A STUDY OF COGNITIVE EVOKED POTENTIALS AND SERUM

LEPTIN LEVELS IN YOUNG OBESE INDIVIDUALS

Dissertation submitted to

The Tamil Nadu Dr. MGR Medical University

In partial fulfillment of the regulations for the award of the degree of

M.D. PHYSIOLOGY

Branch V

INSTITUTE OF PHYSIOLOGY & EXPERIMENTAL MEDICINE

Madras Medical College and Rajiv Gandhi Government General Hospital

CHENNAI –600003

THE TAMIL NADU DR. MGR MEDICAL UNIVERSITY

CHENNAI –600032

MAY 2019

CERTIFICATE

This is to certify that the dissertation entitled “A STUDY OF

COGNITIVE EVOKED POTENTIALS AND SERUM LEPTIN LEVELS IN

YOUNG OBESE INDIVIDUALS.” by Dr.UDHAYA BHARATHI.G, for M.D

Physiology is a bonafide record of the research done by her during the period of

the study (2016-2019) in the Institute of Physiology and Experimental Medicine,

Madras Medical College, Chennai- 600 003.

DEAN Madras Medical College, Chennai – 600003.

DIRECTOR & PROFESSOR Institute of Physiology and Experimental Medicine, Madras Medical College, Chennai-600003

GUIDE

CANDIDATE

ACKNOWLEDGEMENT

I express my profound gratitude to Dr.R.JAYANTHI, M.S., Mch., Dean ,

Madras Medical College & RGGGH, Chennai, for permitting me to do this study

and utilizing all the needed resources for this dissertation work.

I sincerely express my grateful thanks to Prof. Dr.C.THIRUPATHI,

M.D., D.C.H., Director & Professor, Institute of Physiology & Experimental

Medicine, Madras Medical College, Chennai, for his support and advice

throughout the study.

I sincerely express my grateful thanks to Dr.MAYILVAHANAN, MD.,

Director & Professor, Institute of Internal Medicine, Madras Medical College, and

RGGGH, Chennai, for providing me with needed subjects and guiding me in this

study.

I extend my sincere thanks to Prof. Dr.P.SATHYA, M.D., D.G.O.,

Professor, Institute of Physiology and Experimental Medicine, Madras Medical

College, without whom it would have been totally impossible to accomplish this

work. I also sincerely thank her for her valuable guidance and motivation

throughout my study and for being a constant source of inspiration.

I extend my sincere thanks to Prof. Dr.A.PARIMALA, M.D., DCP.,

Professor, Institute of Physiology, Madras Medical College, Chennai, for her

valuable suggestions and motivation throughout my study.

I extend my sincere thanks to Prof. Dr.R.VIJAYALAKSHMI, M.D.,

Professor, Institute of Physiology, Madras Medical College, Chennai, for her

valuable suggestions and motivation throughout my study.

I extend my sincere thanks to Dr.J.RATNA MANJUSHREE, M.D.,

D.C.H., Associate Professor, Institute of Physiology, Madras Medical College,

Chennai, for her valuable suggestions and motivation throughout my study.

I extend my thanks to Dr.K.RAMA DEVI, M.D., Professor and Director,

Institute of Biochemistry for helping me to do the lab test in their department.

I extend my thanks to Dr.VEENA JULITTE, M.D., Assistant Professor

of the Department, Institute of Biochemistry for helping me to do the lab test in

their department.

I express my sincere thanks to Dr.V.GOWRI, M.D., Dr.K.AANANDHA

SUBRAMANIUM, M.D., Dr.D.INDUMATHI, M.D., Dr.SYED SAFINA,

M.D., Dr.J.ANITHA PONMALAR, M.D., Dr.V.SUMATHI, M.D.,

Dr.R.BHUVANESHWARI, M.D., Dr.S.ANBUSELVI, M.D.,

Dr.THENMOZHI, M.D., Dr.I.J.V.PRADEEP VAIZ.T, M.D., Assistant

Professors ,Institute of Physiology and Experimental Medicine, Madras Medical

College, Chennai for their guidance and support.

I express my sincere thanks to my Seniors Post Graduates and my Co Post

Graduates in department of Physiology, Madras Medical College, Chennai.

I sincerely thank Dr.K.SOWMIYA., M.D., for her immense help and

support throughout the study.

Above all I would be unfair if I fail to mention my special gratitude to my

dear parents, my lovable husband and my precious son, who are the pillars of my

career and without whom it would have been impossible to accomplish this work.

I dedicate this work to my supportive family.

I thank God Almighty for helping me throughout this endeavor.

CONTENTS

I. LIST OF TABLES

II. LIST OF GRAPHS

III. LIST OF PHOTOGRAPHS AND FIGURES

IV. ABBREVIATIONS

CHAPTER

NO. TITLE PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 12

3 AIM AND OBJECTIVES 60

4 MATERIALS AND METHODS 61

5 RESULTS 74

6 DISCUSSION 85

7 CONCLUSION 94

8 SUMMARY 95

BIBLIOGRAPHY

ANNEXURES

(i) ETHICAL COMMITTEE APPROVAL

(ii) CONSENT FORM

(iii) PROFORMA

(iv) MASTER CHARTS

LIST OF TABLES

Table No. Title Page

No 1. Classification of obesity in Adults according to WHO 2

2. Consequences of obesity 6

3. Distribution of percentage of obese in various states in India 14

4. Classification of obesity based on WHO Asia Pacific Guidelines 19

5. WHO classification of obesity with BMI (Asian population) 20

6. Risk of Obesity-Associated Metabolic Complications 21

7. Abdominal Depots 27

8. Role of Proteins secreted by adipose tissue 28

9. Bariatric Surgeries for Obesity 32

10. Baseline Characteristics Of Study And Control Group (Mean ± SD) 74

11. Descriptive analysis of Gender in study (obese) N=40 and control (non-obese) N=40 groups 74

12. Descriptive measurements of the study participants 75

13. Comparison of the BMI between the two study groups 76

14. Comparison of the measures of obesity (Waist Circumference, Hip Circumference and Waist Hip Ratio) between the two study groups

76

15. Comparison of the Serum Leptin levels between the two study groups 78

16. Comparison of the P300 Latency and P300 amplitude between the two study groups 78

17. Correlation table of P300 latency and BMI for both study and control groups 79

18. Correlation table of P300 latency and Waist circumference for both study and control groups 79

19. Correlation table of P300 latency and Hip Circumference for both study and control groups 80

20. Correlation table of P300 latency and Waist Hip ratio for both study and control groups 80

21. Correlation table of P300 amplitude and BMI for both study and control groups 80

22. Correlation table of P300 amplitude and Waist circumference for both study and control groups 81

Table No. Title Page

No

23. Correlation table of P300 amplitude and Hip Circumference for both study and control groups 81

24. Correlation table of P300 amplitude and Waist Hip ratio for both study and control groups 81

25. Correlation table of Serum Leptin and BMI for both study and control groups 82

26. Correlation table of Serum Leptin and Waist circumference for both study and control groups 82

27. Correlation table of Serum Leptin and Hip Circumference for both study and control groups 82

28. Correlation table of Serum Leptin and Waist Hip ratio for both study and control groups 83

29. Correlation table of Serum Leptin and P300 Latency for both study and control groups 83

30. Correlation table of Serum Leptin and P300 amplitude for both study and control groups 83

LIST OF GRAPHS

Graph No. Title Page No.

1 Descriptive Analysis Of Gender 75

2 Comparison of the Body Mass Index between the two study groups 76

3 Comparison of the Waist Circumference, Hip Circumference between the two study groups 77

4 Comparison of the Waist- Hip Ratio between the two study groups 77

5 Comparison of the serum Leptin levels between the two study groups 78

6 Comparison of the P300 Latency And P300 Amplitude between the two study groups 79

LIST OF PHOTOGRAPHS

Photograph No. Title

1 Recording of P300 in Neurostim machine

2 Recording of P300 in Clinical Physiology Research Lab at RGGGH

3 Sample collection

4 DRG Leptin ELISA Kit

LIST OF FIGURES

Figure No. Title Page

No. 1 Structure of Leptin 7

2 Causes of obesity 18

3 Normogram for determining BMI 20

4 Complications of obesity 23

5 Light microscopy of Human White Adipose Tissue Between

26-27 6 Light microscopy of Human Brown Adipose Tissue

7 No Clear Boundary Between Two Adipose Tissues

8 Roux-En-Y Gastric Bypass 32

9 Adjustable Gastric Banding 33

10 Biliopancreatic Diversion With Duodenal Switch 33

11 Obesity and Cognition 34

12 Causes of Cognitive Decline in Obesity 35

13 Components of Event Related Potential 37

14 Relationship between Age and P300 Latency 39

15 Role of Leptin 49

16 Peripheral Actions Of Leptin 51

17 Leptin Signal Transduction Pathways in the Hypothalamus 54

18 Interconnection between obesity, elevated Leptin, and Leptin resistance 55

19 Ob/Ob Mice 56

20 Role of Leptin in Cognition 58

21 Subject Selection 63

22 Measurement of Waist circumference (cm) 65

23 Measurement of Hip circumference (cm) 65

24 Electrode Placement 67

25 P300 wave generation by target stimuli and waves generated by background stimuli

Between 67-68

26 Standard Curve of Human Leptin 72

ABBREVIATIONS

1. BMI – Body Mass Index

2. ELISA – Enzyme Linked Immunosorbent Assay

3. IR – Insulin Resistance

4. DM – Diabetes Mellitus

5. CVD – Cardio vascular disease

6. ERP – Event related potentials

7. TNF α – Tumour necrosis factor alpha

8. CRP – C Reactive protein

9. SAA – Serum Amyloid A protein

10. PAI – Plasminogen activator inhibitor

11. MCR 4 – Melanocortin 4 receptor

12. POMC – Pro opio melanocortin

13. ACTH – Adrenocortico trophic hormone

14. PI 3K – Phoshoinositol 3 kinase

15. ras - MAPK – ras mitogen activated protein kinase

16. AOD-1/SREBP-1c – Adipocyte determination and differentiation factor – 1

/ sterol regulatory element binding protein 1c

17. IRS 1 & 2 – Insulin receptor substrates 1 & 2

18. WAT – White adipose tissue

19. BAT – Brown adipose tissue

20. GLUT 4 – Glucose transporter 4

21. FFA – Free fatty acids

22. IL -6 – Interleukin 6

23. WHO – World Health Organisation

24. WC – Waist Circumference

25. HC – Hip Circumference

26. WHR – Waist – Hip Ratio

27. BF% – Body Fat Percentage

28. DEXA – Dual-energy X-ray absorptiometry

29. CHD – Coronary Heart Disease

30. PCOS – Polycystic Ovarian Syndrome

31. CKD – Chronic Kidney Disease

32. OCP – Oral Contraceptive Pills

33. STEPS – Stepwise Approach to surveillance

34. TMB – Tetramethylbenzidine

35. CURES – Chennai Urban Rural Epidemiology Study

36. SSRI – Selective serotonin reuptake inhibitors

37. CO – Combined obesity

38. CT – Computed Tomography

39. MRI – Magnetic Resonance Imaging

40. TNF-β – Tumor Necrosis Factor beta

41. IGF-1 – Insulin – like growth factor 1

42. FDA – Food and Drug Administration

43. EP – Evoked Potentials

44. LEPR – leptin receptor

45. ARP – Agouti-related peptide

46. NPY – Neuropeptide Y

47. CART – Cocaine and Amphetamine-Related Transcript

48. JAK2/STAT3 – Janus-activated kinase 2 / signal transducers and

activators of transcription 3

49. SOCS3 – Suppressors of cytokines signaling family

50. LTP – long-term potentiation

CERTIFICATE – II

This is to certify that this dissertation work titled “A STUDY OF

COGNITIVE EVOKED POTENTIALS AND SERUM LEPTIN LEVELS IN

YOUNG OBESE INDIVIDUALS” of the candidate Dr. G. UDHAYA

BHARATHI with registration Number 201615004 for the award of M.D Degree

in the branch of PHYSIOLOGY. I personally verified the urkund.com website

for the purpose of plagiarism Check. I found that the uploaded thesis file contains

from introduction to conclusion pages and result shows _2%_ percentage of

plagiarism in the dissertation.

Guide & Supervisor sign with Seal

Introduction

1

INTRODUCTION

“Increasing prevalence of childhood obesity has placed the health of an entire

generation at risk” ----- Toms Vilsack

Our generation is the most unfortunate generation that is deemed to witness

the demise of the next generation largely due to obesity epidemic and health

related catastrophe.

Nowadays in developing countries like India, rising prevalence of obesity

has become a major health concern. According to WHO, though obesity is the

most common problem in the present era, it is often a neglected problem in both

developed and developing countries1. The world health statistics report 2012 of

the WHO states that one among six adults worldwide is obese2. The mortality due

to obesity or overweight is about 3.4 million per year. India occupies the third

most obese country in the world. Death due to overweight and obesity is much

higher when compared to underweight3. There is a shortening of life expectancy

by around 2-5 years in the individuals suffering from obesity. The causes of

increase in prevalence of abdominal obesity in India may be the rapid

urbanization which has resulted in intake of high-calorie diets and low physical

activity. In India in the year 2008, 1.3% males and 2.5% females aged more than

20 years are obese3.

Sedentary life style, physical inactivity and western diet are the major

reasons for overweight and obesity.

2

“Obesity is not because it runs in the family: it’s mainly because no one runs in

the family”.

Definition of obesity

WHO defines obesity as “A condition with excessive fat accumulation in

the body to an extent that health and wellbeing are adversely affected” .The

definition of obesity is limited to body mass index (BMI).

According to WHO 1998, it was proposed that thresholds shown in the

below table shall be used to classify BMI in adults

Table No: 1 Classification of obesity in Adults according to WHO

Causes of obesity

Introduction of western diet is the main reason for obesity in adolescent

and young adults in developing countries like India. Saturated fats and simple

carbohydrates which are present in western diet are the main reasons for the

BMI Classification

<18.50 Underweight

18.50 – 24.99 Normal Range

≥25 Overweight

25.00 - 29.99 Pre Obese

30.00 - 34.99 Class I Obese

35 - 39.99 Class II Obese

≥ 40.00 Class III Obese

3

emergence of many non-communicable diseases like cardiovascular problems,

diabetes, hypertension, osteoarthritis, Stroke, obstructive sleep apnoea,

hepatobiliary diseases, endocrine disorders and various forms of cancer4. Apart

from this, decreased cognition is also associated with increased BMI5.

