Serum Ghrelin Levels in Hypothyroid Patients

SERUM GHRELIN LEVELS IN HYPOTHYROID PATIENTS

Dissertation submitted to

THE TAMILNADU Dr. MGR MEDICAL UNIVERSITY

CHENNAI—600032.

In partial fulfilment of the requirement for the award of the degree of

DOCTOR OF MEDICINE IN

BIOCHEMISTRY BRANCH XIII

DEPARTMENT OF BIOCHEMISTRY

COIMBATORE MEDICAL COLLEGE

COIMBATORE-641014.

MAY 2020

UNIVERSITY REGISTRATION NO – 201723653

BONAFIDE CERTIFICATE

This is to certify that the dissertation entitled “SERUM GHRELIN

LEVELS IN HYPOTHYROID PATIENTS” is a bonafide original work done by Dr. P.SUMATHI in partial fulfilment of the requirements of M.D

Biochemistry [Branch-XIII] examination of The Tamilnadu Dr.M.G.R

Medical University to be held in May 2020.

HOD & GUIDE: DEAN:

Dr.S.MANIMEKALAI M.D., Dr.B.ASOKAN M.S.,M.Ch., PROFESSOR AND HOD, Coimbatore Medical College & Hospital, Department of Biochemistry, Coimbatore-14. Coimbatore Medical College, Coimbatore-14.

DECLARATION

I Dr.P.SUMATHI solemnly declare that the dissertation titled

“SERUM GHRELIN LEVELS IN HYPOTHYROID PATIENTS”

is done by me at Coimbatore Medical College& Hospital, Coimbatore

during the period from March 2018- February 2019 under the guidance

and supervision of Prof.Dr.S.MANIMEKALAI, M.D., Professor &

HOD, Department of Biochemistry, Coimbatore Medical College,

Coimbatore600014. This dissertation is submitted to The Tamil Nadu

Dr. M.G.R. Medical University towards the partial fulfilment of the

requirements for the award of M.D Degree [Branch-XIII] in

Biochemistry.

Place: Coimbatore. Dr.P.Sumathi, Date: Post Graduate Student, Department of Biochemistry.

CERTIFICATE II

This is to certify that this dissertation work titled “SERUM GHRELIN

LEVELS IN HYPOTHYROID PATIENTS” of the candidate

Dr.P.SUMATHI with Registration Number 201723653 for the award of

M.D.DEGREE in the branch of BIOCHEMISTRY. I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to limitation pages and result shows 15% (FIFTEEN) of plagiarism in the dissertation.

Guide & Supervisor sign with seal.

ACKNOWLEDGEMENT

I express my sincere thanks to the respected Dean Dr.B.ASOKAN,M.S,MCh, for allowing me to undertake this study in our hospital. I am extremely grateful to Prof. Dr.S.MANIMEKALAI M.D., Professor and Head of the Department of biochemistry for permitting me to carry out my study and for her constant encouragement and guidance.

I am extremely thankful to Dr. ELANGO, M.S, Professor and Head of the Department, Department of Surgery, for granting permission to obtain blood samples from the patients.

I take immense pleasure in expressing my sincere thanks to Associate Professor Dr.A.VEENA JULIETTE M.D., for her constant encouragement and guidance. I personally thank to Prof. Dr.N.DHEEBALAKSHMI M.D., for her valuable opinion & guidance to perform this work.

I thank Assistant Professor Dr.G.EZHIL M.D., for her continuous motivation and valuable guidance throughout my work.

I whole heartedly thank my parents, my husband & daughters M.S.HARSHINI & M.S.SHRI HARINI, my colleagues, and staff of our Central Lab for their support for this work.

I thank Dean& Nodal Officer of the Multidisciplinary Research Unit, of Coimbatore Medical College for allowing me to utilize the ELISA equipment for the analytical process.

I owe my sincere thanks to all the patients for their kind co-operation throughout the study. ABBREVIATIONS

BMI Body Mass Index

HT Hypertension

GH Growth Hormone

DM Diabetes Mellitus

GIT Gastro Intestinal Tract

CNS Central Nervous System

BBB Blood Brain Barrier

IP Intraperitoneal

ICV Intracerebroventricular

CHO Cholesterol

TG Triglycerides

LDH Lactate Dehydrogenase

CLIA Chemiluminiscence Immune Aassay

T3 Tri IodoThyronine

T4 Thyroxine

TSH Thyroid Stimulating Hormone

TRH Thyrotropin Releasing Hormone

HPT Hypothalamic-Pituitary-Thyroid axis

D1 Deiodinase 1

Dio1 Deiodinase 1 gene

D2 Deiodinase 2 Dio2 Deiodinase 2 gene

D3 Deiodinase 3

Dio3 Deiodinase 3 gene

DIT Di Iodo thyronine

MIT Mono Iodo Thyronine

GOAT Ghrelin Octo Acyl Transferase

NIS Sodium/ Iodide Symporter

NPY Neuropeptide Y

AgRP Agouti Related Protein

GHS-R Growth Hormone Secretogogue Receptor

TR Thyroid hormone receptor

TPO Ab Thyroid peroxidase antibodies

TAb Thyroglobulin Antibodies

EGF Epidermal Growth Factor

IGF Insulin like Growth Factor

AMPK 5- adenosine monophosphate-activated protein kinase

α-MSH Alpha-melanocyte-stimulating hormone

ARC Arcuate nucleus

BDNF Brain-derived neurotrophic factor

MC4R Melanocortin 4 receptor

MCT8 Monocarboxylate transporter 8

OATP1C1 Organic anion transporting polypeptide 1c1 PVN Paraventricular nucleus

POMC Pro-opiomelanocortin rT3 Reverse T3

SF1 Steroidogenic factor-1

UCP1 Uncoupling protein 1

UCP2 Uncoupling protein 2

VMN Ventromedial nucleus.

TABLE OF CONTENTS

S. No CONTENT PAGE NO

1. INTRODUCTION 1-5

2. AIM & OBJECTIVES 6

3. REVIEW OF LITERATURE 7-48

4. MATERIALS & METHODS 49-58

5. STATISTICAL ANALYSIS 59

6. RESULTS 60-77

7. DISCUSSION 78-81

8. CONCLUSION 82

9. LIMITATIONS OF STUDY 83 10. BIBLIOGRAPHY ANNEXURE A) PROFORMA B) CONSENT FORM 11. C) ETHICAL COMMITTEE APPROVAL CERTIFICATE D) URKUND DIGITAL RECEIPT E) MASTER CHART

INTRODUCTION

HYPOTHYROIDISM

Hypothyroidism is the clinical presentation , when thyroid gland is not able to synthesise adequate amount of thyroid hormones, triiodothyronine (T3) and thyroxine(T4), to maintain normal blood levels and to the needs of peripheral tissues(1).

Primary hypothyroidism is disease in the thyroid gland itself that is unable to produce adequate thyroid hormones. Many patients present with primary hypothyroidism. Permanent Loss or destruction of the thyroid cells, through autoimmune destruction, like Hashimoto disease or irradiation injury, is under Primary Hypothyroidism(1).

Hypothyroidism is rarely secondary, caused by disease in the pituitary gland or hypothalamus resulting in inadequate production of thyroid-stimulating hormone (TSH)(2).

Transient or progressive loss of hormone biosynthesis is associated with compensatory thyroid gland enlargement.

Central or secondary hypothyroidism is caused by inadequate stimulation of a normal thyroid gland, is the result of hypothalamic axis or pituitary axis defects in the TSH molecule(3). Transient or temporary hypothyroidism can be identified as a course of subacute thyroiditis(4).

Prevalence of hypothyroidism in India is 11% showed in FIG-1.

Global distribution of thyroid disorders are depicted in FIG – 2.

FIG – 1- PREVALENCE OF HYPOTHYROIDISM

FIG – 2- GLOBAL DISTRIBUTION OF THYROID DISORDERS

99% of hypothyroidism cases are due to Primary hypothyroidism and remaining 1% due to TSH deficiency. Calculation of incidence of hypothyroidism varies depending on the population, geographical area, sex, age etc(5) .

Subclinical hypothyroidism is defined as increased level of serum

TSH concentration with a normal serum T4 concentration(6). Subclinical hypothyroidism can lead to overt hypothyroidism(7). Clinical manifestations of subclinical hypothyroidism are associated with early or mild stage of Hypothyroidism.

The incidence of hypothyroidism is more in women, in the elderly, and in some race and ethnic groups, some geographical areas(8) .

Neonatal screening programme is done for congenital hypothyroidism. This identifies hypothyroidism in almost 1 in 3000 newborns(9).

The presence of normal or increased thyroid hormone production, but decreased thyroid hormone activity at the tissue level produces clinical signs & symptoms of the disease. These conditions include abnormal peripheral metabolism of thyroid hormone and target tissue resistance to thyroid hormones.

