Classification of Diabetic neuropathy
The simplest definition with regard to what constitutes diabetic neuropathy for
day to day clinical practice. approved at international consensus meeting for the
outpatient management of neuropathy is "the presence of symptoms and/ or signs of
peripheral nerve dysfunction in people with diabetes after exclusion of other
causes".135,
136, 137
The exclusion of other causes is particularly important because
several reports, especially the baseline data from the ongoing Rochester Diabetic
Neuropathy Study, have shown that nondiabetic neurologic diseases may easily be
overlooked and misclassified in the all-encompassing term diabetic neuropathy.137
A more detailed definition of diabetic neuropathy had previously been agreed
on at the 1988 San Antonio Consensus Conference: "a demonstrable disorder, either
clinically evident or subclinical, that occurs in the setting of diabetes mellitus without
other causes for peripheral neuropathy. It includes manifestations in the somatic and
autonomic parts of the peripheral nervous system." Because the precise
etiopathogenesis of neuropathy remains mysterious and classification of neuropathy
based on pathogenetic grounds is not possible, due to these the diverse manifestations
are generally classified according to clinical presentation.136, 137
A number of classifications exist which include purely clinical and descriptive
classification which was originally proposed by Boulton and Ward136,
137
(Table
2.2.4.1) and based on potential reversibility together with clinical description
51
proposed by Thomas138 (Table 2.2.4.2). Watkins and Edmonds have recently
suggested a classification for diabetic neuropathies that separate them into three
groups139 (Table 2.2.4.3):
1. Progressive neuropathies are associated with increasing duration of
diabetes
and
with
other
diabetic
complications.
These
are
predominantly sensory; however autonomic involvement is also

common. The onset is gradual and there is no recovery.
2. Reversible neuropathies have rapid onset, often occurring at the
presentation of diabetes itself. They are not related to diabetes duration
or other diabetic complications. There is usually complete recovery.
3. Pressure palsies occur more frequently in the diabetic state but are not
specific to diabetes. There is no association with duration of diabetes
or other complications.
Table 2.3.2.1: Clinical Classification of Diabetes Neuropathies by Bolton
and Ward
1) Chronic, progressive distal symmetric polyneuropathy
A. Mixed sensorimotor
B. Predominantly sensory
C. Predominantly autonomic
2) Acute axonal polyneuropathy
A. acute, painful, weight loss, poor control ( cachexic)
B. acute, painful, weight good control (insulin neuritis)
3) Proximal motor neuropathy ( amyotrophy)
4) Mononeuropathies
A. Mononeuropathy multiplex
B. Cranial
C. Truncal
5) Entrapment neuropathies
52
Table 2.3.2.2: Clinical Classification of Diabetic Neuropathy by Thomas
1) Rapidly Reversible
A. Hyperglycemic neuropathy
2) Persistent Symmetric Polyneuropathies
A. Distal somatic sensorimotor ( mainly larger fiber)
B. Autonomic
C.Small fiber
3) Focal/Multifocal Neuropathies
A. Cranial
B. Thoracoabdominal radiculopathies
C. Focal limb
D. Amyotrophy
E. Compression/entrapment
4) Mixed forms
Table 2.3.2.3: - Clinical classification of Diabetic Neuropathies by Watkins and

Edmonds
1) Progressive neuropathies
A. Chronic sensory-motor neuropathy
a. Distal symmetrical
b. Predominantly sensory
c. Autonomic involvement
d. Gradual onset
e. Progressive with increasing duration of diabetes
f. Related to glycemic control
g. Related to other microvascular complication
2) Reversible neuropathies
A. Mononeuropathies
a. Proximal motor neuropathy ( amyotrophy)
b. Cranial nerve palsies ( III, IV, VI)
c. Truncal radiculopathies
B. Acute painful neuropathies
a. Associated with poor glycaemic control
b. Associated with rapid glycaemic control
c. Sudden onset
d. Spontaneous recovery
e. Not related to other microvascular complication
f. Not related to duration of diabetes
3) Pressure palsies
A. Carpel tunnel syndrome
53
Table 2.3.2.4: Classification of Diabetic Neuropathy140
Generalised
Focal and
Multifocal
Typical : Distal
Symmetric
Polyneuropathy
Compression
Acute
sensory
Chronic
sensorimotor
Small fibre
neuropathies
Large fibre
neuropathies
Median:Carpal
Tunnel
Ulnar: Cubital
tunnel
Fibular: Fibular
head
Latral femoral
Cutaneous
inguinal
ligament
Atypical
Ischemic
Insulin
neuritis/
treatment
neuropathy
Chronic
inflammattory
demyelinating
polyneuropath
y (CIDP)
Mononeuritis
Diabetic
amyotrophy
Cranial Nerver
palsy (3.6.7)
Focal limb
neuropathies
Cervical
radiculoplexus
Neuropathies
Thoracolumbar
Radiculoneulrop
athy
Lumbosacral
Radiculoplexus
Neuropathies
Autonomic
neuropathy
Cardiovascular
Gastrointestinal
Genitourinary
Hypoglycemic
Unawareness and
associated
autonomic
failure
Sudomotor
54
Miscellaneous
Mixed
Polyneuropathy
after
ketoacidosis
Polyneuropathy
with glucose
impairment
Neuropathy
with weight loss
Diabetic
Cachexia
2.3.3
Epidemiology of Diabetic neuropathy
i. Prevalence
Estimates of prevalence of DN vary widely from 5% to nearly 60% and every
so often 100% if patients with asymptomatic abnormalities of nerve conduction are
included. A case control study conducted by Booya et al has establish neuropathy in
10% of diabetic patients at the time of diagnosis and overall in 50% of patients with a
25-year history of the disease. 141
Prevalence diabetic neuropathy depend on the criteria used for its estimation
and a type of population studied. Dyck et al conducted population-based study on
visceral autonomic neuropathy which was based upon symptoms had prevalence of
5.5% .142 In contrast, another community-based study conducted by Ziegler reported
prevalence of DAN, as defined by abnormal heart rate variability (HRV) tests, of
16.7%.143, 144,145
ii.
Role of risk factors

The European Diabetes Complications Prospective Study (EURODIAB),
included 1,101 patients with type 1 diabetes with mean of 7.3 years of duration of
diabetes concluded that smoking, glycosylated haemoglobin (HbA1C), diabetes
duration and components of the metabolic syndrome (including hypertension, obesity,
triglycerides and cholesterol) were associated with an increased risk of
polyneuropathy. Various recent studies reported that, duration of diabetes, age, long
term poor glycemic control, high blood pressure, high triglycerides levels, low HDL,
high HbA1C, peripheral arterial disease, high alcohol intake, increased height,
smoking, obesity, retinopathy, renal failure, low socioeconomic status, co-morbid
diseases, missed doses of Hypoglycaemic agents > 5 times/month, treatment with
55
insulin are the various factors studied and proved that raises the likelihood of
neuropathy146-151.
2.3.4
Pathophysiology
Diabetic neuropathies are a heterogeneous group of conditions that involve
different parts of the somatic and autonomic nervous systems. They can be focal or
diffuse, proximal or distal. The pathogenesis of DN is also heterogeneous in nature
which includes different causative factors, persistent hyperglycemia, microvascular
insufficiency, oxidative stress, nitrosative stress, defective neurotrophism and
autoimmune-mediated nerve destruction.152, 153,154,155 Despite of recent advances, the
precise pathogenesis of diabetic peripheral neuropathy remains obscure. However,
consensus is that neuropathy in diabetes mellitus is a multifactorial disease.
The diabetic state produces impaired neurotrophism, axonal transport and gene
expression through at least four major pathways. 1) Excess glucose is diverted away
from glycolysis by the polyol pathway that depletes NADPH and cellular antioxidant
capacity. 2) Glucose also may become oxidized and form AGEs that alter
extracellular matrix, activate receptors that produce ROS intermediates, and alter
intracellular protein function. 3) PKC becomes activated either directly by glycolytic
intermediates or indirectly as a second messenger for stress hormones, leading to
increased vascular disease, inflammation, and oxidative stress. 4) Partial glycolysis
causes accumulation of glycolytic intermediates and leads to escape of fructose-6phosphate along the hexosamine pathway that increases vascular disease and further
ROS generation.156
Other metabolic factors includes altered fatty acid metabolism with depletion
of prostagladin precursors especially linolenic acid and PGE1 which play a major role
in regulating tissue Na+-K+ ATPase activity, and reduced concentration of nerve
56
growth factors.157 However, Tesfaye et al have demonstrated vascular endoneurial
hypoxia resulting from arteriovenous shunting among epineural vessels with the
proliferation of new leaky neural vessels among diabetic patients.158
Recent studies in patients with impaired glucose tolerance provide important
insights into the role of the degree of glucose dysmetabolism in the development of
neuropathy. The deleterious effect of hyperglycemia is confirmed by the occurrence
of neuropathy associated with impaired glucose tolerance. Accumulation of polyols,
which is observed in animal model of diabetes, also occurs in humans, but whether
the accumulation of polyols in nerves leads to neuropathy is not established. In 2006,
a study in an adolescent diabetic cohort showed that AKR1B1 polymorphisms might
influence the decline of nerve function.159, 160
Leinninger GM et al proposed the potential role in diabetic neuropathy of
mitochondria of sensory neurons located in dorsal root ganglia. These mitochondria
are especially vulnerable because in the hyperglycemic neuron they are the origin of
production of reactive oxygen species which can damage their DNA and membranes.
Deregulation of fission and fusion proteins that control mitochondrial shape and
number can impair cell functions and might lead to degeneration.161
King RH et al reported that advanced glycation end products resulting from
hyperglycemia act on specific receptors inducing monocytes and endothelial cells to
increase the production of cytokines and adhesion molecules. Advanced glycation end
products have been shown to have an effect on matrix metalloproteinase, which might
damage nerve fibers.162
Ziegler D et al supports a role for oxidative stress in the pathogenesis of
diabetic neuropathy in animal models, which has led to clinical trials of antioxidants
such as -lipoic acid, a powerful antioxidant that scavenges hydroxyl, superoxide and
57
peroxyl radicals and regenerates glutathione. In these trials, -lipoic-acid
administration improved nerve conduction velocity and had some positive effects on
neuropathic symptoms.163
Figure 2.3.4.1: Pathogenesis of Diabetic neuropathy
58
i.
Steps involved in Pathogenesis of diabetic neuropathy (DN):The mechanisms pathogenesis underlying DN depend on aetiology. Diabetes,
being the most common etiological factor, is also the most studied in terms of
pathogenesis of diabetic neuropathy.164 The pathophysiology of diabetic neuropathy
includes increased oxidative stress yielding advanced glycosylated end products
(AGEs), polyol accumulation, decreased nitric oxide/impaired endothelial function165
impaired (Na+/ K+) - ATPase activity166 and homocysteinemia.167 In addition, not
only are nerve cells more likely to be destroyed in a hyperglycemic environment but
repair mechanisms are also defective. Reduced levels of neurotrophic agents,
including nerve growth factor and insulin like growth factor, have been noted in
experimental diabetes. 168
Table 2.3.4.1: Possible pathological mechanisms of
diabetic neuropathy
59
Figure 2.3.4.2: Pathophysiological factors in diabetic neuropathy
ii. Oxidative Stress/Protein Glycosylation

Diabetes results in increased products of oxidation. In hyperglycemia, glucose
combines with protein, yielding glycosylated proteins, which can become damaged by
free radicals and combine with fats, yielding AGEs that damage sensitive tissues. In
addition, glycosylation of antioxidant enzymes can render the defense system less
efficient. Significant evidence points to increased oxidative stress in diabetic PN,
either because of enhanced production of reactive oxygen species (ROS) or defective
scavenging of free radicals.
Housman et al conducted a study to compared markers of oxidative stress in
189 people with diabetes (105 with PN, 22 with PN plus CAN and 22 with no PN or
CAN) with 85 controls. In his study, markers of oxidative stress included the plasma
8-iso-prostaglandin F2a, superoxide anion generation, and lag time to peroxidation by
peroxynitrite. Subjects with PN or PN plus CAN demonstrated significant elevations
of all three markers, as well as significant decreases in the protective antioxidant
vitamins C and E. 169, 170, 171
60
The effect of pro-oxidants has been examined in experimental diabetic
neuropathy. In one study, rats exposed to two pro-oxidant interventions either the
drug primaquine or a vitamin E-deficient diet demonstrated decreased nerve
conduction velocity (NCV), nerve growth factor in the sciatic nerve and
neuropeptides compared to diabetic rats not exposed to additional oxidative stress.172
Figure 2.3.4.3: Oxidative stress generated in diabetic neuropathy
iii. Polyol accumulation

Glucose is able to passively diffuse without insulin into certain types of cells,
including nerve cells. Once inside the cell, glucose is converted to sorbitol and other
polyols by the enzyme aldose reductase. Because polyols do not passively diffuse out
61
of cells, they concentrate within cells such as neurons, creating an osmotic gradient
that allows excess sodium and water to follow.173
It is now believed that, in addition to osmotic effects, polyol-pathway linked
metabolic changes are involved. Fructose is also a by product of polyol-pathway
activation via the sorbitol dehydrogenase-driven conversion of sorbitol to fructose.
High fructose levels result in increased AGE precursors another source of oxidative
stress.174
iv. The sorbitol pathway:
Accumulation of sorbitol and fructose in nerve cells has been shown to decrease
(Na+/ K+)-ATPase activity. In addition, free carnitine and myo-inositol content in the
caudal nerves of diabetic rats were significantly decreased with polyol accumulation.
Providing the rats with an aldose reductase inhibitor decreased the depletion of both
myo-inositol and carnitine in caudal nerves and preserved (Na+/ K+)-ATPase activity
in the sciatic nerve adding further evidence that metabolic derangements are
associated with polyol-pathway hyperactivity.175
Is there a link between abnormal glucose metabolism through the sorbitol
pathways and reversible abnormalities in nodal (nodes of Ranvier) function. It has
been established that the enzyme aldose reductase is localized in the nodes of
Ranvier, but to understand the effects of sorbitol pathway on nerve conduction, it is
first necessary to understand another biochemical abnormality of diabetic peripheral
nerve.
62
Figure 2.3.4.4: The Sorbitol Pathway
Figure 2.3.4.5:- Schematic depiction of glucose

metabolism in normal and diabetic nerve208.
v. Nitric Oxide Deficiency/Impaired Endothelial

Function: The Arginine Connection
Vascular factors have also been implicated in the pathogenesis of diabetic PN.
Nerve blood flow is diminished in experimental diabetic neuropathy, and numerous
studies indicate it may be mediated by alterations in nitric oxide metabolism. One
such study examined nerve blood flow and nitric oxide synthase (NOS) activity in the
microvasculature serving peripheral nerves in diabetic rats.176 Hyperglycemia resulted
in a significant diminution of nerve blood flow compared to controls. N-nitro-L-
63
arginine, an inhibitor of NOS, also resulted in decreased nerve blood flow. L-arginine
reversed the effects of NOS inhibition and restored blood flow to the nerves.
An animal study also found disruptions in neuronal nitric oxide synthase
(nNOS) in experimental diabetes. Decreased nNOS expression was associated with
increased neuropathic pain.177 Nitric oxide plays an important role in controlling
(Na+/K+)-ATPase activity a diminution of which has been implicated in the
pathogenesis of PN. Experimental analysis revealed hyperglycemia results in an
excess of endothelial superoxide radicals that result in reduced stimulation of NO on
(Na+/K+) ATPase activity, this effect is inhibited by L-arginine.178 Another animal
study, however, did not find a relationship between alter NO activity and the
development of sensory PN.179
2.3.5
Cardiovascular autonomic neuropathy (CAN)
One of the most ignored of all serious complications of diabetes is CAN, which
encompasses damage to the autonomic nerve fibres that innervate the heart and blood
vessels intern results in the abnormalities of heart rate control and vascular dynamics.
Autonomic dysfunction can affect daily activities of individuals with diabetes and
may invoke potentially life-threatening outcomes.180-181
CAN occurs when peripheral autonomic fibres which include both sympathetic and
parasympathetic of the cardiovascular system get affected, which leads consequential
in neuro-humoral regulation disturbances. The sympathovagal balance (both tonic and
phasic) modulates the function of three of the main CVS structures: the sinus node
(heart rate), the ventricles (end-systolic and end-diastolic volumes) and the blood
vessels, including microcirculation (total peripheral resistance). CAN is the most
clinically important and well studied form of DAN and it is also one of the most
64
unnoticed of all serious complications of diabetes. This encompasses damage to the
autonomic nerve fibers that innervate the heart and blood vessels which in turn results
in abnormalities of heart rate control and vascular dynamics. Autonomic dysfunction
can influence daily activities of individuals with diabetes and may raise potentially
life frightening outcomes.
Advances in technology built on decades of research and clinical testing due to
this now it is possible that identify early stages of CAN with the use of careful
measurement of autonomic function and to provide therapeutic choices that are based
on symptom control and that might abrogate the underlying disorder.
2.3.5.1 Epidemiology of CAN
Diabetes affects more than 23 million people in the U.S. and an estimated 250
million worldwide. Diabetic neuropathies, including CAN are a common chronic
complication of T1DM and T2DM confer high morbidity and mortality to diabetic
patients. The reported prevalence of CAN varies greatly depending on the criteria
used to identify CAN and the population studied. CAN prevalence range from as low
as 2.5% of the primary prevention cohort in the Diabetes Control and Complications
Trial (DCCT) 182 to as high as 90% of patients with long-standing T1DM who were
potential candidates for a pancreas transplantation.183 In a large cohort of patients with
T1DM and T2DM, Ziegler et al.184 reported that using predefined heart rate variability
(HRV) tests and spectral analysis of the R-R intervals 25.3% of patients with T1DM
and 34.3% of patients with T2DM had CAN and he also commented that age, sex, and
other risk factors may also influence CAN development.
Little information exists as to frequency of CAN in representative diabetic
populations. This is further complicated by the differences in the methodology used
and the lack of standardization. Fifteen studies using different end points report
65
prevalence rates of 1% to 90%.185 The heterogeneous methodology makes it difficult
to compare epidemiology across different studies. CAN may be present at diagnosis
and its prevalence increases with age, duration of diabetes and poor glycemic control.
CAN also cosegregates with distal symmetric polyneuropathy, microangiopathy and
macroangiopathy.
Table 2.3.5.1.1: Prevalence of CAN reported by various studies179
Study
Type of
Diabetes
Sample
Size
AFT
Prevalence
(%)
Ewing et al,
1980
T1DM,
T2DM
61
Valsalva, Handgrip,
Postural BP
54
Dyrbergel,
1981
T1DM
75
HR variation,
Valsalva
17. 27
Obrien et al,
1991
T1DM
506
HR variation,
Valsalva
17
Ziegler et al,
1992
T1DM
647
HRV analysis
25.3
T2DM
524
HRV analysis
34.3
Kennedy et al,
1995
T1DM
290
HR variation,
Valsalva
90, 88
DCCT, 1998
T1DM
1,441
HR variation,
Valsalva,
Postural BP
2.6-23 (OR )
A.K Basu,
2010
T2DM
50
HR, Valsalva,
30:15,
Postural BP,
Handgrip
54
66
2.3.5.2 Clinical Manifestations of CAN
i. Resting Tachycardia
Abnormalities in HRV are early findings of CAN. Resting tachycardia and a
fixed heart rate are characteristic late findings in diabetic patients with vagal
impairment. Resting heart rates of 90 to 100 bpm and occasional heart rate increments
up to 130 bpm occur. The highest resting heart rates have been found in patients with
parasympathetic damage, occurring earlier in the course of CAN than sympathetic
nerve function. In those with evidence for combined vagal and sympathetic
involvement, the rate returns toward normal but remains elevated. A fixed heart rate
that is unresponsive to moderate exercise, stress or sleep indicates almost complete
cardiac denervation. Thus, heart rate may not provide a reliable diagnostic criterion of
CAN in the absence of other causes unless it increases more than 100 bpm.186
Figure 2.3.5.2.1: The autonomic innervations of the

heart and the effects of diabetes.
67
ii. Exercise intolerance
Autonomic dysfunction impairs exercise tolerance reduces response in heart
rate and blood pressure (BP) and blunts increases in cardiac output in response to
exercise.187, 188,189,190 Diabetic patients who are likely to have CAN should be tested
for cardiac stress before undertaking an exercise course. Patients with CAN need to
rely on their perceived exertion and not heart rate to avoid the hazardous levels of
intensity of exercise.191
Presently, there is inadequate evidence to recommend routine screening of
asymptomatic diabetic patients with an exercise ECG test. Emerging data support the
usefulness of stress testing in identifying diabetic patients with preclinical coronary
artery disease and particularly patients with high risk features and co-morbidities such
as long standing disease, CAN, multiple chronic renal failures, resting ECG
abnormalities and peripheral artery disease.192
iii. Intraoperative and Perioperative (Cardiovascular Instability)
Perioperative cardiovascular morbidity and mortality is increased 2 to 3 fold in
patients with diabetes. Compared with nondiabetic subjects, diabetic patients
undergoing general anaesthesia may experience a greater degree of decline in heart
rate and BP during induction of anaesthesia and less of an increase after tracheal
intubation and extubation. Vasopressor support is needed more often in diabetic
individuals with CAN than in those without CAN.193 The normal autonomic response
of vasoconstriction and tachycardia does not completely compensate for the
vasodilating effects of anaesthesia. There is an association between CAN and more
severe intraoperative hypothermia that results in decreased drug metabolism and
impaired wound healing. Reduced hypoxic induced ventilatory drive requires
preoperative CAN screening for loss of HRV.194, 195
68
iv. Orthostatic Hypotension
Orthostatic hypotension is defined as a fall in BP (i.e. >30 mm Hg systolic or
>10 mm Hg diastolic BP) in response to a postural change from supine to standing.196
Symptoms include weakness, faintness, dizziness, visual impairment, and even
syncope after a change from a lying to a standing posture. Orthostatic hypotension
may become disabling but the BP fall may also be asymptomatic.197 Orthostatic
symptoms can be misjudged as hypoglycemia and can be infuriated by a number of
drugs, including vasodilators, diuretics, phenothiazines, predominantly tricyclic
antidepressants and insulin.
A change from lying to standing normally results in activation of a
baroreceptor, centrally mediated sympathetic reflex which results in an increase in
peripheral vascular resistance and cardiac acceleration. In patients with diabetes,
orthostatic hypotension is usually attributable to damage the efferent sympathetic
vasomotor fibers, mostly in the splanchnic vasculature.198 In addition ,a decrease in
total vascular resistance contributes to pathogenesis of this disorder. In individuals
with orthostatic hypotension, there may be a reduced norepinephrine response relative
to the fall in BP. Reduced cardiac acceleration and cardiac output may also be
important, as well as low blood volume or reduced red cell mass. Other factors such
as postprandial blood pooling, the hypotensive role of insulin and treatment of kidney
or heart failure with diuretics, leading to volume depletion possibly will worsen the
orthostatic symptoms.199
69
v.
Progression of CAN
Although much remains to be learned about the natural history of CAN, previous
reports can be coalesced into a few observations that provide some insights with
regard to progression of autonomic dysfunction.
It can be detected at the time of diagnosis200.
Neither age nor type of diabetes are limiting factors in its emergence, it has
been found both in young people with newly diagnosed T1DM and in older
people newly diagnosed with T2DM200-204. Poor glycaemic control plays a
central role in development and progression.205
Intensive therapy can slow the progression and delay the appearance of
abnormal autonomic function tests.206
Subclinical autonomic neuropathy can be detected early using autonomic
function tests200.
Autonomic features that are associated with sympathetic nervous system
dysfunction (eg orthostatic hypotension) are relatively late complications of
diabetes.
There is an association between CAN and diabetic nephropathy that
contributes to high mortality rates.200 Even with consensus regarding these
general observations much remains unclear.
Some individuals with symptoms associated with autonomic neuropathy die
suddenly and unexpectedly.207
Clinical signs and symptoms of autonomic dysfunction do not always
progress. This underscores the need for performance of quantitative autonomic
function tests to identify individuals at risk for premature death.208
Type 1 and type 2 diabetes may have different progression paths.
70
The relationship between autonomic damage and the duration of diabetes is
still not so clear, although numerous studies like Vinik et al support an
association.
Prevalence and mortality rates may be higher among individuals with type 2
diabetes which may be due to longer duration of glycaemic abnormalities
before diagnosis.200
2.3.6 Diabetic peripheral neuropathy (DPN)