Greater predisposition to abdominal obesity and accumulation of visceral

fat have been noted in Asian Indians and this has been termed as “Asian Indian

phenotype”6,7. In countries like India, the rise in obesity prevalence could be

attributed to the increasing urbanization, use of mechanized transport, increasing

availability of processed and fast foods, increased television viewing, adoption of

less physically active lifestyles and consumption of more “energy dense, nutrient-

poor” diets8-10.

Anthropometric measures in obesity

Several methods are used to measure obesity. They include both direct and

indirect techniques.

INDIRECT TECHNIQUES

1. Body mass index (quetelet’s index)

Simplest anthropometric measures are used in the estimation of body fat.

BMI = Weight (kg)/ height2 (m) 3

2. Waist circumference and Hip circumference

Measurement of Waist and hip circumference is done twice with an

inextensible anthropometric tape. The subject should be standing erect in relaxed

manner with arms by the sides and feet close together. For all measurements the

4

tape was positioned at a level parallel to the floor. All measurements were in

centimeters (cm) to the nearest 0.1cm11.

Waist circumference is an approximate index of intra-abdominal fat mass

and total body fat. It is a globally used parameter and is the simplest way to

quantify central obesity.

A non-stretchable measuring tape is used to measure the waist

circumference in centimeters. Measurement of waist circumference is done at the

end of normal expiration, at the point where the horizontal girth is the least

between the costal margins and the upper border of the iliac crest2.Men with a

waist circumference ≥ 102 cm and Women with a waist circumference ≥ 88 cm

has an increased risk of complications3.

Hip Circumference is measured at the widest part of the hip at the level of

the greater trochanter.

3. Waist - Hip ratio

“Waist Hip Ratio (WHR) is the ratio of waist circumference to hip

circumference.” A high WHR > 1.0 in men and > 0.85 in women indicates

abdominal fat accumulation3. It is an index of central obesity

4. Skin fold thickness

Skin fold thickness measures the subcutaneous fat that correlates with total

body fat. Mid triceps, biceps, supra iliac and supra scapular regions are the areas

where we measure the skin fold thickness by using skin calipers. Sum of these

5

measurements more than 40 mm in males and more than 50 mm in females

indicates obesity3.

5. Body fat percentage

“Body fat percentage is the total weight of body fat divided by body

weight”. A body fat percentage ≥ 23% in males and ≥ 29% in females indicates

obesity. Body Fat Percentage (BF %) can be calculated from BMI in adults using

the equation given below.

BF% = (0.23 x Age) + (1.2 x BMI) - (10.8 x Sex) - 5.4

Where, BMI is in Kg/m2; Sex=0 for female and 1 for male; Age is in yrs;

DIRECT TECHNIQUES

1. Radiological Methods

Precise measurement of abdominal fat is done using CT, MRI and Dual-

energy X-ray absorptiometry - DEXA scan12.

2. Other methods

The direct techniques involve a number of procedures that include

densitometry, measurement of total body potassium, estimation of total body

water, neutron activation techniques and electrical impedance.

6

Obesity and its consequences

Obesity causes many health hazards ranging from mild morbidity to mortality.

Table No: 2 Consequences of obesity

Greatly increased Moderately increased Mildly increased

Type 2 diabetes CHD PCOS

Gall bladder disease Hypertension Cancers

Dyslipidaemia Osteoarthritis (Knee) Reproductive hormone abnormality

Obstructive sleep apnoea

Hyperuricaemia and Gout Impaired fertility

Insulin resistance Low back ache

Breathlessness Increased risk of anaesthesia complications

Obesity and adipokines

The adipokines like adiponectin, retinol binding protein, leptin, IL -6,

TNF- α, and PAI-1 are related to the occurrence of obesity and metabolic

consequences of the same.

Leptin

Leptin is a hormone which was identified in 1994 by J.M.Friedman. The

location of Ob (Lep) gene in humans is chromosome 7. Molecular weight of

Leptin in humans is 16-kDa and it is made up of 167 amino acids13. Leptin which

is a hormone mainly produced by the adipocytes of white adipose tissue14. Leptin

is also produced by the brown fat, bone marrow, syncytiotrophoblasts of the

placenta, ovaries, epithelial cells of the mammary glands, skeletal muscle and

fundic glands of the stomach especially the lower part.

7

Figure 1 Structure of Leptin

Role of Leptin in Obesity

Although leptin reduces appetite, there is leptin resistance in obesity like

that of resistance of insulin in type 2 diabetes, there is a higher circulatory levels

of leptin in obese individuals as there is an increase in percentage of body fat in

such persons15. Although there is an increase in the circulatory levels of leptin in

obese persons, because of the leptin resistance, they are unable to suppress hunger

and control weight gain. Consumption of a fructose rich diet from birth causes

reduction in mRNA expression for receptors of leptin and decrease in circulatory

levels of leptin. There also occurs an increase in triglyceride levels and a trigger

of resistance to both insulin and leptin when there is consumption of high fructose

diet for long term16.

Leptin, an appetite suppressor, which controls food intake, also stimulates

oxidative stress, inflammation, thrombosis, arterial stiffness, angiogenesis and

8

atherogenesis. These leptin-induced effects may be the predisposing factor for

the development of cardiovascular diseases. Elevated leptin concentrations have

also been related to Chronic Kidney Diseases (CKD) incidence and progression

as well as with insulin resistance, T2DM, micro- and macrovascular diabetic

complications.

Cognition

The word cognition is derived from the Greek word ‘gnosis’ which means

knowledge.

Cognitive function is attributed to “Acquisition, processing, integration,

storage plus recovery of information”.17

Cognition and Obesity

Obese individuals tend to have higher levels of adipokines released by fat

cells. These cells decrease white matter in the brain, which is responsible for

nerve connections throughout the brain. Decreased neuronal functioning and

decreased vascular supply to brain may lead to brain atrophy and consequently

loss of normal brain functioning.18

Increased BMI increases central adiposity and waist circumference which

results in white matter changes, disturbances of blood-brain barrier integrity and

brain atrophy. Hippocampal dysfunction is associated with increased body weight

which is mainly due to western diet.19

9

Intake of western diet

Blood brain barrier integrity impaired

Disrupts memory19

Leptin and Cognition

Apart from working as a regulator of body weight, fat storage and energy

homeostasis, leptin exerts it’s effects in cognitive function also20. Leptin acts

within areas such as the cerebral cortex and hippocampus to influence neuronal

survival and promotion and thereby promotes learning, memory and other forms

of cognition21. Resistance to leptin caused by the higher circulating levels results

in reduced transport of leptin across the blood brain barrier and thus, there occurs

a reduction in downstream intracellular signaling20. The result of this resistance to

leptin impairs cognitive performance. .

Cognitive evoked potentials

Cognitive Evoked Potentials - Р300 which is the response of the brain,

recorded under the conditions of the identification of the significant distinguishing

stimulus, it facilitates the inspection of cognitive functions and memory in the

healthy persons and patients with different manifestation of cognitive

impairments.22

The P300 component is a positive deflection occurring between 250 and

500ms after stimulus onset and is thought to reflect attentional processes. This

10

component is often recorded using an oddball paradigm, in which the subject is

presented with frequent and infrequent stimuli and asked to count the infrequent

stimuli.23An auditory cognitive Event Related Potential (ERP) is generated by

playing a baseline series of frequently occurring rhythmic auditory stimuli for a

subject and then presenting secondary auditory stimuli (rare stimulus) at random.

The subject mentally counts the secondary auditory “oddball” (rare) stimuli, and

this specific intellectual function generates a discrete waveform of cognitive

evoked response called the P300 component since its latency is about 300 ms

(millisecond) after the stimulus24.

P300 amplitude is related to the amount of attentional resources devoted to

the task, whereas P300 latency indexes stimulus classification speed. Event

related potentials (P300) that reflect cortical activities related to cognitive

functions. P300 latency represents the information processing time; longer latencies

represent slower processing. P300 amplitude is associated with the attentional

system and working memory, with higher amplitudes representing more preserved

functions25.

Leptin, cognition and obesity

Obesity is linked with low neurocognitive performance. Higher the

concentrations of leptin and C-reactive protein, lower is the P3 amplitudes. This

showed a significant negative association between serum leptin and P300

amplitude. However, leptin is regarded as a sole predictive factor of status of

neurocognitive function in obese individuals. Event Related Potentials (ERP) are

11

useful markers of executive dysfunction and provide some information about the

neurophysiological basis.26

Serum leptin level is assessed and correlated with that of controls of

normal BMI. Increased serum leptin in obese persons which is a main factor for

lower cognitive performance is to be measured and correlated with P300 amplitude

and latency in this study.

With the above material I have proposed a study on the topic “A Study of

Cognitive evoked potentials and Serum Leptin in young Obese individuals”.

Review of Literature

12

REVIEW OF LITERATURE

Review of literature deals with obesity, obesity and cognition, leptin and

cognition, leptin as a cognition marker and the leptin level variation in obese and

non obese young individuals.

Hippocrates, a Greek physician said that “Corpulence is not only a disease

itself, but the harbinger of others. Sudden death is more common in those who are

naturally fat than in the lean”27.

Bray GA et al, 1998 postulates that the definition of obesity is limited to

BMI and related to increase in adipocyte cell size and/or cell number28.

In 1985, in a Consensus Development Conference on Health Implications

of Obesity held by The National Institute of Health, obesity was concluded to be a

disease29.

“Obesity is characterized by excessive accumulation of fat in the body to

an extent that it may have an adverse effect on health, leading to decreased

expectancy of life and/or increased health problems” (WHO,2000)30

Obesity and Overweight

“Obesity is generally defined as a body mass index (BMI) of 30 kg/m2 and

higher”.

“Overweight is defined as a BMI between 25 and 30 kg/m2”.

13

Overweight is more common among men than women but obesity is more

common among women. Overweight and Obesity occupies the fifth position in

causing deaths globally3.

Prevalence of Obesity

The prevalence varies between every country, and between regions within

countries. Obesity and socio-economic status are inversely related, particularly

among women. The prevalence of obesity seems to be increasing nowadays,

especially in developing countries. Prevalence of obesity in populations mean the

fraction of people who have an excess storage of body fat. In adult men with an

average weight, the percentage of the body fat is in the order of 15-20%. In

women, this percentage is higher (i.e) 25-30%.This differences in weight between

individuals are only partly due to variations in body fat. To differentiate between

overweight and normal weight people, the weight or indices based on height and

weight (such as the body mass index, BMI) are used widely31. Different body

morphology or types of fat distribution are independently related to the

complications of obesity32. Since 1980, the prevalence of obesity has doubled in

adults and the incidence has tripled among overweight children and adolescents 33.

The National Health and Nutrition Examination Survey (NHANES) done between

2003 and 2004 states that 66.3% of adults are overweight and 32.2% are obese34.

In the United States, obesity is ranked second next to the use of tobacco as the

leading preventable cause of death 35.

14

In India

In India, in the year 2008,1.3% of males and 2.5% of females aged > 20yrs

are obese3.In 1991-1995 cross sectional surveys conducted in India revealed a

considerably higher prevalence of obesity in urban (7.1% in men and 16.4% in

women) when compared with rural (0.7% in men and 2.2% in women)36. It clearly

demonstrates that urbanization is one of the most important cause for increased

prevalence of overweight and obese.

At an alarming rate, the prevalence of obesity is increasing worldwide and

is now a problem for both developed and developing countries. Even in India,

where one third of the population falls below the poverty line, has experienced

steady growth in the urban middle class population to 200 million, of which 40 to

50 million are overweight37.

In Tamilnadu

According to the NFHS-3 data, conducted in the three states namely

Jharkhand, Maharastra and Tamilnadu, the percentage of obese in both the

genders are highest in Tamilnadu when compared with the other two states38.

Table No: 3 Distribution of percentage of obese in various states in India

STATES PERCENTAGE OF

OBESE IN MEN BMI ≥ 25 kg/ m2

PERCENTAGE OF OBESE IN WOMEN

BMI ≥ 25 kg/ m2 Tamil Nadu 19.8 % 24.4% Maharashtra 15.9 % 18.1% Jharkhand 5.3 % 5.9%

15

Also in another study named The Chennai Urban Rural Epidemiology

Study (CURES) conducted in Chennai city in Tamil Nadu reported age

standardized prevalence of different types of obesity.

Generalized obesity - 45.9 %

Abdominal obesity - 46.6 %

Isolated generalized obesity - 9.1 %

Isolated abdominal obesity - 9.7 %

Causes of obesity

No single factor is responsible for obesity. Multifactoral causes such as

genetic, endocrinal, environmental, psychological, and managerial factors are

responsible.39

Age:

Both the genders, till the age of 50-60 years, weight increases with age.

Those who have gained excessive weight during infant period gain more weight in

later life3.

Gender:

Women generally have a higher rate of obesity than men, although men

have higher rates of overweight. Men gain more weight at the age of 29-35 years

and women at the age of 45-49 years3.

16

Socio-cultural factors

Educational level and income:

In industrialized countries, there is a higher prevalence of overweight in

those with lower socioeconomic status3.

Marital status:

Usually after marriage, weight increases, which leads to overweight40.

Urbanisation

The causes for increase in obesity in urban areas are due to mechanization,

increasing availability of processed fast foods and carbonated drinks, improved

motorized transport, increased viewing of television and video games and

adoption of less physically active lifestyles 2,24.

Biological factors

Parity:

BMI increases with increasing number of children, on average of about 1kg

per pregnancy3.

Behavioural factors

Dietary intake:

Higher prevalence of obesity is mainly due to increased consumption of

fatty food3.

17

Smoking:

Smoking lowers body weight and cessation of smoking increases body

weight3.

Alcohol consumption:

Moderate alcohol consumption is sometimes associated with higher body

mass index3.

Physical activity:

Sedentary lifestyles in conjunction with Physical inactivity is the main

route cause of overweight and obesity31.

Genetic factors

Obesity in any parent determines a higher severity of their

offspring obesity and metabolic co-morbidities, more importantly when obesity is

present in the mother or in both parents41.