Congenital primary hypothyroidism may be the consequence of several inborn defects involving key steps needed for development and function of the thyroid gland and associated with

2 different clinical features. Thyroid dysfunction is associated with changes in body weight, food intake, and energy expenditure(10).

GHRELIN

Ghrelin is a 28-amino acid acyl peptide mainly produced by the stomach and identified in 1999(11) as a natural ligand of the GH secretagogue receptor type 1a (GHS-R1a). It was originally recognized as the stimulus for the release of growth hormone, with multiple actions in the body. Its best-known function involves energy metabolism.

Ghrelin is an orexigenic hormone which regulates energy intake by stimulating the appetite, resulting in adipogenesis (the formation of fat cells) and reduced lipolysis (the breakdown of lipids)(11).

Circulating total Ghrelin levels are decreased under conditions of positive energy balance, such as obesity, whereas an increase in total

Ghrelin concentrations is observed in negative energy balance states, such as anorexia nervosa or diet-induced weight loss(12).

At the hypothalamic level, ghrelin stimulates GH release and regulates appetite and energy balance. Increased levels are seen in catabolic conditions and decreased levels are found in obesity. An inverse correlation between serum ghrelin levels and resting energy expenditure

(REE) has been recorded in healthy women.

Thyroid disease is associated with changes in appetite, food intake, and Resting Energy Expenditure (REE). Hypothyroid patients gain weight inspite of their decreased appetite.

Ghrelin is involved in the regulation of food intake, fat storage and energy balance and it has orexigenic effects in humans in whom it stimulates appetite and increases food intake(13). Previous studies have reported decreased levels of ghrelin in hyperthyroidism.

As alterations in body weight and appetite are hallmarks of thyroid disorders, it seems rational to assess the changes in the serum ghrelin levels in different states of thyroid dysfunction and explore the role of ghrelin in appetite changes in these patients.

AIM & OBJECTIVES

AIM AND OBJECTIVES

AIM OF THE STUDY

To evaluate the role of Ghrelin in the appetite changes in

Hypothyroid patients.

OBJECTIVE OF THE STUDY

 To assess the appetite behaviour of hypothyroid patients.

 To assess the level of Serum Ghrelin level in Hypothyroid patients

and Euthyroid individuals.

 To compare the Serum Ghrelin level in Hypothyroid patients and

Euthyroid individuals.

REVIEW OF LITERATURE

Hypothyroidism

History

Hypothyroidism was described for the first time in London (1870).

It was named Myxedema in 1888. It was found out that cretinism,

Myxedema & Post Thyroidectomy changes were a result of loss of function of thyroid gland(14). Kendall isolated Thyroxin hormone in

1914. Harrington synthesized Thyroxine in 1926. However, synthesis of

Thyroxine was done in large scale only in 1949. Later it became a universally accepted therapy for hypothyroidism.

THYROID GLAND

Anatomy of Thyroid Gland (14)

Thyroid gland has a midline isthmus lying horizontally just below the Cricoid cartilage, right & left lateral lobes that extend superiorly together in front of neck giving the appearance of a butterfly. The gland is enclosed by pre tracheal fascia under the strap neck muscles which makes the gland move up with deglutition (FIGURE-3,3A).

FIGURE-3: ANATOMY OF THYROID GLAND

FIGURE-3A: ANATOMY OF THYROID GLAND

Histology of Thyroid (14)

Thyroid gland is divided by thin fibrous septa into pseudolobules, which are composed of follicles or acini densely surrounded by capillary networks (FIGURE-5). Follicular walls are lined by cuboidal epithelium.

The peptide sequences of T4 and T3 are synthesized stored and as proteinaceous colloidal material called Thyroglobulin that fill the lumen of the follicles (FIGURE-4&5).

Embryology of Thyroid Gland

Thyroid gland develops from the ectoderm of the pharyngeal floor with some contribution from the lateral pharyngeal pouches. Thyroglossal duct, which extends from the foramen caecum near the base of the tongue to the isthmus of the thyroid arises as descent of the midline thyroid anlagen. The posterior aspect of the thyroid gland becomes associated with the parathyroid glands & the para follicular C cells, which are derived from ultimo-bronchial body during development and become incorporated into its substance. While they undergo malignant transformation, the C cells are the source of the calcitonin & leads to medullary thyroid carcinoma. At about 10-12 weeks of gestation, the foetal thyroid begins to concentrate & organify Iodine. Maternal TSH and

T4 do not cross the placenta, but the maternal TRH crosses the placenta.

The major source of thyroid hormone in the foetal life is T4 from the foetal

FIGURE-4: THYROID CELL

FIGURE-5: MICROSCOPIC PICTURE OF

THYROID FOLLICLE CELL thyroid itself. The functional unit is foetal pituitary – thyroid axis which is distinct from that of the mother.

Physiology of Thyroid Gland (15)

Thyroid secretes the hormones Thyroxin (T4), Triiodothyronine

(T3) & Calcitionin. Thyroid follicles secrete only the first two hormones termed as “Thyroid Hormones”. Calcitonin is chemically & biologically different, secreted from the parafollicular C cells. It regulates calcium metabolism along with Parathormone (PTH). Iodine enters the thyroid in the form of inorganic or organic iodide which is oxidized by peroxidase enzyme. Subsequent reactions result in the formation of thyroxin. The only source of T4 is thyroid gland. Thyroid secretes 20% of T3; extra glandular tissues produce the remaining amount by peripheral conversion of T4 into T3.

CHEMISTRY AND SYNTHESIS OF THYROID HORMONE (15)

Both T4 and T3 are product of 2 molecules of tyrosine with iodine containing derivatives of Thyronine. Thyroxine (T4) is 3, 5, 3‟, 5‟ –

Tetraiodothyronine and T3 is 3, 5, 3‟ – Triiodothyronine. Thyroid hormones are synthesized & stored in thyroid follicles as part of

Thyroglobulin, a glycoprotein synthesized in thyroid cells. There are 5 steps in synthesis of thyroid hormones (FIGURE-6).

1. IODIDE UPTAKE / IODIDE TRAPPING: Iodine from peripheral circulation is taken into follicles by active transport Na + I – symporter.

FIGURE-6: SYNTHESIS OF THYROID HORMONE IN FOLLICLE

Iodine content of follicle regulates the iodide trap. Minimal storage activates & large storage inhibits this trap. This process is mediated by

TSH. Drugs like Perchlorate, Thiocyanates and Nitrates inhibit this trapping.

2. OXIDATION AND IODINATION: Iodide trapped by follicular cells is transported by active transporter “Pendrin” across the apical membrane

& oxidized by thyroid peroxidise enzyme in the follicular membranes & forms iodinium ions (I+) or hypoiodous acid (HOI) or enzyme linked hypoiodate (E-OI) with the help of H2O2. These various forms of iodine bind with Thyroglobulin and form Monoiodothyronine (MIT) and

Diiodothyronine (DIT).

3. COUPLING: Pairs of iodinated tyrosine residues form T3 and T4 by coupling. Coupling is an oxidative reaction which is catalysed by the same thyroid peroxidise. TSH regulates this coupling process as well.

4. STORAGE AND RELEASE: Tyrosine residues are stored as

Colloids. These are taken back into follicular cells by endocytosis and undergo lysosomal proteolysis to T4 and T3. At rest, follicles filled with colloids are flat / cuboidal cells whereas TSH stimulated follicles are columnar cells with lack of colloids (FIGURE-4).

5. PERIPHERAL COVERSION OF T4 TO T3: Conversion occurs in

Kidney & liver. One third of T4 undergoes conversion & most of T3 in plasma is derived from liver. Target organs take up T3 for metabolic

10 functions except brain & pituitary which take up T4 and convert in to T3 by their own cellular mechanisms (FIGURE-11).

Relation between T3 and T4 (15)

Physiologically Thyroid secretes higher amount of T4 compared to

T3. Normally T4 is the major circulating form as it is bound with plasma proteins 15 times more than T3. T3 is five times more potent than T4. T3 acts very faster than T4. Peak effect of T3 comes earlier (1-2 days) whereas peak effect of T4 comes later (6-8 days). T3 is more tightly bound to the nuclear receptors than T4. About one third of T4 is converted to T3 in peripheral tissues, liver and kidney, by D1 type of 5’ Deiodinase (D1 type 5’ DI) and released in to circulation. T3 is also generated within the cells like skeletal muscles, brain, pituitary and heart, by another enzyme type called type 2 deiodinase (D2 type 5’ DI). T4 is converted to metabolically active T3 or inactive reverse T3 (r T3). T4 & T3 are metabolized in liver by conjugation with glucuronate and sulfate. Enzyme inducers such as Phenobarbitone, Carbamazepine & Phenytoin increase the metabolic clearance of the hormones without decreasing the proportion of free hormones in the circulation. Finally, T3 is the active form. T4 is a transport form i.e precursor of T3. Normal daily secretion of

T3 is 10 - 30 mcgm. T4- 60-90 mcgm. T3 and T4 are bound with 3 plasma proteins – Thyroxin binding globulin (TBG) & Thyroxin binding prealbumin (Transthyretin) & Albumin. Plasma t ½ of T3 is 1-2 days; of

T4 is 6-7 days. The half-life is increased in hypothyroidism and shortened in hyperthyroidism due to enhanced and blunted metabolism respectively.