The definition of diabetic peripheral neuropathy is "the presence of symptoms
and/ or signs of peripheral nerve dysfunction in people with diabetes after exclusion
of other causes". DPN is a one of the long term complication of diabetes. Exposure to
high blood glucose levels over an extended period of time causes damage to the
peripheral nerves that go to the arms, hands, legs, and feet.
Peripheral
neuropathy,
also
called distal
symmetric
neuropathy or
sensorimotor neuropathy due to nerve damage in the arms and legs. Feet and legs are
likely to be affected before hands and arms. Symptoms of peripheral neuropathy may
include:
Numbness or insensitivity to pain or temperature
Tingling, burning or prickling sensation
Sharp pains or cramps
Extreme sensitivity to touch, even light touch
Loss of balance and coordination
These symptoms are often worse at night. Peripheral neuropathy may also cause
muscle weakness and loss of reflexes, especially at the ankle, leading to changes in
the way a person walks.
71
Foot deformities, such as hammertoes and the collapse of the midfoot may occur.
Blisters and sores may appear on numb areas of the foot because pressure or injury
goes unnoticed. If an infection occurs and is not treated promptly, the infection may
spread to the bone and the foot may then have to be amputated. Many amputations are
preventable if minor problems are caught and treated in time.
Figure 2.3.6.1: Most commonly affected areas in Diabetic Peripheral Neuropathy
2.3.6.1 Epidemiology of DPN

Diabetic peripheral neuropathy (DPN) is a common complication estimated to
affect 30% to 50% of individuals with diabetes. Chronic sensorimotor distal
symmetric polyneuropathy is the most common form of DPN.
Epidemiological data indicates that the prevalence of DPN is higher in type 2
than type 1 diabetes. Kastenbauer et al had observed an evidence of peripheral
neuropathy in nearly one third of patients with T1DM and more than half of patients
with T2DM.209 However, another population-based cohort studies have shown that
72
66% of T1DM and 59% of T2DM had objective evidence of DPN.147 Schmader K E et
al has been estimated among 2024% of diabetic patients having peripheral
neuropathy.210 In a recent MONICA/KORA survey from Augsburg, Germany, the

prevalence of painful polyneuropathy was found to be 13.3% in diabetic subjects,
8.7% in individuals with impaired glucose tolerance, 4.2% in individuals with
impaired fasting glucose and 1.2% in individuals with normal glucose tolerance.211
whereas, studies conducted in the Middle East Region (MER) report higher rates of
painful DPN ranging from 35% to 65%.212 Low P A et al reported that that 50% of
diabetic patients with polyneuropathy have asymptomatic CAN, while 100% of
patients with symptomatic CAN will have polyneuropathy. 214
Table 2.3.6.1.1: Prevalence of DPN reported by various studies213, 214, 215
Study
Type of
study
Type of
Diabetes
Sample
Size
Type of
assessment
Prevalence
(%)
Young MJ,
1993
Clinic
based
T1DM &
T2DM
(Known
cases)
6,487
VPT
32.1
Ashok at al,
2002
Clinic
based
T2DM
( Known
cases)
1,000
VPT
19.1
Arindam dutta
at al, 2005
Clinic
based
T2DM
(New
cases)
100
NSS, NDS,
VPT,
NCV,CMAP
29
Predeepa et al,
2008
Population
based
T2DM
( known
and New
cases)
1,629
VPT
26.10
A Mythilli et
al, 2010
Clinic
based
T2DM
(Known
cases)
100
DNS, DNE,
VPT, SWM
71
73
2.3.6.2 Symptoms of Diabetic peripheral neuropathy
Diffuse neuropathy or generalized polyneuropathy is typically characterized
by the predominant fibre types involved: small-fiber sensory, large- fibre sensory,
large and small fibre sensory or sensorimotor polyneuropathy.216
DPN often presents with neuropathic pain but can also present with decreased
balance or a change in gait. The International Association for the Study of Pain has
described neuropathic pain as pain initiated or caused by a primary lesion or
dysfunction in the nervous system. 217 However, a recent review proposed redefining
neuropathic pain as pain arising as a direct consequence of a lesion or disease of the
somatosensory system.218 Neuropathic pains is different in quality and onset from
nociceptive pain, which patients can often link to a specific musculoskeletal or soft
tissue injury.
The neuropathic pain from DPN usually has a gradual and insidious onset and
appears to arise denovo. Three distinct types of pain have been described in patients
with DPN. Dysesthesic pain is an unpleasant abnormal sensation, whether
spontaneous or evoked and often presents as severe burning or itching sensations.
Paresthetic pain presents as a pins and needles sensation or an electric or knife like
shooting pain. Muscular pain presents as a deep, dull, aching or cramping pain that
may be described as night cramps.
Each of these types of pain may have a different pathogenesis and anatomical
distribution. Although most patients have a mixed or pansensory neuropathy,
occasionally patients present with selective involvement. Involvement of small
sensory fibres often causes severe superficial burning pain, whereas involvement of
large sensory fibres is more often associated with paresthesias, loss of ankles jerks,
and decreased balance. (Table 2.3.6.2.2)
74
In general, pain from DPN is worse at rest and in the evenings, possibly
because the nervous system is not distracted in the evening by a multiplicity of
other inputs (eg, visual, locomotory or thinking) that it is constantly processing during
the day. Patients with diabetes whose pain is worse with walking or standing need to
be evaluated for a concomitant disorder. It is classically symmetrical and distal. Since
DPN is felt to be a dying back of the nerves, it affects the most distal extremities
first, resulting in the stocking and glove distribution (Figure 2.3.6.2.1). Patients
with DPN may experience allodynia, which is pain due to a stimulus that does not
normally provoke pain (eg, severe pain caused by a bed sheet touching bare toes) or
hyperalgesia, which is an increased response to a stimulus that is not normally painful
(eg, pain occurring with light touch).255 Patients may also complain of other abnormal
sensations, such as my shoes feel too tight, or describe a feeling of walking on
pebbles or like their feet are in ice water.140
Figure 2.3.6.2.1: Stocking and glove distribution in patient with diabetic

peripheral neuropathy
.
75
Table 2.3.6.2.2: Clinical Features of Small and Large Fibre Diabetic
Peripheral Neuropathy
Clinical features
Small fiber
Pain predominates, often burning and superficial

Allodynia ( Pain from normal stimuli such as bed sheets)
Defective warm thermal sensation
Defective autonomic dysfunction
Decreased sweating ( dry feet)
Impaired vasodilatation ( cold hands/feet)
Large fiber
May involve sensory or motor nerve

Most distal nerves affected first stocking-glove pattern
More neurologic signs than symptoms
Impaired vibratory perception
Depressed or absent deep tendon reflexes
Pain is deep described as gnawing, like a toothache or even cramping
2.3.6.3 Clinical assessment of DPN

The physical examination in a patient with suspected DPN begins with
inspection of the lower extremities. Observation may reveal dry and cracked skin
and/or cold skin (despite good pulses) in patients with DPN. These abnormalities
result from autonomic components of DPN, including loss of sweat glands and
shunting of blood away from the skin. A characteristic finding on neurologic
examination is loss of deep tendon reflexes. Ankle jerks are initially decreased and
then lost. Knee jerks may be preserved until more advanced stages of the disease
when the hands become affected.
The 128 Hz tuning fork is the instrument of choice to check for the presence
or absence of vibratory sensation in the feet. Initially, patients show deficits in
76
vibration perception of the great toes, but over time the deficits move proximally to
the metatarsalphalangeal joints, dorsum of the foot, ankle, and the mid-tibial region.
Use of the Semmes-Weinstein monofilament has helped define degrees of
sensory loss in the feet and hands of patients with DPN. To use the device properly,
the examiner should place the tip of the monofilament perpendicular to the plantar
surface of the great toe. Examiners should not allow the filament to slide across the
skin or make repetitive contact to the site. Areas of callus must be avoided for an
accurate exam. With their eyes closed, patients should be able to sense the
monofilament by the time the monofilament buckles.216 (Figure 2.3.6.3.1).
Figure 2.3.6.3.1:- Semmes-Weinstein monofilament examination.252
The thicker (higher number) the monofilament, the more force is required to
cause the buckle. Patients without neuropathy should be able to sense the 3.61
monofilament (equivalent to 0.4 g of linear pressure).
The inability to sense monofilaments of 4.17 (equivalent to 1 g of linear
pressure) or higher is considered consistent with neuropathy (large fiber modality).
The inability to sense a monofilament of 5.07 (equivalent to 10 g of linear force) is
77
consistent with severe neuropathy and loss of protective sensation. Unfortunately,
most physicians are only familiar with or have access to the 5.07 monofilament and
there is a widespread misconception that DPN may be ruled out when a patient can
feel this size device. Small fibre nerve loss can be detected with pinprick, although a
cold perception gradient loss (using hot and cold water-filled test tubes or the flat side
of a tuning fork) may be easier to define than loss of pinprick perception. The last
sensory modality to become abnormal in this distal gradient loss neuropathy is loss of
joint position sense (proprioception).
Unfortunately, this is an all too common finding in many patients with longstanding DPN. Foot deformities are common findings in advanced cases of DPN.
Although primarily a sensory disorder, DPN often includes an accompanying motor
disorder that leads to the hammer toe or claw foot deformities. 216
2.3.6.4 Causes of DPN and Laboratory Testing
The consensus definition of DPN requires ruling out other disorders that cause
peripheral neuropathy before attributing the neurologic findings to diabetes. (Table
2.3.6.4.1) Evaluation of the patient should include basic laboratory tests, such as a
thyroid-stimulating hormone, serum protein electrophoresis, and vitamin B12 level, to
look for other causes of polyneuropathy. Patients with diabetes mellitus also
commonly develop other diseases such hypothyroidism, B12 deficiency, celiac
disease and uremia, all of which may cause or contribute to peripheral neuropathy.
It should also be noted that prediabetes (previously called impaired glucose
tolerance or impaired fasting glucose) may also present with DPN. In fact,
neurologists will frequently order a glucose tolerance test when a patient presents with
an idiopathic peripheral neuropathy.216-219
78
Table 2.3.6.4.1: - Causes of Diabetic peripheral neuropathy
Diabetes
Alcoholism
Non-alcoholic liver diseases
Critical illness
Autoimmune
neuropathies
(eg.
chronic
inflammatory
demyelinating
polyneuropathy)
HIV disease
Drugs (eg. Isoniazid, cisplatin)
Toxin ( eg. arsenic)
Hypothyroidism
B12 deficiency
Uremia
Lymphoma
Multiple myeloma
Malabsorption ( eg. celiac disease)
Hereditary
Lyme disease
Amyloidosis
Connective tissue disorders ( eg rheumatoid arthritis)
2.3.7. Diagnosis of Diabetic neuropathy

The diagnosis DN is often difficult to establish in individuals because clinical
symptoms usually appear late in the course of the disease and may be nonspecific.
Early detection of Autonomic Neuropathy would propose the need for an aggressive
approach in the management of diabetes mellitus with the help of simple autonomic
function test. Good clinical history and complete physical examination are the basis of
assessment.
79
A detailed history includes documentation of symptoms of DN and review of
diabetes history, disease management, daily glycemic records and previous HbA1C
levels. Asking about similar symptoms in family members may help in distinguishing
familial neuropathies. Medication history including use of over the counter products
and herbal or homeopathic products and environmental exposures should be assessed.
A careful history also includes differential diagnosis (Table 2.2.9.1) because diabetes
is not the one on the list of causes of peripheral neuropathy. 220-231
Table 2.3.7.1: Differential diagnosis of Diabetic Neuropathies219
Types
Syndromes
Congenital
Charcot-Marie-Tooth Syndrome
Traumatic
Entrapment Syndromes
Inflammatory/infiltrative
Sarcoidosis, Leprosy, Lyme disease, HIV, Refsums

disease, Amyloidosis, Periarteritis Nodosa
Neoplastic
Carcinoma, Pariarteritis syndrome, Myeloma,

leukemias, Lymphomas
Metabolic/Endocrine
Diabetes, Uremia Pernicious anaemia,

Hypothyroidism, Acute Intermittent Porphyria
Vascular
Diabetes, vasculitis
Toxic
Alcohol, Heavy metals (lead, Mercury, Arsenic),

Hydrocarbons, drugs (pyridoxine toxicity),
chemotherapeutics.
Autoimmune
Diabetes, Phospholipids antibody syndrome, GuillainBarre Syndrome, Chronic inflammatory Demyelinating

Polyneuropathy ( CIDP), Multifocal Motor
Neuropathy
80
Table 2.3.7.2:- Diagnostic tools for Diabetic autonomic neuropathy185, 222-228
System
involved
Cardiovascular
system
Types of
Autonomic
neuropathy
Methods of Assessment
Cardiovascular Heart Rate variability analysis and

Autonomic
cardiovascular reflex tests
Neuropathy
parasympathetic tests :HR response to standing,
Valsalva ratio, Deep breathing test
Sympathetic tests:- postural hypotension, handgrip tests
Digestive
system
Gastrointestinal
Renal system
Bladder
dysfunction
Endoscopy
Scintographic measurement of oesophageal bolus transit
time for oesophageal dysfunction
Scintography
Isotope breath tests
Ultrasonography for gastroparesis, hydrogen breath test
for diabetic diarrhoea
Barium enema for constipation and digital examination
of the rectum, anorectal manometry
Endoanal ultrasonography
Colon transit tests
Proctoscopy and sigmoidoscopy for fecal incontinence
Renal function test
Cystometry
Uroflow
simultaneous pressure/flow studies
Sphincter electromyography and urethral pressure
Reproductive
system
Impotence
Comprehensive history sexual, medical, drug use, risk

factors, psychological factors and hormonal level.
Measurement of nocturnal penile tumescence
Tests to assess penile, pelvic, and spinal nerve function
cardiovascular autonomic function tests
measurement of penile and brachial blood pressure
Sudomotor
functions
Sudomotor
dysfunction
Quantitative Sudomotor Axon Reflex Test (QSART)

Thermoregulatory Sweat Test, Sympathetic Skin
Response or Neuropad/ Indicator Plaster method or
Quantitative Direct and Indirect Reflex Test (QDIRT)
81
2.3.7.1: Diagnostic assessment for CAN Ewings criteria
As the clinical importance of diabetic autonomic neuropathy has become
recognized the need has grown for simple objective tests to confirm its presence or
absence. DJ Ewing and cleark proposed to give a practical guide to those tests which
we consider reliable, reproducible, simple and non-invasive. These criteria have so far
been fulfilled only in tests based on cardiovascular reflexes.232 Though tests using
cardiovascular reflexes are most often done on diabetics, they are equally applicable
in the diagnosis of autonomic damage caused by other disorders.
In the early 1970s, Ewing et al. proposed five simple noninvasive cardiovascular
reflex tests that have been applied successfully in many studies (work reported in
1985. Simple noninvasive cardiovascular reflex tests have now become the gold
standard by which an autonomic neuropathy is diagnosed objectively and by which
other tests are judged.232
These tests based on the responses of the heart rate and blood pressure to a variety
of stimuli. The first three reflect cardiac parasympathetic integrity, while the other
two start to give abnormal results with more severe sympathetic nerve damage.
The diagnosis of CAN should be based on the results of a battery of autonomic
tests rather than one single test. All of the tests described here for the assessment of
cardiovascular autonomic function can be performed at community setting. Those
patients with cardiovascular autonomic dysfunction who have system specic
symptoms will need to be referred to a specialist for rened testing.232, 233,234
82
A. Cardiovascular reflex tests ( Autonomic function tests)
Parasympathetic tests
a. Heart rate response to deep breathing
Normally the heart rate varies continually but this depends on an intact
parasympathetic nerve supply. The variation is abolished with atropine but
uninfluenced by propranolol and will be more pronounced at slow heart rates and
during deep breathing.235 Diabetics with autonomic neuropathy may have a noticeable
reduction heart-rate variation. Heart-rate variation can be studied during quiet
breathing, deep breathing or after a single deep inspiration. Deep breathing at six
breaths a minute is the most convenient and reproducible technique236, 237.
The patient sits quietly and breathes deeply at six breaths a minute (five
seconds in and five seconds out) for one minute. An electrocardiogram is recorded
throughout the period of deep breathing, with a marker used to indicate the onset of
each inspiration and expiration. The maximum and minimum R-R intervals during
each breathing cycle are measured with a ruler and converted to beats a minute. The
result is then expressed as the mean of the difference between maximum and
minimum heart rates for the six measured cycles in beats a minute. The test has the
advantage of being objective, simple to perform and requires very little cooperation
from the patient. Heart-rate variation has also been measured as the ratio of the heart
rate at expiration to that at inspiration so, it is called E: I ratio.237, 238
b. Heart rate response to standing.

During the change from lying to standing a characteristic immediate rapid
increase in heart rate occurs which is maximal at about the 15th beat after standing.
Then, a relative overshoot bradycardia occurs maximal at about the 30th beat.238 This
83
response is mediated by the vagus nerve.239 Diabetics with autonomic neuropathy
show only a gradual or no increase in heart rate after standing.
The test is performed with the patient lying quietly on a couch while the heart
rate is recorded continuously on an electrocardiograph. The patient is then asked to
stand up unaided and the point at starting to stand is marked on the electrocardiogram.
The shortest R-R interval at around the 15th beat and the longest R-R interval
at around the 30th beat after starting to stand are measured with a ruler. The
characteristic heart-rate response is expressed by the 30:15 ratio. This test is simple
and objective requires little patient co-operation, is reproducible, and does not depend
on either age or the resting heart rate.
c. Valsalva maneuver.
In healthy subjects, the reex response to the Valsalva maneuver includes
tachycardia and peripheral vasoconstriction during strain, followed by an overshoot in
blood pressure and bradycardia after release of strain. The response is mediated
through alternating activation of parasympathetic and sympathetic nerve fibres.
Pharmacological
blockade
studies
using
atropine,
phentolamine
(an-
adrenergic antagonist), and propranolol (a nonspecic adrenergic blocker) conrmed

dual involvement of autonomic nerve branches for the response to this maneuver by
demonstrating the drugs varied effects of attenuation or augmentation of the
hemodynamic response to the maneuver at specic times during the response. In
patients with autonomic damage from diabetes, the reex pathways are damaged. This
is seen as a blunted heart rate response and sometimes as a lower than normal decline
in blood pressure during strain, followed by a slow recovery after release.240 In the
standard Valsalva maneuver, the supine patient, connected to an ECG monitor,
forcibly exhales for 15 s against a xed resistance (40 mmHg) with an open glottis.
84
The response to performance of the Valsalva maneuver has four phases and in
healthy individuals can be observed as follows:
Phase I: Transient rise in blood pressure and a fall in heart rate due to
compression of the aorta and propulsion of blood into the peripheral
circulation. Hemodynamic changes are mostly secondary to mechanical
factors.
Phase II: Early fall in blood pressure with a subsequent recovery of blood
pressure later in the phase. The blood pressure changes are accompanied by
an increase in heart rate. There is a fall in cardiac output due to impaired
venous return causing compensatory cardiac acceleration, increased muscle
sympathetic activity, and peripheral resistance.
Phase III: Blood pressure falls and heart rate increases with cessation of
expiration.
Phase IV: Blood pressure increases above the baseline value (overshoot)
because of residual vasoconstriction and restored normal venous return and
cardiac output.
The Valsalva ratio is determined from the ECG tracings by calculating the
ratio of the longest R-R interval after the maneuver (reecting the bradycardic
response to blood pressure overshoot) to the shortest R-R interval during or shortly
after the maneuver (reecting tachycardia as a result of strain).
85
Sympathetic tests
a. Systolic blood pressure response to standing.

Blood pressure normally changes only slightly on standing from a sitting or
supine position. The response to standing is mediated by sympathetic nerve bers. In
healthy subjects, there is an immediate pooling of blood in the dependent circulation
resulting in a fall in blood pressure that is rapidly corrected by baroreex
mediated peripheral vasoconstriction and tachycardia. In normal individuals, the
systolic blood pressure falls by 10 mmHg in 30 s. In diabetic patients with autonomic
neuropathy, baroreex compensation is impaired. A response is considered abnormal
when the diastolic blood pressure decreases more than 10 mmHg or the systolic blood
pressure falls by 30 mmHg within 2 min after standing. A task force of the American
Academy of Neurology (AAN) and the American Autonomic Society dened
orthostatic hypotension as a fall in systolic blood pressure of 20 mmHg or diastolic
blood pressure of 10 mmHg accompanied by symptoms. 240
Figure 2.3.7.1.1: Baroreflex mechanism
86
b. Diastolic blood pressure response to sustained handgrip.

In this test, sustained muscle contraction will be measured by using a handgrip
dynamometer which causes a rise in systolic and diastolic blood pressure and heart
rate. This rise is caused by a reex arc from the exercising muscle to central command
and back along efferent fibres. The efferent fibres innervate the heart and muscle,
resulting in increased cardiac output, blood pressure, and heart rate.
The dynamometer is rst squeezed to isometric maximum, then held at 30%
maximum for 5 min. The normal response is a rise of diastolic blood pressure 16
mmHg, whereas a response of 10 mmHg is considered abnormal.241 Patients with
DAN are more likely to exhibit increase in diastolic blood pressure.
c. Response to tilting
The hemodynamic response to standing is a commonly performed measure of
autonomic function. Passive head-up tilting provides a more precise level of
standardization to the orthostatic stimulus and reduces the muscular contraction of the
legs, which can reduce lower leg pooling of blood. A tilt angle of 60 is commonly
used for this test. The tilt may be maintained for 1060 min or until the patients
orthostatic symptoms can be reproduced.
The orthostatic stress of tilting evokes a sequence of compensatory
cardiovascular responses to maintain homeostasis. As for the stand response, the
normal tilted reex consists of an elevation in heart rate and vasoconstriction. If reex
pathways are defective, blood pressure falls markedly with hemodynamic pooling. An
abnormal response is dened similarly to that associated with standing.242
87
Table 2.3.7.1.1: - Normal values of various cardiovascular reflex tests
Type of tests
Cardiovascular
Variables
Normal Borderline Abnormal
reflex tests
Valsalva
Valsalva
Maneuver
Ratio
Parasympathetic
HR response to
(30:15
tests
standing
1.11-1.20
1.10
ratio)
1.04
1.01-1.03
1.00
E:I ratio
1.21
1.20
15
11-14
10
16
11-15
10
16
11-15
10
10
11-29
30
Deep Breathing
Delta HR
tests
(beats/min)
Cold Presser
test
Increased
DBP
(mm of
Hg)
Increased
DBP
Sympathetic
tests
1.21
Handgrip test
(mm of
Hg)
Orthostatic
hypotension
Fall in
SBP (mm
of Hg)
88
2.3.7.2 Diagnosis assessment for CAN - HRV analysis
A. Historical view
Heart rate variability (HRV) describes variations in both instantaneous heart rate
and RR intervals. Beat-to-beat fluctuations reflect the dynamic response of the cardiovascular control systems to a host of naturally occurring physiological perturbations.
In particular, arterial and venous blood pressures are altered continuously as a result
of the cyclic variation in intrathoracic pressure associated with respiratory
movements, and also because of the fluctuations in peripheral vascular resistance
resulting from regional blood flow autoregulation. The sympathetic and
parasympathetic nervous systems maintain cardiovascular homeostasis by responding
to beat to beat per turbations that are sensed by baroreceptors and chemoreceptors.243
Although oscillations in heart rate and blood pressure were identified over 100
years ago, the notion that certain frequencies may be indicative of either sympathetic
or parasympathetic tone is considerably newer and has led to great clinical interest in
describing changes in a range of physiological and pathological conditions, such as
heart failure, diabetes, hypertension, sepsis and brain death.244, 245
The first recorded measurements of oscillations in the cardiovascular system were
described by Mayer in 1876. He observed pronounced oscillations in blood pressure
in anaesthetised and spontaneously breathing rabbits at 0.1 Hz. A variety of animal
and human research has established two clear frequency bands in heart rate and blood
pressure signals. These bands include oscillations associated with respiration between
0.2 to 0.4 Hz (high frequency) and bands with a lower frequency range, below 0.15
Hz.
89
The latter has often been subdivided into the low-frequency range below0.09 Hz
as well as a mid-frequency range (0.09-0.15Hz). In 1981 Akselrod introduced power
spectrum analysis of heart rate fluctuations in order to quantify beat-tobeat cardiovascular control. Power spectrum density analysis provides the basic
information of how power (variance) is distributed as a function of frequency. In
1996, the Task Force of the European Society of Cardiology and the Northern
American Society of Pacing and Electrophysiology published guidelines regarding
standardization of nomenclature, specification of methods of measurement, definition
of physiological and pathophysiological correlates, description of clinical applications
and identification of different areas for future research.246
The clinical relevance of heart rate variability was first appreciated in 1965 when
Hon and Lee noted that foetal distress was preceded by alterations in inter
beat intervals before any change occurred in the heart rate itself.246 The association of
higher risk of post infarction mortality with reduced heart rate variability was first
shown by Wolf in 1977.247 The clinical importance of heart rate variability became
appreciated in the late 1980s, when it was demonstrated that low heart rate variability
was a strong and independent predictor of mortality after an acute myocardial
infarction.248
B. Measurement of heart rate variability
i. Time domain methods
Variations in heart rate may be evaluated by a number of methods. Perhaps the
simplest to perform are the time domain measures. With these methods either the
heart rate at any point in time or the intervals between successive normal complexes
are determined. In a continuous electrocardiographic (ECG) record, each QRS
complex is detected, and the so called normal to normal (NN) intervals (that is all
90
intervals between adjacent QRS complexes resulting from sinus node depolarizations)
or the instantaneous heart rate is determined. Simple time domain variables that can
be calculated include the mean NN interval, the mean heart rate, the difference
between the longest and shortest NN interval, the difference between night and day
heart rate, etc.
ii. Statistical methods and geometric methods
From a series of instantaneous heart rates or cycle intervals, particularly those
recorded over longer periods, traditionally 24 h, more complex statistical timedomain measures can be calculated. These may be divided into two classes, (a) those
derived from direct measurements of the NN intervals or instantaneous heart rate, and
(b) those derived from the differences between NN intervals. These variables may be
derived from analysis of the total electrocardiographic recording or may be calculated
using smaller segments of the recording period. The latter method allows comparison
of HRV to be made during varying activities, e.g. rest, sleep, etc.
The simplest variable to calculate is the standard deviation of the NN
interval (SDNN), i.e. the square root of variance. Since variance is mathematically
equal to total power of spectral analysis, SDNN reflects all the cyclic components
responsible for variability in the period of recording. As discussed further in this
document, short-term 5-minrecordings and nominal 24 h long-term recordings seem
to be appropriate options.
Other commonly used statistical variables calculated from segments of the total
monitoring period include SDANN, the standard deviation of the average NN interval
calculated over short periods, usually 5 min, which is an estimate of the changes in
heart rate due to cycles longer than 5 min, and the SDNN index, the mean of the 5
91
min standard deviation of the NN interval calculated over 24 h, which measures the
variability due to cycles shorter than 5 min.
The most commonly used measures derived from interval differences
include RMSSD, the square root of the mean squared differences of successive NN
intervals, NN50, the number of interval differences of successive NN intervals greater
than 50 ms, and pNN50t he proportion derived by dividing NN50 by the total
number of NN intervals and geometric measures are HRV triangular index, TINN,
DiVerential index and Logarithmic index. All these measurements of shortterm variation estimate high frequency variations in heart rate and thus are highly
correlated.
iii. Frequency domain methods
Various spectral methods for the analysis of the tachogram have been applied since
the late 1960s. Power spectral density (PSD) analysis provides the basic information
of how power (i.e. variance) distributes as a function of frequency. Methods for the
calculation of PSD may be generally classied as non-parametric and parametric. In
most instances, both methods provide comparable results. The advantages of the nonparametric methods are: (a) the simplicity of the algorithm employed (Fast Fourier
Transform(FFT)) and (b) the high processing speed, whilst the advantages of
parametric methods are: (a) smoother spectral components which can be distinguished
independently of pre selected frequency bands, (b) easy post-processing of the
spectrum with an automatic calculation of low and high frequency power components
and easy identication of the central frequency of each component, and (c) an
accurate estimation of PSD even on a small number of samples on which the signal is
supposed to maintain stationary.
92
iv. Spectral components
Spectral components are distinguished in a spectrum calculated from short term
recordings of ECG for 2 to 5 min (Table 2.2.9.2.2)
Table 2.3.7.2.1: Selected time-domain measures of HRV
Variable
Units
Description
Statistical measures
SDNN
ms
Standard deviation of all NN intervals.
SDANN
ms
RMSSD
ms
Standard deviation of the averages of NN intervals in all 5