Secondary causes

Medications

Psychiatric/Neurological

Antipsychotics: Clozapine, Olazapine, Risperidone

Antidepressants: Selective serotonin reuptake inhibitors (SSRI), Tricyclic

antidepressants

Lithium

Antiepileptics: Valproate, Gabapentin,Carbamazepine

18

Anti diabetic drugs

Insulin, Sulfonylureas, Thiazolidinediones

Others

Hormonal contraceptives, Corticosteroids, Progestational agents,

Antihistamines, β blockers, α blockers

Figure 2 Causes of Obesity

Syndromes of obesity.

1. Bardet –Biedl Syndrome

2. Carpenter Syndrome.

3. Cohen Syndrome

4. Prader – Willi Syndrome

5. Borjeson – Forssman – Lehmann Syndrome

6. Down Syndrome

7. MOMO syndrome42

19

Table No:4 Classification of obesity based on WHO Asia Pacific Guidelines

Types BMI Waist Circumference

Generalized obesity ≥ 25 kg/m2 with or without abdominal obesity

Abdominal obesity ≥ 90 cm for men and ≥ 80 cm

for women with or without Generalized obesity

Isolated generalized obesity ≥ 25 kg/m2 < 90 cm in men and < 80 cm in

women Isolated abdominal

obesity < 25 kg/m2 ≥ 90 cm in men or ≥ 80 cm in women

Combined obesity (CO): Individuals with both Generalized Obesity and

Abdominal obesity.

Non obese subjects: Individuals without either Generalized Obesity or

Abdominal Obesity2.

MEASUREMENT OF OBESITY

BMI is a useful marker for overall obesity but it is not a direct measure of

adiposity. Calculation of BMI is done by dividing the weight in kilograms by

square of height in meters.

20

WHO CLASSIFICATION OF OBESITY WITH BMI IN THE ASIAN

POPULATION

In Asia, because of the high prevalence of co-morbidities, particularly

diabetes and hypertension there is a decreased range for normal BMIs (i.e., 18.5 to

22.9 kg/m2 rather than 18.5 to 24.9 kg/m2)43.

Table No: 5 WHO Classification of Obesity with BMI (Asian Population)

NORMOGRAM FOR DETERMINING THE BODY MASS INDEX

Figure 3 Normogram for determining BMI

21

Alternate measures to measure the adiposity

Waist Circumference, Waist-to-Height ratio, and Waist-to-Hip ratio are

more useful indices for defining abdominal obesity. Of this Waist Circumference

showing the strongest relationship than the other indices.44

Huxley et al., also strongly proposed waist circumference and the waist –

hip ratio superior to BMI in predicting cardiovascular disease risk45.

Women with waist hip ratio of > 0.85 and men with the same > 1 fall into

the abnormal range. It indicates abdominal fat accumulation3.

The WHO expert report released in December 2008 proposed the following

cut off values.

Table No 6: Risk of Obesity-Associated Metabolic Complications31

Gender

Increased risk of health problems

Greatly increased risk of health problems

Level 1 (alerting zone) Level 2 (action level)

Men ≥94 cm(~ 37 inches) ≥102 cm( ~ 40 inches)

Women ≥80 cm(~32 inches) ≥88 cm(~35 inches)

The most popular among all the measures is the waist/hip circumference

ratio.46 Waist/ Height ratio may be a better predictor of morbidity because the

waist is positively associated with disease and height unrelated to body

composition or fat distribution, is inversely associated with disease31.

22

Deurenberg et al. established that the body fat percentage in adults can be

accurately estimated by using this equation:

Body fat%=1.2 (Body Mass Index) + 0.23 (age)-10.8 (gender)-5.4

where gender=l for men and gender=0 for women47.

There occurs a very close correlation of waist circumference with

abdominal fat tissue. Thus the most important risk factor for adult onset type of

diabetes is abdominal obesity even when controlled for other parameters like age,

history of smoking and family history of diabetes48.

A study done by Gothenberg et al. in 2010 concludes that development of

diabetes in the future can be predicted by the waist hip ratio. This study also states

that waist hip ratio is a better predictor of risk of angina pectoris, myocardial

infarction, stroke and sudden death. Waist hip ratio predicts the above said

conditions independent of the BMI49.

Triceps skin fold thickness reflects body fat and is widely used in

pediatrics because it is non-invasive and very easy to perform. It is less accurate

than the measurement of weight and height and it under/over estimates body mass

by as much as 10%.

Other methods

In recent days, abdominal obesity can be quantified most accurately in a

sophisticated manner with imaging techniques like CT or MRI, DEXA scan

(under water weighing) and electrical impedence. Of these CT and MRI provide a

23

precise measurement of depots of specific adipose tissue and directly assess the

intra- abdominal visceral fat deposition 50. Measuring the total body water, total

body potassium, body density are the other methods but it is complex one to

measure.

PATHOGENESIS OF OBESITY

An adequate balance is required between the consumption of energy

through diet and the expenditure of energy through bodily functions and exercise.

When there is an imbalance between these two obesity results. Thus obesity is the

result of excess intake of energy, reduced expenditure of energy or a combination

of both. Excess body fat is usually distributed as subcutaneous fat in the regions

like thighs, buttocks and breasts in women and in men, there is a relative excess of

body fat stored in the abdominal cavity and as abdominal subcutaneous fat.51

Figure 4 : Complications of Obesity

24

Adipose tissue

Adipose organ contributes to the regulation of body's homeostasis. The

adipose organ is a multidepot organ with a complex shape52.

Preadipocytes are the precursors of adipose tissue which consists of the

adipose cell that stores the lipid and stromal compartment made up of vascular

tissue, macrophages and the preadipocytes. A cascade of transcription factors

specified to derive the adipose tissue from the preadipocytes of mesenchymal

origin mediate the differentiation process precisely orchestered in a series of

steps53.

In females, there is more developed mammary and gluteofemoral

subcutaneous adipose tissue than in males. Around 8 to 18 % of body weight in

lean adult men and 14 to 28 % of body weight in women is constituted by the

adipose tissue54.

Factors expressed by Adipose tissue in modulation of adipogenesis.

Insulin-like growth factor-1

Transforming growth factor-β

Tumor necrosis factor-α

Macrophage colony-stimulating factor,

Angiotensin-2

Autotaxin–lysophosphatidic acid

Leptin

Resistin55

25

Adipose tissue a true endocrine organ

Adipocytes

Adipose tissue, a type of loose connective tissue made up of an extensive

network of blood vessels with collagen fibers, fibroblasts, and immune cells that

surround lipid-laden cells, known as adipocytes.

Apart from energy depots which is the storage area for triglycerides during

feeding, adipose tissue also releases fatty acids during fasting. It secretes

adipokines which acts at both the local (paracrine / autocrine) and systemic

(endocrine) level56. Fat (or triglyceride) is the primary form in which potential

chemical energy is stored in the body. Adipocytes have a maximum volume that

cannot be further expanded. This maximum volume, also referred to as “critical

cell size,” is genetically determined and specific for each depot40.

The amount of triglyceride in adipose tissue is the cumulative sum of the

differences between energy intake and energy expenditure.

Types of adipose tissue:

White and Brown Adipose tissue

The colors of the adipose tissue are white and brown. The white parts are

made mainly by white adipocytes and the brown parts mainly by brown

adipocytes. The relative amounts of white and brown parts are genetically

determined and depend on several factors like age, sex, environmental

temperature, and nutritional status. There is no clear anatomical boundary

26

between brown and white adipose tissues. Many other parts of the adipose organ

are mixed with brown adipocytes widespread within the white depot57. In humans

the principal form of adipose tissue is white adipose tissue (WAT), whose

adipocytes have an eccentric nucleus with a single lipid droplet.

Active regulation of homeostasis of the whole body is done by the WAT

which is also regarded as a vital endocrine organ 58.WAT produces several factors

known as adipokines, which controls several important functions such as glucose

and lipid metabolism, blood coagulation, blood pressure, and steroid hormone

modulation.

Brown adipocytes produce and secrete many substances, such as autocrine,

paracrine, and endocrine factors59,60. Brown adipose tissue (BAT) is concerned

with adaptive thermogenesis. In contrast to white adipose tissue, BAT expends

stored energy as heat which it accomplishes through a unique mitochondrial

protein namely uncoupling protein (UCP- 1) or thermogenin that uncouples fuel

oxidation from ATP generation by breaking the hydrogen ion gradient across

inner mitochondrial membrane61.

When the energy balance becomes positive, white part of the adipose organ

gets increase. White adipocytes undergo hypertrophy followed by hyperplasia.

Obesity induced by a high-fat diet is hypertrophic, whereas obesity induced

by hypothalamic lesions caused by administration of monosodium glutamate is

hyperplastic.41

Figure 5: Light microscopy of Human White Adipose Tissue.

Figure 6: Light microscopy of Human Brown Adipose Tissue

Figure 7: No clear boundary between two adipose tissues

27

MAJOR ABDOMINAL DEPOTS IN HUMANS

Adipose tissue that is found in specific locations are referred to as adipose

depots.

Table No: 7 Abdominal Depots

DEPOT APPROXIMATE SIZE (kg) COMMENTS

1.Subcutaneous : ( anterior + posterior) 1-20 The most variable of the

abdominal depots

2. Intra-abdominal :

2 a. Omental 0.5 – 3 Visceral depots; drain mostly to portal vein

2 b. Mesenteric 0.5 – 2 Visceral depots; drain mostly to portal vein

2 c. Perirenal 0.5 – 2 Retroperitoneal

The visceral fat depots like the omental and mesenteric fat are those which

require the most important attention and focus among all other intra abdominal fat

depots 62.

Functions of adipose tissue

1. Store energy in the form of lipids (Main role)

2. Insulation and cushioning of the body.

3. Reservoir of nutrients.

Collins S et al, 1995 described adipose tissue as the body’s largest energy

reservoir63. It is basically required for glucose homeostasis to be normal.

Proteins secreted by adipose tissue

For regulating various body functions it also synthesis and secrete proteins.

28

Table No: 8 Role of Proteins secreted by adipose tissue

PROTEINS SECRETED BY ADIPOSE TISSUE ROLE

Leptin Adenosine Acylation stimulating protein

Friedman,Halaas et al 64 Kather et al 65

Sniderman et al 66

Regulation of fuel flux

Adiponectin TNF-α

Weyer et al 67

Moller et al 68

Regulation of insulin action

Angiotensin converting enzyme Angiotensinogen PGI2

Gorzelniak et al 69

Van Harmelen et al 70

Fink et al 71

Regulation of vasomotor tone

TNF-β PGI2 IGF-1

Alessi et al 72 Negrel et al73 Wabitsch et al74

Regulation of cell turn over

Interleukin – 6 Adipsin TNF – α

Hirano et al75

Esterbauer et al76 McDermott et al77

Regulation of inflammation

17 β – OH steroid Cytochrome P 450 dependent aromatase

Crobould et al 78

Bulun et al79

Steroid conversion, reproduction, bone

mass

Plasminogen activator inhibitor-1 PGI2

Crandall et al80 McCarty et al81

Regulation of coagulation

Agouti signal protein Voisey et al82 Others

Adipocytokines

Adipose tissues produce a wide range of proteins termed Adipocytokines.

Adipose tissue synthesizes and secretes “adipokines”. They are leptin, resistin and

adiponectin. Additionally, adipocytes secrete proinflammatory cytokines, such as

29

tumor necrosis factor (TNF)-alpha and interleukins, and proteins involved in

coagulation and vascular function83. The various adipokines and cytokines

secreted by the visceral adipocytes have a notable role in the development of

systemic complications of obesity.

Regulators of fat deposition

Numerous factors like rate of blood flow, activity of the sympathetic and

parasympathetic nervous systems, hormone delivery and presence of various

substrates in the plasma regulate the functioning of the adipose tissue in the body.

The metabolic activity within the adipose tissue is found to be dynamic and shows

complexities. The amount of stored triacylglycerol (TG) in the adipocytes denotes

a measure of balance between deposition and mobilization of fat.

MANAGEMENT OF OBESITY:

For the management of obesity multidisciplinary approach is followed.

Weight loss is the most effective treatment for obesity-related physical and

psychological comorbidities. Efforts should be needed for prevention strategies

and raising awareness about the complications of obesity among young

adults.84Treatment includes dieting, drugs and weight loss surgery. Dieting and

drugs seem to be effective for overweight or mildly obese individuals but there is

a high rate of failure in the morbidly obese cases. Bariatric surgery to be the most

effective treatment for morbidly obese persons to achieve the desirable long-term

results of substantial weight loss.40

30

LIFESTYLE MANAGEMENT:

Diet, Pharmacological, and Behavioral Treatment

The US Food and Drug Administration (FDA), as well as an expert panel

convened by the National Heart, Lung, and Blood Institute, have strongly insisted

that weight loss medications be used only as an adjunct in addition to a lifestyle

modification that includes diet, physical activity, and behavior therapy85.But

disadvantage of lifestyle modification is weight regain after treatment termination.

Patients regain 30 to 35% of their lost weight on average, in the year following

treatment. Approximately 3 to 5 year after therapy, 50% or more of participants

have returned to their baseline weight86,87. Most obese people, left untreated for

3 to 5 year, would probably gain 0.5 to 1 kg per year88.

DIET THERAPY:

High-carbohydrate, high-fiber and low-fat diets result in decreasing serum

cholesterol concentrations and thus the risk for coronary heart disease.40

PHYSICAL ACTIVITY THERAPY

Daily physical activity plays a fundamental role in energy balance, weight

control, and overall health. Exercise program can attenuate some of the health

risks associated with diabetes, hypertension, and osteoarthritis. Energy imbalance

involving increased energy intake (eating too many calories), decreased energy

expenditure (not enough physical activity), or a combination of both leads to

weight gain and obesity. At least 2.5 hours of moderate-intensity physical activity

or exercise per week, a gradual progression to 3.3 to 5 hours per week facilitates

long-term maintenance of weight loss89.

31

BEHAVIOURAL THERAPY:

Make the patients aware of their behaviours and to change their

behavioural patterns (e.g) where and when food was eaten rather than changing

the actual caloric intake or energy balance. Key strategies included self-

monitoring, goal setting, stimulus control, cognitive restructuring. Problem

solving, self-reinforcement, stress management, contingency management, social

support and ongoing contact. Attendance at treatment sessions leads to initial

success of behavioural treatment in reducing weight short termly90.