Only source of T4 is Thyroid gland(16).

Clinical Scenarios with altered concentration of TBG

S.NO INCREASED TBG DECREASED TBG 1. New born Phenytoin 2. OCP/Estrogen/Tamoxifen Acromegaly 3. Biliary cirrhosis Androgens 4. Chronic active hepatitis Nephrotic syndrome 5. Acute Intermittent Porphyria Large doses of glucorticoids 6. Pregnancy Chronic liver disease

THYROID HORMONE RECEPTOR (THR/TR):

Thyroid receptor hormones are two types- thyroid hormone receptor α (TRα) and thyroid hormone receptor β (TRβ). Each receptor coded by different gene, α receptor gene on chromosome 17 and β receptor gene on chromosome 3.

These genes are alternately spliced to generate three main homologous nuclear receptor isoforms (TRα1, TRβ1, and TRβ2). The three main isoforms are bind high affinity with T3 and regulate thyroid hormone- mediated transcription(10).

TRα is main isoform regulating T3 activity in the heart, skeletal muscle, bone and brain;

TRβ is the main isoform regulate the activity of T3 in the liver.

Adipose tissue expresses both TRα and TRβ.

TRβ1 is expressed in most of all tissues, while TRβ2 is express mainly in the hypothalamus, pituitary, cochlea, and retina.

Many coactivator and corepressor proteins affect the action of thyroid receptors and hence thyroid hormones produce different effects on the body. Since T3 is less tightly bound to plasma protein than is T4 but more strongly to thyroid hormone receptors T3 acts more rapidly and more potently than T4(10).

MECHANISM OF ACTION

All the cellular activities of the tissues in our body are affected by the thyroid hormones (T3)(15). Most of the actions of thyroid hormones are exerted through modulation of gene expression. The intracellular receptors are located in the cytosol or in the nucleus. Mostly T3 binds more avidly to TR in the nuclei than T4. The hormone receptor complex binds to DNA through zinc fingers and exerts its effect by acting on the gene expression on the target cell and regulate cell function.

STEPS INVOLVED IN THE ACTION OF HORMONES

THROUGH THEIR EFFECT ON GENE EXPRESSION

(FIGURE-7)

1. TRANSPORT:

The secreted hormones are carried to the target tissue with the help of the serum binding protein.

2. INTERNALIZATION:

Thyroid hormones are lipophilic, they can easily diffuse across the plasma membrane.

3. RECEPTOR – HORMONE COMPLEX FORMATION:

This is by the binding of the hormone to the specific receptor inside the cell.

FIGURE-7: ACTION THROUGH THE EFFECT OF GENE

EXPRESSION BY BINDING OF HORMONES WITH

INTRACELLULAR RECEPTORS(15)

4. CONFORMATIONAL CHANGE:

Activation of receptor is by the conformational change occurring in the receptor proteins.

5. GENE TRANSCRIPTION:

The activated receptor-hormone complex diffuses into the nucleus and binds to the specific region on the DNA known as hormone responsive element (HRE).This initiates Gene transcription.

6. Binding of the receptor-hormone complex to DNA alters the rate of transcription of the required gene.

7. The resultant mRNA diffuses into the cytoplasm and promotes the translation process at the ribosome. Thus new proteins are formed which have specific responses.

This ultimately results in:

- Increased synthesis of enzymes and specific structural or functional proteins. The thyroid hormones have anabolic and metabolic actions.

- Increased synthesis of Na+ - K+- ATPase is responsible for the calorigenic function of thyroxine. During increased Na+ transport there is a lot of energy consumption which leads to increased metabolic rate.

- Increase in the number and activity of mitochondria and hence the rate of ATP synthesis. When there is extremely high concentration of

15 thyroid hormones, uncoupling of oxidative phosphorylation occurs. This results in production of large amount of heat with lesser ATP.

Normal Resting Oxygen Utilization in human ranges from 225-250 ml/min. In hypothyroid state it is about 150ml/min and to about

400ml/min in the hyperthyroid state.

The measure of calorigenic action of thyroxine partly depends on the level of circulating catecholamine. So increased metabolic rate is associated with increased utilization of many other hormones and vitamins; so it is advisable for patients with thyroid disorders to consume more vitamins.

ACTION OF THYROID HORMONES ON VARIOUS SYSTEMS(15)

1. EFFECTS ON GROWTH & TISSUE DEVELOPMENT:

Thyroid hormones are important for normal body growth and development.

(i) Thyroid hormones exert their effect directly by increasing synthesis of proteins and enzymes and indirectly by increasing production of growth hormone and somatomedins. These hormones play a major role in skeletal maturation & body growth.

(ii) Role in tissue differentiation and maturation.

(iii) Role in development of Nervous tissue:

In the Nervous System for Axonal and Dendritic development and for normal myelination, T3 is to be necessary. In case of Congenital

Hypothyroidism, the child will have a striking feature of mental retardation. Early detection and hormonal replacement therapy helps to prevent severe mental retardation.

2. EFFECTS ON THE METABOLIC RATE:

Thyroid hormones stimulate the metabolic function and thereby increase the basal rate of oxygen consumption and heat production in the body except brain, retina, gonads, lungs and spleen.

3. EFFECTS ON RESPIRATORY SYSTEM:

Thyroid hormones stimulate O2 utilization of tissues by a) Increase in the resting respiratory rate, minute ventilation and ventilator responses to hypercapnia and hypoxia; these actions maintain normal Po2 and Pco2. b) Increase in Oxygen carrying capacity of blood by increasing the red blood cell mass to a small extent.

4. EFFECTS ON METABOLISM:

CHO Metabolism:

T4 & T3 cause an overall increase in enzymes there by lead to:

 Increased glucose absorption from GIT.

 Glucose metabolism acceleration resulting in rapid uptake of

glucose by the cells, increased glycolysis, increased

gluconeogenesis and insulin secretion.

Fat Metabolism:

Thyroid hormone produces the following effects:

 Increase in Fatty acid level by mobilization of fat from adipose

tissue.

 Decrease in the quantity of cholesterol, phospholipids, and

triglycerides in plasma.

 Decrease in plasma cholesterol due to increased excretion in bile.

Protein Metabolism:

If the synthesis and secretion of thyroid hormones are normal, positive nitrogen balance (increase in RNA and protein synthesis) is maintained. Excess thyroid hormones lead to negative nitrogen balance, with catabolic effect. This causes muscle weakness and creatinuria in the hyperthyroid patients.

Effects on Vitamin Metabolism:

Vitamins are the important components of some enzymes and act as Coenzymes. Thyroid hormones increase the quantity of enzymes and thereby increase the need for vitamins. Hence, in hyperthyroidism vitamin deficiency is one of the common features.

5. EFFECTS ON WATER & ELECTROLYTE BALANCE

Thyroid hormones play a significant role in the regulation of water and electrolyte balance via Sodium Iodide transporter.

6. EFFECTS ON CARDIOVASCULAR SYSTEM:

The thyroid hormones increase the cardiac output and ensure adequate oxygen delivery to the tissues. The thyroid hormones produce the following effects on the cardiovascular system: i) Tachycardia

Increased Heart rate (even at rest and during sleep) is one of the clinical sign to assess the function of the thyroid gland. ii) Force of Cardiac contraction

The force of cardiac contraction is increased by the thyroid hormones. Adrenergic stimulation causes inotropic effects on the heart.

The cardiac contractile force is enhanced by the myocardial calcium uptake and adenylyl cyclase activity. iii) Cardiac output

When the blood volume is increased, heart rate and the force of contraction of the heart increase which lead to increased cardiac output. iv) Effect on Blood Pressure:

Systolic BP: Increased due to increased strength and rate of heart beat.

Diastolic BP: Decreased due to peripheral vasodilatation.

Pulse Pressure: Increased, but there is no change in mean arterial pressure. v) Vasodilatation and increased blood flow to tissues:

The increased blood flow to tissues as a result of vasodilatation occurs by two mechanisms.

 Indirect Mechanism:

Thyroid hormones cause rapid utilization of O2 and increased production of heat and CO2. So vasodilatation occurs in skin, muscle and heart. Cutaneous vasodilatation helps in dissipation of excessive heat produced.