min segments of the entire recording.
The square root of the mean of the sum of the squares of
differences between adjacent NN
Intervals.
SDNN index
ms
SDSD
ms
NN50 count
pNN50
Mean of the standard deviations of all NN intervals for all 5

min segments of the entire recording.
Standard deviation of differences between adjacent NN
intervals.
Number of pairs of adjacent NN intervals differing by more
than 50 ms in the entire recording.
Three variants are possible counting all such NN intervals
pairs or only pairs in which the first or
the second interval is longer.
NN50 count divided by the total number of all NN intervals.
Geometric measures
Total number of all NN intervals divided by the height of the
histogram of all NN intervals
HRV
triangular
index
TINN
ms
Differential
index
ms
Logarithmic
index
Measured on a discrete scale with bins of 7.8125 ms (1/128

s).
Baseline width of the minimum square difference triangular
interpolation of the highest peak of the
histogram of all NN intervals
Difference between the widths of the histogram of differences
between adjacent NN intervals
measured at selected heights (e.g. at the levels of 1000 and 10
000 samples)
Coefficient of the negative exponential curve k e.-et which is
the best approximation of the
histogram of absolute differences between adjacent NN
intervals
93
Table 2.3.7.2.2: Selected frequency domain measures of HRV
Variable
Units
Description
Frequency range
Analysis of shortterm recordings (5 min)

5 min
total
power
ms2
The variance of NN
intervals over the
approximately 0.4 Hz
ms2
temporal segment
Power in very low
frequency range
Power in low frequency
range
LF power in normalised
units
LF/(Total
PowerVLF)x100
Power in high
frequency range
HF power in
normalised units
HF/(Total
PowerVLF)x100
Ratio LF [ms2]/HF
[ms2]
Analysis of entire 24 h
0.04 Hz
Variance of all NN
intervals
Power in the ultra low
frequency range
Power in the very low
frequency range
Power in the low
frequency range
Power in the high
frequency range
Slope of the linear
interpolation of the
spectrum in a loglog scale
VLF
LF
ms2
LF norm
n.u.
HF
ms2
HF norm
n.u.
LF/HF
Total
power
ULF
ms2
VLF
ms2
LF
ms2
HF
ms2
ms2
0.040.15 Hz
0.150.4 Hz
0.003 Hz
0.0030.04 Hz
0.040.15 Hz
0.150.4 Hz
94
Figure 2.3.7.2.3: Power spectral density of HRV analysis (Frequency domain

analysis)
C. Clinical component of HRV

i. HRV and nervous system
Disorders of the central and peripheral nervous system have effects on HRV.
The vagally and sympathetically mediated uctuations in HR may be independently
affected by nerve disorders. All normal cyclic changes in HR are reduced in the
presence of severe brain damage and depression. The signicance of HRV analysis in
psychiatric disorders arises from the fact that one can easily detect a
sympathovagal imbalance (relative cholinergic and adrenergic modulation of HRV.
249
95
ii.
HRV and ANS

The ANS have sympathetic and parasympathetic components. Sympathetic
stimulation which occurs in response to stress, exercise and heart disease can causes
increase in HR by increasing the ring rate of pacemaker cells in the
hearts sinoatrial node. Parasympathetic activity, primarily resulting from the function
of internal organs, trauma, allergic reactions and the inhalation of irritants, decreases
the ring rate of pacemaker cells and the HR, providing a regulatory balance in
physiological autonomic function. The separate rhythmic contributions from
sympathetic and parasympathetic autonomic activity modulate the heart rate (RR)
intervals of the QRS complex in the electrocardiogram (ECG), at distinct frequencies.
Sympathetic activity is associated with the low frequency range (0.040.15 Hz) while
parasympathetic activity is associated with the higher frequency range (0.150.4 Hz)
of modulation frequencies of the HR. This difference in frequency ranges allows
HRV analysis to separate sympathetic and parasympathetic contributions evident.
This should enable preventive intervention at an early stage when it is most
benecial.250
iii. HRV and blood pressure
Several structural and functional alterations of the cardiovascular system that
are frequently found in hypertensive individuals may increase their cardiovascular
risk beyond that induced by the BP elevation alone.
Electrocardiographic evidence of left ventricular hypertrophy (LVH) and
strain are associated with increased morbidity and mortality. HRV is signicantly
reduced in patients with LVH secondary to hypertension or aortic valve disease.
Cardiac vagal nerve activity is inuenced by the arterial baroreex. The amplitude of
respiratory sinus arrhythmia (HRV) has been found to correlate with baroreex
96
sensitivity which is reduced in hypertension and diabetes. This reduction in baroreex
sensitivity is correlated with cardiac LVH. A method to describe relationships
between short- term BP uctuations and heart-rate variability in resting subjects was
analyzed in the frequency domain. 251, 252
iv. HRV and gender, age
The HRV was more in the physically active young and old women.288
Bonnemeir et al studied the HR variation for healthy subjects from 20 to 70 years and
found that the HRV decreases with age and variation is more in the case of female
than men. 253 Previous studies have assessed gender and age related differences in time
and frequency domain indices and some nonlinear component of HRV. The amount
of HRV is inuenced by physiologic and maturational factors. Maturation of the
sympathetic and vagal divisions of the ANS results in an increase in HRV with
gestational age and during early postnatal life. Ramaekers D et al reported that the
HRV decreases with age.254, 255, 256, 257
v. HRV and smoking
Studies have shown that smokers have increased sympathetic and reduced vagal
activity as measured by HRV analysis. Smoking reduces the HRV. One of the
mechanisms by which smoking impairs the cardiovascular function is its effect on
ANS control.258 Altered cardiac autonomic function, assessed by decrements in HRV,
is associated with acute exposure to environmental tobacco smoke (ETS) and may be
part of the pathophysiologic mechanisms linking ETS exposure and increased cardiac
vulnerability.259
vi. HRV and alcohol
HRV reduces with the acute ingestion of alcohol, suggesting sympathetic activation
and/or parasympathetic withdrawal. Malpas et al have demonstrated vagal neuropathy
97
in men with chronic alcohol dependence using 24 h HRV analysis. ECG indices of
vagal activity have been reported to have signicantly lower indices of cardiac vagal
nerve activity than normal volunteers, in acute alcoholic subjects. 260
D. HRV analysis methods for assessment of CAN

In the past years, earlier detection of autonomic dysfunction294,
295
became
feasible thanks to the spectral analysis study of HRV. This modern technology uses a
mathematical algorithm (fast Fourier transform) to turn a complex biological signal,
such as HRV (result of the sympathovagal balance in the sinus node), into its causing
components, presenting them according to the frequency with which they alter the.
HRV can be assessed either by calculation of indices based on statistical
analysis of R-R intervals (time-domain analysis) or by spectral analysis (frequencydomain analysis) of an array. Spectral analysis involves decomposing the series of
sequential R-R intervals into a sum of sinusoidal functions of different amplitudes and
frequencies by several possible mathematical approaches, such as fast Fourier
transformation or autoregressive models. The result (power spectrum) can be
displayed with the magnitude of variability as a function of frequency.
In other words, the power spectrum reflects the amplitude of the heart rate
fluctuations present at different oscillation frequencies. The power spectrum of HRV
has been shown to consist of 3 major peaks: (1) very-low frequency component
(below 0.04 Hz), which is related to fluctuations in vasomotor tone associated with
thermoregulation; (2) low-frequency (LF) component (around 0.1 Hz), which
represents the so-called 10-s rhythm (Mayer waves) associated with the baroreceptor
reflex; and (3) high frequency (HF) component (around 0.25 Hz), which is related to
respiratory activity. The very-low-frequency heart rate fluctuations are thought to be
98
mediated primarily by the sympathetic system, and the LF fluctuations are
predominantly under sympathetic control with vagal modulation, whereas the HF
fluctuations are under parasympathetic control.260
Because spectral analysis is carried out under resting conditions, it has the
advantage that active cooperation of the patient is not required. Numerous factors may
influence the test results: age, heart rate, respiratory rate, BP, eating, drinking coffee,
smoking, body position, volume status, mental stress, drugs, exercise, and time of
day.261, 262
2.3.7.3 Diagnosis assessment for Diabetic Peripheral Neuropathy

A complete physical examination with shoes and socks removed should be
done to screen and assess vibration perception (using a 128-Hz tuning fork), light
touch (cotton wisp), pressure (10-g monofilament), superficial pain (sterile safety
pin), temperature sensation, ankle reflexes and joint proprioception test. A 10-g
filament, which is widely available, disposable, easy to use and it is very useful in
assessing protective sensation at the distal halluces and identifying the risk of foot
ulceration.
Its sensitivity alone ranges from 20% to 64% and likely improves if multiple
sites on the foot are tested (8 sites recommended). Combinations of more than one test
have nearly 87% sensitivity in detecting DPN. Weakness of small foot muscles
(extensor hallucis longus and extensor digitorum brevis) should be assessed.
Peripheral pulses, resting pulse and blood pressure on lying and standing should be
measured. The feet should be examined for ulcers, calluses, and deformities, and
footwear should be inspected. Indeed, longitudinal studies have shown that a simple
clinical examination is a good predictor of future foot ulcer risk.
99
Therapeutic and Laboratory studies should be ordered as dictated by clinical
findings and might typically include a complete blood count, general chemistries,
HbA1c levels, serum B12, thyroid function, blood urea nitrogen, serum creatinine,
antinuclear antibodies, sedimentation rate, rheumatoid factor, and urine protein
electrophoresis. Nerve-conduction studies evaluate whether the myelin or nerve axon
is affected and helps to pinpoint a specific diagnosis. It demonstrates slowing of
conduction velocity across the compressed segment (ulnar, fibular, tibial nerve) or
increased distal latency compared to nearby nerves (median nerve) in compression
mononeuropathies.
Electromyography can distinguish if weakness is from a nerve or muscle
disorder. Nerve biopsy is reserved for patients with severe and progressive disease
and can helps to identify vasculitis, amyloidosis, sarcoidosis, and several types of
hereditary neuropathies. After thorough evaluation, treatment is geared to the
underlying etiology.263
All patients with diabetes should be screened for DPN at diagnosis of type 2
diabetes and 5 years after the diagnosis of T1DM and at least annually by examining
sensory function in the feet and checking ankle reexes. One or more of the following
can be used to assess sensory function: pinprick, temperature, and vibration
perception (using a 128-Hz tuning fork), or pressure sensation (using a10g monolament pressure sensation at the distal halluces). Any history of neuropathic
symptoms should be elicited and a careful clinical examination of the feet and lower
limbs should be performed. 263, 264
2.3.8 Treatment for Diabetic Neuropathy
The first step in the management of DN is tight glycaemic control and correction of
any associated other metabolic derangements. According to Diabetes Control and
100
Complications Trial (DCCT) committee, strict glycaemic control not only decreased
the incidence of neuropathy but also slowed its progression by 57%.265
i. Treatment for Autonomic neuropathy

Management of Orthostatic Hypotension consists of educating the patient
regarding strategies to avoid or address reversible causes of hypotension by raising
the head end of the bed, increasing salt intake to 1020 g/day, small frequent meals,
two cups of strong coffee, increased fluid intake, wearing clothing such as
compression/elastic stockings that increase venous return and by having
Fludrocortisone 200 mg or Ibuprofen 400 mg thrice daily (better tolerated than
Indomethacin) or Sympathomimetic agents. Antioxidants and Cardio selective Betablockers may be beneficial in CAN.
Proton Pump therapy is conventionally used for patients with esophageal
dysmotility. Fluid consumption immediately after consumption of medications should
be advised in order to avoid pill-induced esophagitis in this patients.266
Diets low in fat and soluble fibre may be beneficial in patients with
gastroparesis, although pharmacotherapy with Prokinetic agents (Metoclopramide,
Erythromycin and Domperidone) is the mainstay of therapy.
Domperidone may now be regarded as the current first-line agent owing to
adverse effects with Metoclopramide and Erythromycin. The management of
diarrhoea, constipation and faecal incontinence is still largely symptomatic once
specific other causes like Celiac disease and Exocrine Pancreatic Insufficiency are
excluded.
Management
of
Erectile
dysfunction
should
include
Psychological
Counselling; however, pharmacotherapy with the PDE5 inhibitors (Sildenafil,
101
Vardenafil and Tadalafil) is the mainstay of therap provided there is no
contraindication to its use. Treatment of bladder dysfunction may be behavioural
(bladder retraining, pelvic floor exercises and fluid intake schedule), pharmacological
(antimuscarinic agents, cholinergic agents, tricyclic antidepressants, alpha adrenergic
agonists and Baclofen), or surgical (selective pudendal nerve block). Treatment of
choice for neurogenic bladder dysfunction with partial or complete urinary retention
or a contractile bladder remains clean intermittent catheterization.267, 268
ii. Treatment for DPN
The ideal therapy should be directed at preventing or arresting the progressive
loss of nerve function and improving symptoms with minimal side effects. However,
once pain develops, current treatment options are not specific for the underlying cause
of nerve damage and are aimed often only at partially alleviating the symptoms due to
significant adverse effects.
Evidence-based treatment guidelines in Western countries268,
269
and in the
MER consistently recommended Pregabalin and Gabapentin as first-line treatments

for painful DPN, with Duloxetine as a second-line treatment. However, various
evidences support the use of Tricyclic Antidepressants as the first-line treatment
unless there are contraindications. Because patients often have multiple comorbidities,
physicians must consider potential adverse effects and possible drug interactions
before prescribing a medication. Thus, treatment must be individualized for each
patient based on efficacy, side effects profiles and drug accessibility including
cost.268-270
Tricyclic antidepressants (TCA): Amitriptyline and Imipramine has been
confirmed in several randomized controlled trials. An updated Cochrane
review of Antidepressants for treating neuropathic pain revealed an overall
102
effectiveness, with a Number Needed to Treat (NNT) of 1.3 for diabetic
neuropathy based on five studies involving TCAs. Although TCAs are
generally affordable and effective, but one in five patients discontinues
therapy because of adverse effects. 271,272
Serotonin-norepinephrine reuptake inhibitors (SNRIs) and Selective serotonin
reuptake inhibitors (SSRIs): SNRIs (Venlafaxine and Duloxetine) are a
promising category of antidepressants for treatment of DPN pain. They are
better tolerated and have fewer drug interactions than TCAs. A 2007 Cochrane
review examined three studies of Venlafaxine for neuropathic pain, revealing
a NNT of 3.1.273
Anticonvulsants: Gabapentin and Pregabalin are recently confirmed in RCT as
first line treatment for DPN pain268-269. They are alpha 2- delta inhibitors
which act on the dorsal horn of the spinal cord to inhibit voltage gated calcium
channels. A 2005 Cochrane review evaluating the use of Gabapentin in painful
neuropathy calculated a combined NNT of 4.3 from five studies of DPN pain
and two studies of postherpetic neuralgia and mixed neuralgia.306 Pregabalin is
the second agent approved by the FDA for the indication of PDN, while,
Duloxetine being the first approved agent. Pregabalin has been evaluated in
three parallel, placebo-controlled studies in the treatment of PDN with the
results of NNT of 4.2. The advantage of Gabapentin and Pregabalin is their
renal excretion and lack of interaction with other medications. Pregabalin is
effective when given twice daily, in contrast to Gabapentin, which is usually
given in three daily doses. Main side effects include drowsiness, dizziness,
peripheral oedema, weight gain, and myoclonic jerks at higher doses.274
103
Other agents: N-methyl-D-aspartate (NMDA) antagonists (Dextromethorphan,
Memantine,
Amantadine),
Opioids
(Morphine
Sulfate,
Tramadol,
Oxycodone), Antioxidant (alpha Lipoic Acid), Anti-Arrhythmic agents

(Mexiletine), Capsaicin cream/patch and Isosorbide Dinitrate spray are other
options for the treatment of DPN.
Combination therapy: If combination therapy is necessary, physicians should
consider the mechanism of action when choosing medications and consider
consulting a pain management specialist or neurologist. It is important to
avoid combining TCAs with SSRIs or SNRIs to avoid Serotonin Syndrome, a
life threatening condition with autonomic and neurologic symptoms.
Combination therapy with Opiates can be considered, where, one study
showed a decreased need for Opiates when combined with Gabapentin.275, 276
104
Material and Methods

3.1 Study design and sampling methods
3.1.1 Study design: A community based cross-sectional study.
3.1.2 Study Setting and duration:
The study was carried out in three Primary Health Center (PHC) areas of
Handignur, Vantamoori and Kinnaye of North Karnataka associated with J. N.
Medical College, Belgaum, during August 2009 to August 2012.
3.1.3 Ethics approval:
The study was approved by the JNMC Institutional Ethics Committee.
Informed consent was taken from all participants before enrolling into the
study.
3.1.4 Inclusion criteria:
Individuals of age 30 years and above.
Individuals with symptoms of T2DM.
Known cases of T2DM (FBG 126mg/dl).
3.1.5 Exclusion criteria: Individuals who declined to provide informed consent.
Pregnant women / who had delivered a baby weighing 4.5Kg / women
who had gestational diabetes.
Individuals with T1DM.
Individuals with cognitive, neurological, psychological and endocrinal
disorders.
Individuals with congenital heart diseases.
105

3.2 Sample Size:
The sample size was calculated based on reported prevalence in rural areas
(lowest being 3.8%) that are geographically and socio-culturally similar to the study area.
Considering absolute error of 1% with 95% confidence level, (inflation factor for cluster
sampling considered as 2) the sample size was estimated to be 3000.51,53
3.2.1 Estimation of sample Size

The sample size of 3000 individuals was calculated as below:
n = 4pq/d2
p = Anticipated prevalence of 3.8% based on previous studies.
q = 100-p
d = absolute error (1%) with 95% Confidence Interval (CI)
n = 4 x 3.8 X (100-3.8) / (1)2
n= 1462.22
Inflation factor for cluster sampling was considered as 2. Hence,
n = 1462.22 X 2 = 2924.44 = 3000
Final sample size n = 3000
106

3.3 Study area and Sampling Method:-
Figure 3.3.1: Population Details
Study area population = 1,16,041

Handignur
Vantamoori
Kinnayae
25,606
(22%)
57,055
(49%)
33,380
(29%)
The study area (Annexure VI: Photograph 1, 2, 3) covered population of 1, 16,041 under
three PHCs (Handignur, Vantamoori and Kinnayae) spread over 18 sub centers which
included 58 villages.
Sampling was done by using multistage cluster sampling method. It included three stages
as:
1st stage PHC level (Flow chart-1)
2nd stage Sub centers level (Flow chart 2, 3 and 4)
3rd stage Village level (Flowchart - 2, 3 and 4)
Sample size estimation formula for different PHC areas
Sample size of PHC area = Population of PHC X 3000/ Population of study area
107

3.4 Sampling distribution plan:
Flow chart 1:- Multistage cluster sampling at three PHC area

Total Screening
Sample size
(n = 3000)
Multistage Cluster
Sampling
(At three PHCs area)
Handignur PHC
area
Population (25606)
Vantamoori PHC
area
Population (33380)
[Screening samples
n1= 662]
[Screening samples
n2= 863]
108
Kinnayae PHC
area
Population (57055)
[Screening samples
n3= 1475]

A. Handignur PHC area:
Multistage cluster sampling was done in Handignur PHC area under four sub
centers and respective villages given below:Flow chart 2: Multistage cluster sampling at Handignur PHC area
Handignur PHC area
Screening samples (n1) = 662
ccc
Multistage Cluster
Sampling
1. Kangrali sub
center (322)
a. Kangrali
(271)
b. Alatga(51)
2. Agasaga sub
center (125)
a. Agasaga(76)
b. Malenatti(19)
c. Chalwaenatti(30)
3. Handignur sub
center (110)
a. Handignur(68)
b. Kurihal (7)
c. Bodkyanatti
(35)
4. Shivapur sub
center (105)
a. Shivapur(25)
b. Ningyanatti
(13)
c. Idalhond(7)
d. Gurmanatti (3)
e. Parshiwad (3)
f. Sutagatti (33)
g. Marangaol
(21)
Screening for type 2 diabetic mellitus:

o 606 participants were done
Screening for autonomic dysfunction:
o 93 diagnosed T2DM participants were done.
Non respondents :- 56 participants (due to migration, personal problem
and death)
109

B.
Vantamoori PHC Area:

Multistage cluster sampling was done in Vantamoori PHC area under five
sub centers and respective villages is given below:Flow chart 3:- Multistage cluster sampling at Vantamoori PHC area
Vantamoori PHC area
Screening samples (n2) = 863
Multistage Cluster
Sampling
Screening for type 2 diabetic mellitus:

o 786 participants were done
Screening for autonomic dysfunction:
o 140 diagnosed as T2DM
participants were done.
Non respondents 77 (due to migration,
personal problem and death)
110
1. Vantamoori (181)
a. Vantamoori (126)
b. Halbhavi (13)
c. Bomanatti (12)
d. Irbhavi (08)
e. H Hosur (22)
2. Bhutramatti (131)
a. Gugranatti (19)
b. G hosur (12)
c. Ukkad (09)
d. Bhutramatti (57)
e. Ramdurg (10)
f. Bennali (10)
g. Darsarwadi (10)
h. Dgodihal (04)
3. Honaga(186)
a. Honaga (172)
b. Jumanal (14)
4. Kakti A(203)
5. Kakti B (162)
a. Sonatti (20)
b. Kakti B (142)