PHARMACOTHERAPY:

Orlistat and sibutramine are the two drugs which is used for long-term use

to achieve and maintain weight loss. Treatment with orlistat showed mean weight

loss of 2.9 kg after 1 year with, and with sibutramine showed weight loss of

4.5 kg after 1year. Other drugs approved for short term use are Diethylproprion,

Phentermine, Benzphetamine and Phendimetrazine40.

32

SURGERY:

Bariatric surgery is the best solution for substantial long-term weight loss

due to lack of effectiveness. Bariatric procedures include restrictive,

malabsorptive, or a combination of both.

Mechanism of Action of Various Contemporary Bariatric Surgeries

Table No: 9 Bariatric Surgeries for Obesity MECHANISM PROCEDURE

Restrictive Vertical banded gastroplasty Gastric banding

Malabsorptive Jejunoileal bypass Biliopancreatic diversion

Combination Roux-en-Y gastric bypass Each patient should be individually evaluated and counseled regarding the

advantages and disadvantages of all procedures in order to match the type of

procedure to the patient’s expectancies and needs. Currently, Biliopancreatic

diversion (BPD) is mostly used as a treatment for hyperobese patients where more

weight loss is desired91.

Figure 8: Roux-en-Y gastric bypass

33

Figure 9: Adjustable gastric banding.

Figure 10: Biliopancreatic diversion with duodenal switch

For all obese persons, there is no single perfect operation. It depends on the

patients’ health condition and weight of the patient.

Obesity- Changes in cognitive functions:

Insulin resistance, gut-brain axis, systemic mediators and central

inflammation processes shows the connections between obesity and the risk of

34

cognitive impairment. The recent hypothesis postulates that the microbial

architecture of the gut is a part of the “gut-brain axis” stating the relation between

the intake of fat rich unbalanced diet and high risk of impairment in cognition.

Stress can also be a potential risk factor promoting abdominal obesity that can

further increase grey and white matter atrophy contributing to impaired cognitive

function18. Patients who are obese show several deficits of brain tissue, especially

in the frontal and occipital lobes, specific areas like anterior cingulated gyrus,

hippocampus and thalamic region. They also show deficits of grey matter in

frontal lobes and post central gyrus while there is enlargement of volume in white

matter of orbitofrontal region with abnormal myelination and neuronal defects in

the frontal lobe92.

Figure 11: Obesity and Cognition

35

Figure 12: Causes of Cognitive Decline in Obesity

COGNITIVE EVOKED POTENTIAL P300

P300 Event Related Potential (ERP) is the brain recording method which is

used to assess human cognition. It reflects a neuroelectrical activity associated

with attention and memory process. Long latency evoked potentials (EPs) are

related to cognitive processing and are referred as cognitive EPs, event related

potentials (ERP), P3, P300 and endogenous EP93. P300 is a large broad positive

component of Event Related Potential (ERP) appearing at about 300ms or more

following a rare task related stimulus. It is also called the P3 wave as it is the third

major positive peak in sensory evoked potential.

36

Recording of the P300 evoked potentials

The wave form of P300 is elicited as an odd ball paradigm. The subject is

instructed to target and count a rare stimulus occurring in a regular rhythmic train

of auditory stimuli. This specific intellectual function generates a discrete

waveform of cognitive evoked response. This is called P300 component because its

latency is about 300 ms (millisecond) after the stimulus24.

The positive and negative waveforms are designated as P and N

respectively. There are two negative (N1 and N2) and two positive waves (P2 and

P3). These are labeled by their average latency in normal individuals, i.e P3

appears around 300ms after the stimulus.

P3 is composed of two components: P3a and P3b.

P3a - Component of the frontal region.

Latency ranging from 220 to 280 milliseconds.

Depicts automatic cognitive processing, working memory and orientation

response.

P3b - Component has a cento parietal topography

Longer latency ranging from 280 to 600 milliseconds.

Reflects immediate memory mechanism triggered when the mental schema

of stimulus environment is updated and refreshed 94.

P3b functional significance is more recognised thanP3a

37

P300 wave occurs only when the subject involved in the target stimuli

detection. P300 wave represent the transfer of information to consciousness a

process that involves many different regions of the brain.

Alterations in both P300 latency and P300 amplitude occur in patients with

cognitive dysfunction. Cognitive decline subjects having smaller amplitude and

longer latency than the age and gender matched controls. Higher amplitudes and

shorter latencies linked with improved cognitive function.

Figure 13: Components of Event Related Potential

P300 AMPLITUDE:

Amplitude is defined as the difference between pre stimulus baseline and

the largest going peak in ERP within the time window determined by stimulus

modality, task conditions and subject age 95. P300 amplitude is measured over the

central electrodes Fz, Cz and Pz, the amplitude increases in the electrodes in

38

progression from frontal to the parietal electrodes96. Amplitude varies with

improbability of the targets.

P300 LATENCY:

Latency is defined as the time from stimulus onset to point of maximum

amplitude within the same time window95. P300 latency indexes classification

speed which is dependent and proportional to the time required to detect and

process the target stimuli. P300 latency is shorter over the frontal electrodes than

the parietal electrodes. Latency varies with the difficulty of discriminating the

target stimulus from the standard stimulus.

Uses

ERP reflects cognitive processes such as attention allocation and activation

of immediate memory. It is used for the assessment of cognitive functions,

especially for decision making in different disorders. The event related potential

clearly indexes fundamental attention and memory related operations.

Target to Target Interval

Target to target interval refers to the speed with which a resource can

redirect itself to process a new stimulus. Thus P300 reflects comparisons of

working memory and conscious awareness that brings about the sequential effects

of the stimulus. The number of non-target stimuli preceding the target stimulus in

a given sequence and the interval between the stimuli are the determinants of the

P300 measures. Also the interval between two target stimuli affects P300. The

39

amplitude of the P300 waveform increases with increase in target to target interval

while the latency decreases.

Factors affecting the Amplitude and Latency of P300 wave

Physiological Variations

Age:

There is a decrease in latency of P300 wave in children with the lowest

values at 15-25 yrs of ages. There is a steady increase in amplitude and decrease

in latency upto the second decade. Conversely after the age of 80 years the

opposite effects occur with prolonged latency and low amplitude. Below is the

graph depicting the relation between age and P300 latency98.

Figure 14: Relationship between Age and P300 Latency

Gender:

The ERP parameters do not statistically differ with respect to gender.99

Intelligence and Personality:

Latency of the wave is related to the ability of the subject to solve the

problems.

40

Circadian Rhythm:

The time of the day does not make any appreciable difference that is

statistically significant except for the minor variations made by the level of

arousal of the individual100.

Food Intake:

Recent food consumption increases the amplitude101.

Seasonal Variation:

Duration of daylight determines the effect of seasonal changes on P300 as

increased arousal during day time increases the level of activity102.

Menstrual Cycle:

The interaction of stimulus with hormones causes increase in amplitude of

P300 during the ovulation period103.

Exercise:

There is an increase in intellectual performance and cognitive functions

after exercise or any energetic activity. Mental performance is facilitated by

increased frequency of physical exercise104.

Attention:

A reduction in amplitude of the wave is seen with decreased alertness,

drowsiness and inattentiveness.

41

Sleep:

Sleep disorders decreases the amplitude and increase the latency of P300.

This is believed to be the effect of variable arousal levels and also the same

effects are seen during the onset of sleep105.

Drugs:

Anticholinergics and antihistaminics increases P300 latency and decreases

its amplitude. L-DOPA reduces P300 latency in Parkinson’s disease patients but no

effect in normal persons. Physostigmine, a cholinergic antagonist decreases P300

latency and increases its amplitude. Benzodiazepine reduces the P300 amplitude.

Intraindividual Variability:

P300 measured in the same individual varies considerably at different times

of measurement.

Clinical conditions showing variations in P300 wave

1. Schizophrenia

Reduced P300 amplitude is the characteristic of schizophrenia. The

amplitude of P300 is inversely related to severity of symptoms particularly

negative symptoms. In schizophrenia decreased P300 amplitude has been found in

comparison to controls because the task requires cognition which is impaired in

these patients106.The lower amplitude is mainly found over the left temporal

region and is not due to the medication taken by the patients 107. The cause for

reduced P300 is due to reduced volume of left-anterior hippocampus, amygdala and

left superior temporal gyrus108.

42

2. Alzheimer’s disease:

In Alzheimers disease, the P300 amplitude is smaller and latency is longer

when compared with normals109.

3. Dementia

P300 latency was delayed and decreased in amplitude. Helpful in

differentiating behavioural abnormalities in dementia and those due to psychiatric

disturbances. P300 is normal in psychiatric disturbances. P300 has extensively used

as objective evaluation of cognitive functions.P300 is a useful technique for

documenting age related cognitive changes.

4. Alcoholism

Alcohol reduces the amplitude of P300 wave and increases its latency.

P300amplitude reduction may be related more to the positive family history for

alcoholism than to the amount or duration of alcohol intake. Amplitude of P300

wave may be a marker for the genetic predisposition to alcoholism. The

underlying explanation usually is that alcoholic patients exhibit arousal,

attentional and memory disturbances

43

5. Bipolar disorder

Patients with bipolar disorder have latency prolongation and amplitude

reduction compared to unipolar disorder. Latency was reduced following

treatment with reboxetine and citalopram treatment.

6. Depression:

In patients with severe depression, P300 amplitude is reduced. The latency

ofP300 may be slightly increased.

7. Attention Deficit Hyperkinetic Disorder:

In attention deficit hyperkinetic disorder P300amplitude at posterior

recording site is reduced and anterior site augmented 110. This abnormality is

reversed to a normal pattern recording after treatment with Methyl phenidate111.

8. Autism:

Autistic children have impaired processing of auditory and visual stimuli.

Both latency prolongation and amplitude reduction are seen in the P300 recorded

from these patients 112.

9. Post-traumatic stress disorder:

Lower P300 amplitudes have been found among patients compared to

controls who got exposed to the traumatic event but did not develop the

disorder113.

44

10. Other disorders in children

Children with dyslexia have P300 waves with longer latency and smaller

amplitude when compared with normal children. Vitamin B12 deficiency result in

neurobehavioral abnormality and even megaloblastic sadness. Cognitive

abnormality occur in 30 – 50% of patients with vitamin B12 deficiency and is

usually associated with features of subacute combined degeneration. In a study on

vitamin B12 deficiency neurological syndrome cognitive impairment as assessed

by MMSE was reported in 50% patients and prolonged P300 latency in 45.5%

patients114. Following treatment with vitamin B12 neuro-behavioural psychometric

tests and P300 improved115.

11. Other disorder

P300 wave latency delayed in hepatic encephalopathy, renal failure or in

hemodialysis.

Neuro Psychology of P3a and P3b:

A distracter stimulus in between the standard sequential stimulus produces

P3a. Short latency P300 in the frontal electrodes and rapid habituation occurs with

perceptually novel stimuli like dog’s barking amidst standard stimuli. This

novelty P300 represents the activity of frontal and hippocampal areas116. P3a is

more related to orienting response when compared to P3b as evidenced by the

decrease in novelty P300 amplitude in response to repetitive stimuli. A “no-go”

P300 is elicited by a non-novel distractor which does not require a response in three

odd ball paradigms. This type of P300 shows maximum amplitude in the central

45

parietal areas indicating that no-go response is the result of inhibition tasks. P3a

has a central maximum and the P3b has a parietal maximum. Both P3a and P3b

show shorter peak latencies in the frontal electrodes when compared to the

parietal electrodes.

Non repeating stimulus involves novel items while repeating stimulus

events engage top-down processing. Thus novelty P300 and P3a differ in the manner

of engaging the attentional process for distracter stimuli117.

Neural origins of P3a and P3b:

In spite of the appreciable advancement in the understanding of P300, in the

past 25 yrs. The neural generators of the P3a are yet to be precisely located. P3a

amplitude is decreased in frontal lobe lesions. But such patients show maximum

amplitude of P3b in the parietal areas. Thus the integrity of frontal and

hippocampal areas plays a role in the generation of normal P3a118. The medial

temporal lobe is the area of brain responsible for the generation of P3b. This P3b

correlates with the relative size of the hippocampus in comparison with the size of

temporal lobe. The integrity of temporo-parietal junction contributes to the scalp

recordings of P300 either through transmission or generation process from the

hippocampus. Thus it is evident that the neural circuitary of the P3a and P3b

involves the frontal and temporo- parietal areas of the brain.

Frontal lobe activity is initiated when the subject attempts discriminating

the target and standard stimuli. Frontal lobe is sensitive to demands of attention

induced by performing a task. Engaging an attentional foci and processing a

46

stimulus generates P3a. Operation of memory in the tempero-parietal junction

following an attentional focus generates P3b119. The changes produced in the

frontal lobe as a result of activation while matching to sample task information is

shunted to infero-temporal structures where the task content updating for stimulus

presentation is indexed. This provides the evidence that activation of frontal and

temporo-parietal areas generates P3a and P3b.

Functional magnetic resonance imaging (fMRI) band magneto

encephalography have been used in recent studies to generate P300. Based on

Combined ERP and fMRI study the frontal lobe and the insula are found to be

involved in the generation of P3a, whereas P3b generation shows the involvement

of parietal and inferior temporal areas. Thus it is evident that there are distinct

attentional subsystems in processing the target and distracter stimuli 120.

Neuropharmacology of P300:

Dual Neurotransmitter Hypothesis:

The neurotransmitters responsible for the genesis of P300 could not be

exactly delineated. But the available research points that dopaminergic activity is

involved in generation of P3a that occurs due to the focal attention and working

memory process of the frontal lobe121. A nor epinephrinergic activity mediating

the tempero-parietal process is involved in the genesis of P3b122.

The locus- coeruleus- norepinephrine (LC-NE) activity of the parietal

cortex underlies the genesis of P3b during Target detection task. This LCNE

47

involvement in P300 generation confirms with attention resource allocation and

arousal related effects123.

The measurement and documentation of P300 in obese people:

There is a growing evidence that obesity is associated with impaired

cognitive function including executive function, attention and memory not only in

patients with co-morbid medical conditions like cerebrovascular pathology,

hypertension and diabetes but also in healthy obese subjects. Tascilar ME et al

after studied 50 children with obesity and 23 age- and sex-matched healthy

control subjects concluded that there is impairment of neural activity associated

with sensory and cognitive information processing in obese children when

compared with non obese children. Prolonged Latency and decreased amplitude

significantly seen in obese group compared to that of non obese controls. Central

nervous IR might be a common denominator of metabolic disorders and cognitive

dysfunction in obese people124. TheP300 latency time is generally accepted as a

measure of speed of cognitive processing, and its amplitude to reflect the number

of neurons allocated to the eliciting task. It is clinically used as an index of

cognitive function. The latency and amplitude of the waveform were recorded.