 Direct Mechanism:

Thyroid hormones directly decrease systemic vascular resistance by dilating the arterioles in the peripheral circulation. vi) Increased Heart Strength:

This is beneficial only when there is mild elevation of thyroid hormones. vii) Normal Arterial pressure:

The mean arterial pressure usually remains normal after administration of thyroid hormones.

7. EFFECTS ON NERVOUS SYSTEM:

 Thyroid hormones play an essential role in the development of

nervous system. The critical period for the development of nervous

system is up to one year of life.

 T4 enhances wakefulness, alertness, responsiveness to various

stimuli, auditory sense, awareness to hunger, memory and learning

capacity.

 The normal thyroid hormone availability is responsible for the

normal emotional tone.

 Thyroid hormone increases the speed and amplitude of peripheral

nerve reflexes.

8. EFFECTS ON GASTROINTESTINAL TRACT:

 Increase in food intake by increasing the appetite.

 Increases the digestive juice secretion rate.

 The motility of GIT is increased.

 Excess of thyroid hormone causes diarrhea.

9. EFFECTS ON REPRODUCTIVE SYSTEM:

In both sexes, thyroid hormones have definite action in the regulation of reproductive functions.

In Males:

1. Lack of thyroid hormones lead to loss of Libido.

2. Excess hormone can cause Impotence.

In Females

Lack of thyroid hormone has varying effects and produces the following dysfunctions.

1. Menorrhagia & Polymenorrhea

2. Irregular periods

3. Amenorrhea

10. EFFECTS ON OTHER ENDOCRINE GLANDS:

Thyroid hormones have significant effects on other parts of endocrine system.

 Pituitary production of growth hormone is increased, but

Prolactin is decreased.

 Adrenocortical secretion of cortisol and clearance is stimulated

but plasma free cortisol level remains normal.

 Oestrogen and Androgen ratio in males is increased.

 Parathyroid hormone and 1, 25-(OH) 2 vitamin D are decreased

as a compensatory effect of thyroid hormone on bone resorption.

11. EFFECTS ON KIDNEY:

Renal plasma flow, Glomerular filtration rate and Tubular transport maximum are also increased by thyroid hormones.

EPIDEMIOLOGY

Thyroid diseases are among the commonest occurring Endocrine

Disorders worldwide(17). Our country too, is of no exception from this disorder. Thyroid disease is common in women, with the prevalence of

3-5% in the general community(18).

It has been estimated that about 42 million people in India suffer from thyroid diseases, in accordance from recent survey in several studies.

Several population based studies have defined the prevalence of subclinical hypothyroidism(19).

SCREENING FOR HYPOTHYROIDISM (20)

Various recommendations have been proposed.

1. American Thyroid Association (ATA):

Men and women >35 years must be screened every 5 years

2. American Association of Clinical Endocrinologist:

Women and older people should be screened.

3. American college of Obstetrics and Gynaecology:

Women with autoimmune disease and family history of thyroid disease screened at 19 years.

4. American college of Physicians (ACP):

Symptomatic thyroid disease in Women > 50 years should be screened.

5. Royal college of Physicians:

Screening in healthy adult population is not justified

6. Indian Thyroid Society:

Routine screening is not indicated.

SCREENING OF HIGH RISK POPULATION (20)

In the following group of patients routine screening for thyroid dysfunction would be beneficial

 Menstrual irregularities

 Infertility

 Dyslipidemia

 Unexplained Hyponatremia,

 Type 1 diabetes

 Carpel tunnel syndrome

 Depression

 Short stature

 Recurrent abortion

 Down’s syndrome

 Pregnancy

 Family history of thyroid disease

 Exposure to radiation

DIAGNOSIS OF THYROID DISORDER

TSH estimation – an effective screening tool for thyroid dysfunction.

If TSH levels are elevated, T3/T4 estimation should be done. If

T3/T4 levels are normal, subclinical hypothyroidism should be suspected.

Free T3/T4 estimation is done to confirm the diagnosis. If

Hashimoto’s Thyroiditis is suspected Anti TPO Antibodies should be estimated to confirm the diagnosis.

REGULATION OF THYROXIN SECRETION (FIGURE-8)

Thyroid hormone secretion is regulated by Hypothalamic -

Pituitary – Thyroid axis (HPT axis). Thyrotropin releasing hormone

(TRH) from hypothalamus stimulates anterior pituitary to secrete TSH, which in turn stimulates thyroid gland and as a result thyroxin is released from thyroid follicles(15). T3 & T4 are then released into circulation. T3 and T4 by the negative feedback mechanism directly control both hypothalamus and anterior pituitary.

THYROTROPIN RELEASING HORMONE (TRH):

TRH is a major positive regulator for pituitary TSH synthesis and release. TRH production starts in fetus as early as 30 days of the Intra

Uterine period. It undergoes rapid degradation in the serum. It reaches pituitary by a pathway consisting of TRH fibres that enter median eminence & release TRH into portal system. TRH also reach pituitary by direct diffusion from hypothalamus or cerebrospinal fluid via sub arachnoid process(15).

The Anterior Pituitary

Anterior lobe contains active cells that produce TSH. TSH cells are part of the lineage that is dependent on home box transcription factor pit-

12. Fetal pituitary TSH synthesis can be detected by 13 weeks but remain low till 18 weeks, then it increases dramatically in pituitary & in serum.

This is followed by increase in the serum total and free T4 levels.

FIGURE-8: SCHEMATIC OF THE REGULATION OF THE

PRODUCTION AND METABOLISM OF THYROID HORMONE

Autoregulation:

Autoregulation of the thyroid gland is done by iodide. If the intake exceeds it leads to inhibition of thyroxine synthesis. This auto regulatory phenomenon is known as the Wolff-Chaikoff effect. When iodide level falls, the production of thyroid hormone returns to normal.

Thus constancy of the plasma concentration of thyroid hormones is maintained.

THYROID STIMULATING HORMONE (TSH):

CHEMISTRY & METABOLISM OF TSH

Human TSH is a glycoprotein that contains 211 amino acid residues. It is made up of two subunits, α and β. The α subunit is encoded by a gene on chromosome 6 and the β subunit by a gene on chromosome

1. The α and β subunits become noncovalently linked in the anterior pituitary thyrotropes(16).

TSH-α is identical to the α subunit of luteinizing hormone, follicle- stimulating hormone, and human chorionic gonadotropin (hCG). The functional specificity of TSH is conferred by the β subunit. The structure of TSH varies from species to species.

The biologic half-life of human TSH is about 60 min. TSH is degraded mostly in the kidneys and to a lesser extent in the liver. Secretion is pulsatile, and mean output starts to rise at about 9:00 PM, peaks at

26 midnight, and then declines during the day. The normal secretion rate is about 110 μg/day. The average plasma level is about 2 μg/mL.

In some patients with benign or malignant tumours of placental origin, plasma hCG levels can rise so high that they produce mild hyperthyroidism.

TSH regulates thyroid gland function through TSH-R, a seven transmembrane G protein – coupled receptor (GPCR). The TSH – R is coupled to the sub unit of Stimulatory G protein (G), activates adenylyl cyclase, leading to increased production of C - AMP. TSH also stimulates phosphatidylinositol turnover by activating phospholipase C.

The functional role of TSH – R is explained by consequences of naturally occurring mutations. Recessive, loss of function mutations produce congenital hypothyroidism and thyroid hypoplasia. Dominant, gain of function mutations lead to sporadic or familial hyperthyroidism.

This is characterized by thyroid cell hyperplasia and goitre. This resembles the changes caused by TSH covalent binding or interacting with thyroid stimulating immunoglobulin (TSI) in Grave’s disease. Activating

TSH-R mutations occur as somatic events, leading to clonal selection and expansion of the affected thyroid follicular cell and independent functioning thyroid nodules. TSH is the dominant hormonal regulator of thyroid gland growth and function, many growth factors secreted in the thyroid gland that regulate the synthesis of thyroid hormone. They are

EGF, transforming growth factor (TGF), epidermal growth factor and insulin like growth factor I (IGF-1). The quantitative roles of these factors are not well studied, but they are interdependent factors in selected diseases. In Acromegaly, increased levels of growth hormone and IGF-1 are associated with goitre and predisposition to Multinodular Goitre

(MG). Some interleukins (ILs) & cytokines are released in circulation with autoimmune thyroid disease, which induce thyroid growth, whereas others lead to apoptosis.

Iodine deficiency upregulates the NIS. It increases blood supply to thyroid gland and increases iodine uptake. Excess iodide uptake in thyroid cells will lead to transient inhibition of thyroid iodide organification called

Wolff Chaikoff effect.

In individuals with normal thyroid, Iodide organification resumes and thyroid gland escapes from this inhibitory effect. In case of any autoimmune thyroid disease the suppressive action of high iodide may persist.