C.
Kinnayae PHC area

Multistage cluster sampling was done in Kinnayae PHC area under
nine sub centers and respective villages is given below:Flow chart 4: Multistage cluster sampling at Kinnayae PHC area
1.
Kinnayae PHC area
(Screening samples (n3) =
1475)
2.
Multistage cluster
Sampling
3.
4.
5.
6.
Screening for type 2 diabetic

mellitus:
o 1, 292 participants were done
Screening for autonomic
dysfunction:
o 241 diagnosed as T2DM
participants were done.
Non respondents 183 (due to
migration, personal problem and
death)
111
7.
8.
9.
Desur (142)
a.
Desur (115) b. Zadshapur (27)
Karle (136)
a.
Janewaadi (13)
b.
Navage(48)
c.
Balanmatti (14)
d.
Bomanwadi (11)
e.
Kutalwadi(12)
f.
Karle (38)
Macche-I(124)
Macche II (165)
a.
Tipu nagar (80)
b.
Macche-II (85)
Santibasatwad(161)
a.
Basaweshvar nagar (15)
b.
Kalenatti (15)
c.
Santibasatwad (131)
Waghwade (139)
a.
Markandya nagar (09)
b.
Walmiki nagar (53)
c.
Waghewade (77)
Kinnayae (127)
a.
Badarwadi (34)
b.
Kinnayae (58)
c.
Ranekunde (35)
Peeranwadi (268)
Khaderwadi (213)
a.
Devendra nagar (21)
b.
Khaderwadi (81)
c.
Rajaram nagar (22)
d.
Hanumanwadi (22)
e.
Brahmnagar (22)
f.
Hunchenatti (45)

3.5 Study Protocol:
1. The identification of localities and households was done with help of Auxiliary
Nurse Midwives (ANMs).
2. The selection of the lane and first house within locality was done by random
selection by employing procedures described in the clustered sampling technique
used for evaluation of universal immunization coverage.277
3. Starting from each 4th house onwards all the houses within the lane were covered
continuously, keeping towards the left. This procedure was continued until the
sample size for each sub center was obtained.
4. During house visit, the objectives of the study were explained to the eligible
household members.
5. Eligible subjects who were unavailable during first visit were approached on
another pre-informed date as per their convenience. Even after two such visits if
the subject was unavailable, then he/she was considered as a non-respondent.
6. The purpose and procedures of the study were explained to the study participants
and the subjects were asked to participate voluntarily. Informed consent was
obtained and participants were enrolled in the study.
7. Personal, face to face interview was taken by using predesigned questionnaire /
investigation proforma (annexure III).
8. Assessment of T2DM, cardiovascular autonomic neuropathy and diabetic
peripheral neuropathy was done by using standard methods.
112

3.6
Personal details and History:

a. Literacy and occupation status: The participants were interviewed with a predesigned questionnaire / investigation proforma (Annexure-III, Annexure VI:
Photograph IV) regarding identification, demographic details, behavioral
components, social and biological variables.
Literacy: - Education was classified based on International Standard

Classification of Education (UNSCO, 1997). Literacy was categorized as
Illiterate, Primary school education, Secondary school education,
Graduates and above. 278
Occupation: - The occupation of study participant was classified as

workers and non-workers as per census of India 2001. Further workers
were subdivided based on their occupation such as Skilled-I to Skilled
IV. (Govt. of India Report, 2004).279,280
Workers : Skilled-I: Agriculture, Skilled II: Laborer, Skilled III:
Associate professional (Self Employee), Skilled IV: Higher
professionals (Govt employee)
Non workers: Homemaker and Elderly persons (who are not
involved in active working) 279, 280
b. Smoking and alcohol:

Smoking pattern such as smokers and nonsmokers was noted. The alcohol
consumption pattern of alcoholic and nonalcoholic was noted.281
113

c. Family History of Diabetes
Detailed family history of T2DM was taken. This was verified either
by blood glucose measurement of the parents or in the person's
absence, by other circumstantial evidences (physician report, diet
modifications, consumption of drugs).
Known cases of T2DM were included in the study. Duration of

diabetes and medication details was noted.
In the present study, if the response was "diabetes status of parents

not known", it was assumed to be "No family history of DM.,282
3.7
Anthropometrical Measurements 283, 284, 285, 286

Anthropometric
measurements
(AnnexureIII)
included
height, weight, waist circumference, hip circumference and WaistHip Ratio

i.
Weight (Kg): Weight was recorded by using a standard weighing

scale (Krups weighing scale) that was kept on firm horizontal
surface. Weight was recorded to the nearest 500gms.
ii.
Height (cm): Height was recorded using a measuring tape to the

nearest 1 cm. participants were requested to stand upright without
shoes with their back against the wall, heels together and looking
forward.
iii.
Body mass index (BMI):- BMI was recorded using Quetelets

equation. [Weight (kg)/height (m2)].
114

Classification of BMI :- 18.9 kg/m2 Underweight, 19-24.9 kg/m2
Normal, 25-29.9 kg/m2 Pre-obese (overweight) and 30kg/m2
Obese
iv.
Waist circumference (cm) : Waist circumference was measured to

the nearest 0.1 cm at the mid-point between the costal margin and
iliac crest using a non-stretchable measuring tape, at the end of
normal expiration with the participant standing erect in relaxed
position, feet 25-30 cm apart.287
v.
Hip circumference (cm):- Hip circumference was measured at the

level of greater trochanters (widest position of hip) to the nearest
0.1cm with a measuring tape, while the participant was standing with
the arms by side and feet together.287
vi.
Waist-Hip ratio: - Waist-Hip ratio was calculated as the ratio of

waist circumference and hip circumference.287
vii.
Central Obesity: - Central/abdominal obesity was considered to be

present when waist circumference 90 cm in males and 80 cm in
females.287
viii.
Truncal obesity:- Waist-Hip ratio of > 1.0 for males and > 0.85 for
females was defined as truncal obesity.288
115

3.8 Biochemical screening method for Type 2 Diabetes mellitus
3.8.1 Fasting blood glucose (FBS) measurements 286, 289
Blood glucose estimation was done for all participants irrespective of
whether they had diabetes or not.
The subjects were asked to be on overnight fasting (for 8 hrs). Next
morning, after confirming fasting, blood glucose was measured.
On a pre-informed date, fasting blood glucose (FBS) was estimated (after
overnight fast) in morning by using a standard digital glucometer (Omni
test Plus B-Brown, Germany).
All those participants, who had fasting blood glucose more than 126 mg/dl
were considered as diabetic as per WHO criteria. FBS was measured by
using standard glucometer as per the WHO recommendation for
epidemiological purposes.290 (Annexure VI: Photograph 5, 6)
3.8.2 Glucometer (standardization/calibration)

Glucometer was standardized by (as per ISO criteria) cross-checking with
the laboratory results (Biochemistry Laboratory of KLES Hospital &
Medical Research Centre, Belgaum). Difference in the glucometer and the
laboratory readings was not above 1% and correlation coefficient was
found to be 0.8.
Glucometer (Omnitest Plus of B brown, Germany, Annexure VI photograph - 5) was calibrated by using following method:a. Date and time on meter was calibrated
b. Code on strip was checked
116

c. Matching of code on strip and meter was done
d. Selection of finger pricking method was done (Capillary
method)
e. Collection of fasting blood sample was done.
f. Fasting blood sample was applied on strip
g. Results were obtained and recorded
h. Calibration was done at ambient temperature and humidity was
checked.
3.8.3 Testing Glucometer accuracy 288:
1. Glucometer accuracy standards were set by referring guidelines of
International Organization for Standardization (ISO) 15197.
2. Clark Error Grid was referred to analyze and display accuracy.
3. Glucometer accuracy was tested by analyzing self blood sample (fasting)
on Glucometer (Omnitest Plus) and self blood sample was also given at
KLE biochemistry laboratory (laboratory test) at same time (9.55am),
results was found given below:
Table 3.8.3.1 : Testing Glucometer accuracy

Sr No.
Biochemical tests
FBS (mg/dl)
Glucometer test method (1st Sample)
94
Laboratory test Glucose Oxidase peroxides
98
(GODPOD) method ( 2nd Sample)
117

3.9 Blood pressure measurement (mmHg)
Blood pressure was measured in the left arm in sitting posture, with the
participant in a relaxed state. Standardized mercury sphygmomanometer
(Diamond deluxe BP apparatus, Pune India) with adult size cuff was
used. The first appearance of (phase I of Korotkoff sounds) sound was
used to define systolic blood pressure (SBP). The disappearance of sound
(phase 5) was used to define diastolic blood pressure (DBP). Study
participant was considered to be hypertensive if he/she was an already
diagnosed case of hypertension and /or on treatment or with a current
SBP of > 140 mmHg or DBP > 90 mmHg (as per JNC VII criteria). 291
3.10 Assessment of Diabetic Cardiac Autonomic Neuropathy HRV analysis

3.10.1 Baseline parameters and Heart Rate Variability analysis (HRV) 292
a. ECG (lead II ) recording was done for 5 min (short term method) by
using
INCO
two
channel
Digital
Physiograph
Model:EPR-2
manufactured by NIVIQUIRE, data acquisition system, version 56.1.1,

Bangalore instrument and chemicals Pvt Ltd., Model town, Ambala city
134003, Haryana (India).
b. Basal Heart rate (HR) Participant was instructed to lie down in
supine position for 10 minutes and avoid talking. HR was recorded in
beats per minute (bpm) using ECG (lead II) connection by Digital
Physiograph.
118

c. Basal Blood pressure (mmHg):- The participants systolic blood
pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were
measured on the contralateral arm by using Omron digital BP apparatus.
(As described by Walker et al 1992 using a semi-automated BP monitor
Omron digital BP monitor model 11 EM-403c, Tokyo Japan).
3.10.2 Protocol for Heart Rate Variability analysis (HRV):
1. Participants were asked to come to primary health center or nearby sub
centers according to their convenience between 9 am to 5 pm.
2. Participants were asked to abstain from smoking, alcohol, caffeine / tea
consumption and strenuous physical exercise 2 hrs prior to tests.
3. Participants were told about precautions to be taken and procedure was
explained to them before recording.
4. ECG (lead II) electrode was connected and basal heart rate and blood
pressure (baseline recording) were recorded in supine posture with
participant lying on the couch.
5. ECG (lead II) was recorded for 5-10 min in supine posture and HRV
parameters were analyzed. (Annexure VI: Photograph 7, 8)
3.10.3 Parameters analyzed (HRV analysis)
a. Time domain methods:a. Basal HR (bpm)
b. R-R interval (ms)
c. SDNN (ms) (standard deviation of NN intervals)
119

d. RMSSD (ms) (square-root of mean squared differences of
successive NN interval)
e. pNN50 (%) (Proportion derived by dividing NN50 by the total
number of NN interval).
f. Triangular Index
b. Frequency domain methods:a. Low frequency (LF Peak) in ms2
b. High frequency (HF peak) in ms2
c. LF (nu)
d. HF(nu)
e. LF/HF ratio
Figure 3.10.1: - Flow chart summarizing individual steps used when recording and
processing the ECG signal in order to obtain the HRV data292
120

3.11
Assessment of Diabetic Cardiovascular Autonomic Neuropathy Ewings

test criteria 293
3.11.1 : General precautions - before conducting autonomic function tests:
1. Before assessment of autonomic function test, participants were
instructed to have a light breakfast in the morning at around 8.00 am.
2. Participants were asked not to take tea/coffee or other beverages on the
day of test, which are likely to influence the autonomic function.
3. Participants were advised to remain unperturbed throughout the test.
4. Uniform protocol was maintained for each test for each participant.
5. After one test, second test was started when baseline parameters were
attained. To achieve that, between the two tests, participants were
allowed to relax for minimum of 10 min.
3.11.2 Baseline parameters:

The baseline parameters of participants recorded were heart
rate and blood pressure, which were taken as control or baseline value.
These parameters were also recorded again after complete rest of 10
minutes between two autonomic function tests.
1. Blood Pressure (BP):- BP was recorded with the help of automatic
blood pressure monitor OMRON model HEM-7111 (manufactured by
OMRON HEALTH CARE Co., Ltd. 24, Yamanouchi Yamanoshitacho, Ukyo-ku, Kyoto, 615-0084, JAPAN).
121

a. Specifications of OMRON BP apparatus ( HEM-7111):
i. Method used oscilloscopic method
ii. Range for pressure: 0 to 299 mm of Hg and for pulse:
40 to 180/min
iii. Accuracy for pressure: 3 mm of Hg and for pulse:
5% of display reading
2. ECG recording: - ECG was recorded with the help of INCO II
channel Digital Physiograph model: EPR2 [manufactured by
NIVIQURE, Bangalore and Instruments & chemicals Pvt.Ltd. Model
town, Ambala City 134003, Haryana (INDIA)]
3. Hardware specification:
Computer interface unit (RS 232C interface circuit)
Signal processing unit
ECG bio-amplifier module for conventional ECG recording
4. Software specifications:
INCO-NIVIQURE digital acquisition system. Ver 52.0. This
NIVIQURE is Windows 97 based ECG/Pulse/EEG/EMG
instrument.
122

3.11.3 AUTONOMIC FUNCTION TESTS 293
All participants underwent through five different autonomic function
tests according to Ewings and Clarke criteria as given below:
1. HR response to standing (30:15 ratio)
2. HR response during deep breathing (DBT)
3. Valsalva manoeuvre (Valsalva ratio) (VM)
4. BP response to sustain handgrip [Handgrip dynamometry test (HGD)]
5. BP response to standing (Postural Hypotension/OTT)
6. Cold pressor test (CPT) (exceptional test)
These tests were divided into two categories depending on the involvement of
sympathetic and parasympathetic divisions.
1. Category 1 (Parasympathetic tests):- These tests were added into category-1 on
the basis that these tests evoke reflex autonomic responses at the subcortical level
with involvement of parasympathetic system.
a. Heart Rate (HR) response to standing (30:15 ratio)
b. Controlled breathing / Deep breathing tests for sinus arrhythmia (Delta HR
and E:I ratio)
c. Valsalva maneuver
2. Category 2 (Sympathetic tests):- These tests were added into category-2 on the
basis of involvement of cortical influences to evoke autonomic responses and
involvement of sympathetic system.
a. Handgrip dynamometry (HGD)
b. Orthostatic Hypotension tests (OTT)
123

c.
Cold pressor test (CPT)
1. Parasympathetic tests293
A. Heart Rate and BP response to standing (orthostatic tolerance test including
30:15 ratio) - In this test autonomic stress is induced by changing the position from
lying down to standing. (Annexure VI photograph 9, 10)
Stimulus
- Change of posture from lying to standing
Afferents
- Baroreceptors and cranial nerve IX & X
Efferent
- Sympathetic (adrenergic), Parasympathetic (Cardiovagal, cholinergic)
Normal response - Initially increase in heart rate followed by decrease in heart rate, fall in
blood pressure
The patient was instructed about the test. The test was conducted after 10 min rest in
supine position. With the subject lying gently in the supine position baseline blood
pressure, heart rate was recorded. The subject was then asked to attain standing
position within 3 seconds singlehandedly for 2 minutes and after that to again come
back to supine position. Blood pressure was recorded at an interval of 30 sec. for 2
min. and also during the recovery phase. 30:15 ratio was calculated from ECG
recording obtained during standing.
Figure 3.11.3.1.1: ECG recording during HR response to standing

124

In this test after standing shortest R-R interval at or around 15th beat and the longest
R-R interval at or around 30th beat were measured and the heart rate response was
expressed as the 30:15 ratio was calculated as the ratio between the longest R-R
interval at or around the 30th beat and the shortest R-R interval at or around the 15th
beat.
Normal values:a) Fall of systolic BP 10 mmHg Normal, SBP = 11-29 mmHg Borderline and
SBP 30 mmHg Abnormal
b) 30:15 ratio: 1.04 Normal, 1.01- 1.03 Borderline and 1.0 Abnormal.
Physiology of the test: - The change of posture from lying to standing puts
hydrostatic stress on the venous return. The venous return decrease due to pooling of
blood in the lower limbs results in decrease in the blood pressure. The decrease in
blood pressure activates the baroreflex resulting in rise of the heart rate (between 10
20 seconds). The rise of heart rate raises the blood pressure towards the resting
values. The recovery of blood pressure results in the decrease in the heart rate later
(25 35 seconds).
Precautions:1.
It was ensured that subject stands up within 3sec from supine position.
Proper instruction was given in this respect.
2.
Subject was told to inform the investigator if he/she feels dizziness or is

uncomfortable during standing.
3.
Subject should not hold anything during standing.
125

4. The time interval for the BP measurements during the test should be
appropriately followed.
5. If subject feels dizzy and not able to stand more, subject should be requested
to sit comfortably.
B. Heart Rate response during deep breathing (DBT): In this test heart rate
changes during respiration is assessed. (Annexure VI photograph 11)
Stimulus
- Deep and regular breathing at the rate of 6 breaths per minute
Afferents
- Central
Efferent
- Parasympathetic (Cardiovagal, cholinergic)
Normal response -Increase in heart rate with inspiration and decrease in heart rate with
expiration
The subject was instructed about the test. The subject in sitting position was asked to
breathe smoothly, slowly and deeply at six breathes a minute (five seconds inspiration
and five seconds expiration) for one minute. The investigator gives a hand indication to
maintain the rate and timing of breathing. After taking baseline recording, ECG recording
was continued for throughout the test. The maximum and minimum R-R interval during
each breathing cycle was measured from electrocardiogram and converted to beats per
minute. The result of deep breathing difference was then expressed as the mean of the
difference between maximum and minimum heart rates for six measured cycles in beats
per minute. And E:I ratio was calculated by ratio of longest R-R interval during
expiration and shortest R-R interval during inspiration averaged over 6 breathing cycles.
126

Calculation:
a) Delta HR difference between the maximal and minimal Heart Rate during
inspiration and expiration respectively, average for 6 cycles.
b) E: I ratio: - Ratio of longest RR interval and shortest RR interval averaged
over 6 cycles.
Normal values:a) Delta HR - 15 bpm - Normal, 11-14 bpm - Borderline, 10 bpmAbnormal
b) E:I ratio - 1.21 Normal
Physiological basis of the test: - The variation of heart rate with respiration often
known as sinus arrhythmia is primarily mediated by the vagal innervations of the
heart. The neuronal output from the respiratory centre influences the gain of the
afferent and efferent outputs at the nucleus tractus solitarius. This type of respiratory
sinus arrhythmia mainly results from spillover of signals from the medullary
respiratory center into the adjacent vasomotor center during inspiratory and
expiratory cycles of respiration. Pulmonary stretch receptors as well as cardiac
mechanoreceptors and possibly baroreceptors also contribute in the regulation of
heart rate variations.
Precautions:
1. Signal should be given properly so that inspiratory and expiratory phase of
each cycle can be maintained for 5 sec.
2. Breathing of subject should be slow, smooth and deep which can be
confirmed from the respiratory tracing during the test.
127

3. If cycles are not appropriately done, it is repeated again in order to get 6
complete cycles.
4.
Test should not be conducted in patients with acute respiratory disease.
D. Valsalva maneuver In this test autonomic stress is induced by raising

intrathoracic pressure against open glottis. (Annexure VI photograph 12)
Stimulus
- Forced expiration through open glottis
Afferents
- Baroreceptors and cranial nerves IX and X.
Efferent
- Parasympathetic (Cardiovagal cholinergic) Sympathetic (adrenergic)
Normal response:
Phase I: rise in blood pressure, decrease in heart rate
Phase II: fall in blood pressure with rise late in the Phase II, increase in heart
rate (HR)
Phase III: fall in blood pressure
Phase IV: increase in blood pressure, decrease in HR
The test procedure was explained to the subject. In this test the subject sitting
comfortably was asked to blow out into mouthpiece connected to mercury
sphygmomanometer and subject was asked to breathe forcefully into mercury
manometer and have been told to maintain the expiratory pressure at 40mm Hg for
15 sec(during maneuver). A small air leak in system is useful to prevent the closure
of glottis during the maneuver. At the end of 15 seconds the pressure was released.
Appropriate care was taken to prevent deep breathing before and after the maneuver.
After taking the baseline ECG, recording was taken during and after the maneuver.
The result of Valsalva ratio was expressed as the ratio of longest R-R interval after
128

the maneuver to shortest R-R interval during the maneuver. Tachycardia ratio was
calculated by the ratio of shortest R-R interval during the maneuver to the longest RR interval before the effort.
Calculations
a) Valsalva ratio: Longest RR interval during Phase IV/Shortest RR interval
during Phase II.
b) Tachycardia ratio: Shortest RR Interval during the maneuver/Longest RR
interval before the maneuver.
c) Bradycardia ratio: Longest RR interval during the maneuver/ longest RR
interval before the maneuver.
Figure 3.11.3.2: ECG recording during Valsalva maneuver
Normal values:
Normal - Valsalva ratio (VR) 1.21, Borderline- 1.11-1.20 Abnormal VR 1.10
Physiological basis of the test: - Valsalva maneuver involves forced expiration

through open glottis. When the forced expiration is started (Phase I), the blood
pressure rises for a few beats along with decrease in heart rate (increase in RR
129

interval). With continued strain (Phase II) at 40 mmHg, the pressure in the thorax is
higher than the pressure in the great veins. As a result the venous return becomes
very low. This leads to drop in the blood pressure. The drop in the pressure is sensed
by the baroreceptors in the aortic arch and carotid sinus. The baroreceptor reflex is
initiated leading to vagal withdrawal and sympathetic stimulation. As a result the
heart rate increase (RR interval decreases). The blood pressure keeps falling during
the early phase II despite increase in heart due to falling stroke volume. However, in
the late phase II, the blood pressure may show increase. On the release of the
respiratory strain (Phase III), the blood pressure drops suddenly for few beats and
then rises again (Phase IV). The rise in blood pressure is due to sudden increase in
the venous return leading to overshoot above the baseline values. Due to
baroreceptor reflex, this rise is associated with the decrease in heart rate (increase in
the RR interval).
It is important to note that changes in the blood pressure during Phase I and
Phase III are purely mechanical events. Rise in heart rate during the phase II is
mediated initially with vagal withdrawal and subsequently by increase in
sympathetic outflow. The decrease in the heart rate in response to overshoot in Phase
IV is mediated by baroreceptor reflex (vagal).
130

2. Sympathetic Tests293
a. Handgrip dynamometry Test (HDT) In this test autonomic stress is evoked by
maximal voluntary contraction. (Annexure VI photograph 13)
Stimulus
- Isometric exercise
Afferents
- Myelinated mechanosensitive and unmyelinated chemosensitive
afferents from muscles

Efferent
- Sympathetic (adrenergic)
Normal Response - Rise of systolic and diastolic blood pressure, rise in heart rate
Apparatus used in this test was Jammar handgrip dynamometer (INCO,

AMBALA, INDIA; range 0-60 kg). The subject was instructed about the test and
demonstrated the procedure to use handgrip dynamometer. After recording baseline
heart rate and blood pressure in sitting position the maximum voluntary contraction
was determined using the dominant hand. The subject was then asked to maintain the
handgrip with dominant hand at 30% of maximum force for 2 minutes. The changes
in heart rate and blood pressure during handgrip were recorded on contralateral arm
at interval of 30 seconds for 2 min and also after the handgrip at the interval of 1 min
for 3 minutes.
Calculation:
a) Highest DBP during the tests
b) Basal DBP
131