Raji et al reported that BMI>30kg/ m2 was associated with atrophy in frontal

lobes, the anterior cingulated gyrus, hippocampus and thalamus when compared

with normal weight individuals125.

ADIPOKINES

The visceral adipose tissue secrete numerous adipokines and cytokines.

48

LEPTIN:

Leptin is an anti-obesity and satiety regulating hormone. Leptin is a

hormone secreted by adipose tissue. The name “leptin” derives it’s origin from the

Greek word “leptos” for thin. It is a 16 kDa protein product of Ob gene consisting

of 167 amino acids. The ''Ob (Lep)'' gene - Ob stands for obese, Lep for leptin.

Cytogenetic location is 7q31.31. In humans it is located on chromosome 7.It was

originally discovered by JM Friedman by positional cloning of ob/ob mice, a

mouse model of obesity. Leptin is one of the most enigmatic of the adipokines.40

Source of Leptin

Leptin is expressed mainly in adipose tissue. In the placenta, skeletal

muscle, gastric fundic mucosa, and mammary epithelium also, low levels of

Leptin have been found. Circulating levels of leptin increased with energy stores

and signaled the brain to inhibit feeding and decrease body weight and fat. It

circulates both in a bound and a free hormone. Plasma leptin is higher in obese

when compared with lean individuals. Leptin falls rapidly during fasting, and

increases after feeding. Its circulating concentrations in directly reflect body fat

stores.126

49

Figure 15: Role of Leptin

LEPTIN RECEPTORS (OB-RS):

The leptin receptor (LEPR) belongs to the cytokine receptor class I

superfamily103. Six different isoforms were formed namely LEPRa, LEPRb,

LEPRc, LEPRd, LEPRe and LEPRf after the alternative splicing of LEPR. Leptin

and growth hormone receptors belong to a family of cytokine receptors coupled to

the JAK–STAT pathway. Leptin Receptors (LEPR) are highly expressed in the

hypothalamus part of brain which plays a role in regulating body weight. Specific

leptin receptors (Ob-R) are expressed in the peripheral tissues also.

The Ob-Ra isoform which is short leptin receptor isoform, plays an

important role in transporting leptin across the blood–brain barrier. Ob-Rb

isoform which are the long leptin receptor isoform is the important one present in

the hypothalamus, which is a site for the regulation of energy homeostasis and

50

neuroendocrine function and mediate signal transduction. Ob-Re is the secreted

form that binds circulating leptin, regulating the concentration of free leptin127.

Leptin and its actions

Leptin acts mainly as a “starvation hormone40. Friedman JM et al, 1998

described leptin as one of the most important central and peripheral signals for the

maintenance of energy homeostasis128. Deficiency of leptin or leptin receptor in

humans causes extreme obesity and implicates leptin mediated signaling in the

regulation of food intake, energy expenditure also in reproductive, thyroid and

immune functions129. Fall in leptin during fasting is a potent signal to the brain to

increase feeding, reduce energy expenditure, and mediate changes in hormone

levels designed to conserve energy.126 Leptin increases insulin sensitivity in

peripheral tissues via a CNS mechanism130-132. Lord GM et al, 1998 postulates

that in addition to its action on CNS, through its peripheral receptors affect many

systemic processes, such as immunity133. Margetic S et al, 2002 states that leptin

has an action on reproduction also134. According to Rahmouni J and Haynes WJ et

al explain its action on cardiovascular functions135. Leptin exerts a permissive

action to restore normal hypothalamic–pituitary–gonadal axis function during

caloric deprivation40. Leptin treatment restores gonadotropin and sex steroid

levels and puberty, confirming its primary role in reproduction126,136,137.

51

Figure 16: Peripheral actions of Leptin

Leptin deficiency

Ob/ob mice, which lack leptin and db/db mice which lack leptin receptors

are obese. Human beings with a homozygous mutation in the leptin gene or the

leptin receptor have morbid obesity. Deficiency of leptin or its receptor leads to

hyperphagia, morbid obesity, insulin resistance, hyperlipidemia, hypothalamic

hypogonadism and immunosuppression. Diet-induced obesity is associated with

elevated leptin levels and blunted response to leptin126. Congenital leptin

deficiency in rodents and humans is associated with hypothalamic hypogonadism

and lack of pubertal development 126,136,137. Leptin deficiency is associated with

deficits in brain weight and neuronal and glial proteins, some of which are

restored by leptin treatment138.

52

Leptin Resistance (LR)

Leptin resistance may result from diminution of leptin transport across the

blood–brain barrier (BBB) or impairment of leptin signal transduction in the

brain40.

Hyperleptinemia is seen in many genetic variants including the Lys109Arg

or Gln223Arg mutation in the leptin receptor (LEPR) gene. Mutations of some

other genes including Pro-opiomelanocortin (POMC) and the melanocortin 4

receptor (MC4R) also result in an obese phenotype with associated

neuroendocrine dysfunction.

Central LR is the diminished leptin receptor quantity and impaired signal

transduction. Peripheral LR is the inability of peripheral leptin to reach the brain.

In humans, both defects coexist. Also, hyperleptinaemia is a strong indicator of

LR139.

Leptin Signaling

Reduction in leptin signaling in the hypothalamic area and elevated

peripheral and central leptin levels are frequently associated with occurrence of

obesity of adult onset and diet induced type58. Leptin concentration depends on

the quantity of stored energy in fat and the status of energy balance. Importantly,

most patients who had amenorrhea prior to leptin therapy developed normal

menses after treatment 140.

Leptin acts through the two groups of arcuate neurons which are located in

the hypothalamus region of the brain. One group expressing Agouti-related

53

peptide and Neuropeptide Y (NPY) and the other group expressing Pro –Opio-

Melano-Cortin (POMC) and Cocaine and Amphetamine-Related Transcript

(CART). Low leptin level stimulates the expression of NPY and AGRP in the

Arcuate nucleus directly whereas increases MCH in the lateral hypothalamus

indirectly. This causes hyperphagia and restoration of body weight.126,141

Concurrently, low leptin disinhibits POMC neurons in the Arc, thereby decreasing

the synthesis and release of the anorectic peptide alpha-MSH126,104. Defect in the

leptin signaling causes severe obesity.

Mechanism of action of Leptin:

Vaisse and Halaas et al, 1996 observed that Ob-Rs produce its effects

predominantly through activation of JAK2/STAT3 (Janus-activated kinase 2/

signal transducers and activators of transcription3) pathway.127 Other than this, it

also activates mitogen-activated protein kinase (MAPK) pathways, insulin

receptor substrate125. The JAK2/STAT3 pathway is the most important one for

mediating leptin’s effects on homeostatic energy regulation58.

Nakashima K et al states that Leptin receptors on ligand binding undergo

homooligomerization142. This leads to JAK2 autophosphorylation and

phosphorylation of tyrosine kinases involved in intracellular cytokine signaling.

Phosphorylated leptin receptor recruits STAT3 which is activated through JAK2

phosphorylation. Activation of STAT3 proteins leads to dimerization and

translocation of the same to the nucleus. There it binds with DNA and initiate

gene transcription. Binding of members of Suppressors of cytokines signaling

54

family (SOCS3) to tyrosine or receptor janus kinase complex mediates negative

feedback on leptin signaling (Dunn SL et al, 2005)143.

According to Bjorbeck C et al only Ob-Rb contains STAT-binding site 144.

Then it binds with Janus Kinase JAK2 primarily (KloekCet al, 2002)145. Zhang

Yet al, 1994 states that the long-form leptin receptor (Ob-Rb) signaling through

JAK2/STAT3 is required for control of feeding and energy expenditure to

maintan normal energy homeostasis. Mutations of this gene result in the obese

phenotype of the db/db mouse and the Zucker rat146. The soluble leptin receptor

isoform Ob-Re may oppose leptin transport by decreasing surface binding and

endocytosis of leptin (Bates SH et al, 2003)147 Disruption of leptin signaling in the

hypothalamus leads to obesity and confirms the central role of leptin in the

maintenance of energy balance (Cohen P et al, 2001)148

Figure 17: Leptin signal transduction pathways in the hypothalamus

55

Leptin Regulation

Leptin level is regulated by many hormones present in the body.

Upregulation of Leptin is done by insulin and cortisol and downregulation by

catecholamines. TNF-α also acts as a paracrine regulator to increase the secretion

of leptin.. Leptin autoregulates its own expression by glucose and fatty acids.

Leptin and a relation with Obesity

Obese humans and animals show resistance to central actions of leptin.

Obesity is associated with rise in leptin levels but diminished leptin sensitivity.

The most important characters of leptin resistance are decrease in number of

leptin receptors, impaired signaling and reduction in the response to available

exogenous leptin. Long term exposure to increased levels of leptin causes

desensitization of the physiological responses of the body to leptin58.

Figure 18: Interconnection between obesity, elevated Leptin, and Leptin

resistance

56

Considine RV et al after a study of serum leptin concentration in 136

normal-weight subjects and 139 obese subjects concluded that serum leptin

concentrations have a strong linear correlation with the percentage of body fat in

the positive direction. When compared to non obese subjects, the obese

individuals had double the amount of ob mRNA15.

Figure 19: Ob/Ob mice

Leptin and a relation with central nervous system

Leptin has an action on the structure and function of neurons and also the

glial cells. Leptin exerts its action over neurogenesis, excitability of neuron and

neuroprotection. Other than this, leptin also has an effect on axon growth,

synaptogenesis, morphology of dendrites, development of oligodendroglial cells

and regulation of beta-amyloid levels. Leptin has the ablity to alter structure in

regions such as the midbrain, hindbrain and the Hippocampus. Leptin is regarded

as neuroprotective substance as it inhibits cell death by apoptosis, improves

survival of the cell, attenuates cell death, provides protection against the cytotoxic

57

effects of glutamine and oxidative stress, and promotes proliferation of progenitor

cells in the hippocampal region. Leptin plays roles on mood and cognitive

disorders. Local injection of leptin into the hippocampus improves memory and

performance related to the memory by facilitation of spatial learning and long-

term potentiation (LTP)-a form of synaptic plasticity in the hippocampus. Leptin

also enhances the functions of NMDA receptor and thus helps in the facilitation of

conversion of short term memory into LTP. By rapid remodeling of the dendrites,

it enhances the LTP at the CA1 synapses of the hippocampus. But the central

resistance to leptin in obese humans prevents them from getting the benefits of the

protective functions of the comparatively high levels of leptin. The adipoinsular

axis involving leptin and insulin significantly regulates the brain function92.

Leptin receptors are expressed throughout the brain, mainly in the hippocampus

and various cortical regions.

Treatment of leptin-deficient humans with recombinant methionyl human

leptin decreases body weight and also increases the volume of the anterior

cingulate gyrus, inferior parietal lobule, and the cerebellum. These brain volume

increases demonstrating that leptin can have sustained effects on the human

brain149.

Leptin is associated with increased brain-derived neurotrophic factor

expression and neurogenesis in the prefrontal cortex. Leptin administrated directly

into the hippocampus of rodents suppressed memory consolidation, specifically

for the spatial location of food150.

58

Leptin as biomarker cognition:

.Leptin has a role in higher cognitive functions. It acts within the brain to

support and promote cognitive function. Leptin alters neuronal/synaptic function

and structure, and influences neuronal survival and proliferation within the areas

of the brain. So there is an improvement in learning, memory and other forms of

cognition. Resistance to leptin impairs cognitive performance. In hippocampus

leptin induces synaptic plasticity by converting short-term potentiation of synaptic

transmission into long-term potentiation (LTP). This process is the basis of

learning and memory formation139. Impairment of this process is associated with

cognitive deficits. The relationship between leptin and cognitive function exists

on multiple levels. Leptin also acts within areas such as the cortex and

hippocampus to influence neuronal function and promote cognition21. Due to

tissue leptin resistance, hyperleptinaemia may act as a pathophysiological marker

for impaired cognitive function.

. Figure 20: Role of Leptin in Cognition

59

From the above discussion it is evident that cognitive decline can result

from obesity. The adipokine, leptin has a definite role in the occurrence of

obesity. Also there are evidences for the association of leptin with cognitive

decline.

With these evidences I have postulated this study with a hypothesis that

serum leptin levels in obese individuals has a definite relationship with the

cognition of those individuals as measured by cognitive evoked potentials (P300).

Aim and Objectives

60

AIM AND OBJECTIVES

AIM:

To study the Cognitive Evoked Potentials and serum leptin level in young

obese individuals.

OBJECTIVE:

1. To estimate the latency and amplitude of P300 evoked potentials in young

obese and non-obese individuals.

2. To estimate the anthropometric measurements (i.e the standing height &

weight), the measures of obesity indices (i.e BMI, Waist and Hip

Circumference, Waist/Hip ratio) in Young obese individuals.

3. To assess and correlate the serum leptin levels in young obese individuals

of varying BMI.

4. To study if there is any decrement in the P300 in the young obese group

compared to the control group.

5. To compare the P300 values with the serum leptin levels in the young obese

group and the control group to evaluate if leptin can be kept as a cognition

marker in young obese individuals.

6. To find out the correlation between the serum leptin levels and obesity

indices, variables of P300 among the study group.

Materials and Methods

61

MATERIALS AND METHODS

This is a cross sectional study conducted during the period of April 2017 to

March 2018 at the Institute of Physiology and Experimental Medicine, Madras

Medical College, after obtaining approval from Institutional Ethics Committee

(IEC), Madras Medical College, Chennai –03. Subjects for this study were

recruited from the Institute of Internal Medicine and The Medical Endocrine

Clinic, Rajiv Gandhi Government General Hospital, Chennai.

Subject Selection:

The study group consists of Forty young obese individuals belonging to

both genders in the age group of 18 to 30 years with a BMI of ≥ 30 (Group I).

Forty subjects in both genders of the same age group with a normal range BMI 18

to 24.9 (Group II) were taken as controls.