CAUSES OF HYPOTHYROIDISM

Primary

 Subtotal or Total Thyroidectomy

 Iatrogenic - External beam radiotherapy for Hodgkin‟s lymphoma.

 Congenital Hypothyroidism: TSHR mutation,

Dyshormonogenesis, Aplasia or ectopic thyroid gland.

 Infiltrative disorders like sarcoidosis, scleroderma, cystinosis,

amyloidosis, hemochromatosis & Reidel’s thyroiditis.

 Autoimmune hypothyroidism: atrophic thyroiditis, Hashimoto’s

thyroiditis.

 Drugs: Sunitinib, Iodine excess (including iodine – containing

contrast media & Amiodarone), Antithyroid drugs, Interferon,

Cytotoxics, Aminoglutethimide, Lithium and P-AminoSalicylic

acid, Deficiency of iodine.

 Transient withdrawal of Thyroxine treatment

 Post treatment or Subtotal Thyroidectomy for Graves’ disease

 Subacute Thyroiditis

 Silent Thyroiditis including Postpartum Thyroiditis

Secondary

Hypothalamic disease: Infiltrative disorders, tumors, trauma, idiopathic.

Hypopituitarism: Tumors, pituitary surgery / irradiation / infiltrative disorders. Isolated TSH deficiency, Genetic forms of combined pituitary hormone deficiencies Sheehan’s syndrome, trauma, Dexarotene treatment.

CLINICAL PRESENTATION OF HYPOTHYROID DISORDERS

Symptoms

Fatigue, lethargy, Dry skin, Tiredness, Weakness, Hair loss&

Constipation.

Signs

Puffiness of face, hands & feet, Diffuse alopecia, Pseudo Myotonic reflexes, Weight gain with poor appetite, Dry coarse skin, Cold extremities, Serous effusions, Difficulty in concentrating and poor memory, Dyspnoea, Peripheral edema, Bradycardia, Menorrhagia,

Hoarseness, Carpal tunnel syndrome.

Clinical Examination

Examination is normal in most of the hypothyroids. Some patients have

1. Typical hypothyroid facies suggestive of overt

hypothyroidism.

2. Skin may be cold, dry, rough & scaly.

3. Peripheral edema of feet and hand typically non pitting

in nature.

4. Nails may be brittle and thickened.

5. Madarosis (loss of hair in the lateral third of the

eyebrows).

6. Sinus bradycardia with diastolic hypertension. Blood

pressure may be normal (or) low in subclinical

hypothyroidism.

7. Local examination-The thyroid gland may be rubbery,

enlarged & firm. It is non-tender, commonly no bruit is

heard. Thyroid may be normal in size also.

8. Memory loss & slow speech.

9. A polyneuropathy like carpal tunnel syndrome with

involvement of peripheral nerves in form of the

paraesthesia.

SICK EUTHYROID SYNDROME / NON THYROIDAL ILLNESS

SYNDROME (NTIS)

Low T4 and T3 with normal (or) low TSH, Low concentration of thyroid hormones in all tissues. Found in starvation, severe systemic illness, cardiac failure, liver failure, infections, malignancy, adrenal hypofunction.

Benefit of thyroxine replacement is controversial.

Treatment – manage the underlying illness.

CLASSIFICATION OF HYPOTHYROIDISM

Primary hypothyroidism

Congenital

Agenesia, dyskinesia, ectopia

Dyshormonogenetic goitre

Iodide transport or utilization defect (NIS or pendrin mutations)

Iodotyrosine dehalogenase deficiency

Organification disorders (TPO deficiency or dysfunction)

Defects in thyroglobulin synthesis or processing

TSH receptor defects and other forms of idiopathic TSH

unresponsiveness

Thyroidal Gprotein (Gs type) abnormalities

(pseudohypoparathyroidism type 1a)

Acquired

Thyroiditis

Autoimmune

Hashimoto’s thyroiditis (goitrous and atrophic forms)

Painless and postpartum thyroiditis (transient, may result in

permanent thyroid failure)

Other thyroiditis

Subacute (transient, rarely result in permanent thyroid failure)

Riedel’s thyroiditis

Thyroid infiltration

Amyloidosis

Hemochromatosis

Sarcoidosis

Cystinosis

Scleroderma

Iodine deficiency, goitrogens in foodstuffs, pollutants

Iodine excess

Iatrogenic

131 I, Surgery

External irradiation for nonthyroidal malignancy

Drugs blocking synthesis or release of thyroxine

Antithyroid drugs (methimazole, carbimazole, propylthiouracil)

Other drugs: lithium, ethionamide, sulfonamides, iodide

Tyrosine kinase inhibitors (TKIs): sunitinib, regorafenib

Cytokines (interferon-α, interleukin-2, others)

Consumptive hypothyroidism

Type 3 deiodinase (D3) expression in large tumor

Central hypothyroidism

Congenital

TSH deficiency or structural abnormality

TRH and TRH receptor defects

Acquired

Pituitary (secondary) or hypothalamic (tertiary) disorders

Bexarotene (retinoid X receptor agonist) treatment

Dopamine excess

Severe illness

CENTRAL EFFECTS OF THYROID HORMONES ON APPETITE

CHANGES:

The HPT axis have a role in energy expenditure & regulation of food intake in humans. Thyroid disease leads to alteration of appetite & body weight, these effects are produced by peripheral action of thyroid hormones. But recent studies shows local regulation of thyroid hormones in CNS play a important role in regulation of appetite(10).

In CNS hypothalamus & brain stem are main area for appetite regulation. Hypothalamus is subdivided into many inter connecting nuclei, they are PVN, VMN, ARC. In this ARC is located near the median eminence, where BBB is not well formed so diffusion of CSF fluid occur easily. ARC nucleus to react firstly to circulating factors involve in appetite & food intake.(FIGURE-9)

Hypothyroidism associated with hypophagia & Hyperphagia seen in hyperthyroid state as a result of thyroid hormones directly act on CNS appetite circuits.

ARC contains two types of energy homeostasis regulatory neuronal populations.

1. Express the POMC which encodes alpha-melanocyte stimulus

hormone (α-MSH)

2. Orexigenic factors – NPY & AgRP

Recent study reported peripheral administration of T3 will increase

the hypothalamic NPYmRNA, suggest T3 may increase appetite

via NPY & reduce hypothalamic POMC expression. (FIGURE-10)

S.No HORMONE EFFECT ON FOOD INTAKE

1. TRH Decrease

2. TSH Decrease

3. T3 Increase

FIGURE-9: SCHEMATIC DIAGRAM OF CENTRAL APPETITE

REGULATION(10) so

FIGURE-10 : EFFECT OF FASTING ON THE HYPOTHALAMO-

PITUITARY-THYROID AXIS(10)

Central Changes in T3 Mediated by D2 & D3:

Deiodinases (thioredoxin enzymes) regulate the T3 &T4

activation and inactivation. This enzyme remove iodine from T4.

Three types of Deiodinases enzymes present in humans. D1

expressed in liver, kidney, thyroid. D2&D3 are highly expressed in

CNS(21).

In hypothalamus activity of D2 & D3 depends on nutritional

status, so tissue specific changes occur in hypothalamus depends

on T3 concentrations. This is the reason for regulation of food

intake & energy expenditure.

In brain conversion of T4 to T3 is catalysed by D2. D2 play main role in thyroid hormone mediated feedback regulation of TRH production &

TSH secretion. (FIGURE-11).

D2 enzymes highly express in Tanycytes, these cells are specialised endothelial cell present in 3rd ventricle. Tanycytes express Two types of

Thyroid specific transporters(10).

1. Monocarboxylate transporter 8 (MCT8)

2. Organic anion transporting polypeptide 1C1 (OATP1C1)

Expression of Dio2 & D2 activity is increased in hypothyroidism.

D2 activity high in ARC & median eminence, where it’s

expressed in tanycytes. This D2 activity is directly contact with

NPY/AgRP neurons of ARC, which also express UCP-2. UCP-1 is

FIGURE-11: EFFECT AND CONSEQUENCES OF FASTING ON

CENTRAL T3 LEVELS, MEDIATED BY D2 AND D3

important for associated non-shivering thermogenesis, it release energy in the form of heat.

UCP-2 is present in inner mitochondrial membrane, it is tissue specific regulated by T3.

T3 is enter into CNS by various mechanism. One of them is directly crossing via BBB. Another mechanism of T3 transport is through CSF via tanycytes in 3rd ventricle. During fasting upregulation of D2 activity in ARC leads to increased UCP-2 activity & mitochondrial proliferation in NPY/AgRP neurons.

D3 is responsible for inactivation of T4 to rT3. In CNS D3 activity is regulated by thyroid hormone concentration, low levels in hypothyroidism and high levels in hyper thyroidism.