Normal values:a) Increase in DBP 16 mmHg Normal
b) Increase in DBP 11-15 mmHg Borderline
c) Increase in DBP 10 mmHg Abnormal
Physiology of the test: - Voluntary muscle activity is associated with sympathetic
outflow to the cardiovascular system to increase the heart rate and blood pressure.
The rise in heart rate is also due to parasympathetic withdrawal and activation of
other central command. The accumulation of metabolites during the isometric
contraction initiates the exercise reflex resulting in sustained sympathetic activity.
Isometric exercise is associated with increased sympathetic outflow leading to rise in
diastolic pressure.
Precautions:
1. Subject should grip on dynamometer with maximal effort for the
measurement of MVC.
2. Investigator should monitor the subject properly throughout the HGT for the
sustained contraction of 30% of MVC.
3. The time interval for the BP measurement during the test and after the test
should be appropriately followed.
4. The subject should avoid the body movement during and after the test.
132

b. Cold pressor test In this test changes in heart rate and blood pressure after
immersing the dominant hand in cold water is tested. Here cold water temperature
acts as a stress. (Annexure VI photograph 14)
Stimulus
- Immersion of dominant hand in cold water (100C)
Afferents
- Nociceptive and cold receptor, pain and temperature pathways
Efferent
- Sympathetic pathways to circulation (adrenergic)
Normal Response
- Rise in blood pressure and heart rate
Apparatus used in this test was plastic tray containing ice, water and thermometer for
measuring the temperature of water. After recording baseline heart rate and blood
pressure subject was instructed about the test. Then the dominant hand of subject
was immersed up to the wrist in ice-chilled water at around 100 C for 2 min(care was
taken to see that subject does not touch the bottom of the cold water bath). Changes
of heart rate and blood pressure during the test were recorded on contralateral arm
every 30 sec for 2 min during the task and after the task every 1min for 3 min. After
the hand was removed from water, it was covered with towel immediately.
Calculation:
a) Highest DBP during test
b) Baseline DBP
Normal values: - Increase in DBP 16 mmHg Normal, 11-15- Borderline and
10 mmHg - Abnormal
133

Physiology of the test - The cold water causes stimulation of cold receptors and pain
receptors in the hand. The information is carried to the brain through spinothalamic
pathways. The reflex involves, rise in sympathetic outflow to the vasculature and
heart resulting in rise in blood pressure and heart rate.
Table 3.11.3.1:- Flow plan for performing tests of cardiovascular autonomic

function
Test
Position
Approximate
Apparatus required
time of tests
(min)
Heart rate response to
Sitting
Valsalva Manoeuvre
Heart rate variation
Aneroid manometer,
electrocardiograph
Sitting
Electrocardiograph
Sitting
Handgrip
during deep breathing

Blood-pressure response
to sustained handgrip
dynamometer,
sphygmomanometer
Immediate heart rate
Lying to
response to standing
standing
Blood-Pressure respond
Lying to
to standing
standing
134
Electrocardiograph
sphygmomanometer
Table 3.11.2.2.:- Cardiovascular autonomic function test with reference values

(Parasympathetic tests) 293
Heart rate tests
Variables
Normal
Borderline
Abnormal
Valsalva Maneuver
Valsalva Ratio
1.21
1.11-1.20
1.10
HR response to
standing
(30:15 ratio)
1.04
1.01-1.03
1.00
E:I ratio
1.21
1.20
Delta HR
(beats/min)
15
11-14
10
Deep Breathing tests
Table 3.11.3.3: Cardiovascular autonomic test with reference values

(Sympathetic tests) 293
Heart rate tests
Cold Presser test
Variables
Increased DBP
Normal
Borderline
Abnormal
16
11-15
10
16
11-15
10
10
11-29
30
(mmHg)
Increased DBP
Handgrip test
(mmHg)
Orthostatic
hypotension
Fall in SBP
(mmHg)
135

3.12: Cardiac Autonomic Neuropathy Scoring Ewing test criteria 293, 294
1. Categorization as per Ewings and Clarke criteria: - The participants were
subjected to five non-invasive autonomic function tests as recommended by
Ewings criteria and scored categorized as:
1. Normal: all five tests normal or one borderline.
2. Early involvement: one of the three heart rate tests abnormal or two
borderlines.
3. Definite involvement: two or more of the heart rate tests abnormal.
4. Severe involvement: two or more of the heart rate tests abnormal plus one
or both blood pressure tests abnormal or both borderlines.
The scoring were added and CAN was classified as per Ewing criteria as
follows
1. 0 1 = no CAN
2. 2 3 = early CAN
3. 4 6 = definite CAN
4. Above 6 = severe CAN
2. Categorization as per Bellavere criteria 293, 294
As per Bellavere criteria only following tests were used for scoring
1. Deep breathing test (Delta HR)
2. Valsalva maneuver (Valsalva Ratio)
3. Lying to standing (30:15 ratio)
The scoring were added and CAN was classified as per Bellavere criteria as:
0 1 = no CAN, 2 3 = early CAN 4 6 = definite CAN
136

3.13 Tests for assessment of Diabetic Peripheral Neuropathy (DPN) 295
1. Diabetic Neuropathy Symptom Score (DNS):

Subjects were instructed about the test. Subject was questioned
regarding the presence or otherwise of symptoms, either positive or negative
suggesting the presence of neuropathy. The questionnaire was the DNS score
which was an adapted version of the earlier version- Neuropathy symptom
score (NSS). The questions should be answered yes (positive - 1 point) if a
symptom occurred more times a week during the last 2 weeks or no
(negative- no point) if did not.
The DNS score has the following items: (i) unsteadiness in walking, (ii)
pain, burning or aching at legs or feet, (iii) prickling sensations in legs or feet,
and (iv) numbness in legs or feet. Presence is scored 1, absence 0, maximum
score 4 points. 0 - absence of polyneuropathy and 1-4 indicated presence of
polyneuropathy. (Annexure -IV,)
2. Diabetic Neuropathy Examination Scores (DNE):

Subjects were instructed about the procedure and test. This score was
based on a thorough neurological examination, similar to its earlier versionthe Neuropathy Disability Score (NDS). The DNE score consisted of eight
items, two testing muscle strength, one tendon reflex and five sensations. The
maximum score is 16. A score of > 3 points is considered abnormal.
137

Muscle strength: quadriceps femoris, tibialis anterior, reflex: ankle reflex,
sensation:-pinprick sensitivity, touch sensitivity, vibration perception and joint
position sensation was tested. Only the right leg and foot was tested.
The scores for each item include 0 - normal and 1 - mild/moderate deficit;
muscle strength: MRC scale 3-4; reflex: decreased but present; sensation: decreased
but present and 2- severely disturbed/ absent; reflex- absent; sensation- absent.
Maximum score was 16 points. A score of > 3 indicated presence of polyneuropathy.
(Annexure- V, Annexure VI- Photographs 15 to 20)
3.12 Statistical analysis

Data analysis was done by using Software Package of Social Sciences (SPSS)
trial version 16. Differences were considered significant at P 0.05 level with
confidence interval of (CI) 95%.
i.
The trends in the prevalence By descriptive statistics

a. Prevalence of T2DM in the study area
b. Prevalence autonomic dysfunction among the T2DM participants
in the study area.
c. Prevalence of diabetic autonomic neuropathy among the T2DM
participants in the study area
d. Distribution of
T2DM participants by sociodemogrpahic and
anthropometric characteristic
138

ii.
Comparison of numerical outcome like Age, Height, Weight, BMI,

Waist-Hip ratio, FBS, SBP and DBP in diabetic and non-diabetic
participants were assessed by using student unpaired t-test.
iii.
To study the association between various potential risk factors and

prevalence of T2DM, CAN and DPN
a. Association between various risk factors and prevalence of T2DM,
CAN and DPN was assessed by using chi-square test and logistic
regression analysis, considering diabetes as a dichotomous
outcome (dependant variable) and age, sex, occupation, literacy,
diet, BMI, truncal obesity, central obesity, smoking, alcohol
consumption, family history of diabetes and duration of diabetes as
independent variables.
b. Odds ratio were calculated to know the strength of association of
various risk factors with the outcome (dependant variable).
139
Results
A total of 3000 participants with 30 years of age meeting the eligibility criteria
were enrolled in the study. Among the total, 2684 were screened with response rate of
89.28% and data was analyzed. The results are presented under following headings:
4.1
Socio-demographic and Anthropometric characteristics of study

participants
4.2
Prevalence of Type 2 Diabetes mellitus (T2DM)
4.3
Association between various potential risk factors and prevalence of

T2DM
4.4
Risk factors of T2DM
4.5
Prevalence of CAN HRV analysis
4.6
Sympathovagal status of T2DM participants HRV analysis
4.7
Association between various potential risk factors and prevalence of

CAN HRV analysis
4.8
Risk factors of CAN HRV analysis
4.9
Prevalence of CAN Ewings and Bellavere criteria
4.10
Sympathovagal status of T2DM participants Ewings test criteria
4.11
Association between potential risk factors and prevalence of CAN

Ewings tests criteria and Bellavere criteria
4.12
Risk factors of CAN Ewings criteria
4.13
Distribution of T2DM participants by various cardiovascular reflex tests
4.14
Prevalence of DPN
4.15
Association between potential risk factors and prevalence of DPN
4.16
Risk factors of DPN DNE and DNS scoring method
140
Results
4.1: Socio-demographic and Anthropometric characteristic of study participants
Of the 2684 screened participants (mean age of 52.268.84 years), majority
belonged to age group of 40-49 year (32%), 60 years (31.4%) and 50-59 years (29.7%)
whereas the participants with age group of 30-39 years constituted a very less portion
(6.82 %). There were more females (63.2%) than males (Table 4.1.1) Nearly half (45.1%)
of the participants were illiterate and around two thirds of them (36.1%) had secondary
school education while very few had completed primary school education (14.6%) and
graduation (4.69%). (Table 4.1.2) Majority of participants were farmers (Skilled I 42.1%) and not involved in active work (Housewives and older people 32.9%).
Majority were consuming mixed diet (71.7%). Nearly two-thirds of the participants were
overweight (35%) and nearly half of them had both central and truncal obesity (47.2%).
(Table 4.1.3) Among the males, approximately half of the participants were smokers
(55%) and alcoholics (47%). Nearby, one fourth of the participants had family history of
diabetes (23.4%). (Table 4.1.4) The mean Age (P =0.002), Weight (P<0.001), BMI (P
<0.001), SBP (P<0.001) and Fasting blood sugar (P <0.001) were significantly higher in
T2DM participants as compared to non-diabetics. (Table 4.1.5)
4.2: Prevalence of T2DM

Out of 2684 screened, 474 (299 previously known cases and 175 newly detected)
were identified as T2DM with a prevalence of 17.7%. Among the three PHCs, Kinnaye
(18.7%) had highest prevalence followed by Vantamoori (17.8%) and Handignur
(15.3%). (Figure 4.2.1) Among the sub-centers, Khaderwadi (22%) had a highest
prevalence rate. (Figure 4.2.2)
141
Results
4.3: Association between Socio-demographic, anthropometric potential risk factors
and prevalence of T2DM
The prevalence of T2DM significantly increased with age (P< 0.001), highest
being in the age group of 60 years (21.2%). Prevalence was slightly higher in females
(18.3% Vs 17.03%) than males, which was not statistically significant. (Table 4.3.1)
Significant association was found between occupation (P< 0.001) and T2DM. Prevalence
was lowest among the agricultural workers (11.7%) and high among the non- workers
(23.4%). (Table 4.3.2) Statistically significant differences were also observed with type
of diet (P = 0.031), obese (P < 0.001), truncal obesity (P= 0.042), history of smoking (P <
0.001), alcohol consumption (P < 0.001) and with family history of diabetes (P < 0.001).
(Table 4.3.3, Table 4.3.4)
4.4: Risk factors of T2DM

Out of 11 factors analyzed, 8 potential risk factors had significant association with
prevalence of T2DM in bivariate analysis. After controlling potential confounding factors
by multivariate regression analysis, a total of 6 risk factors of T2DM were identified.
Age and occupation of the participants were significant constitutional risk factors
of T2DM. In comparisons with all the age groups, the age group 60 years had 6.62
times higher risk of developing T2DM. Nearly three and half fold of higher risk of
occurrence of T2DM was seen in skilled III (OR 3.58 CI 1.69-7.57) category while
Skilled IV (OR 2.44 CI 1.07-5.58) had 2.44 fold risk of getting of T2DM as compared to
other occupational categories. (Table 4.4.1)
142
Results
The participants consuming mixed diet (CI 0.92-1.79) were 1.29 times more
likely to have T2DM than vegetarians. Body Mass Index (BMI) was found to be an
individual significant risk factor for developing T2DM. Overweight (OR 14.70 CI 9.5222.72) and obese (OR 9.90 CI 3.24-30.30) had multiple times higher risk of developing
T2DM. For those having truncal obesity the chance of having T2DM was 1.14 times
higher (CI 1.05-1.89) than those without truncal obesity. (Table 4.4.2)
Smokers (OR 1.39 CI 0.76-2.31) and alcoholics (OR 2.23 CI 1.24-4.01) were at
higher risk in the development of T2DM in relation with non-smokers and non-alcoholic.
Family history was found to be important risk factor with 35 time greater chance of
developing T2DM as compared to those without family history of diabetes. (Table 4.4.3)
143
Results
Table 4.1.1: Distribution of study participants by age and gender
Characteristics
Category
Total number
Percentage
Age ( in years)
30-39
183
6.8
40-49
859
32.0
50-59
798
29.7
60
844
31.4
Male
987
36.8
Female
1697
63.2
Total
2684
100
Gender
Mean age : 52.268.84 years
144
Results
Table 4.1.2: Distribution of study participants according to literacy status and
occupation
Characteristics
Literacy
Category
Total number
Percentage
Illiterate
1211
45.1
Primary school
498
18.6
Secondary school
849
31.6
Graduation and
126
4.69
Skilled I
1137
42.4
Skilled II
283
10.5
Skilled III
93
3.46
Skilled IV
287
10.7
Non workers
884
32.9
2,684
100
above
Occupation
Total
145
Results
Table 4.1.3: Distribution of study participants by diet pattern, BMI, central and
truncal obesity
Characteristics
Category
Total number
Percentage
Veg
760
28.3
Mixed diet
1924
71.7
18.5-18.9
84
3.13
Mean BMI
19-24.9
1377
51.3
(25.103.83)
25-29.9
940
35
30
283
10.5
Yes
1266
47.2
No
1418
52.8
Yes
1268
47.2
No
1416
52.8
Total
2684
100
Diet
BMI
Central obesity
Truncal obesity
146
Results
Table 4.1.4: Distribution of study participants by smoking habits, alcohol

consumption and family history of diabetes
Characteristics
Category
Total number
Yes
547
20.4
No
2137
79.6
Yes
465
17.3
No
2219
82.7
Family History of
Yes
629
23.4
DM
No
2055
46.6
Total
2,684
100
Smoking
Alcohol
147
Percentage
Results
Table 4.1.5: Comparison of anthropometric & biochemical variables between
diabetic and non-diabetic participants
Variable
Category
Mean SD
P value
Age*
Non-diabetic
52.018.8
0.002*
T2DM
53.438.5
Non-diabetic
156.48.0
T2DM
154.78.0
Non-diabetic
59.67.9
T2DM
67.278.5
Non-diabetic
24.433.3
T2DM
28.254.3
Non-diabetic
0.880.06
T2DM
0.880.07
Non-diabetic
100.211.4
T2DM
128.235.1
Non-diabetic
125.410.5
T2DM
129.013.2
Non-diabetic
87.75.6
T2DM
85.58.4
Height
Weight*
BMI*
Waist-Hip Ratio
FBS*
SBP*
DBP
< 0.001*
< 0.001*
< 0.001*
0.075
< 0.001*
< 0.001*
0.390
*Significantly higher among T2DM participants (P < 0.05)
148
Results
Figure 4.2.1: Prevalence of T2DM in the study area
%
T
2
D
M
20
18
16
14
12
10
8
6
4
2
0
18.7
17.8
17.7
15.3
Handignur
Vantamoori
Kinnayae
Total prevalence
Handignur Vantamoori
Kinnayae
Total
prevalence
Study area
Figure 4.2.2: Sub-center wise prevalence of T2DM

25
21.8
17.7
20
15
10
15.316.2
18
22
21.221.9
20.7
18.717.7
18.4
18.217.8
18
17.7
16.4
15.9
14.7
13.4
10.2
5.6
Handignur phc
subcenters
Vantamoori PHC
subceners
149
Overall prevalance
Kinnaye PHC prevalance
Waghewade
Santibasatwad
Peeranwadi
Macche-II
Macche -I
Khadarwadi
Kinnyae PHC subcenters
STUDY AREA
Desur
Karle
Kinnayae
Vantamoori PHC prevalance
Kakati B
Kakati A
Honaga
Bhutramatti
Vantamoori
Handignur PHC prevalance
Shivapur
Kangrali
Agasaga
0
Handignur
%
T
2
D
M
Results
Table 4.3.1: Association between age, gender and prevalence of T2DM
Potential risk
factors
Sub- category
Age (Years)*
Number of study
participants
Total number
NonDiabetics
T2DM
30-39
160(87.43)
23(12.5)
183(6.82)
40-49
715(83.52)
144(16.7)
859(32)
50-59
661(83.77)
128(16.2)
789(29.7)
60
665(78.79)
179(21.2)
844(31.4)
2 = 12.486, df =3, P < 0.001*
Gender
Male
819(83)
168(17.02)
987(36.8)
Female
987(81.5)
224(18.49)
1211(45.1)
2 = 0.438, df =1, P = 0.438
Total
2210(82.33)
474(17.7)
2684(100)
Figures in parentheses indicate the percentage of respective frequency, df = degree of

freedom and 2 = chi-square value
* Potential risk factors significantly associated with prevalence of T2DM (P<0.05)
150
Results
Table 4.3.2: Association between literacy, occupation and prevalence of T2DM
Potential risk
factors
Sub-category
Number of study
participants
NonDiabetics
Literacy
Total
number
Diabetic
Illiterate
987(81.5)
224(18.49) 1211(45.1)
Primary
407(81.7)
91(18.27)
498(18.6)
Secondary
713(84)
136(16.01)
849(31.6)
Graduation
103(18.25)
23(18.25)
126(4.69)
and above
2 = 2.315, df = 1, P = 0.510
Occupation*
Skilled I
1004(88.3)
133(11.7)
1137(42.4)
Skilled II
56(80.2)
56(19.8)
283(10.5)
Skilled III
73(78.5)
20(21.5)
93(3.46)
Skilled IV
229(79.8)
58(20.2)
287(10.7)
Non workers
207(23.4)
207(23.4)
884(32.9)
2 = 51.051, df = 4, P < 0.001*
Total
2210(82.34)
474(17.7)
2684(100)

151
Results
Table 4.3.3: Association between diet, BMI, central obesity, truncal obesity and
prevalence of T2DM
Potential
predictors
Sub-category
Diet*
Number of study
participants
Total number
NonDiabetics
T2DM
Veg
645(84.9)
115(15.1)
760(28.3)
Mixed diet
1565(81.3)
359(18.7)
1924(71.7)
2 = 4.662, df = 1, P = 0.031*
18.9
79(94)
5(6)
84(3.13)
Mean BMI
19-24.9
1274(92.5)
103(7.5)
1377(51.3)
(25.103.83)
25-29.9
731(77.8)
209(22.2)
940(35)
30
126(44.5)
157(55.5)
283(10.5)
BMI*
2 = 3.979, df = 3, P < 0.001*

Central obesity
Yes
1045(82.5)
221(17.5)
1266(47.2)
No
1165(82.2)
253(17.8)
1418(52.8)
2= 0.068, df =1, P = 0.794

Truncal obesity*
Yes
1024(80.8)
244(19.2)
1268(47.2)
No
1186(83.8)
230(16.2)
1416(52.8)
2 = 1.259, df = 1, P = 0.042*
Total
2210(82.34) 474 (17.7)
2684(100)

152
Results
4.3.4: Association between smoking, alcohol consumption and family history of
diabetes with prevalence of T2DM
Potential
predictors
Sub-category
Number of study
participants
NonDiabetic
Diabetics
Total number
Smoking*
Yes
404(73.9)
143(26.1)
547(20.4)
No
1806(84.5)
331(15.5)
213(79.6)
2 = 33.994, df = 1, P < 0.001*

Alcohol*
Yes
340(73.1)
125(26.9)
465(17.3)
No
1870(84.3)
349(15.7)
2219(82.7)
2 = 32.891, df = P < 0.001*

Family History
Yes
221(35.1)
408(64.9)
629(23.4)
No
1989(96.8)
66(64.9
2055(46.6
of DM*
2 = 1.259, df = 1, P < 0.001*

Total
2210(82.34)
474(17.7)
2684(100)

153
Results
Table 4.4.1:- Socio-demographic risk factors of T2DM
Risk
factors
SubCategory
Age*
Univariate analysis
Multivariate analysis
OR
95%CI
P
value
OR
95%
CI
P value
30-39
40-49
1.41
1.09-1.81
0.007*
0.86
0.439
50-59
1.34
1.05-1.70
0.020*
0.97
60*
1.87
1.17-2.98
0.008*
6.62
0.601.25
0.871.39
3.4212.82
0.867
< 0.001*
2 = 12.486, df = 3, P < 0.001
Occupation*
Skilled I
SkilledII
SkilledIII*
SkilledIV*
NonWorkers
0.89
0.53-1.51
0.678
1.44
0.338
2.06
1.22-3.49
0.007*
3.58
1.08
0.61-1.91
0.788
2.44
1.11
0.62-1.97
0.721
1.72
0.683.03
1.697.57
1.075.58
0.753.93
2 = 12.51.051, df = 4, P < 0.001

*Risk factors of T2DM, CI = confidence interval and OR = odds ratio
154
< 0.001*
0.034*
0.200
Results
Table 4.4.2: Socio-demographic and anthropometric risk factors of T2DM
Risk
factors
SubCategory
Diet*
Veg
Univariate analysis
OR
95%CI
P value
OR
95%
P value
CI
1
1.29
0.921.79
0.134
0.031*
Non-veg
18.9
BMI*
1.29
1.02-1.62
2 = 4.662, df = 1, P = 0.031*
1
-
19-24.9
4.36
3.29-5.78
< 0.001*
4.48
2.966.80
< 0.001*
25-29.9
15.38
11.3620.83
< 0.001*
14.70
9.5222.72
< 0.001*
30
19.61
7.752-50
9.90
3.2430.30
< 0.001*
< 0.001*
2= 3.979, df = 3, P < 0.001*

Truncal
Obesity*
No
Yes
1.23
1.01-1.50
0.042*
1.41
1.051.89
0.022*
2 =4.140, df = 1, P =0.042*
* Risk factors of T2DM, CI = confidence interval and OR = odds ratio
155
Results
Table 4.4.3: Socio-demographic risk factors of T2DM
Risk
Sub-
factors
Category
Univariate analysis
OR
95%CI
P value
OR
95%
P value
CI
Smoking*
No
Yes
1.94
1.55-2.42
<0.001*
1.39
0.76-
0.331
2.31
2 = 33.994, df =1, P < 0.001
Alcohol*
No
Yes
1.97
1.56-2.50
<0.001*
2.23
1.24-
0.007*
4.01
2 = 32.891, df = 1, P < 0.001*
Family
No
Yes
35.96
27.60-
<0.001*
35.67
26.4-
<0.001*
History of
DM*
46.85
48.42
2 = 1.259, df = 1, P < 0.001*
* Risk factors of T2DM, CI = confidence interval, OR = odds ratio
156
Results
4.5: Prevalence of CAN and DPN
A total of 474 T2DM participants (299 previously known cases and 175 newly
detected cases) were identified (being prevalence of 17.7%) and were screened for the
assessment of autonomic dysfunction by using various screening methods (HRV analysis,
cardiovascular reflex tests and peripheral neuropathy examination). Hence, the data of
474 T2DM were analyzed to estimate the prevalence and related risk factors of CAN and
DPN. The results are presented accordingly.
4.5.1: Prevalence of CAN - HRV analysis

4.5.1.1: Time Domain Method
The overall prevalence of CAN was 63.7% among the T2DM participants.
Furthermore, out of the three PHCs, Kinnayae had highest prevalence as compared to
Vantamoori (63.6%) and Handignur (54.8%) (Figure 4.5.1)
4.5.1.2: Frequency Domain Method
Overall prevalence of CAN was 60.8% among the T2DM. In addition, between
three PHCs, Kinnayae (64.2%) had highest prevalence followed by Vantamoori (61.4%)
and Handignur (49.5%). (Figure 4.5.1)
4.6 : Sympathovagal status in T2DM participants