Inclusion criteria:

Subjects in the age group of 18 to 30 years having a BMI of ≥ 30 and forty

subjects of the same age group with a normal range BMI of both genders were

included in the study.

Exclusion criteria:

Subjects with:

1. Age <18 and >30 years

2. BMI <18.5 and >40 kg/m2

3. Type I and Type II DM [according to the American Diabetes Association

Criteria]

62

4. H/O hypertension [SBP < 140 mmHg and DBP < 85 mmHg]

5. Ischemic Cardiovascular disease

6. Renal / Hepatic disease

7. Any Acute or Chronic infections

8. HIV /AIDS

9. Any endocrine dysfunction

10. Alcohol or drug abuse/ Smoking

11. Malignancy

12. Any haematological disorder

13. Chronic Medication therapies (such as on antidepressants, anticonvulsants,

hypoglycemic drugs, anti hypertensives, lipid lowering agents, OCPs and

corticosteroids) and Hormonal therapy

14. Pregnancy

15. Psychiatric patients

Applying the above inclusion and exclusion criteria, a total of 80 subjects

participated in the study.

Methodology:

Forty subjects of the age group between 18-30 yrs with BMI ranging more

than 30 of both sexes, fulfilling the above criteria were selected from the

department of endocrinology of the Rajiv Gandhi Government General Hospital

and were taken as the study group.

63

Forty normal individuals of the age group between 18-30 years with body

mass index of 18.5 - 24.99 of both sexes fulfilling the above criteria were taken as

the control group.

Informed consent was obtained from the participants. A detailed history

was recorded before including a subject into the study. A meticulous and thorough

physical examination was done and the subjects were screened with a set of

laboratory investigations so as to exclude the conditions that may interfere with

the parameters of the study.

Figure 21: Subject selection

Measurement of Anthropometric Indices:

Weight of the subject:

The subjects were instructed to wear light clothing and to stand erect with

their arms relaxed at their side, with both feet closed together without shoes. By

• 47 CASES• 43 CONTROLS

90 SUBJECTS SCREENED(18-30 YEARS)

• 7 CASES• 3 CONTROLS

EXCLUDED AS THEY DIDN'T MEET CRITERIA

• 40 CASES• 40 CONTROLSSELECTED SUBJECTS

64

using a portable standard weighing machine, weight in kilograms measured to

nearest 1 kg was recorded.

Height of the subject:

The subjects were instructed to stand erect with both feet close together

without shoes and the vertical height to the nearest 1 cm was measured.

BMI:

Calculation of BMI was done using Quetlet’s index.

BMI = Weight (kg)/ height2 (m)

Waist circumference

Before checking Waist and Hip Circumference the subject was instructed

strictly to relax the abdominal muscles and stand erect keeping both arms by the

sides and feet close together so that the body weight is distributed evenly across

the feet. The waistline is exposed by removing the clothing.

The WHO Stepwise Approach to surveillance (STEPS) protocol was

followed while measuring the waist circumference. Measurement is done at the

end of normal expiration. Approximate midpoint between the top of iliac crest and

lower margin of the last palpable rib is the point of measurement of WC. A

measuring tape resistant to stretch providing a constant tension of 100 gm is used

for measurement such that it is parallel to the floor while measuring.

65

Figure 22: Measurement of Waist Circumference (cm)

Hip circumference:

The widest portion of the buttocks is measured to record the hip

circumference as per the protocol of WHO Stepwise Approach to surveillance

(STEPS)

.

Figure 23: Measurement of Hip Circumference (cm)

WAIST TO HIP RATIO

Waist circumference (cm) = ------------------------------ Hip circumference (cm)

66

ELECTROPHYSIOLOGICAL ASSESSMENT

P300 EVENT RELATED POTENTIAL

The participants in the study were informed to have a hair wash on the day

of recording the event related potential P300. After explaining the procedure in

detail and clarifying the doubts, an informed consent was obtained both in verbal

and written form. P300 auditory event related potential was measured in the patient

and control group at the research lab attached to institute of physiology and

experimental medicine. An initial trial was given for the patient so that they can

identify the target stimuli easily. The event related potential is recorded using the

MEDICAID Physiopac 2 channel neurostim machine. The event related potential

P300 is recorded while performing an odd ball paradigm.

The subjects will be presented with two types of auditory stimuli in the

testing, a non-target stimuli and a target stimuli through head phones in both of

their ears. The auditory stimuli were of pure tone type presented at the rate of

1.25s, a total of 100-150 stimuli are given. Each stimuli duration was 100 ms and

with an intensity of 70 db. The non-target stimuli and the target stimuli presented

in the ratio of 80% and 20%. The stimuli that the participants did not respond to

will be rejected and the rest of the waves for the target stimuli will be taken up for

calculating the P300. Initial demonstration is given before the test is done.

P300 is assessed in terms of its latency and amplitude. Latency is taken as

the time from the stimulus application to the onset of response it is usually

between 250-400 milliseconds. Amplitude is the measure between the prestimulus

67

baseline and the largest positive peak in event related potential in the latency

window of 250 – 400 milliseconds. The amplitude is usually of 10 μV value in

normal individuals. It is the P3b component of the P3 wave that is considered

extensively for measuring cognitive evoked potential, so this was measured at Pz.

The reference, ground and active electrodes were placed according to the 10-20

international system of electrode placement. Reference electrode was kept at

mastoids, ground electrode at forehead and active electrode at vertex. Surface

electrodes were attached by using Ten20 conductive paste and micropore tape.

Figure 24: Electrode Placement

Figure 25: P300 wave generation by target stimuli and waves generated by background stimuli

Photograph-1: Recording of P300 in Neurostim Machine

68

Photograph-2: Recording of P300 in Clinical Research Lab at RGGGH

Measurement of Serum leptin levels:

Sample collection and storage:

The DRG Diagnostics Leptin sandwich Elisa kit which is available

commercially was used to assay the serum leptin levels. Manufacturer’s

instructions were followed in the Handling of sample, storage and preparation.

After obtaining the informed consent, venepuncture of the median cubital

vein of the subject was performed with universal aseptic precautions to collect 5

ml of blood sample. The sample was centrifuged to separate the serum which was

stored at -20° Celsius. The samples were then transported under cold chain

maintenance to the Institute of Biochemistry, Madras medical college, Chennai-03

for assessing serum leptin levels.

69

Photograph-3: Sample Collection

Principle of the test:

The DRG ELISA kit used in this study is a solid phase enzyme linked

immunosorbent assay (ELISA) for leptin. It works according to the principle of

sandwich ELISA. The kit consists of 96 microtitre wells. The wells are coated

with a monoclonal antibody. Antibodies are specific to leptin which binds with a

unique antigenic site on the leptin molecule. Subjects sample containing

endogenous leptin is incubated in the coated microtitre wells. A specific

biotinylated monoclonal anti leptin antibody is then added to those microtitre

wells to form a sandwich complex and incubated. Then after washing off the

unbound material a streptavidin peroxidase complex is added to detect the

unbound leptin. Addition of the substrate produces a colour with an intensity

proportional to the concentration of leptin in the sample.

70

Reagents Required:

1. Microtitre wells (96 wells) Plate with Break Apart Wells coated with

monoclonal anti-leptin antibody

2. Standard (0-5) with concentrations 0-2-5-25-50-100 ng/ml

3. Control (Low and High)

4. Assay Buffer leptin Calibrators

5. Antiserum – Monoclonal biotinylated anti-leptin antibody

6. Enzyme Complex – Streptavidin conjugated to horseradish peroxidase

7. Substrate Solution – Tetramethylbenzidine (TMB)

8. Stop solution – 0.5M Sulphuric acid

9. Wash Solution

Other materials required

Microtitre plate calibrated reader (450 ± 10 nm)

Calibrated variable precision micropipettes

Distilled water, absorbent paper, timer, semi logarithmic graph paper

Photograph-4: DRG Leptin ELISA Kit

71

Assay procedure:

Before use all reagents and specimens should come to room temperature.

Standard controls or samples about 15 μL each were added with new tips that are

disposable into corresponding wells that were labeled

Assay buffer about 100 μL was dispensed into each well. Then it was

mixed for 10 seconds and incubated for 2 hours at room temperature. Well

contents were shaken out briskly. About 300 μL of wash solution was dispensed

into each well to wash the wells. Wells were washed 3 times with diluted wash

solution. Wells were stroke on absorbent paper to remove residual droplets.

Antiserum about 100 μL each was dispensed and incubated at room

temperature for 30 minutes. Well contents were shaken out. Wash procedure

repeated and wells were stroke on absorbent paper. 100 μL of Enzyme Complex

each was added and incubated at room temperature for 30 minutes. Then contents

were shaken out. Wash procedure repeated and wells were stroke on absorbent

paper.

Substrate solution about 100 μL was dispensed and incubated at room

temperature for 15 minutes. Stop Solution 50 μL each was dispensed to stop

enzymatic reaction. Within 10 minutes using ELISA microtitre plate reader,

absorbance was found at 450 ± 10 nm for all the wells.

72

Calculation of Results

Calculation of the absorbance (optical density) values was done for the

samples and standard controls. A standard curve was plotted for the mean

absorbance value of each concentration of the standard controls with

concentration in the x axis against the absorbance in the y axis. This standard

curve is used to find out the concentration of the substance in the sample.

Normal values in lean men are 2 to 5.6 ng/ml and in lean women are 3.6 to

11.1ng/ml.

Serum leptin values thus obtained for the obese subjects were compared

and correlated with those of the subjects with normal BMI.

Figure 26: Standard Curve of Human Leptin

73

Results

Statistical Analysis Plan:

All the data obtained from all the Cognitive evoked potentials and serum

leptin levels were analyzed using SPSS version 21.

The analysis includes both the comparison of the values of the same

variable between the case and the control group and the correlation between the

variables within the group. The following statistical plans were used.

COMPARISON BETWEEN GROUPS:

CHI - SQUARE TEST:

For comparing the BMI, event related potential P300 latency, amplitude, the

serum leptin values between the case and the control group Chi- Square test was

used.

CORRELATION BETWEEN VARIABLES WITHIN THE GROUP

PEARSON’S CORRELATION:

For correlation of variables within the group that is correlation between

a) BMI and the event related potential P300 latency and amplitude

b) The event related potential P300 latency, amplitude and serum leptin level

c) BMI and serum leptin levels

Pearson’s correlation was used. The correlation was done for the above

said variables both in the case and the control group.

Results

74

RESULTS

Data obtained from conducting the cognitive evoked potentials and serum

leptin levels were statistically analyzed. Mean and standard deviation of the

variables were determined for the control and young obese groups. Chi square test

was employed for statistical analysis as the test of significance at 95% confidence

interval.

* P value < 0.05 was considered as significant

** P value < 0.01 was considered as highly significant

***P value < 0.001 was considered as very highly significant

Table No: 10 Baseline Characteristics of Study and Control Group

(Mean± SD)

BASELINE PARAMETERS S.No. Variable Obese Non- Obese

1. Age 24.83 ± 3.86 24.88 ± 3.97 2. Height 155.05 ± 5.62 163.48 ± 7.63 3. Weight 81.50 ± 6.78 60.55 ± 6.19

Table No: 11 Descriptive analysis of Gender in study (obese) N=40 and

control (non-obese) N=40 groups

Gender Group Frequency Percentage

Male Study group 13 32.5%

Control group 15 37.5%

Female Study group 27 67.5%

Control group 25 62.5%

75

Graph No : 1 Descriptive analysis of Gender

Table no: 12 Descriptive measurements of the study participants

TABLE NO: 12

Variables GROUP I

(Obese subjects) Mean ± SD

GROUP II ( Non – Obese)

Mean ± SD

BMI 33.97 ± 1.92 22.72 ± 0.86

Waist Circumference (cms)

119.85 ± 9.44 83.45 ± 1.80

Hip Circumference (cms) 131.30 ± 11.42 104.95 ± 2.93

W/H Ratio 0.91 ± 0.02 0.80 ± 0.02

P300 latency 354.61 ± 12.53 335.15 ± 6.45

P300 amplitude 6.69 ± 2.40 9.22 ± 1.61

Serum Leptin (ng/ml) 24.45 ± 7.57 7.80 ± 3.47

32.50%37.50%

67.50%62.50%

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

OBESE NON OBESE

PERC

ENTA

GE

GENDER DISTRIBUTION

MALE

FEMALE

76

Table No: 13 Comparison of the BMI between the two study groups

Variables Groups Mean SD P Value

BMI (kg/m²)

Group I (Study group)

33.97

1.92 <0.0001***

Group II (Control group)

22.72 0.86

***P value < 0.001 very highly significant

Graph No: 2 Comparison of the BMI between the two study groups

Table No: 14 Comparison of the measures of obesity (Waist Circumference,

Hip Circumference and Waist Hip Ratio) between the two study groups

Variables Group Mean SD P Value Waist Circumference

(cms) Group I 119.85 9.44 <0.0001*** Group II 83.45 1.80

Hip Circumference (cms)

Group I 131.30 11.42 <0.0001*** Group II 104.95 2.93

Waist/ Hip Ratio Group I 0.91 0.02 <0.0001*** Group II 0.80 0.02 ***P value < 0.001 very highly significant

33.97

22.72

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

cases controls

BMI I

N K

g/m

2

BMI

bmi

77

Graph No: 3 Comparison of the Waist Circumference, Hip Circumference

between the two study groups

Graph No: 4 Comparison of the Waist- Hip Ratio between the two study

groups

119.85

83.45

131.3

104.95

0

20

40

60

80

100

120

140

cases controls

CEN

TIM

ETER

S

WAIST AND HIP CIRCUMFERENCE

WaistCircumference(cms)hip circumference(cms)

0.91

0.8

0.74

0.76

0.78

0.8

0.82

0.84

0.86

0.88

0.9

0.92

cases controls

WAIST HIP RATIO

waist hip ratio

78

Table No: 15 Comparison of the Serum Leptin levels between the two study

groups

Variables Group Mean SD P Value

Serum Leptin (ng/ml)