Local regulation of thyroid hormone in CNS may regulate appetite. Balance between D2 & D3 expression in CNS may control hypothalamic thyroid hormone concentration(10).

GHRELIN

Ghrelin is an acylated peptide hormone. Ghrelin was first described in 1999 as the endogenous ligand of released growth hormone secretagogue receptor (GHSR)(11). It is a 28-amino acid peptide, in that the serine 3 residue is n-octanoylated (FIGURE-12). Ghrelin has been isolated from the secretory granules of X/A-like cells in the submucosal layer of the stomach; it is also detected in the hypothalamic arcuate nucleus and regulates energy balance(22). The X/A-like cells contain round, compact, electron-dense granules filled with ghrelin(23). Ghrelin is also synthesized in the placenta, kidney, heart, and thyroid(24). Ghrelin was named after the Proto-Indo-European root “ghre” meaning “to grow” as ghrelin exhibits potent growth hormone (GH)-releasing activity(25).The hormone name is based on its role as a growth hormone- releasing peptide [ghrelin].

Discovery of Ghrelin hormone:

Before the Ghrelin hormone was identified, its receptor (Ghrelin receptor-Growth hormone secretagogue receptor (GHSR)) was discovered by Howard et al(26) in1996 and reported later in 1999; its natural ligand was not known. Three years later (1999), its natural ligand,

Ghrelin was identified by Kojima et al from rat stomach(23).

FIGURE-25: OREXIGENIC ACTION OF GHRELIN

Ghrelin, the "hunger hormone", also known as lenomorelin (LNN), is a peptide hormone produced by ghrelinergic cells in the gastrointestinal tract. Ghrelin functions as a neuropeptide in the central nervous system(27). Besides regulating appetite, it also plays a significant role in regulating energy homeostasis. When the stomach is empty, ghrelin is secreted. When the stomach is stretched, ghrelin secretion inhibited. It acts on hypothalamic brain cells both to increase hunger, and to increase gastric acid secretion and gastrointestinal motility to prepare the body for food intake(28).

Ghrelin is the most potent circulating orexigen, and its plasma levels are elevated prior to the meals and it stimulates feeding (Wren et al 2001)(29). Ghrelin is encoded by the GHRL gene.

Orexin is a neuropeptide, which regulate the wakefulness, appetite and arousal. Orexin also known as Hypocretin. Orexin producing neurons are present in periforncial area & lateral hypothalamus in human brain; only 10000 -20000 neurons present in our brain(30).

Two types of Orexin peptides- Orexin A and Orexin B & Two types of Orexin receptors- OX1 and OX2 presents in humans. These peptides binds to G-protein coupled receptors for their action. Orexin A binds to

OX1 and OX2 receptors, but Orexin B binds only OX2 receptor.

FUNCTION OF OREXIN

1. Brown fat activation – Orexin neurons regulate brown adipose

tissue activity via sympathetic nervous system to enhance energy

expenditure.

2. To promote wakefulness(30).

3. Orexin increases craving of food. Recent studies show Orexin

producing cells are inhibited by Leptin, activated by Ghrelin and

Hypoglycemia(30).

4. Lipid metabolism- OXA increases Lipogenesis, inhibits Lipolysis,

& stimulates secretion of Adiponectin.

5. Mood Elevator- Increased level of Orexin A produce happiness in

humans, decrease level produce sadness.

The ghrelin gene contains preproghrelin - coding exons (exons1-4).

During processing, a 23 amino acid secretion- signal peptide is cleaved from the 117 amino acid preprohormone (N-terminus), resulting in 94 amino acid proghrelin(FIGURE-13). This peptide is further cleaved and gives rise to 28 amino acid ghrelin peptide and a 66 amino acid C-terminal propeptide. After cleavage from proghrelin, the ghrelin peptide can be post translationally octanoylated at its third residue serine(FIGURE-14), by enzyme ghrelin O-acyl transferase (GOAT). This modified form is ghrelin. A non octanoylated form of ghrelin (acylated ghrelin, des-acyl ghrelin, des-ghrelin) circulates at a higher level in the blood. It does not

FIGURE-14: SCHEMATIC ON THE POST-TRANSLATIONAL

PROCESSING AND ACYLATION OF GHRELIN.

bind with its receptor (GHSR 1a), and considered as biologically inactive

(FIGURE-15)(11).

GOAT:

GOAT, enzyme responsible for octanoylation of ghrelin is a member from the membrane- bound O-acyl transferase (MBOAT) family of enzymes, encoded by the MBOAT4 gene(27). It is a hydrophobic, membrane-bound enzyme with 8 domains, localised to the endoplasmic reticulum. It is coexpressed with ghrelin in the cells of stomach and in the pancreatic islets. This enzyme is an attractive and specific target for the modification of octanoylation; also it could provide a target for development of drugs to prevent weight gain, obesity, insulin resistance. It also plays an important role in the regulation of metabolism, absorption of lipid in the gut and energy intake. This c-ghrelin (has 66 amino acids) cleaved to give obestatin (FIGURE-16, 17)(31).

Relation between ghrelin and obestatin:

Obestatin is C- terminally amidated and also it has opposite effects to ghrelin hormone on food intake. It has multiple roles in adipogenesis, inducing sleep, pancreatic homeostasis and cancer(24).

When octanoic acid (caprylic acid) is linked to serine post translationally at the third position by the enzyme ghrelin

Oacyltransferase (GOAT), ghrelin becomes active. This enzyme is

FIGURE-15: FORMATION OF GHRELIN FROM PREPRO-GHRELIN.

FIGURE-16: FROM GHRELIN GENE TO GHRELIN PEPTIDE.

FIGURE-17: FORMATION OF GHRELIN & OBESTATIN

located on the cell membrane of ghrelin cells in the pancreas and stomach(27).

Ghrelin cells are also found in oxyntic glands (20%), pyloric glands, small intestine. They also produce another food intake limiting hormone Nesfatin-1. They are also present in lungs, pancreatic islets, gonads, adrenal cortex, placenta and kidney. They have gastrin receptors.

Other names of ghrelin cells are A-like cell, X cell, X/A- like cell

(rat), Epsilon cell (pancreas), P/D sub 1cell (humans).

Growth Hormone Secretagogue Receptor: (FIGURE-18,19)

GHSR 1a is a classical, 7- transmembrane domain, G protein coupled receptor(24).

The ghrelin hormone secretogogue receptor (GHSR1a) is involved in many functions of ghrelin including

1. Stimulation of growth hormone release

2. Regulation of motility and secretion of gastro intestinal

system

3. Increase in hunger

4. Regulation of immune function

5. Role in sleep and memory.

These receptors are present in high density in the hypothalamus, pituitary, throughout the gastrointestinal tract and on the vagus nerve.

FIGURE-18: TYPES OF GHS RECEPTOR

FIGURE-19: GHSR SIGNALING

Another form is GHSR 1b isoform which is inactive because it does not bind ghrelin and ghrelin does not activate signalling via this receptor.

It is over expressed in lung cancer. Although its function is not clear, it acts as a negative regulator of GHSR 1a by reducing its cell surface expression and constitutive signalling. Its over expression in many cancers indicates its clinical significance(32).

The non-octanoylated form is DESACYL GHRELIN; it does not activate the GHSR receptor, but it is involved in other actions such as cardiac function, appetite stimulation, antagonising ghrelin, inhibition of hepatic glucose output. Synthesis of Ghrelin, its posttranslational modification, action& degradation are depicted in FIGURE-20.

GHRELIN AND LEPTIN:

Ghrelin hormone discovered 7 years after Leptin hormone. Leptin is also a hormone involving in appetite regulation(33). It has opposite effect in appetite regulation in relation to ghrelin (FIGURE-21).

FIGURE-20: COMPONENTS OF GHRELIN SYSTEM

FIGURE-21: GHRELIN & LEPTIN LEVELS IN RELATION TO MEALS

MECHANISM OF ACTION

Ghrelin regulates the process of energy homeostasis. It influences both energy input, by adjusting hunger signals, and energy output, by adjusting the proportion of energy leading to ATP production, fat storage, glycogen storage, and short-term heat loss(34). The end result of these processes maintains body weight (FIGURE-22,23). Ghrelin have a multiple site of action in our body, showed in FIGURE-24.

The receptor-binding pharmacophore of ghrelin shows to consist of the first seven amino acids at the N-terminus, plus the fatty-acid moiety.

Ghrelin peptide undergoes a post-translational modification at serine-3 residue is covalently linked to a medium-chain fatty acid, especially octanoic acid, through an ester bond. This type of acylation is most unique to ghrelin and is required for the peptide to bind to its classical receptor, the GHS-R1a. Most biological actions of ghrelin, especially those involving endocrine and anabolic effects, require acylated ghrelin (active form)(29).