Mean RR, SDNN, RMSSD, pNN50, and HRV triangular index and High
Frequency (H.F) component were reduced (parasympathetic dysfunction) and LF/HF
ratio was increased (sympathovagal dysfunction) amongst T2DM participants. (Table
4.6.1)
157
Results
4.7 : Association between various potential risk factors and prevalence of CAN
4.7.1: Time domain methods
The proportion of CAN in T2DM participants increased significantly with
increase in the age (P < 0.001). Prevalence of CAN was highest amongst participants in
the age group of 50-59 years (88.3%) and 60 years (87.2%).
Males had higher
prevalence (70.8% Vs 59.8%) than females, which was found statistically significant
(P=0.012). There were no significant differences in the prevalence of CAN within the
participants who were smokers, alcoholics, having family history of diabetes, overweight,
obese and with truncal obesity. (Table 4.7.1.1) Participants consuming mixed diet had
higher prevalence (68.2% Vs 54.8%) than vegetarians, the relationship between diet (P =
0.017) and prevalence of CAN was statistically significant. Prevalence of CAN increased
significantly with increase in the duration of diabetes (P < 0.001). All the participants
with duration of diabetes 6-10 years had CAN abnormality. (Table 4.7.1.2) There were
no significant differences in the prevalence of CAN within the participants with respected
to smokers, alcoholics, family history of diabetes, obese and having central and truncal
obesity
4.7.2: Frequency Domain Method
Prevalence of CAN was higher in the age group of 50-59 (88.3%) and 60
(83.8%) than other age groups. Significant relationship was found between the age
groups (P < 0.001) and prevalence of CAN. (Table 4.7.2.1) Participants consuming
mixed diet had higher prevalence (75.7% Vs 24.3%) than vegetarians, which was
statistically significant (P = 0.017). Prevalence of CAN in relation to duration of diabetes
showed a rising trend. Longer duration of diabetes (P <0.001) were found to be
158
Results
significantly associated with the presence of CAN.(Table 4.7.2.2) There were no
significant differences in the prevalence of CAN within the participants with respected to
gender, smokers, alcoholics, family history of diabetes, obese and with central and
truncal obesity.(Table 4.7.2.1, Table 4.7.2.2)
4.8 : Risk factors of CAN HRV analysis

Out of 11 factors analyzed, only 4 potential risk factors (age, gender, diet and
duration of diabetes) had significant association with prevalence of CAN in bivariate
analysis. After controlling potential confounding factors by logistic regression analysis, a
total of 4 risk factors of CAN were identified.
4.8.1: Time domain method

As the age increases, risk of developing CAN also increases. In comparison with
the age group of 30-39 years, age group of 60 years (OR 71.22 CI 15.-62.45.45, P
<0.001) and 50-59 years had higher risk of developing CAN (OR 25 CI 13.7-45.45 P <
0.001) . Females were 0.64 times (OR 0.64 CI 0.42-0.99 P = 0.012) more likely to have
CAN than males. Participants consuming mixed diet (OR 0.58 CI 0.37-0.91 P = 0.017)
were at higher risk for having CAN as compared to vegetarians. All the Participants with
duration of diabetes 5 years (CI 686-11260, P < 0 .001) had 2779 times higher risk for
getting CAN. (Table 4.8)
159
Results
4.8.2: Frequency domain method
In comparison with age group of 30-39 years and 40-49 years, the age group of
60 years had 111.1 fold (OR 111.1 CI 15.-62.45.45, P < 0.001) and 50-59 years (OR
25.6, CI 14.3-47.2, P < 0.001) had 25.6 fold of higher risk of developing CAN.
Participants consuming mixed diet had 0.21 times higher chance for developing CAN.
Participants with duration of diabetes 5 years (OR 1896, CI 422-8504, P < 0.001) were
1896 times more likely to have CAN than to newly diagnosed T2DM. (Table 4.8)
160
Results
Figure 4.5.1: Prevalence of Cardiovascular autonomic neuropathy in study area
HRV analysis
% CAN(Frequency Domian Analysis)
61.4
% CAN(Time Domain Analysis)
64.7
63.6
67.2
60.8
63.7
54.8
49.5
Handignur
Vantamoori
Kinnayae
Overall prevalance
Table 4.6.1:- Parameters of HRV analysis reflect autonomic status in T2DM

participants
TEST
Methods
Tests parameters
Time domain analysis
Heart rate variability

analysis (HRV)
Frequency domain analysis
161
MeanSD
Mean RR(ms)
796.3537.50
SDNN(ms)
93.4831.91
RMSSD(ms)
14.67.7
pNN50 (%)
7.64.9
Mean HR(bpm)
82.047.0
HRV Triangular
index
16.19.7
HF peak(ms2)
578.9418.17
LF peak(ms2)
767.0522.98
HF(n.u)
23.634.4
LF(n.u)
47.872.3
LF/HF ratio
2.090.37
Results
Table 4.7.1.1: Association between age, gender, smoking, alcohol consumption,
family history of diabetes and prevalence of CAN Time domain method
Potential risk
factors
Age*
(Years)
Total Number
(n=474)
Parasympathetic
dysfunction
(CAN)
30-39
23 (4.9)
Abnormal
2 (8.7)
40-49
144 (30.4)
31 (21.5)
50-59
128 (27)
113 (88.3)
60
179 (37.8)
156 (87.2)
SubCategory
2 = 2.169, df = 3, P < 0.001*

Male
168 (35.4)
70.8
Female
306 (64.6)
59.8
Gender*
2 = 5.707, df = 1, P = 0.017*
No
331 (69.8)
214(64.7)
Yes
143 (30.2)
88(61.5)
Smoking
2 =0.057, df = 1, P = 0.847
Alcohol
No
349 (73.6)
218(62.5)
Yes
125 (26.4)
302(63.7)
2 = 2.887, df = 1, P = 0.089
Family History of
DM
No
95(20)
57(60.0)
Yes
379(80)
245(64.6)
= 0.709, df = 1, P = 0.400
Figures in parenthesis indicate the percentage of respective frequency, df = degree of
*potential risk factors significantly associated with prevalence of CAN (p< 0.05)
162
Results
Table 4.7.1.2: Association between diet, BMI, central obesity, truncal obesity and
duration of diabetes and prevalence of CAN- Time domain method
Potential risk factors
Parasympathetic
Sub-Category Total Number
dysfunction
(n=474)
(CAN)
Abnormal
Diet*
Veg
115 (24.3)
62 (53.9)
Mixed
359 (75.7)
240 (66.9)
2 = 6.308, df = 1, P = 0.012*
BMI
18.9
5(1.1)
2(40.0)
19-24.9
103 (21.7)
60(58.3)
25-29.9
209 (44.1)
140(67.0)
30
157 (33.1)
100(63.7)
2 =3.513, df = 3, P = 0.319
Central obesity
No
253(53.4)
162 (64.0)
Yes
221(46.6)
140 (63.3)
2 = 0.024, df = 1, P = 0.877
No
230 (48.5)
153 (66.5)
Yes
244 (51.5)
149(61.1)
Truncal obesity
2 = 1.525, df = 1, P = 0.217
Duration of DM*
(Years)
Newly detected
175 (36.9)
6 (3.4)
5Years
38(8)
36 (94.7)
6-10
123 (25.9)
123 (100)
11
138 (29.1)
137 (99.3)
60
179 (37.8)
156 (87.2)
2 = 4.364, df = 4, P < 0.001*

*Potential risk factors significantly associated with prevalence of CAN (p< 0.05)
163
Results
Table 4.7.2.1: Association between various sociodemographic variables and
prevalence of CAN Frequency domain method
Potential risk
factors
Sub-Category
Total number
Sympathovagal
dysfunction
(CAN)
Abnormal
30-39
23(4.9)
1(4.3)
40-49
144(30.4)
24(16.7)
50-59
128(27)
113(88.3)
60
179(37.8)
150(83.8)
Age*
(Years)
2 = 2.286, df = 3, P < 0.001*

Gender
Male
168(35.4)
111(66.1)
Female
306(64.6)
177(57.8)
2 = 0.263, df =1, P =0.079

Smoking
No
331(69.8)
205(61.9)
Yes
143(30.2)
83(58.0)
2 = 0.634, df = 1, P = 0.426
Alcohol
consumption
No
349(73.6)
208(59.6)
Yes
125(26.4)
80(64.0)
2 = 0.748, df = 1 , P = 0.387
Family
History of DM
No
95(20)
56(58.9)
Yes
379(80)
232(61.2)
2 = 0.164, df =1, P = 0.686

164
Results
Table 4.7.2.2: Association beween various socio-demographic and anthropoemetric
variables and prevalence of CAN Frequency domain method
Potential risk
factors
Sub-Category
Total number
Sympathovagal
dysfunction
(CAN)
Abnormal
Diet*
Veg
115(24.3)
59(51.3)
Mixed
359(75.7)
229(63.8)
2= 5.693, df = 1, P =0.017*
BMI
18.9
5(1.1)
2(40.0)
19-24.9
103(21.7)
61(59.2)
25-29.9
209(44.1)
132(63.2)
30
157(33.1)
93(59.2)
2 = 1.663, df = 3, P 0.645
Central
No
253(53.4)
151(59.7)
obesity
Yes
221(46.6)
137(62.0)
2 = 0.263, df = 1, P = 0.608
Truncal
No
230(48.5)
142(61.7)
obesity
Yes
244(51.5)
146(59.8)
2 = 0.180, df 1, P = 0.672
Newly detected
175(36.9)
2(1.1)
Duration of
5Years
38(8)
32(84.2)
DM*
6-10
123(25.9)
120(97.6)
(Years)
11
138(29.1)
134(97.1)
2 = 4.159, df = 3, P < 0.001*

165
Results
Table 4.8.: Sociodemogrpahic and anthropometric risk factors for CAN HRV
analysis
Risk
factors
Age*
(Years)
Duration
of DM*
(Years)
SubCategory
Time domain analysis
OR
95%CI
30-39
40-49
Frequency domain analysis
P value
OR
95%CI
2.88
0.45-4
4.40
0.718.21
50-59
25
13.745.45
25.6
14.347.2
60
71.22
15.62333.33
111.1
14.71000
Newly
diagnosed
5 Years
and above
2779
68611260
1896
4228504
Veg
< 0.001*
< 0.001*
< 0.001*
< 0.001*
0.017*
Diet*
0.017*
Mixed
0.58
0.370.91
0.21
0.1-0.44
Male
0.012*
Gender*
Female
0.64
0.420.99
0.079
0.60
0.380.93
OR = odds ratio, CI = confidence interval, * Risk factors of CAN (P < 0.05)
166
P value
Results
4.9 : Prevalence of CAN Cardiovascular reflex tests (Ewings tests criteria)
4.9.1 Ewing tests criteria

Overall prevalence of CAN was 56.8%. Out of three PHC areas, Kinnayae
(60.6%) had a higher prevalence than Vantamoori (49.3%) and Handignur (43%) In
addition, prevalence of early CAN (14%) was higher in participants from Handignur
PHC as compared to Kinnayae (12%) and Vantamoori (7.1%) (Figure 4.9.1.1)
4.9.2
Bellavare criteria
The total prevalence of CAN was 59.9 %. In three PHC centers, the highest
prevalence was found in Kinnayae (67.8%) as compared to Vantamoori (60.7%) and

Handignur (53.8%). Further, participants from Handignur (14%) showed higher
prevalence of early CAN than Kinnayae (10.8%) and Vantamoori (5.7%). (Figure
4.9.2.1)
4.10 Sympathovagal status among T2DM participants

Parameters reflecting parasypathetic status ( 30:15 ratio, Delta HR & Valsalva
ratio) and sympathetic status (postural hypotension, fall in SBP and fall in DBP) were
reducded, indicating the sympathovagal dysunction among the T2DM participants.
(Table 4.10.1)
167
Results
4.11. Association between various potential risk factors and prevalence of CAN
4.11.1: Bellavere criteria
Prevalence of CAN increased significantly with increase in the age (P < 0.001),
highest being in the age group of 50-59 years (58.2%) and 60 years (84.4%). There was
no statistically significant difference in the prevalence of CAN among the participants
with respect to gender, smoking, alcohol consumption, family history of diabetes, obese
and truncal obesity. (Table 4.11.1.1, Table 4.11.1.2) High prevalence was found to be in
the participants consuming mixed diet (63.8% Vs 47.8%) than vegetarians, which was
statistically significant (P = 0.005). Prevalence of CAN increased significantly with
longer duration of diabetes (P < 0.001). Participants with duration of diabetes 11 years
(97.8%) and 6-10 years (91.1%) had higher prevalence of CAN as compared to newly
diagnosed cases of T2DM (Table 4.11.1.2)
4.11.2: Ewing tests criteria
There was increase in the prevalence of CAN with increase in the age (P < 0.001).
(Figure 4.11.1) This association of different age groups with prevalence of CAN was
found to be statistically significant. Gender, smoking, alcohol consumption, family
history of diabetes, central and truncal obesity did not shown any significant relation with
the prevalence of CAN.(Table 4.11.2.1) Participants consuming mixed diet (63.8% Vs
47.8%) had higher prevalence than vegetarians, which was statistically significant (P =
0.005). There was significant association between duration of diabetes and CAN (P
<0.001) showing progressive increased in CAN with increase in duration of diabetes. The
prevalence of CAN in relation with duration of diabetes showed a rising trend. (Table
4.11.2.1 Table 4.11.2.2, Figure 4.11.2)
168
Results
4.12: Risk factors of CAN
Total of 11 factors were analyzed, out of which only 3 potential risk factors (age,
diet and duration of diabetes) had significant association with prevalence of CAN in
bivariate analysis. After controlling potential confounding factors by multivariate
regression analysis, only 2 risk factors of CAN were identified.
Participants with age group of 60 years were found to have 3.998 times more
likely to develop the CAN as compared to all other age groups. Risk of developing
diabetes increased with increase in duration of diabetes. Participants with duration 11
years had 237.420 fold, 6 -10 years had 148.97 fold and 5 had 50.438 fold of higher
risk for the development of CAN than newly diagnosed. (Table 4.12.1)
4.13: Distribution of T2DM participants by various cardiovascular reflex tests

Most of the T2DM participants had abnormal deep breathing tests (Delta HR)
(60.3%) than other parasympathetic tests whereas majority of participants showed
sympathetic dysfunction in cold pressor test (72.8%) and handgrip test (55.3%) as
compared to other sympathetic tests. (Table 4.13.1)
169
Results
Figure 4.9.1: Prevalence of CAN Ewing test criteria
70
Handignur PHC
Kinnayae PHC
60
Vantamoori PHC
Overall prevalance
50
59.360.6
56.8
43
%
40
C
A 30
N
32.333.6 30.2
27.4
20
14
7.1
10
12 11
10.8
0
0 2.1
Normal
Early CAN
Definite CAN
Severe CAN
Figure 4.9.2:- Prevalence of CAN Bellavere criteria
70
61.8
60.7
60
59.9
53.8
50
%
C 40
A 30
N 20
Normal (%)
33.6
32.3
27.4
14
10.8
5.7
10
30.2
Definate CAN(%)
9.9
0
Handignur PHC Vantamoori PHC Kinnayae PHC
STUDY AREA
170
Early CAN (%)
Overall
prevalance
Results
Figure 4.11.1:- Association between age groups and prevalence of CAN
90
80
82.1
79.7
78.3
68.8
70
56.8
% CAN
60
50
Normal
40
Early CAN
30
Definate CAN
20
severe CAN
17.4
13
12.5
8.7
10
8.67.6
4.7
1.4
8.48.4
1.1
0
30-39
40-49
50-59
60
Overall CAN
Age (Years)
Figure 4.11.2: Association between duration of diabetes and prevalence of CAN
Normal
120
% CAN
80
Early CAN
97
100
87.8
Definate CAN
78.9
severe CAN
60.5
56.8
60
40
20
17.7
1.12.3
18.4
15.8
5.3
9.8
1.6 0.8
0.72.2 0
6 to 10
11
0
Newly
Detected
Duration of Diabetes (Years)
171
Overall CAN
Results
Table 4.10.1: Autonomic function test parameters depicting sympathovagal status
among T2DM participants (n=474)
TEST
Sub category
Parameters
MeanSD
Valsalva Ratio
Valsalva ratio
1.180.12
30:15 ratio (HR

Response to
standing
Basal HR
72.016.4
30:15 ratio
1.010.10
Basal SBP
133.546.2
Basal DBP
85.236.6
fall in SBP(mmHg)
18.510.1
E:I ratio
1.150.08
Delta HR
10.225.7
Basal HR
70.37.0
Basal SBP
1264.7
Basal DBP
78.025.3
Difference in DBP
10.335.8
Basal SBP
1274.6
Basal DBP
80.144.5
Difference in DBP
9.284.5
Orthostatic
Hypotension
Deep Breathing
Autonomic
function tests
Handgrip test
Cold presser test
172
Results
Table 4.11.1.1: Association between age, gender, smoking, alcohol and family
history of diabetes and prevalence of CAN Bellavere criteria (n=474)
Potential
risk
factors
SubCategory
Total
number
Normal
Early
CAN
Definite
CAN
30-39
23(4.9)
18(78.3)
3(13)
2(8.7)
40-49
144(30.4)
99(68.8)
23(16)
22(15.3)
50-59
128(27)
11(8.6)
8(6.2)
109(85.2)
60
179(37.8)
15(8.4)
13(7.3)
151(84.4)
Male
168(35.4)
41(24.4)
18(10.7)
109(64.9)
Female
306(64.6)
102(33.3)
29(9.5)
175(57.2)
Nonsmoker
331(69.8)
96(29)
33(10)
202(61)
Smoker
143(30.2)
47(32.9)
14(9.8)
82(57.3)
Alcohol Nonalcoholic 125(26.4)

consumption
Alcoholic
349(73.6)
33(26.4)
13(10.4)
79(63.2)
110(31.5)
34(9.7)
205(58.7)
Age*
Gender
Smoking
Family
History of
DM
No
95(20)
32(33.7)
7(7.4)
56(58.9)
Yes
379(80)
40(10.6)
228(60.2)
111(29.3)
p value
(2value)
(df)
< 0.001*
(2.346)
(3)
0.128
(4.104)
(1)
0.696
(0.724)
(1)
0.564
(1.145)
(1)
0.527
(1.283)
(1)
Figures in parenthesis indicates the percentage of respective frequency, chi-square value

(2), degree of freedom (df)
* Potential risk factors significantly associated with prevalence of CAN
173
Results
Table 4.11.1.2: Association between diet, BMI, central, truncal obesity, duration of
diabetes and prevalence of CAN Bellavere criteria (n=474)
Potential
predictors
SubCategory
Total
number
Normal
Early
CAN
Definite
CAN
P value
(2value)
(df)
Diet*
Veg
115(24.3)
48(41.7)
12(10.4)
55(47.8)
Mixed
359(75.7)
95(26.5)
35(9.7)
229(63.8)
0.005*
(10.483)
(1)
18.9
5(1.1)
2(4)
3(1.1)
19-24.9
103(21.7)
32(31.1)
9(8.7)
62(60.2)
25-29.9
209(44.1)
58(27.8)
18(8.6)
133(60.2)
30
157(35.1)
51(32.5)
20(12.7)
86(54.8)
No
253(53.4)
77(30.4)
25(9.9)
151(59.7)
Yes
221(46.6)
66(29.9)
22(10)
133(60.2)
No
244(51.5)
79(32.4)
22(9)
143(58.6)
Yes
230(48.5)
64(27.8)
25(10.9)
141(61.3)
Newly
detected
5Years
175(36.9)
138(78.9)
30(17.1)
7(4)
38(8)
2(5.3)
6(15.8)
30(78.9)
6-10
123(25.9)
2(1.6)
9(7.3)
112(91.1)
11
138(29.1)
1(0.7)
2(1.4)
135(97.8)
BMI
Central
obesity
Truncal
obesity
Duration of
DM*
0.635
(4.308)
(3)
0.991
(0.18)
(1)
0.505
(1.367)
(1)
<0.001*
(3.862)
(3)

*Potential risk factors significantly associated with prevalence of CAN
174
Results
Table 4.11.2.1:- Association between age, gender, smoking, alcohol consumption,
family history of diabetes and prevalence of CAN Ewing test criteria (n=474)
Potential
predictors
SubCategory
Total
number
Normal
Early
CAN
Definite
CAN
Severe
CAN
P value
(2value)
(df)
30-39
23((4.9)
18 (78.3)
3(13)
0(0)
2 (8.7)
2(1.4)
18 (12.2)
Age*
40-49
Gender
Smoking
144 (30.4) 99 (68.8) 25 (17.4)
50-59
128(27)
17(8.6)
9(7)
6(4.7)
102 (79.7)
60
179 (37.8)
15(8.4)
15(8.4)
2(1.1)
147 (82.1)
Male
168 (35.4) 41 (24.4) 18(10.7)
3(1.8)
106 (63.1)
Female
306 (64.6)
34(11.1)
7(2.3)
163 (53.3)
No
331(69.8)
96(29)
38(11.5)
8(2.4)
89 (87.1)
Yes
143(30.2)
47 (32.9)
14(9.8)
2(1.4)
80(55.9)
No
349(73.6)
110
(31.5)
39(11.2)
8(2.3)
192(55)
Yes
125(26.4)
33 (26.4) 13(10.4)
2(1.6)
77(61.6)
No
95(20)
32 (33.7)
9(9.5)
1(1.1)
53(55.8)
Yes
379(80)
111
(29.3)
43(11.3)
9(2.4)
216 (57.6)
102
(33.3)
Alcohol
Family
History of
DM
< 0.001*
(2.419)
(3)
0.183
(4.857)
(1)
0.737
(1.259)
(1)
0.623
(1.762)
(1)
0.711
(1.378)
(1)

175
Results
Table 4.11.2.2:- Association between diet, BMI, central obesity, truncal obesity and
duration of diabetes and prevalence of CAN Ewing test criteria (n=474)
Potential
Risk
factors
SubCategory
Total
number
Normal
Early
CAN
Definite
CAN
Severe
CAN
P value
(x2value)
(df)
Veg
115 (24.3)
48(41.7)
12(10.4)
0(0)
55(47.8)
Mixed
359 (75.7)
95(26.5)
18.9
5(1.1)
2(40)
0(0)
1(20)
2(40)
19-24.9
103(21.7)
32(31.1)
9(8.7)
4(3.9)
58(56.3)
25-29.9
209(44.1)
58(27.8)
21(10)
4(1.9)
126(60.3)
30
157(33.1)
51(32.5)
22(14)
1(0.6)
83(52.9)
No
253(53.4)
77(30.4)
25(9.9)
6(2.4)
145(57.3)
Yes
221(46.6)
66(29.9) 27(12.2)
4(1.8)
124(56.1)
No
230(48.5)
64(27.8)
25(10.9)
7(3)
134(58.3)
Yes
244(51.5)
79(32.4)
27(11.1)
3(12)
135(55.3)
175 (36.9) 138 (78.9) 31(17.7)
2(1.1)
4(2.3)
Diet*
BMI
Central
obesity
Truncal
Obesity
Duration of
DM*
Newly
detected
0.007*
(12.112)
(1)
40(11.1) 10(2.8) 214(59.6)
5Years
38(8)
2(5.3)
6(15.8)
7(18.4)
23(60.5)
6-10
123 (25.9)
2(1.6)
12(9.8)
1(0.8)
108(87.8)
11
138 (29.1)
1(0.7)
3(2.2)
0(0)
134(97.1)
0.084
(15.277)
(3)
0.848
(0.800)
(1)
0.416
(2.843)
(1)
< 0.001*
(4.408)
(3)

176
Results
Table: - 4.12.1: Sociodemogrpahic and anthropometric risk factors for CAN
Risk
factors
SubCategory
CAN
OR
95% CI
P value
30-39
40-49
1.144
0.835
50-59
2.055
0.3124.235
0.40910.316
60
3.998
0.94816.861
2 = 2.419, df = 3, P < 0.001*
0.05*
Newly
diagnosed
5
50.438
< 0.001*
6-10
148.970
11
237.420
11.05 230.09
30.456728.65
30.1521869
Age*
Duration
of DM*
(Years)
0.382
< 0.001*
< 0.001*
2 = 4.408, df = 3, P < 0.001*

Diet
Veg
Mixed
1.955
0.8584.454
0.111
2 = 12.112, df = 1, P = 0.007*
Chi-square value (2), degree of freedom (df), OR= odds ratio