Group I 24.45 7.57 < 0.0001***

Group II 7.80 3.47

***P value < 0.001 very highly significant

Graph No: 5 Comparison of the Serum Leptin levels between the two study

groups

Table No: 16 Comparison of the P300 Latency and P300 Amplitude between

the two study groups

Variables Group Mean SD P Value

P300 Latency Group I 354.61 12.53 <0.0001 *** Group II 335.15 6.45

P300 Amplitude Group I 6.69 2.40 <0.0001*** Group II 9.22 1.61

***P value < 0.001 very highly significant

24.45

7.8

0

5

10

15

20

25

30

cases controls

ng/m

l

S.LEPTIN

S.LEPTIN

79

Graph No: 6 Comparison of the P300 Latency and P300 Amplitude between the

two study groups

Table No: 17 Correlation table of P300 latency and BMI for both study and

control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 latency 354.61± 12.53 r = 0.0720 p= <0.0001*** BMI 33.97 ± 1.92

Control group (n=40)

P300 latency 335.15 ± 6.45 r = 0.0712 p= <0.0001*** BMI 22.72 ± 0.86

***P value < 0.001 very highly significant

Table No: 18 Correlation table of P300 latency and Waist circumference for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 latency 354.61± 12.53 r = 0.1658 p= <0.0001*** Waist

Circumference 119.85 ± 9.44

Control group (n=40)

P300 latency 335.15 ± 6.45 r = 0.1755 p= <0.0001*** Waist

Circumference 83.45 ± 1.80

***P value < 0.001 very highly significant

354.61

6.69

335.15

9.220

50

100

150

200

250

300

350

400

P300 LATENCY P300 AMPLITUDE

P300 LATENCY AND P300 AMPLITUDE

CASES

CONTROLS

80

Table No: 19 Correlation table of P300 latency and Hip Circumference for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 latency 354.61± 12.53 r = 0.1283 p= <0.0001*** Hip

Circumference 131.30 ± 11.42

Control group (n=40)

P300 latency 335.15 ± 6.45 r = 0.0758 p= <0.0001*** Hip

Circumference 104.95 ± 2.93

***P value < 0.001 very highly significant

Table No: 20 Correlation table of P300 latency and Waist Hip ratio for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 latency 354.61± 12.53 r = 0.0334 p= <0.0001*** Waist Hip ratio 0.91 ± 0.02

Control group (n=40)

P300 latency 335.15 ± 6.45 r = 0.0613 p= <0.0001*** Waist Hip ratio 0.80 ± 0.02

***P value < 0.001 very highly significant

Table No: 21 Correlation table of P300 amplitude and BMI for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 amplitude 6.69 ± 2.40 r = - 0.1076 p= <0.0001*** BMI 33.97 ± 1.92

Control group (n=40)

P300 amplitude 9.22 ± 1.61 r = -0.2119 p= <0.0001*** BMI 22.72 ± 0.86

***P value < 0.001 very highly significant

81

Table No: 22 Correlation table of P300 amplitude and Waist circumference for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 amplitude 6.69 ± 2.40 r = - 0.0715 p= <0.0001*** Waist

Circumference 119.85 ± 9.44

Control group (n=40)

P300 amplitude 9.22 ± 1.61 r = -0.3547 p= <0.0001*** Waist

Circumference 83.45 ± 1.80

***P value < 0.001 very highly significant

Table No: 23 Correlation table of P300 amplitude and Hip Circumference for

both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 amplitude 6.69 ± 2.40 r = - 0.0444 p= <0.0001*** Hip

Circumference 131.30 ± 11.42

Control group (n=40)

P300 amplitude 9.22 ± 1.61 r = -0.2461 p= <0.0001*** Hip

Circumference 104.95 ± 2.93

***P value < 0.001 very highly significant

Table No: 24 Correlation table of P300 amplitude and Waist Hip ratio for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

P300 amplitude 6.69 ± 2.40 r = - 0.0399 p= <0.0001*** Waist Hip ratio 0.91 ± 0.02

Control group (n=40)

P300 amplitude 9.22 ± 1.61 r = - 0.0266 p= <0.0001*** Waist Hip ratio 0.80 ± 0.02

***P value < 0.001 very highly significant

82

Table No: 25 Correlation table of Serum Leptin and BMI for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = 0.2979 p= <0.0001*** BMI 33.97 ± 1.92

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = 0.2977 p= <0.0001*** BMI 22.72 ± 0.86

***P value < 0.001 very highly significant Table No: 26 Correlation table of Serum Leptin and Waist circumference for

both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = 0.1087 p= <0.0001*** Waist

Circumference 119.85 ± 9.44

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = 0.2860 p= <0.0001*** Waist

Circumference 83.45 ± 1.80

***P value < 0.001 very highly significant Table No: 27 Correlation table of Serum Leptin and Hip Circumference for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = 0.0785 p= <0.0001*** Hip

Circumference 131.30 ± 11.42

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = 0.1474 p= <0.0001*** Hip

Circumference 104.95 ± 2.93

***P value < 0.001 very highly significant

83

Table No: 28 Correlation table of Serum Leptin and Waist Hip ratio for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = 0.0696 p= <0.0001*** Waist Hip ratio 0.91 ± 0.02

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = 0.0672 p= <0.0001*** Waist Hip ratio 0.80 ± 0.02

***P value < 0.001 very highly significant

Table No: 29 Correlation table of Serum Leptin and P300 Latency for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = 0.2423 p= <0.0001*** P300 latency 354.61± 12.53

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = 0.1376 p= <0.0001*** P300 latency 335.15 ± 6.45

***P value < 0.001 very highly significant Table No: 30 Correlation table of Serum Leptin and P300 amplitude for both study and control groups

Group Parameter Mean ± SD Pearson’s correlation

Study group (n=40)

Serum Leptin 24.45± 7.57 r = -0.1825 p= <0.0001*** P300 amplitude 6.69 ± 2.40

Control group (n=40)

Serum Leptin 7.80 ± 3.47 r = -0.4021 p= 0.02* P300 amplitude 9.22 ± 1.61

***P value < 0.001 very highly significant *p Value significant at the level of p<0.05

84

The above results after statistical analysis shows that

1. Serum leptin values are higher in the study group when compared to the

control group and the values are also statistically significant.

2. Also there is a statistically significant increase in P300 latency and decrease in

amplitude in the study group in comparison with the control group.

3. P300 latency shows a statistically significant positive correlation with the

measures of obesity viz. BMI, WC, HC and WHR.

4. Whereas there is a negative correlation between the P300 amplitude and

measures of obesity which is also statistically significant.

5. Serum leptin values correlates positively with BMI, WC, HC, WHR and P300

latency while it negatively correlates with the P300 amplitude.

All the above results are highly significant statistically.

Discussion

85

DISCUSSION

The goal of my current study was to evaluate the cognitive evoked

potentials in young obese individuals and also to estimate Serum Leptin level in

young obese individuals. BMI, measures of obesity, P300 latency, P300 amplitude

and serum leptin levels were estimated and compared in both the obese and the

non-obese subjects.

The study was conducted at the Institute of Physiology and Experimental

medicine, Madras Medical College for the participants were recruited from the

Department of Endocrinology of Rajiv Gandhi Government General Hospital,

Chennai. The study was aimed at including forty young obese individuals of the

age 18 to 30 years with BMI ≥ 30 of both sexes and forty age matched normal

controls with BMI of 18.5 -24.9.

To prevent confounding bias both the groups were age matched. The mean

age of the case and control group were 24.83 ± 3.86 and 24.88 ± 3.97

respectively.

Measures of obesity

The BMI of the controls was 22.72 ± 0.86 and that of the obese was 33.97

± 1.92. The P value was less than 0.0001, very highly significant. The waist

circumference and hip circumference of the obese group was increased

significantly when compared to the controls. The waist circumference of the obese

group was 119.85 ± 9.44 and that of controls was 83.45 ± 1.80. The P value was

86

less than 0.0001, very highly significant. The hip circumference of the obese

group was 131.30 ± 11.42 and that of controls was 104.95 ± 2.93. The P value

was less than 0.0001, very highly significant. The waist hip ratio of the obese

group was 0.91 ± 0.02 and that of controls was 0.80 ± 0.02. The P value was less

than 0.0001, very highly significant.

Cognitive evoked potentials:

In both cases and controls P300 latency and P300 amplitude were recorded.

The mean of the P300 latency for the case group was 354.61 ± 12.53 whereas the

mean latency for the control group was 335.15 ± 6.45. By using chi- square test

the calculated p value was less than 0.0001 which shows that there was extreme

statistical significance between the case and the control group. The P300 latency in

the case group (obese) was prolonged than that of the control group (non-obese).

The shorter the latency, the superior the cognitive performance. So the latency

prolonged in the case group shows that the cognitive performance is affected in

the obese individuals.

The mean of the P300 amplitude in the case and the control groups were

6.69 ± 2.40 and the 9.22 ± 1.61 respectively. The p value calculated to find the

statistical significance between the groups was less than 0.0001. This p value

denotes that there is extreme statistical significance between the amplitudes of the

study and the control group. The P300 amplitude is larger in the control group than

in the case group. Larger the amplitude, better the cognitive performance. The

87

finding in this study is that the amplitude in the case group is less than that of the

control group.

Prolonged latency and reduced amplitude show that the cognitive

performance of the case group (obese group) is inferior to the cognitive

performance of the control group that there is cognitive dysfunction in the obese

individuals.

This finding in this study is consistent with the findings in the studies of

Tascilar ME et al124 that prolonged latency and decreased amplitude of P300

evoked potentials is observed in obese individuals when compared with that of

non obese controls. This change showed that there is a cognitive decline in obese

individuals.

Serum Leptin levels:

On assessing by ELISA method, we could find that the serum leptin level

in the control group (7.80 ± 3.47) and that of the obese group (24.45 ± 7.57)

showed a great difference. The obese group showed a very significant high level

of serum leptin with a P value of less than 0.0001, very highly significant. This

finding in the present study is supported by the findings of Masoud Y Al Maskari

et al who concluded in their study that serum leptin concentrations were increased

in relation to increased body fat151. Leptin receptor tolerance could be a cause for

such an elevated level of leptin in obesity. Hyperleptinemia is a precondition for

leptin resistance.

88

Correlation of P300 latency with the measures of obesity

As the measures of obesity i.e BMI, waist circumference, hip

circumference, waist hip ratio increases P300 latency also prolonged in my study.

In both cases and controls BMI, WC, HC, WHR are positively correlated with

latency of P300. The correlation was done using the Pearson’s correlation test. The

p- value was calculated by using chi-square test.

For BMI r- value was found to be 0.0720 for BMI in cases and 0.0712 in

controls. This suggests a strong positive linear relationship between the two

variables. The P value calculated for the r- value in both the cases and controls are

less than 0.0001. It shows that it is extremely statistically significant.

For waist circumference the r- value was found to be 0.1658 in cases and

0.1755 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

For hip circumference the r- value was found to be 0.1283 in cases and

0.0758 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

For waist hip ratio the r value was found to be 0.0334 in cases and 0.0613

in controls. The P value calculated for the r- value in both the cases and controls

are less than 0.0001. It shows that it is extremely statistically significant.

From the positive correlation obtained it can be understood that whenever

the measures of obesity such as BMI, WC, HC, and WHR increases the P300

89

latency also increases. This is comparable with studies of Jennifer H. Fergenbaum

et al who states that increased waist circumference associated with fivefold

increased risk of lowered cognitive performance compared to those without an

increased waist circumference152.

Correlation of P300 amplitude with the measures of obesity

As the measures of obesity i.e BMI, waist circumference, hip

circumference, waist hip ratio increase, P300 amplitude decreased in my study. In

both cases and controls, BMI, WC, HC, WHR are negatively correlated with

amplitude of P300. The correlation was done using the Pearson’s correlation test.

The p - value was calculated by using chi-square test.

For BMI r- value was found to be -0.1076 in cases and -0.2119 in controls.

This suggests a strong negative linear relationship between the two variables. The

P value calculated for the r- value in both the cases and controls are less than

0.0001. It shows that it is extremely statistically significant.

For waist circumference the r- value was found to be -0.0715 in cases and -

0.3547 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

For hip circumference the r- value was found to be -0.0444 in cases and -

0.2461 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

90

For waist hip ratio the r value was found to be -0.0399 in cases and -0.0266

in controls. The P value calculated for the r- value in both the cases and controls

are less than 0.0001. It shows that it is extremely statistically significant.

From the negative correlation obtained, it can be understood that whenever

the measures of obesity such as BMI, WC, HC, and WHR increase, the P300

amplitude decreases. The above data suggest that there is association between

obesity and cognition. As the measures of obesity increases then there is a

cognitive decline. These findings are similar to that of a study done by Shaheen E

Lakhan153.

Perpiñá et al., 2016, Fagundo et al., 2016 in their study suggested that

executive function impairments are larger in obese individuals with higher

BMIs154,155. Shields et al., 2017 in his studies reported that obesity-induced

activation of innate immunity directly causes low-grade inflammation in obese

persons. The immune system involved in inflammation in obesity is the main

reason to impair executive function156. But Ariza et al., 2012, Delgado-Rico et al.,

2013 suggested that there is no significant differences in executive function

between obese and non obese groups157,158.

Correlation of Serum Leptin with the measures of obesity

As the measures of obesity i.e BMI, waist circumference, hip

circumference, waist hip ratio increases Serum Leptin increases in my study. In

both cases and controls BMI, WC, HC, WHR are positively correlated with

91

Serum Leptin. The correlation was done using the Pearson’s correlation test. The

p - value was calculated by using chi-square test.

For BMI r- value was found to be 0.2979 for BMI in cases and 0.2977 in

controls. This suggests a strong positive linear relationship between the two

variables. The P value calculated for the r- value in both the cases and controls are

less than 0.0001. It shows that it is extremely statistically significant.

For waist circumference the r- value was found to be 0.1087 in cases and

0.2860 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

For hip circumference the r- value was found to be 0.0785 in cases and

0.1474 in controls. The P value calculated for the r- value in both the cases and

controls are less than 0.0001. It shows that it is extremely statistically significant.

For waist hip ratio the r value was found to be 0.0696 in cases and 0.0672

in controls. The P value calculated for the r- value in both the cases and controls

are less than 0.0001. It shows that it is extremely statistically significant.

From the positive correlation obtained it can be understood that whenever

the measures of obesity such as BMI, WC, HC, and WHR increases Serum Leptin

level also increases.

In obese individuals, serum leptin level increases but resistance to serum

leptin occurs. This is the most common reason for obese individuals with

92

increased serum leptin levels. These results are highly consistent with the findings

of Considine RV et al. They strongly suggested due to insensitivity to leptin

production endogenously even though there is an increase in Serum Leptin the

percentage of body fat is higher in these individuals15.