The mechanisms by which nutrients suppress ghrelin levels are studied. We find that ingested calories suppress ghrelin with an efficacy order in rats and humans of carbohydrates > proteins > lipids. The relatively weak suppression of this orexigenic hormone by lipids could represent one mechanism promoting highfat diet-induced weight gain(35).

FIGURE-22: GHRELIN& LEPTIN ACTION IN HYPOTHALAMUS

FIGURE-23: HYPOTHALAMIC PATHWAY INVOLVED IN FOOD INGESTION

FIGURE-24: SITE OF ACTION

Gastric-CNS connection is an essential part of energy homeostasis, and several communication pathways are studied, including the gastric intracellular mTOR/S6K1 pathway mediating the connection among ghrelin, nesfatin and endocannabinoid gastric systems, both afferent and efferent vagal pathways. Ghrelin achieves its orexigenic (FIGURE-25) action through hypothalamic AMPK pathway. The important effect of ghrelin is its ability to stimulate food intake via activation of hypothalamic neuro circuits(13).

Ghrelin and synthetic ghrelin mimetics (growth hormone secretagogues) increase body weight and fat mass by triggering receptors in the arcuate nucleus that include the orexigenic neuropeptide Y (NPY) and agouti-related protein (AgRP) neurons (FIGURE-26,27). Nakazato

M., Murakami N.,et al observed that intracerebroventricular (icv) injection of ghrelin increased food intake in rats, but unable to increase ghrelin levels when NPY and AgRP neurons were blocked(13).

Ghrelin-responsiveness of these neurons is both leptin- and insulin- sensitive. Ghrelin reduces the mechanosensitivity of gastric vagal afferents, so they are less sensitive to gastric distension.

FIGURE-25: OREXIGENIC ACTION OF GHRELIN

FIGURE-26: MAIN HYPOTHALAMIC PATHWAYS INVOLVED IN

GHRELIN INDUCED FOOD INTAKE

NPY-Neuro peptide Y

AGRP-Agouti related peptide

POMC-Pro-opiomelanocortin

CART-cocaine- and amphetamine- regulated transcript

CRF-corticotrophin-releasing hormone

FIGURE-27: GHRELIN ACTION NPY/AgRP m RNA LEVEL

FUNCTION OF GHRELIN

The main function of Ghrelin is to know the current energy state of the body (FIGURE-28)(11). Ghrelin helps balance the energy in our body by the following ways:

1. Promotion of secretion of growth hormone by activation of growth

hormone Secretagoue receptor (FIGURE-29).

2. Decreasing utilization of fat in adipose tissue leading to adiposity

through stimulate expression of fat storage related proteins in

adipocytes(36).

3. Stimulation of gastric emptying(28).

4. Promotes the proliferation of intestinal cells and inhibits apoptosis

during oxidative stress.

5. Enhances anti-inflammatory action. It is useful in treatment of

mucosal injury of stomach due to their regenerative function.

6. In pancreas, inhibits glucose induced insulin release(37).

7. Ghrelin levels are low in obesity except in Prader-Willi syndrome

associated obesity. Lack of sleep enhances ghrelin and decreases

leptin level leading to obesity due to excessive production of

hunger(38).

8. Cognitive adaptation to changing environments(13).

9. The process of learning.

FIGURE-28: MAIN PHYSIOLOGICAL FUNCTIONS OF GHRELIN

FIGURE-29: SECRETION OF GROWTH HORMONE BY GHRELIN

10. Function on immunity- promotes proliferation of Lymphocytes in

primary lymphoid organs (bone marrow, thymus)(39).

11. Decreasing fertility via inhibiting the action of gonadotropin

releasing hormone.

12. Ghrelin hormone produced by the fetal lung and promotes lung

growth(40). One study reports a correlation between ghrelin levels

and birth weight by using cord blood levels(41).

13. Sleep - Inverse relationship between the plasma level of ghrelin and

hours of sleep(30).

14. Serum ghrelin levels are increased in stressful conditions even

when adrenal hormone level is normal.

15. GOAT is expressed in chondrocytes, and its levels are influenced

by the degree of chondrocyte differentiation. So acylated ghrelin

derived from chondrocytes plays an important role in chondrocyte

cell biology(42).

Degradation of ghrelin

The half-life of acyl-ghrelin in Rat- 30 mins & in Humans 240 mins. This variability of ghrelin degradation is due to the enzymes that inactivate ghrelin: butyryl cholinesterase in humans and

Carboxylesterases in rats(43).

THERAPEUTIC APPLICATIONS OF GHRELIN:

1. Growth hormone deficiency.

2. Eating disorders- Anorexia nervosa, Bulimia nervosa, Prader-willi

syndrome.

3. Gastro intestinal disease- anti inflammatory.

4. Cardio vascular disease: Heart failure, dilated cardiomyopathy.

5. Osteoporosis.

6. Aging.

7. Chronic wasting syndrome - Cachexia, AIDS, post-operative

patients.

MATERIALS & METHODS

MATERIALS AND METHODS

After obtaining approval from the Institutional Ethics Committee of Coimbatore Medical College and Hospital, the study was conducted in the Department of Biochemistry Central Lab, CMCH.

STUDY POPULATION

CASES

The study sample comprised 50 unrelated south Indian

Hypothyroid patients of mean age .

Inclusion Criteria: Newly Diagnosed Hypothyroid patients, Age between

18 to 55 yrs for cases & controls.

Exclusion Criteria: Who are already consuming hypothyroid drugs,

Patients with chronic illness like DM, HT, Heart disease, Patients with auto immune gastric diseases, Treatment with drug affecting gastric secretion, BMI <20kg/m2 or >30kg/m2, Euthyroid healthy controls having gastric disease.

CONTROL SUBJECTS

Controls were recruited from Master health Check-up. Apparently healthy individuals matched for age and sex were included. All of them were free from symptoms and signs of Hypothyroid dysfunction.

Sample collection and Processing

3 mL of blood was collected into a plain tube by Venipuncture after overnightfasting. The blood was allowed to clot and Serum was separated by centrifugation. 1mL of serum was stored in one micro centrifuge tubes

at -20֠ C for analysis of Ghrelin level. The levels of Glucose,total cholesterol, triglycerides and high density lipoprotein, Liver function test were measured in XL 640 fully automated analyzer by colorimetric methods using commercially available kits within 6 hrs of blood collection.

ESTIMATION OF SERUM GHRELIN LEVEL

Kit Used

Human GHRL (Ghrelin) ELISA From Elabscience

Methodology

Competitive ELISA Method

Principle

The ELISA kit uses the Competitive ELISA principle. The microplate of the kit has precoated with Human GHRL. Human GHRL in the sample or standard competes with fixed amount of Human GHRL on the solid phase supporter for sites on the Biotinylated Detection

Antibody specific to human GHRL. Excess conjugate & unbound sample or standard are washed from plate, Avidin conjugated to Horseradish

Peroxidase (HRP) are added to well & incubated, TMB substrate solution

50 is added to each well. The enzyme –substrate reaction is terminated by adding stop solution & colour changes is measured spectrophotometrically at wavelength of 450nm.

CONTENTS OF THE KIT (FIGURE-30)

1. Wash buffer

2. Lyophilized anti-Ghrelin polyclonal antibody

3. Standard

4. TMB substrate

5. Stop solution

6. Sample diluent

The concentration of human GHRL in the samples is determined by compare with OD of the samples to the standard curve.

Reagent preparation

1. Bring all reagents to room temperature (18~25℃) before use. Preheat the Microplate reader for 15 min before OD measurement.

2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with deionized or distilled water to prepare 750 mL Wash Buffer.

3. Standard working solution: Centrifuge the standard for 1 min. Add

1.0 mL of Reference Standard &Sample Diluent, let it stand for 10 min and turn it upside down for several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a stock solution of

FIGURE-30: CONTENTS OF THE KIT

FIGURE-31: DILUTION METHOD 10 ng/mL. Then make serial dilutions as needed. The recommended dilution gradient is as follows: 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0 ng/mL.

Dilution method: Take 7 EP tubes, add 500 μL of Reference Standard &

Sample Diluent to each tube(FIGURE-31).

Pipette 500 μLof the 10 ng/mL stock solution to the first tube and mix up to produce a 5 ng/mL stock solution.

Pipette 500 μL of the solution from former tube to the latter one in order according to this step. The illustration below is for reference.

Note: the last tube is regarded as blank. Don’t pipette solution to it from the former tube.

4. Biotinylated Detection Ab working solution: Calculate the required amount before experiment (50 μL/well). Inactual preparation, more account of 100~200 μL should be prepared. Centrifuge the stock tube before use, dilute the 100× Concentrated Biotinylated Detection Antibody to 1×working solution with Biotinylated Detection Antibody Diluent.