*significant risk factors of CAN
177
Results
4.13.1: Distribution of T2DM participants by various cardiovascular reflex tests
(n = 474)
Test Type
Cardiovascular reflex
tests
Normal
Borderline
Abnormal
Effect of deep
breathing on heart rate
136
(28.7)
52(11)
286(60.3)
Effect of Valsalva
Manoeuvre on heart
rate
182
(38.4)
62(13.1)
230(48.5)
Parasympathetic
tests
Heart rate response to

standing (30:15 ratio)
161 (34)
49(10.3)
264(55.7)
Effect of sustain hand

grip on BP
109(23)
103(21.7)
262(55.3)
Sympathetic tests
Postural Hypotension
175
(36.9)
38(8)
123(25.9)
Effect of cold pressor

on BP
63(13.3)
66(13.9)
345(72.8)
Figures in parentheses indicate the percentage of respective frequency
178
Results
4.14 Prevalence of DPN
4.14.1: DNS scoring method
Based on DNS score, the total prevalence of DPN in the study area was 58%.
Among three PHCs, the higher prevalence was found in Kinnayae (63.1%) as compared
to Vantamoori (58.6%) and Handignur (44.1%). (Figure 4.14)
4.14.2: DNE scoring method
Based on DNE score, overall prevalence of DPN was 59.1%. Between three
PHCs, Kinnayae (63.5%) had highest prevalence followed by Vantamoori (60.7%) and
Handignur (45.2%). (Figure 4.14)
4.15 : Association between sociodemogrpahic, anthropometric potential risk factors
and prevalence of DPN
The age of the participants showed significant association with prevalence of
DPN (P < 0.001). (Figure 4.15.1.1) Prevalence of DPN was higher in the age group of
60 years (84.9%) and 50-59 years (82.8%) than other age groups. Males had slightly
higher prevalence of DPN than females (66.7 Vs 54.9%), which was significantly
associated with prevalence of DPN (P = 0.005) (Table 4.15.1.1). There was significant
relationship between alcoholics and prevalence of DPN. In addition, Alcoholics had
higher prevalence (66.4% Vs 55%) than non-alcoholics. Participants with smoking,
family history of diabetes, mixed diet, overweight and with truncal obesity did not
showed any significant differences in the prevalence of DPN. Prevalence of DPN
increased significantly with increase in the duration of diabetes (P < 0.001), being highest
179
Results
in the participants with duration of diabetes >11 years (98.6%) and 6-10 years (97.6%).
(Figure 4.15.1.2, Table 4.15.1.2)
4.15.2: DNE score method
The age of the participants showed significant association with the prevalence of
DPN (P < 0.001). (Figure 4.15.2.1, Table 4.15.2.1) In comparison with the age group of
30-39 years and 40-49 years, the age group of 50-59 years (85.9%) and 60 years
(84.4%) had higher prevalence of DPN. The relationship between gender (P = 0.005) and
prevalence of DPN was found to be significant. Males had slightly higher prevalence of
DPN (66.7 Vs 54.9%) than females.(Table 4.15.2.1) There was no significant differences
in the prevalence of CAN within smokers, alcoholics, family history of diabetes,
consuming mixed diet, overweight, obese and with truncal obesity. Longer duration of
diabetes (P < 0.001) and presence of DPN were found to be significantly associated.
Prevalence of DPN was higher in the participants with duration of diabetes >11 years
(98.6%) and 6-10 years (97.6%) as compared to the newly diagnosed. (Figure 4.15.2.2,
Table 4.15.2.2)
4.16: Risk factors of DPN

Out of 11 factors analyzed, only 3 potential risk factors (age, gender and duration of
diabetes) had significant association with prevalence of DPN in bivariate analysis. After
controlling potential confounding factors by multivariate regression analysis, only 1 risk
factor of DPN was identified.
180
Results
Prevalence of DPN increases significantly with increase in the duration of
diabetes. (P < 0.001) (Figure 4.15.1.2) Participants with duration of diabetes 11 years,
6-10 years and 5 years had 829.12 fold, 459.88 fold and 45.86 fold of higher chance of
developing DPN respectively than newly diagnosed. (Table 4.16.1)
4.16.2: DNE score method
There was significant increase in the prevalence of DPN with increase in the age.
(P < 0.001) In comparison with age group of 30-39 years,40-49 years and 60 years, age
group of 50-49 years (AOR 224.25 CI 1.376-223.3, P = 0.004) had highest risk for
development of DPN. (Figure 4.15.2.1) Longer duration of diabetes (P < 0.001) showed
significant association with prevalence of DPN. (Figure 4.15.2.2) Participants with
duration of diabetes 11 years, 6-10 years and 5 years had 521.8 fold, 168.3 fold and
82.31 fold of higher risk of having DPN respectively as compared to newly diagnosed.
(Table 4.16.1)
Figure 4.14: Prevalence of Diabetic Peripheral Neuropathy (DPN) in the study area
DNS(%)
DNE(%)
70
60
50
40
30
20
58.6 60.7
63.1 63.5
58 59.1
Vantamoori PHC
Kinnayae PHC
Overall prevalance
44.1 45.2
10
0
Handignur PHC
181
Results
Table 4.15.1.1:- Association between age, gender, smoking, alcohol consumption,
family history of diabetes and prevalence of DPN- DNS method (n=474)
Diabetic peripheral
neuropathy (DPN)
Potential risk
factors
Sub Category
Total number
Age*
30-39
23((4.9)
2(8.7)
40-49
144(30.4)
15(10.4)
50-59
128(27)
106(82.8)
60
179(37.8)
152(84.9)
Gender*
2 = 2.424, df =3, P < 0.001*

Male
168(35.4)
Female
306(64.6)
2 = 7.994, df = 1, P = 0.005*
No
331(69.8)
Smoking
Alcohol
consumption*
Family History of
DM
Yes
143(30.2)
Abnormal
112(66.7)
163(53.3)
189(57.1)
86(60.1)
2 = 0.379, df = 1, P = 0.538
No
349(73.6)
192(55)
Yes
83(66.4)
125(26.4)
2 = 4.898, df = 1, P = 0.027*
No
95(20)
Yes
379(80)
50(52.6)
225(59.4)
2 = 1.415, df =1, P = 0.234
Figure in parentheses indicate the percentage of respective frequency

2 = Chi-square value and df = degree of freedom
*Potential risk factors significantly associated with prevalence of DPN,
182
Results
Table 4.15.1.2:- Association between diet, BMI, central obesity, truncal obesity,
duration of diabetes and prevalence of DPN DNS method (n=474)
Potential Risk
factors
SubCategory
Total
Number
Diabetic peripheral
neuropathy (DPN)
Abnormal
Diet
Veg
115(24.3)
61(53)
Mixed
359(75.7)
214(59.6)
2= 1.542, df = 1, P < 0.214

BMI
18.9
19-24.9
25-29.9
5(1.1)
103(21.7)
209(44.1)
2(40)
56(54.4)
131(62.7)
30
157(33.1)
86(54.8)
2 = 3.771, df = 3, P < 0.287

Central obesity No
Yes
253(53.4)
146(57.7)
221(46.6)
129(58.4)
2 = 0.021, df = 1, P = 0.884
Truncal obesity No
Yes
230(48.5)
139(60.4)
244(51.5)
136(55.7)
2 = 1.072, df = 1, P = 0.300
Duration of
DM*
New
175(36.9)
4(2.3)
38(8)
20(52.6)
6-10
123(25.9)
116(94.3)
11
138(29.1)
135(97.8)
5Years
2 = 3.799, df = 3, P < 0.001*

Figures in the parentheses indicate the percentage of respective frequency,
*potential risk factors significantly associated with prevalence of DPN,
183
Results
Figure 4.15.1.1:- Association between age groups and prevalence of DPN -DNS
method
90
82.8
84.9
80
70
% 60
50
D 40
P
30
N
DPN(%)
20
10
8.7
10.4
30-39
40-49
0
50-59
60
Age (Years)
Figure 4.15.1.2: Association between duration of diabetes and prevalence of DPNDNS method
120
94.3
100
% 80
D 60
P
N 40
97.8
52.6
DPN(%)
20
2.3
0
Newly
Detected
6 to 10
Duration of Diabetes (Years)
184
11
Results
Table 4.15.2.1: - Association between age, gender, smoking, alcohol consumption
and prevalence of DPN DNE method
Potential risk
factors
SubCategory
Total
number
Diabetic peripheral neuropathy

(DPN)
Abnormal
Age*
30-39
40-49
23((4.9)
144(30.4)
1(4.3)
18(12.5)
50-59
128(27)
110(85.9)
179(37.8)
151(84.4)
60
2 = 2.432, df = 3, P < 0.001*

Gender*
Male
168(35.4)
112(66.7)
Female
306(64.6)
168(54.9)
2 = 6.209, df = 1, P = 0.033*
Smoking
No
331(69.8)
196(59.2)
Yes
143(30.2)
84(58.7)
2 = 0.009, df = 1, P = 0.923
Alcohol
consumption
No
349(73.6)
112(66.7)
Yes
125(26.4)
168(54.9)
2= 2.304, df = 1, P = 0.129
Family History No
of DM
Yes
95(20)
50(52.6)
379(80)
230(60.7)
2 = 2.038, df = 1, P = 0.153
Figures in parentheses indicate the percentage of respective frequency,

*Potential risk factors significantly associated with prevalence of DPN
185
Results
Table: 4.15.2.2 - Association between diet, BMI, central, truncal obesity, duration of
diabetes and prevalence of DPN DNE method
Potential
predictors
SubCategory
Total
number
Diabetic peripheral neuropathy

(DPN)
Abnormal
Diet
Veg
115(24.3)
60(52.2)
Mixed
359(75.7)
220(61.3)
2= 2.988, df = 1 , P < 0.084

BMI
18.9
5(1.1)
3(60)
19-24.9
103(21.7)
57(55.3)
25-29.9
209(44.1)
130(62.2)
30
157(33.1)
90(57.3)
2= 1.640, df =3, P = 0.650

Central
obesity
No
253(53.4)
150(59.3)
Yes
221(46.6)
130(58.8)
2= 0.011, df = 1, P = 0.918
Truncal
obesity
No
230(48.5)
140(60.9)
Yes
244(51.5)
140(57.4)
2= 0.597, df = 1, P = 0.440
Duration of New
DM*
5Years
175(36.9)
2(1.1)
38(8)
22(57.9)
6-10
123(25.9)
120(97.6)
11
138(29.1)
136(98.6)
2= 4.073, df = 3, P < 0.001*

Figures in parentheses indicate the percentage of respective frequency,
*Potential risk factors significantly associated with prevalence of DPN
186
Results
Figure 4.15.2.1: Association between age groups and prevalence of DPN-DNE
scoring method
100
85.9
90
84.4
80
%
D
P
N
70
60
50
40
DPN(%)
30
20
10
12.5
4.3
0
30-39
40-49
50-59
60
Age (Years)
Figure 4.15.2.2: Association between duration of diabetes and prevalence of DPN

DNE scoring method
120
97.6
100
% 80
D 60
P
N 40
98.6
57.9
DPN(%)
20
1.1
0
Newly
Detected
6 to 10
Duration of Diabetes ( Years)
187
11
Results
Table 4.16.1: - Sociodemogrpahic and anthropometric risk factors for DPN
Risk
factors
SubCategory
DNS scoring method
OR
95%
P value
CI
DNE scoring method

OR
95%CI P value
30-39
40-49
1.146
0.04231.58
0.936
55.418
1.376223.3
0.033*
50-59
6.004
0.217165.99
0.290
224.25
5.741875.9
0.004*
60
11.803
0.446312.49
2= 2.424, df = 3, P < 0.001*
0.140
193.560
Newly
diagnosed
45.863
11.04 190.10
< 0.001*
82.316
15.606- <0.001*
434.188
6-10
459.886
106.171992
< 0.001*
168.3
253.79111.6
11
829.121
157.284371
2
= 3.799, df = 3, P <0.001*
< 0.001*
521.8
Male
Female
0.421
Age*
(Years)
Duration
of DM*
(Years)
5.7980.003*
646.1
2= 2.432, df = 3, P < 0.001*
-
<0.001*
417.51- <0.001*
652.3
2
= 4.073, df =3, P < 0.001*
1
Gender
0.1601.108
2
= 7.994, df = 1, P = 0.005*
0.421
0.529
0.1680.277
1.669
2= 6.209, df =1, P = 0.033*
*Risk factors for DPN (P<0.05), 2 = Chi-square value and df = degree of freedom,
OR = odds ratio
188
Discussion
Diabetes mellitus has now assumed epidemic magnitude in several countries of
the world. According to International Diabetes Federation, present population with
diabetes in India is 65.5 million and around 72 million people will be affected by this
disease up to year 2030, due to which India would rank first in its share of the global
burden of diabetes.50 The prevalence of diabetes mellitus differs not only between the
rural-urban segments of the population in India but also across the Indian States. This is
because different Indian states are at dissimilar stages of demographic transition.
Villages in Karnataka have undergone a drastic change in living standards and
lifestyles during the last 20-25 years, partly on account of the incursion of money in
recent years from people working abroad in the Gulf States and other affluent countries.
The change in disease profiles brought about by this sudden affluence and its differential
impact on different social classes, largely remain unstudied. This was the trigger for our
exploration into the prevalence of diabetes and related states in a small rural community
in the northern part of the Karnataka. The present study showed a relatively high
prevalence of 17.7% T2DM in rural population of North Karnataka, indicating that this is
a high risk population for the development of diabetes mellitus. These results are
comparable to those of a recent study conducted by Chythra R et al in costal Karnataka
region on 1,351 individuals that showed the prevalence of T2DM to be 16%. Various
studies conducted between 2000 and 2010 in rural population of south-east region have
reported an increase in the prevalence of diabetes (4%-16%) in South Indian states.296, 297
As per the World Health Organization, Diabetes Mellitus (DM) is a
heterogeneous metabolic disorder characterized by common features of chronic
hyperglycemia with disturbance of carbohydrate, fat and protein metabolism. DM is a
189
Discussion
major cause of morbidity and mortality the world over. The incidence of diabetes mellitus
is increasing day by day all over the world. This increase may be largely attributed to the
decreasing physical activity and increasing obesity levels. DM is anticipated to continue
as a major public health problem owing to its serious complications.50, 278
Before commencing any kind of management measures, it is important to assess
the factors that contribute to the occurrence of the disease. By intervening with the
occurrence of these factors, the progression of diabetes and its complications can be
controlled. The chance of occurrence of T2DM in susceptible individuals can be
prevented by reducing the exposure to specific pre-disposing factors. The present study
attempted to identify the association of various risk factors with the prevalence of T2DM
in a rural population of North Karnataka region.298
DM in the elderly is emerging as one of the most important public health
problems of the 21st century. A change in body composition with accumulation of fat in
the abdomen is the key factor in the causation of diabetes in the ageing population. The
size and strength of skeletal muscles, a major tissue involved in glucose metabolism, also
declines with age leading to muscle weakness and a reduction in physical activity. These
changes lead to marked reduction in energy expenditure and abdominal fat accumulation
causing insulin resistance. Results of the present study showed that advancing age is a
contributing factor for the progression of T2DM. This may be due to prolonged exposure
to different types of stress, increasing obesity, changing food habits and environmental
influences. However, the higher prevalence among the younger adults in the age group of
30-39yrs, the most productive age group of the community, is alarming and calls for
focus on prevention of diabetes among younger population. A similar study conducted by
190
Discussion
Sudha S Deo et al on 1,022 individuals of Malwan area situated in Sindhudurg district of
Maharashtra state showed that age 61 had a prevalence of 20% and age 31-40 had
3.1%.299 Diagnosis and management of diabetes in the elderly requires special attention
since age, genetics, body composition and lifestyle factors all interact. Increasing
evidence suggests that postprandial hyperglycemia is more sensitive to diagnose diabetes
in elderly people than in the young. Age related changes in body function and cognition
demand special caution in the selection of hypoglycemic drugs in the elderly.300, 301
In the first half of the last century, the prevalence of T2DM was higher among
women than among men, but this trend has changed with more men than women being
diagnosed with T2DM now. This change in the gender distribution of T2DM is mainly
caused by a more sedentary lifestyle being adopted by men, resulting in increased
obesity. However, recent data have also shown that men develop diabetes at a lower
degree of obesity than women, a finding that supports the view that the pathogenesis of
T2DM differs between men and women. Observations of sex differences in body fat
distribution, insulin resistance, sex hormones, and blood glucose levels further support
this concept. Results of the present study showed that gender was not shown any
significant association with prevalence of diabetes. Similar findings were reported in a
multi-centric study conducted by Khatib N M et al in the year of 2010 in India.298
However, some studies have reported a higher prevalence in females others have found a
higher prevalence among males.302 This is possibly due to the presence of coexisting risk
factors in the two genders. Alternatively, gender may not be a risk factor in T2DM.303
Diabetes mellitus is not considered a professional illness and is not reported to be
associated with types of occupations. The lifestyle of workers adopted in different
191
Discussion
occupations can enhance the onset of the disease. In many cases, workers with long
workdays, multiple jobs, shift work, anxiety and experiencing stress on daily basis will
have difficulty in adopting a healthy life style. These have been evidenced as harmful to
peoples health and making them susceptible to chronic health problems.304 Results of
the present study showed a significant association of occupational work stress and
physical inactivity with prevalence of T2DM. Skilled III workers such as self-employed
and non-workers i.e. homemakers, elderly persons had a significantly higher prevalence
of T2DM. A recent study conducted by Agardh E, et al in the year 2011 305 showed that
occupational stress and physical inactivity can be the risk factors for diabetes. This
association of diabetes with occupation could be due to the combined effect of physical
inactivity and work related stress. 305
Low educational status and lack of health care facilities in the rural areas delay
the diagnosis of diabetes until severe complications develop. More than 70% of people
remain undiagnosed in rural areas.306 Results of present study showed that literacy was
not associated with prevalence of T2DM. Chaturvedi et al have reported similar results in
a cohort of industrial workers.307 Contrary to this, some western studies have reported a
decrease in prevalence with increase in educational status. Illiteracy may result in lesser
awareness about the disease and lesser opportunity for prevention and control.
Additionally, higher educational status may influence the lifestyle factors; physical
inactivity may influence the trends of disease.307
Dietary habits can be linked with obesity, insulin resistance and atherosclerosis.
Dietary management will be helpful for control of disease and its complications. Present
study showed that dietary habits have significant association with prevalence of T2DM.
192
Discussion
People with mixed dietary habits had 1.29 times greater odds for developing T2DM
compared with those with vegetarian diet.308, 309
The past two decades have seen an explosive increase in the number of people
diagnosed with diabetes mellitus worldwide, particularly T2DM, which is found to be
associated with modern lifestyle, abundant nutrient supply, reduced physical activity and
obesity. In obese individuals, adipose tissue releases increased amounts of non-esterified
fatty acids, glycerol, hormones, pro-inflammatory cytokines and other factors that are
involved in the development of insulin resistance. 310, 311, 312,313 The present study showed
that BMI is a significant risk factor for development of T2DM and there was increase in
the prevalence of the T2DM with increase in BMI. Participants with BMI 30 Kg/m2
(OR 9.98) had high risk for developing T2DM. Hence, early identification of high BMI
would be helpful for primary prevention and early diagnosis of diabetes. Kahn S,
conducted a study in the year 2006 to observe the mechanisms linking obesity to insulin
resistance and T2DM and reported BMI as an independent risk factor for development of
diabetes.314
Obesity is a primary risk factor for metabolic diseases, which include coronary
heart disease, hypertension, and T2DM.313,
314
Results of the present study showed
positive association between truncal obesity and prevalence of T2DM (OR 1.41).
However, there was no significant association between central obesity and prevalence of
T2DM. A recent study conducted by Chythra R et al in a similar geographical region on
1,351 individuals reported that truncal obesity was significantly associated with
prevalence of diabetes.297
193
Discussion
Study of parental transmission of diabetes provides insight into the relative
contributions of underlying maternal and paternal influences. Results of the present study
showed significant effect of parental influences on prevalence of diabetes. Participants
having family history of diabetes had a 35.97 times greater risk of developing T2DM.
Earlier studies have reported relatively higher risk with maternal history of diabetes
compared to paternal315,
316.
When both parents were diabetic, the risk of developing
diabetes will be even higher. However, in the present study no such effect was observed,
probably because there were only a few participants with both diabetic parents. Family
history of T2DM could act through environmental factors (diet, stress, physical activity,
socioeconomic status) as well as a genetic mechanism through gene expression. Family
history of diabetes could be an important public health tool in predicting development of
diabetes. 317
Smokers are insulin resistant and exhibit several aspects of insulin resistant
syndrome. Smoking has also been shown to deteriorate glucose metabolism which may
lead to the onset of T2DM. There is also some evidence which suggests that smoking
increases diabetes risk through a mechanism independent of BMI. Smoking has been
associated with a risk of chronic pancreatitis and pancreatic cancer, suggesting that
tobacco smoke may be toxic to the pancreas. Results of the present study showed that
smoking was significantly associated with prevalence of T2DM. This finding concurs
with those of several other cohort studies. 318
Alcohol consumption by diabetics can worsen blood sugar control. Long term
alcohol use can lead to hypoglycemia and also accumulation of certain acids that may
lead to serious health consequences. The present study showed a significant association
194
Discussion
between alcohol consumption and risk of development of diabetes. This is probably due
to the development of insulin resistance among heavy alcohol drinkers due to increased
obesity, especially abdominal/ truncal obesity because of heavy drinking.319 Literature
shows varied association between alcohol consumption and risk of diabetes. 319,320
The autonomic nervous system (ANS) is an extensive neural network. The most
important role of ANS is of regulating the milieu interior by controlling homeostasis and
visceral functions. Diabetes mellitus is most uniquely associated with autonomic
dysfunction. Prolonged hyperglycemia may leads to degradation of the microvasculature
and can cause the specific form of autonomic dysfunction. Morbidity and mortality of
diabetes is due to development of both macrovascular and microvascular complications.
Microvascular complications common among diabetics include retinopathy, nephropathy,
and neuropathy. According to several studies the most significant clinical form of
diabetic neuropathy is Cardiovascular Autonomic Neuropathy (CAN) and Diabetic
Peripheral Neuropathy (DPN). 128
The assessment modality for determining diabetic neuropathy is an important
factor for establishing the presence of disease. In the present study, the assessment
modality for determining autonomic dysfunction among T2DM participants was based on
short term HRV analysis, different cardiovascular reflex tests (Ewings tests battery) and
clinical examination for the assessment of diabetic peripheral neuropathy. The
assessment modality used in present study was as per the recommendations provided by
Task Force of European Society of Cardiology, San Antonio conference on diabetic
neuropathy held by the American Diabetes Association and the American Academy of
195
Discussion
Neurology.292, 293 Since, this was a large epidemiological study in rural population, these
modalities were the most feasible for the assessment.
Prevalence of CAN varies greatly depending on the criteria used for the
assessment and the type of population studied. Very few studies have been done on the
prevalence of CAN in rural population. Results of the present study showed high
prevalence of CAN among T2DM participants in this region. Various studies from year
1982 -2010 have reported the prevalence of CAN but these have used only one single
assessment modality for estimating the prevalence of CAN.321,322 In addition, as present
study has used three standard clinical and subclinical methods for assessment of CAN,
we are more likely to estimate the true prevalence of CAN in this region.
In the present study, using predefined heart rate variability (HRV) tests and
spectral analysis of the R-R intervals, found that 63.7% of individuals had
parasympathetic dysfunction and 60.8% of individual had sympathovagal dysfunction
among T2DM participants. The results are comparable to a large cohort study conducted
by Ziegler in patients with T1DM and T2DM using predefined heart rate variability
(HRV) tests and spectral analysis of the R-R intervals. In that study, 25.3% of patients
with T1DM and 34.3% of patients with T2DM had abnormal findings.324 In present
study by using various cardiovascular reflex tests, the estimated prevalence of CAN was
56.8%. Whereas 60.3% had parasympathetic neuropathy, 55.3% had sympathetic
neuropathy among T2DM participants. These findings are comparable to a recent study
conducted by Mehta et al in Jaipur that has reported a prevalence of CAN in 58% of
cases, all of them having parasympathetic neuropathy and 20% with sympathetic
neuropathy.323 CAN also accounts for silent myocardial infarction and shortens the
196
Discussion
lifespan by resulting in death in 25% 50% of diabetic patients within 510 years of
CAN.135
Diabetic neuropathy is probably a heterogeneous disorder not fully explained by a
single pathogenic mechanism and its risk factors remain obscure. However, age, duration
of disease and degree of glycemic control appear to be strongly associated with diabetic
autonomic neuropathy.325 In T2DM, poor blood glucose control (chronic hyperglycemia)
plays an important role both in the initial pathophysiology (oxidative stress,
microcirculation dysfunction due to nitric oxide loss and Schwann cell lesion due to
accumulation of free radicals) as well as in its progression (axonal degeneration and
neuronal apoptosis) of diabetic neuropathy. Results of the present study showed that risk
factors such as age and duration of diabetes had significant association with prevalence of
CAN.326, 327
In addition, present study also showed that participants with age 60 years and
duration of diabetes 11 years had high risk for developing CAN. Findings of this study
are in agreement with those of Voulgari et al. who report that in T2DM patients CAN is
independently associated with longer duration of diabetes.328 Another study conducted
by Knuiman et al. on 179 individuals with insulin-dependent diabetes mellitus (IDDM),
found that age at diagnosis (younger) and duration of diabetes (longer) were important
time related risk variables for developing CAN.329, 330, 331 Chronic hyperglycemia and
poor glycemic control in diabetic individuals with advanced age and longer duration of
disease can lead to production of advanced glycation end-products, disturbances in
sorbitol pathway, other metabolic disturbances and increased peripheral resistance due to
197
Discussion
loss of elastic properties. These will cause nerve damage leading to various types of
autonomic dysfunction.
Recent studies have shown that pathogenesis of T2DM differs between men and
women. Observations of gender differences in body fat distribution, insulin resistance,
sex hormones and blood glucose levels further support this concept. Results of the
present study showed that females were significantly associated with parasympthathetic
dysfunction and had higher risk for developing CAN. These results are similar to the
study conducted by Robert P. Nolan on 155 female and 106 male participants which
showed similar correlation of gender with decrease in HRV component. 330
Risk of neuropathy increases for diabetics with poor nutrition habits, such as
consuming higher fatty foods and being overweight. Present study showed that T2DM
participants consuming mixed diet showed abnormal heart rate variability and higher
chances for developing CAN. Similar results were reported by studies conducted by
Knuiman M W who had screened 1218 people and showed that diet was involved in
development of diabetic complication.329
Present study further showed that BMI, truncal and central obesity, smoking,
alcohol consumption and family history of diabetes did not show any significant
association with prevalence of CAN among T2DM participants.326, 327 These results were
dissimilar compared to the study conducted by Christiansen J S on juvenile insulin
dependent diabetes in the year of 1978 which showed that smoking and alcohol were
possible risk factors in pathogenesis of diabetic complication. Similarly, a review study
conducted by Vilink I
333
Aaron in the year 2007 showed that alcohol consumption and
obesity may be linked with diabetic complication.332-335
198
Discussion
According to American Diabetes Association (ADA), American Academy of
Neurology (AAN) & Task Force European Society of Cardiology, parameters such as
RMSSD, pNN50, Valsalva Ratio, Delta HR & 30:15 ratio reflect the parasympathetic
activity and LF/HF ratio, postural hypotension, fall in DBP in sustained handgrip reflect
the sympathovagal activity.326, 327 Results of the present study showed that the RMSSD,
pNN50, High frequency (H.F), Valsalva Ratio, Delta HR & 30:15 ratios were reduced
among T2DM participants suggesting parasympathetic dysfunction. Results of present
study were comparable to the recent study conducted by Deepak et al (on 30 diabetics
and 30 age and sex matched controls) reported that the time domain parameters were
reduced in diabetes.336
The parasympathetic dysfunction may be due metabolic
disturbances, formation of advance glycation end products and poor glycemic control
which intern lead to axonal degeneration of vagus nerve.333 It is a well-known fact that
long nerves are prone for neuropathy in DM and vagus is the longest nerve in the body.
In addition, present study also showed that values of Low frequency (L.F.), fall in SBP,
and DBP were decreased among T2DM participants suggesting the sympathetic
dysfunction. These observations are similar to those reported in a study conducted by
Salim et al on 50 diabetics and 50 age and sex matched controls.337 The sympathetic
influence on heart rate is mediated by release of epinephrine and norepinephrine.
Activation of -adrenergic receptors results in cyclic AMP mediated phosphorylation of
membrane proteins and increases in I caL and in If. The end result is a speeding up of the
slow diastolic depolarization. The sympathetic dysfunction may be due to metabolic
disturbances and nerve damage among diabetics.
199
Discussion
The present study showed LF/HF ratio was increased among T2DM participants
which indicate the sympathovagal dysfunction. This depicts the cardiovascular autonomic
neuropathy in T2DM. Mudassir Mirza et al conducted study on 200 diabetic patients (age
and sex matched) reported similar observations.338
Diabetic peripheral neuropathy is one of the most common complications of
diabetes mellitus and it may be the first presenting symptom in T2DM. The prevalence of
DPN varies from 5-100%. Results of the present study showed that 58% and 59.1% of
the participants with T2DM had clinical evidence of diabetic peripheral neuropathy while
27.8% was reported by a study conducted by Franklin et al.339 Ashok S and his
colleagues observed a prevalence of neuropathy was 5.4% in patients with T2DM at the
time of diagnosis.340 This difference in the prevalence of peripheral diabetic neuropathy
in various studies is probably due to differences in modalities used for assessing the
prevalence of diabetic peripheral neuropathy. Present study used clinical examination
which includes Diabetic neuropathy examination (DNE) and Diabetic neuropathy
symptom score (DNS).
Results of present study showed that the prevalence of DPN increases with
advancement in age and with duration of diabetes which suggest that the age and duration
of diabetes are risk factors for the prevalence of DPN. In addition, present study further
showed that participants with age 60 year, duration of diabetes 11 years had a high
risk of developing DPN. This observation was comparable to study conducted by Fargol
Booya et al341 on 110 patients in Iran which reported that age and duration of diabetes are
the contributing factor for developing DPN. Results of another study conducted by R
Predeepa and Rema M in urban south Indian population are also comparable with our
200
Discussion
observations.342 Further, present study also showed that gender was significantly
associated with prevalence of DPN whereas diet, BMI, truncal and central obesity,
smoking, alcohol consumption and family history of diabetes did not show any
significant association. These findings are comparable to study reported by MJ Young et
al
343
on prevalence and associated risk factors of diabetic peripheral neuropathy in the
United Kingdom (Multicentric study).