Correlation of Serum Leptin with the cognitive evoked potentials

As the Serum Leptin level increases, P300 latency increases and P300

amplitude decreases in my study. In both cases and controls P300 latency are

positively correlated with Serum Leptin and P300 amplitude are negatively

correlated with Serum Leptin. The correlation was done using the Pearson’s

correlation test. The p - value was calculated by using chi-square test.

For P300 latency the r- value was found to be 0.2423 in cases and 0.1376 in

controls. The P value calculated for the r- value in both the cases and controls are

less than 0.0001. It shows that it is extremely statistically significant.

For P300 amplitude the r -value was found to be -0.1825 in cases and -

0.4021in controls. The P value calculated for the r- value in the cases are less than

0.0001. It shows that it is extremely statistically significant. The P value

calculated for the r- value in the controls are 0.02, less than 0.05. It shows that it is

statistically significant.

Findings of the present study show that there is a strong relationship

between serum leptin and P300 amplitude and latency. Even though there is an

increase in serum leptin levels prolonged latency and decreased amplitude is seen

93

which is due to endogenous leptin resistance. G Paz-Filho et al 92 in their studies

demonstrated the effect of leptin in cognition. Due to its action on neuron

structure, function and glial cells it improves cognition. But because of leptin

resistance in obese individuals there is a cognitive decline. In this study leptin

replacement shows the improvement in cognition. It clearly explains that due to

leptin resistance in obesity there is a cognitive decline and human brain depends

on leptin for its proper development and function. If leptin replacement is given

then there is a possibility of improvement of cognition in those persons.

Thus the above discussion clearly suggest a possibility that the subjects

with obesity and high levels of serum leptin are prone for cognitive decline as a

obesity related complication in the future.

Conclusion

94

CONCLUSION

The conclusions derived from the present study are:

Serum Leptin levels are increased in obese individuals.

Prolonged P300 latency and reduced P300 amplitude is noted in obese

individuals.

Leptin levels are positively correlated to the BMI, and other measures of

obesity and latency of P300 and negatively correlate with amplitude of P300.

Leptin can be regarded as a biomarker in obesity to indicate the cognitive

decline.

Leptin also has a significant role in the pathogenesis of obesity and its

metabolic complications.

Hence P300 evoked potentials should be done in obese individuals and also

serum leptin levels to be measured in such individuals as it proves to be a

circulating biomarker for cognitive decline related to obesity.

This helps for early identification of obesity related cognitive decline so as

to enable us to intervene appropriately for necessary treatment and

prevention of further damages.

LIMITATIONS OF THE STUDY

Due to limitations in time and cost the sample size in both study and

control groups were kept to the minimum. Also there is unequal representation of

the genders and so the effects of difference in gender is not analysed and specified

in the discussion. This study should be done on a larger gender and age matched

sample to get a more clear picture of the role of leptin as a biomarker of cognitive

decline related to obesity.

Summary

95

SUMMARY

A study was conducted to study the cognitive evoked potentials and

circulating Serum Leptin levels in young individuals with obesity to find out the

relation of serum leptin levels to the decline in cognition related to obesity. Forty

subjects with a diagnosis of obesity and forty subjects who are non obese were the

participants of this study. Measurement of obesity was done using standard

protocols. The serum leptin levels assayed by sandwich ELISA method. Cognition

assessed by recording cognitive evoked potentials- P300 latency and amplitude.

The results of the study and control groups were statistically analysed. There was

a significant difference in all study parameters between the study and control

groups. Measures of obesity, serum leptin levels and P300 latency was significantly

increased in the obese group than compared to the non obese group while there

was a decrease in P300 amplitude in the study group than the controls.

On correlating the serum leptin levels with the measures of obesity and P300

latency in the obese group there was a positive correlation between them which

was statistically significant while there was a significant negative correlation for

P300 amplitude.

Thus the link and association of leptin with obesity and decline in

cognition in obese individuals was well established and thus serum leptin proves

to be a biomarker of cognitive decline in obesity.

96

These findings point towards the need for estimation of serum leptin levels

and assessment of cognitive evoked potentials in the obese individuals for earlier

identification of cognitive decline and prevention of further decline.

Early diagnosis of cognitive decline using the noninvasive P300 evoked

potentials could help in the prevention of adverse effects by early intervention by

life style modification with alterations in the dietary pattern and physical activities

so as to reduce weight.

FUTURE DIRECTIONS

The obese individuals could be followed up after life style modification

and dietary habits to assess if there is any improvement in cognitive function.

Other neuropsychological tests could be given to assess the specific cognitive

functions in obese individuals

I suggest this as a direction for further studies in this field.

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Annexures

ANNEXURES

INFORMED CONSENT FORM Title of the study: “A Study of Cognitive Evoked potentials and serum Leptin levels in young Obese individuals” Name of the Participant: Name of the Principal Investigator: Dr. UDHAYA BHARATHI.G

Name of the Institution: Institute of Physiology and Experimental Medicine, Madras Medical College and Govt. General Hospital, Chennai – 3. Documentation of the informed consent: I _____________________________ have read the information in this form (or it has been read to me). I was free to ask any questions and they have been answered. I am over 18 years of age and, exercising my free power of choice, hereby give my consent to be included as a participant in “A Study of Cognitive Evoked Potentials and Serum Leptin levels in young Obese individuals” 1. I have read and understood this consent form and the information provided to me. 2. I have had the consent document explained to me. 3. I have been explained about the nature of the study. 4. I have been explained about my rights and responsibilities by the investigator. 5. I have been informed the investigator of all the treatments I am taking or have taken in the past ________ months including any native (alternative) treatment. 6. I have been advised about the risks associated with my participation in this study. 7. I agree to cooperate with the investigator and I will inform him/her immediately if I suffer unusual symptoms. 8. I have not participated in any research study within the past________month(s). 9. I am aware of the fact that I can opt out of the study at any time without having to give any reason and this will not affect my future treatment in this hospital. 10. I am also aware that the investigator may terminate my participation in the study at any time, for any reason, without my consent. 12. I hereby give permission to the investigators to release the information obtained from me as result of participation in this study to the sponsors, regulatory authorities, Govt. agencies, and IEC. I understand that they are publicly presented. 13. I have understood that my identity will be kept confidential if my data are publicly presented. 14. I have had my questions answered to my satisfaction. 15. I have decided to be in the research study. I am aware that if I have any question during this study, I should contact the investigator. By signing this consent form I attest that the information given in this document has been clearly explained to me and understood by me, I will be given a copy of this consent document. Name and signature / thumb impression of the participant (or legal representative if participant incompetent) Name ______________________ Signature_________________ Date_____________ Name and Signature of the investigator or his representative obtaining consent: Name ______________________ Signature_________________ Date_____________

PATIENT PROFORMA Name:

Age/ Sex:

Address:

OP No.:

Occupation:

History of diabetes mellitus:

History of Hypertension:

History of associated illness:

a. Thyroid disorders & cancers

b. Renal & hepatic diseases

c. Swellings in & around the neck region

History of smoking and alcohol abuse:

History of taking chronic medications or hormonal therapies:

Investigations: EXAMINATION: General Physical examination: Anthropometric measurements: Ht:

Wt:

WC:

HC:

W/H Ratio:

Vitals:

Temperature:

Pulse rate:

Blood pressure:

Systemic examination:

Cardiovascular system:

Respiratory system:

Gastrointestinal system:

Central nervous system:

MASTER CHART - CASES

S. NO

AGE (YRS)

SEX (M/F)

HT (cm)

WT (Kg)

BMI (Kg/m2)

Waist Circumference

(cms)

Hip Circumference

(cms) Waist Hip Ratio

P300 Latency (milliseconds)

P300 Amplitude (µv)

SERUM LEPTIN (ng/ml)

1 29 F 152 74 32.03 112 120 0.93 348.52 7.4 19 2 23 F 158 82 32.85 135 150 0.90 368.22 5.7 31 3 30 F 153 76 32.47 128 140 0.91 354.22 4.21 26 4 30 F 140 68 34.69 115 130 0.88 370.22 3.8 15 5 28 M 162 89 33.91 127 135 0.94 368.82 6.78 15 6 29 M 151 73 32.02 135 150 0.90 354.52 8.4 22 7 30 M 159 70 30.85 113 120 0.94 338.42 11.2 10 8 21 F 162 84 32.01 128 140 0.91 364.22 5.8 39 9 30 F 163 86 32.37 136 145 0.94 346.22 10.12 28

10 27 F 150 78 34.67 112 120 0.93 368.42 4.6 32 11 28 F 163 82 30.86 125 140 0.89 344.22 7.4 29 12 30 M 153 80 34.17 112 120 0.93 342.22 8.6 17 13 25 F 145 72 34.24 119 134 0.89 368.22 6.2 19 14 23 F 153 78 33.32 121 135 0.90 332.62 12.6 37 15 30 M 153 81 34.6 130 145 0.90 370.42 2.6 32 16 23 M 154 81 34.15 130 140 0.93 364.22 5.6 34 17 28 F 165 88 32.32 111 120 0.93 370.42 6.77 17 18 21 M 158 86 34.45 125 140 0.89 346.22 8.25 18 19 30 F 145 75 35.67 135 150 0.90 343.22 5.4 13 20 28 F 154 83 35.00 120 137 0.88 338.42 8.4 29 21 21 F 157 84 34.08 111 120 0.93 334.42 9.6 19 22 26 F 153 79 33.75 125 137 0.91 370.22 3.6 32 23 22 F 149 76 34.23 113 125 0.90 370.22 5.4 27 24 20 F 160 88 34.38 111 120 0.93 363.42 3.44 27 25 23 F 158 79 31.65 110 115 0.96 354.24 4.6 33 26 19 M 152 80 34.63 112 120 0.93 344.44 8.8 19 27 21 M 150 80 35.56 130 145 0.90 363.42 6.6 33 28 19 M 149 68 30.63 130 145 0.90 369.62 3.12 17 29 25 F 160 90 35.16 104 117 0.89 333.34 9.6 24 30 26 F 152 92 39.82 116 120 0.97 364.62 3.8 34 31 29 F 156 88 36.16 128 140 0.91 343.42 7.6 22 32 19 M 156 90 36.98 110 115 0.96 352.22 6.2 28 33 19 M 153 86 36.74 113 125 0.90 356.22 9.6 31 34 24 F 163 96 36.13 130 145 0.90 352.22 8.2 26 35 23 F 154 86 36.26 116 120 0.97 359.22 4.6 22 36 22 F 160 84 32.81 104 117 0.89 370.24 2.8 30 37 22 F 162 87 33.15 110 125 0.88 348.22 7.8 18 38 26 F 149 72 32.43 115 130 0.88 340.22 7.2 11 39 19 F 158 80 32.05 109 120 0.91 338.22 8.6 27 40 25 M 158 89 35.65 128 140 0.91 354.22 6.8 16

MASTER CHART - CONTROLS

S. NO

AGE (YRS)

SEX (M/F)

HT (cm)

WT (Kg)

BMI (Kg/m2)

Waist Circumference

(cms)

Hip Circumference

(cms) Waist Hip Ratio

P300 Latency (milliseconds)

P300 Amplitude (µv)

SERUM LEPTIN (ng/ml)

1 24 F 156 59 24.2 87 108 0.81 332.22 6.6 11 2 23 M 156 52 21.3 82 104 0.79 339.47 10.5 5 3 30 F 150 52 23 83 110 0.75 333.34 8.9 7 4 25 M 156 58 23.8 81 102 0.79 323.64 7.9 7 5 28 F 170 68 23.5 85 106 0.80 342.67 6.44 13 6 29 F 158 57 22.8 83 107 0.78 348.22 7.8 13 7 21 F 159 57 22.6 82 106 0.77 324.66 11.8 7 8 27 M 170 68 23.5 86 107 0.80 324.56 10.2 10 9 19 M 163 56 21.1 82 106 0.77 327.66 10.6 6

10 22 M 174 70 23.1 83 104 0.80 340.22 8.8 6 11 26 F 172 68 23 84 103 0.82 340.42 8.2 8 12 30 F 168 65 23 86 109 0.79 344.56 11.4 1 13 27 M 168 66 23.4 81 100 0.81 338.46 8.4 10 14 25 F 176 68 22 86 107 0.80 332.44 8.4 9 15 18 F 172 68 23 86 104 0.83 340.12 7.9 15 16 21 M 165 64 23.7 83 105 0.79 336.46 8.7 11 17 26 M 172 67 23.1 81 104 0.78 328.21 9.6 3 18 19 M 152 55 23.9 83 105 0.79 331.22 10.6 9 19 24 F 160 52 20.8 82 100 0.82 340.12 9.4 9 20 24 F 155 57 23.7 81 102 0.79 326.22 7.8 6 21 27 F 167 61 22.5 86 109 0.79 338.23 9.8 9 22 28 F 169 63 22.5 87 109 0.80 336.56 10.4 8 23 30 M 166 61 22.5 83 107 0.78 338.44 11.6 1 24 30 M 156 53 22 82 102 0.80 336.44 10.4 2 25 21 F 155 56 23.3 84 109 0.77 346.56 5.6 15 26 25 F 172 66 22.7 82 105 0.78 336.54 9.6 5 27 29 F 173 64 22 83 98 0.85 334.24 10.5 4 28 29 F 165 61 22.5 85 100 0.85 337.44 9.2 5 29 19 F 152 54 23.4 83 105 0.79 338.4 9.6 7 30 22 M 172 66 22.7 83 103 0.81 323.12 8.8 14 31 22 M 158 54 21.6 87 103 0.84 328.76 11.6 8 32 26 M 162 59 22.4 82 104 0.79 339.22 10.6 8 33 30 F 170 68 23.5 83 107 0.78 334.12 5.8 12 34 30 F 165 63 23 83 105 0.79 336.22 8.6 5 35 27 F 158 57 22.8 82 105 0.78 336.42 9.4 7 36 27 F 167 65 23.3 84 104 0.81 345.22 6.4 10 37 19 F 170 68 23.5 82 101 0.81 335.24 10.4 6 38 19 M 152 48 21.3 83 106 0.78 325.54 9.8 7 39 29 F 168 60 21.4 82 110 0.75 330.12 9.2 6 40 18 F 150 48 21.3 85 107 0.79 334.34 11.4 7