In summary, we found a higher prevalence of T2DM, CAN and DPN in rural
population of North Karnataka. Age, occupation, smoking, alcohol consumption, family
history of diabetes, diet, BMI and truncal obesity are relative risk factors for T2DM.
Further, advanced age and duration of diabetes appear to be significant risk factors for
developing CAN and DPN.
201
Summary
India leads the world with the largest number of diabetic subjects earning the
dubious distinction of being termed the diabetes capital of the world. Within India,
inter-regional disparities in burden of type 2 diabetes mellitus (T2DM) are expected
because of differences in lifestyles and demographic patterns. Diabetes Mellitus is
emerging as a major public health problem owing to its serious complications. However,
there are very few population based studies conducted in rural settings reporting the
prevalence of T2DM and its complications. Further, assessment of the various factors
contributing to the occurrence of the disease will enable the development of strategies
aimed at limiting the progression of the disease. Diabetes mellitus is well known for its
chronic complications of neuropathy, retinopathy and nephropathy that are collectively
termed as tripathy. Diabetic neuropathy is a common complication of diabetes. Diabetic
Autonomic Neuropathy (DAN) is one of the major complications of Diabetes Mellitus
(DM) and its presence is associated with worsening prognosis and poorer quality of life.
Heart Rate Variability (HRV) and cardiovascular reflex tests are sensitive tools that could
be used for early detection of complications among DM patients. The risk of developing
autonomic dysfunction in DM depends on several factors. Hence, this study was
undertaken to determine the prevalence and autonomic dysfunction in T2DM individuals
of rural population of North Karnataka.
A cross sectional study was conducted to find out the prevalence and autonomic
dysfunction in type 2 diabetes individuals residing in three primary health center areas of
Handignur, Vantamoori and Kinnayae in Belgaum district of North Karnataka associated
with J.N. Medical College, Belgaum
202
Summary
Screening for T2DM of 3000 selected participants aged 30 years was done
according to WHO criteria. Fasting blood glucose was estimated by using standard
glucometer. A total of 474 participants were detected as having T2DM. These T2DM
participants were assessed for autonomic dysfunction by using autonomic function tests as
per Ewing test criteria and for diabetic peripheral neuropathy by using Diabetic Neuropathic
Examination (DNE) score and Diabetic Neuropathic Symptom (DNS) scoring system.
The trends in the prevalence of T2DM, CAN and DPN were determined by using
descriptive statistics. Chi-square test and logistic regression analysis was used to study the
association of various risk factors with prevalence of T2DM, CAN and DPN. Odds ratio
was calculated to find out the strength of association of various risk factors with prevalence
of T2DM, CAN & DPN. Differences were considered significant at p < 0.05 level.
A higher prevalence of 17.7% of T2DM relative to the hypothesized prevalence of
3.8% was observed in the study area. Age over 60 years, mixed diet, BMI > 30 kg/m2,
truncal obesity, smoking, alcohol consumption and family history of diabetes were
significantly associated with prevalence of T2DM. Present study also showed a relatively
high prevalence of CAN and DPN in the study area. In addition, it was also observed that
advanced age and duration of diabetes were significantly associated with prevalence of
CAN and DPN.
The higher prevalence of T2DM, CAN and DPN observed in this region may partly
be attributed to the sensitivity of the battery of assessment tools used in the study. High
prevalence of CAN and DPN in T2DM individuals supports the general perception that the
rates are increasing over the years and are reaching epidemic proportions. Factors such as
age, diet, BMI, truncal obesity, smoking, alcohol consumption and family history of
203
Summary
diabetes appear to increase the risk for developing T2DM. Age and duration of diabetes
appear to increase the risk for developing CAN and DPN.
A relatively high prevalence of T2DM, CAN and DPN was observed in the rural
population of North Karnataka. High prevalence of T2DM, CAN and DPN even in rural
community suggests the probable impact of socioeconomic transition on the occurrence of
DM. Age and duration of diabetes appear to increase the risk for development of CAN. The
present study showed reduction in values of HRV and cardiovascular reflex test parameters
which seem not only to carry negative prognostic value but also to precede the clinical
expression of diabetic autonomic neuropathy. Early HRV and autonomic function test
analysis can be used as screening tools for assessment of CAN in community settings.
There is a need to institute screening and awareness programs for early detection, even in
rural areas, so as to prevent the development of the long term complications.
204
Conclusion
A relatively high, higher than the hypothesized, prevalence of T2DM, CAN and
DPN were noted in the rural population of North Karnataka. The observations from the
present study may be useful in local adaptations in planning, implementation and
evaluation of the national health programs such as the National Programs for control of
Diabetes, Cardiovascular diseases and Stroke (NPDCS). High prevalence of T2DM,
CAN and DPN even in rural community suggests the impact of socioeconomic transition
on the occurrence of DM.
The parameters chosen in the present study for assessing CAN (HRV analysis,
cardiovascular reflex test) and DPN (DNS and DNE scoring methods) are in accordance
with the recommendation of Task force of the European society of cardiology and ADA
& AAN. These parameters are sensitive measures of parasympathetic and sympathetic
status of heart. The present study showed reduction in values of HRV and cardiovascular
reflex test parameters which seem not only to carry negative prognostic value but also to
precede the clinical expression of diabetic autonomic neuropathy.
Advancing age and increasing duration of diabetes appear to be risk factors for
development of CAN. HRV analysis and autonomic function tests can be used as
screening tools for assessment of CAN in community settings. There is need to institute
screening and awareness programs for early detection of diabetic complication so as to
prevent long term complications. The findings of this study suggest the need for the
promotion of preventive measures to prevent or delay the development of chronic
complications of diabetes through good glycaemic control, regular monitoring, lifestyle
modification, practice of exercise and yoga to maintain the normal balance of
205
Miscellaneous
A. Limitations of the study

Measurement of HbA1c would have provided a better assessment of the
glycaemic status of the individuals. The dose response relationship of smoking and
alcohol consumption would have been useful for exploring the association with
development of diabetic neuropathy in a more comprehensive manner. Use of vibration
perception for the assessment of diabetic peripheral neuropathy may be considered for
use as a simple screening tool in a community setting.
B. Scope for future studies
This cross sectional study was conducted in a rural population residing in a
geographical region surrounding a major urban centre like Belgaum. Determining the
prevalence of T2DM, CAN and DPN in a larger representative sample of the district or
even of the north Karnataka region will provide a more accurate estimate of the
prevalence in the rural population. There is considerable variation in the reported
prevalence of diabetic neuropathy in different regions as studies differ considerably with
regard to definition, method of assessment and patient selection, despite being considered
one of the most common long-term complications of diabetes.
Diabetic autonomic neuropathy has a slow and insidious onset and hence patients
may suffer from the condition unknowingly for many years. Early diagnosis with simple
screening tools such as those employed in the present study and designing intervention
strategies are of prime importance in preventing potentially serious consequences of
diabetic complications. Establishing population based periodic screening program
206
Miscellaneous
through the public health care system will enable not only early detection, but, also
monitoring for trends over time.
There is need for research to explore the underlying mechanisms of diabetic
neuropathy in T2DM of early onset in particular. Molecular studies may help in
understanding the pathogenesis and development of treatment options for diabetic
neuropathy.
207
ABSTRACT
Introduction: Diabetes Mellitus is emerging as a major health problem owing to its serious
complications. Within India, inter-regional disparities in burden of type 2 diabetes mellitus are
expected because of varying lifestyles and demographic patterns. DM is also well known for its
chronic complication such as tripathy which include neuropathy, retinopathy and nephropathy.
Diabetic neuropathy [particularly, Cardiac autonomic neuropathy and Diabetic peripheral
neuropathy] is most common clinical complication of DM. Its presence is associated with
worsening prognosis and poorer quality of life. The risk of developing autonomic dysfunction in
DM depends on several factors. It is important to assess these factors for developing
interventions that can decrease and or arrest the progression of the disease.
Objectives:
Primary: To estimate the prevalence and autonomic dysfunction assessed by heart rate
variability analysis and autonomic function tests among T2DM individuals of the rural
population residing in areas of three primary health centers associated with Jawaharlal Nehru
Medical College, Belgaum.
Secondary:
1. To estimate the prevalence of diabetic peripheral neuropathy among type 2 diabetes
mellitus individuals.
2. To determine the association of various risk factors such as age, gender, literacy,
occupation, diet, body mass index, central and truncal obesity, smoking, alcohol
consumption and family history of diabetes with prevalence of T2DM, CAN and DPN.
Material and Methods: A cross sectional study was conducted to find out the prevalence and
autonomic dysfunction in T2DM individuals of age above 30 years residing at three primary
I
health center areas associated with Jawaharlal Nehru Medical College, Belgaum. 2684 selected
participants (using multistage cluster sampling method) were screened for type 2 diabetes mellitus
as per WHO criteria. Participants identified as T2DM were screened for autonomic dysfunction by
using various cardiovascular reflex tests (Ewing tests criteria). Diabetic peripheral neuropathy was
assessed by using diabetic neuropathic examination score (DNE) and diabetic symptom scoring
system (DNS).
Results and Discussion: - Overall prevalence of T2DM was found to be 17.7%. Risk factors such
as age, diet, BMI, truncal obesity, smoking, alcohol consumption and family history of diabetes
appear to increase the risk for developing T2DM. Furthermore, advance age and duration of
diabetes appear to be risk factors for developing CAN and DPN in the T2DM participants.
Abnormal heart rate variability among T2DM participants is associated with abnormal heart rate
variability, indicating the autonomic dysfunctions.
Conclusion: - High prevalence of T2DM, CAN and DPN even in rural community suggests the
impact of socioeconomic transition on the occurrence of the disease. HRV analysis and
autonomic function tests can be used as screening tools for assessment of CAN in community
settings. There is need to institute screening and awareness programs for early detection of
diabetic complication so as to prevent long term consequences. The findings of this study
suggest the need for the promotion of preventive measures to prevent or delay the development
of chronic complications of diabetes through good glycaemic control, regular monitoring,
lifestyle modification, practice of exercise and yoga to maintain the normal balance of
Key words: autonomic function tests; type 2 diabetes mellitus; cardiovascular autonomic
neuropathy; diabetic peripheral neuropathy; heart rate variability; diabetic symptom score; diabetic
neuropathy examination score.
II
Annexure
ANNEXURE-Ia
INFORMED CONSENT FORM - ENGLISH
Introduction
We are requesting you to enroll yourself in the study titled A CROSSSECTIONAL
AUTONOMIC
STUDY
TO
DETEMINE
DYSFUNCITON
IN
THE
TYPE-II
PREVALENCE
DIABETIC
OF
MELLITUS
INDIVIDUALS IN RURAL POPULATION RESIDING AT THREE PRIMARY

HEALTH CENTER (PHC) AREAS ASSOCIATED TO JAWAHARLAL
NEHRU
MEDICAL
COLLEGE,
BELGAUM
conducted
by
Mr.Sanjay
D.Bhalerao, Lecturer in physiology, under the guidance of Dr.S.S.Goudar, Professor

and Head, Dept of physiology, JNMC, Belgaum.
You have been requested to participate in the research because you are into
the study group/control group. The purpose of this study is to assess the autonomic
dysfunction in type 2 diabetes mellitus individuals by using HRV analysis,
cardiovascular reflex tests and biochemical investigations.
Procedure
If you select to participate in this study, you will be given a set of
Questionnaire to which you are supposed to answer to the best of your knowledge.
Some measurements will be taken and some tests will be done, after explaining to you
the nature and procedure of those tests.
247
Annexure
Possible benefits/ risks
There are no extra risks involved in becoming a participant in our research.
Voluntary participation / withdrawal
Your participation in the study is completely voluntary. You are free to
withdraw your consent and discontinue your participation in this project at any time.
Confidentiality/privacy
All the information collected about you during the course of this study will
be kept confidential to the extent permitted by the law. You will be identified in
research records by a coded number, information which identifies you personally will
not be released without your written permission.
Institutional/sponsor policy
There is no commitment from the researchers involved in the research plan to
provide any compensation for research related injury.
Financial incentives for participation
Your participation is voluntary and you will not be paid any remuneration for
your participation in the study or for your expenses.
Contact details
If you have any queries/questions regarding the study, you may contact the
study investigators Mr.Sanjay D.Bhalerao (Mobile No. 9844491356) Lecturer,
Department of Physiology, Jawaharlal Nehru Medical College any time. In case you
have any questions about your rights as a study participant you can contact Dr V. D.
Patil, the principal JNMC, Belgaum (0831- 2471350)
248
Annexure
Authorization to publish results
The researchers may use the information gathered from this study for
presentation in scientific journals. However your identity will not be disclosed in such
presentation or publication.
CONSENT STATEMENT
I am making a voluntary decision to allow myself to participate in this study.
My signature below indicates that I have read (or have been read) the information
provided above, that I have been given the opportunity to ask questions and the said
questions have been answered to my satisfaction. I have received a copy of this signed
consent form.
Signature or left thumbprint of participant Or legally authorized Representative
Participants name ____________Participants signature/thumb print______________
Experimenters name_____________ Experimenters signature_________________
Witness name___________________ Witness signature___________________
Guardians name_________________ Guardians signature________________
Date _________________________
249
Annexure
ANNEXURE Ib INFORMED CONSENT FORM - KANNADA
250
Annexure
251
Annexure
ANNEXURE Ic INFORMED CONSENT FORM MARATHI
A CROSS-SECTIONAL STUDY TO DETEMINE THE PREVALENCE

OF AUTONOMIC DYSFUNCITON IN TYPE-II DIABETIC MELLITUS
INDIVIDUALS IN RURAL POPULATION RESIDING AT THREE
PRIMARY HEALTH CENTER (PHC) AREAS ASSOCIATED TO
JAWAHARLAL NEHRU MEDICAL COLLEGE, BELGAUM
252
Annexure
253
Annexure
254
Annexure
255
Annexure
ANNEXURE II ETHICAL CLEARANCE CERTIFICATE
256
Annexure
ANNEXURE III INVESTIGATION PROFORMA
257
Annexure
ANNEXURE IV- DNS QUESTIONNAIRE (DNS SCORING)
1. Are you suffering of unsteadiness in walking? Need for visual control,

increase in the dark, walk like a drunken man, lack of contact with floor.
2. Do you have a burning, aching pain or tenderness at your legs or feet?
Occurring at rest or at night, not related to exercise, excluding
intermittenent claudication.
3. Do you have prickling sensations at your legs and feet? Occurring at rest
or at night, distal > Proximal, stocking glove distribution.
4. Do you have places of numbness on your legs or feet? Distal > Proximal,
stocking glove distribution.
The questions were answered either Yes (positive: 1 point) if a symptom
has occurred during the last 2 weeks or No (negative: no point) if it did not.
Maximum score is 4 and minimum 0.
258
Annexure
ANNEXURE V DNE QUESTIONNAIRE (DNE SCORING)
1. Quadriceps femoris: extension of the knee

2. Tibialis anterior: dorsiflexion of the foot
3. Reflex: Triceps surae (ankle reflex)
4. Sensation: index finger; Sensitivity to pinpricks
5. Sensation: big toe: Sensitivity to pinpricks, Sensitivity to touch, Vibration perception,
Sensitivity to joint position (only right leg and foot are tested)
6. Scoring from 0 to 2: O = Normal 1 = mild/ moderate deficit: Muscle strength:
Medical Research Council Scale 3-4, Reflex: decreased but present, sensation:
decreased but present; 2 = severely disturbed/absent Muscle strength: Medical
Research Council Scale 0-2, Reflex: absent, Sensation: absent , Maximum score: 16
points.
7. A score of > 3 indicates presence of polyneuropathy
259
Annexure
ANNEXURE VI - PHOTOGRAPHS
Photograph 1: Vantamoori PHC area
Photograph 3: Handignur PHC area
Photograph 2: Kinnayae PHC area
Photograph 4: Personal face to face interview

(History Taking)
260
Annexure
Blood glucose testing &HRV analysis
Figure 5: Glucometer
Photograph 7: HRV analysis tools Kit
Photograph 6:- Fasting Blood Glucose testing
Photograph 8:- HRV recording (Time

and frequency domain methods)
261
Annexure
Cardiovascular reflex tests
Photograph 9: 30:15 ratio
Photograph 11: Deep breathing test
Photograph 10: HR and BP response

to lying to standing
Photograph 12: Valsalva maneuver
Photograph 13: Handgrip test
Photograph 14: Cold pressor test
262
Annexure
Diabetic Peripheral Neuropathy Examination
Photograph 15:- Sensitivity to touch
Photograph 16: Sensitivity to pinpricks
Photograph 17: Vibration perception

examination lower extremities
Photograph 18: Vibration perception

examination upper extremities
Photograph 19: Ankle Jerk (reflex)

examination
Photograph 20: Examination of muscle

strength, dorsiflexion and extension
263
Annexure
ANNEXURE VII -PUBLICATIONS
264
Annexure
265
Annexure
266
Annexure
267
Annexure
268
Annexure
269
Annexure
270
Annexure
271
Annexure
272
Annexure
273
Annexure
274
Annexure
275
Annexure
276
Annexure
277
Annexure
278
Annexure
279
Annexure
280
Annexure
281
Annexure
282
Annexure
ANNEXURE VIII- POSTER PRESENTATION
283
Annexure
POSTER PRESENATION
284
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A CROSS-SECTIONAL STUDY TO DETERMINE THE PREVALENCE OF

AUTONOMIC DYSFUNCTION IN TYPE-II DIABETIC MELLITUS INDIVIDUALS IN

For the award of the degree of Doctor of Philosophy

Under the Guidance of

DR. SHIVAPRASAD S. GOUDARMD, MHPE

MELLITUS INDIVIDUALS IN RURAL POPULATION RESIDING AT THREE

A piece of writing or other work reflecting such unauthorized use or imitation.

The deliberate or reckless representation of anothers words, thoughts or ideas as ones

Lecturer, Dept of Physiology

KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

(Accredited A Grade by NAAC)

KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

(Accredited A Grade by NAAC)

KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

(Accredited A Grade by NAAC)

Professor, Department of Physiology, Jawaharlal Nehru Medical College, Belgaum.

Dr. (Mrs.) N.S. Mahantshetti M.D (Pead)

KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

(KLE DEEMED UNIVERSITY)

(Accredited A Grade by NAAC)

We hereby declare that KLE ACADEMY OF HIGHER EDUCATION AND

KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,

who motivated me to register PhD program and

Principal, J. N. Medical College,

Need for the study

Objectives of the study

2.1 Diabetes mellitus

2.2 Diabetes and Autonomic dysfunction

2.3 Diabetic neuropathy

2.3.1 Diabetic autonomic neuropathy

2.3.2 Classification of diabetic neuropathy

2.3.5 Cardiovascular autonomic neuropathy

2.3.5.2 Clinical manifestation

2.3.6 Diabetic peripheral neuropathy

2.3.6.2 Symptoms of DPN

2.3.6.3 clinical assessment of DPN

2.3.6.4 Causes of DPN and Laboratory testing

2.3.7.1 Diagnosis assessment of CAN-AFT

2.3.7.2 Diagnosis of CAN- HRV analysis

3. Clinical component of HRV

4. Diagnosis of CAN by HRV analysis

2.3.7.3 Diagnosis assessment of DPN

2.3.7 Diagnosis of diabetic neuropathy

Experimental designs and sampling methods

Sample size estimation

Study area and sampling distribution plan

Personal detail and History

Biochemical screening methods for Type 2 DM

Assessment of CAN by HRV analysis

Autonomic function tests

CAN scoring methods

Tests for diabetic peripheral neuropathy

Sociodemogrpahic and anthropometric

characteristics of study participants

Association between various risk factors and

Risk factors of T2DM

Prevalence of CAN and DPN

Sympathovagal status among T2DM HRV

prevalence of CAN- HRV analysis

Prevalence of CAN Ewings and Bellavare

prevalence of CAN - Ewings tests and

4.13 Distribution of T2DM participants by various

cardiovascular reflex tests