5. Concentrated HRP Conjugate working solution: Calculate the required amount before experiment (100 μL/well). In actual preparation, more account of 100~200 μL should be prepared. Dilute the 100×

Concentrated HRP Conjugate to 1× working solution with Concentrated

HRP Conjugate Diluent.

Assay procedure

1. Add 50 μL standard or sample to each well. Immediately add 50

μL Biotinylated Detection Ab to each well. Incubate for 45 min at 37℃.

2. Aspirate and wash 3 times.

3. Add 100 μL HRP Conjugate to each well. Incubate for 30 min at 37℃.

4. Aspirate and wash 5 times.

5. Add 90 μL Substrate Reagent. Incubate for 15 min at 37℃.

6. Add 50 μL Stop Solution. Read at 450 nm immediately.

7. Calculation of results.

Typical standard curve and data below is provided for reference

ESTIMATION OF THYROID FUNCTION TESTS

In Thyroid Function Tests following tests are done,

 T3 -Triiodothyronine

 T4 - Thyroxine

 TSH – Thyroid Stimulating Hormone



1.T3 ESTIMATION

Kit Used

MAGLUMI T3 CLIA Kit from Shenzhen

Methodology

Chemiluminiscence Immuno Assay CLIA

Principle

Competitive chemiluminescenceimmunoassay.

Use ABEI to label anti-T3 monoclonal antibodies ,use purified T3 antigens to coat magnetic microbeads. Thesample ,ABEI Label, buffer are mixed thoroughly & incubated at 37,the solution of the magnetic microbeads coated with T3 antigens is added & incubated at 37*. The sample & the magnetic microbeads coated with T3 antigens com pete with

ABEI label, form immune complex. After precipitation in magnetic field discard the supernatant, then perform wash cycle. The starter 1+2 is added to initiate the chemiluminescent reaction. The light signal is measured by photomultiplier within 3 seconds as RLU, which is inversely proportional to concentration of T3.

KIT COMPONENTS

1. MAGNETIC MICROBEADS- coated with purified T3 antigen,

BSA, 0.09% NaN3.

2. CALIBRATOR LOW& HIGH – phosphate buffer, BSA & T3

antigen, 0.09%NaN3.

3. BUFFER – 0.1% ANS phosphate buffer, BSA, 0.09%

4. ABEI LABEL – anti T3 monoclonal antibody labelled with ABEI,

BSA, 0.09% NaN3.

NORMAL RANGE – 0.69-2.15 ng/ml.

3. T4 ESTIMATION

Kit Used

MAGLUMI T4 CLIA Kit from Shenzhen

Methodology

Chemiluminiscence Immuno Assay CLIA

Principle

Competitive chemiluminescence immunoassay.

Use ABEI to label anti-T4 monoclonal antibodies , use purified T4 antigens to coat magnetic microbeads. The sample , displacing solution,

ABEI Label, buffer & the magnetic microbeads are mixed thoroughly & incubated at 37*. The sample & the magnetic microbeads coated with T4 antigens compete with ABEI label, form immune complex. After precipitation in magnetic field discard the supernatant, then perform wash cycle. The starter 1+2 is added to initiate the chemiluminescent reaction.

The light signal is measured by photomultiplier within 3 seconds as RLU, which is inversely proportional to concentration of T4.

KIT COMPONENTS

1. MAGNETIC MICROBEADS- coated with purified T4 antigen,

BSA, 0.09% NaN3.

2. CALIBRATOR LOW& HIGH – phosphate buffer, BSA & T4

antigen, 0.09%NaN3.

3. Displacing solution - 6 ml

4. BUFFER – 0.1% ANS phosphate buffer, BSA, 0.09% NaN3.

5. ABEI LABEL – anti T4 monoclonal antibody labelled with ABEI,

BSA, 0.09% NaN3.

NORMAL RANGE – 5.2-12.7 ng/ml.

3. TSH ESTIMATION

Kit Used

MAGLUMI TSH CLIA Kit from Shenzhen

Methodology

ChemiluminiscenceImmunoAssay CLIA

Principle

Sandwich chemiluminescence immunoassay.

Use ABEI label an anti-TSH monoclonal antibody, FITC to label another monoclonal antibody. The sample ABE label , FITC Label & solution of magnetic beads are mixed thoroughly & incubate at 37*, forming sandwich of immune-complexes. After precipitation in a magnatic field, decant supernatant then perform wash cycle. After that starter 1+2 is added initiate a chemiluminescent reaction. The light signal is measured by a photomultiplier within 3 seconds as RLU, proportional to the concentration of TSH.

KIT COMPONENTS

1. MAGNETIC MICROBEADS- TRIS buffer, 0.09% NaN3, coated

with sheep anti-FITC polyclonal antibody.

2. CALIBRATOR LOW& HIGH – phosphate buffer, BSA & TSH

antigen, 0.09%NaN3.

3. FITC Label- anti-TSH monoclonal antibody labelled with FITC,

BSA, 0.09% NaN3.

4. ABEI LABEL – anti TSH monoclonal antibody labelled with

ABEI, BSA, 0.09% NaN3.

NORMAL RANGE – 0.3-4.5uIU/ml.

The other Biochemical Parameters are done by XL 640 Fully automated machine using commercially available kits.

1. Glucose – Glucose Oxidase Peroxidase Method (GOD-POD),

Normal Range- 75-100mg/dl.

2. Urea - Urease-GLDH, kinetic method, Normal Value- 15-43mg/dl

3. Creatinine - Jaffe´s kinetic – Normal value – 0.7-1.2 mg/dl

4. Cholesterol - CHOD-PAP – Normal Range- <200 mg/dl

5. Triglycerides - GPO-Trinder method – 40-165 mg/dl

6. HDL – Direct method - Male- 35-79 mg/dl, Female – 42-88mg/dl

7. Total Bilirubin - Diazo (Walter & Gerarde) method – 0.1-2 mg/dl

8. Direct Bilirubin - Diazo (Walter & Gerarde) – 0.2-0.5 mg/dl

9. Asparatate Transaminases- IFCC without PDP- upto 31IU/L

10. Alanine Transaminases – IFCC without PDP- upto 45 IU/L

11. Calcium – arsenaso III – 8.6-10.2 mg/dl

12. Phosphorous – Ammonium molybdate UV- 2.5-4.5 mg/dl

13. Total protein – Biuret method – 6-8.5 g/dl

14. Albumin – BCG method -3.5-5.2 g/dl

15. LDH – DGKC method – 220-450 U/L

STATISTICAL ANALYSIS

Data entry was made in the Microsoft Excel software in codes and analysis was done with SPSS-20 computer package.

Categorical variables are expressed as percentages whereas continuous variables are expressed as mean ± standard deviation. Mean &

Standard deviation were estimated from the sample of both study groups.

Association between categorical variable was found by chi-square test and relationship between continuous variable was assessed by

Student’s t-test.

P value <0.05 was considered as statistically significant.

RESULTS

TABLE 1 - DISTRIBUTION OF STUDY POPULATION

Group Frequency Percentage

Cases 50 50

Control 50 50

Total 100 100

Distribution of the study participants

Cases Control

50% 50%

TABLE 2 - GENDER DISTRIBUTION OF STUDY POPULATION

Gender Cases Control

wise P distribution Frequency Percentage Frequency Percentage Value of the study participants Male 3 6.0 3 6.0

Female 47 94.0 47 94.0 1.0

Total 50 100.0 50 100.0

Gender-wise distribution of the study participants

50 40 30 20

Frequency 10 0 Cases Control Female 47 47 Male 3 3

TABLE 3 - BMI OF STUDY POPULATION

Std. BMI Minimum Maximum Mean P value Deviation

Cases 23.0 29.0 26.14 1.72 <0.001 Control 22.0 26.0 24.18 1.06

Mean BMI

26.5 26 25.5 25 24.5

24 Mean BMI level BMI Mean 23.5 23 Cases Control Mean BMI 26.14 24.18

TABLE 4 – COMPARISION OF SERUM GHRELIN LEVEL IN

CASES & CONTROLS

Serum Std. Minimum Maximum Mean P value Ghrelin Deviation

Cases 1.38 9.86 4.32 2.15 <0.001 Control 8.98 11.03 10.01 0.53

Mean serum Ghrelin Level

2 Mean Serum Ghrelin level level Ghrelin Serum Mean

0 Cases Control Series1 4.32 10.01

TABLE 5 - COMPARISION OF THYROID PROFILE IN CASES &

CONTROLS

Thyroid Std. Minimum Maximum Mean Profile Deviation

T3 0.00 2.00 1.15 0.49

Cases T4 0.00 38.00 6.29 5.03

TSH 13.00 100.00 49.73 30.01

T3 1.08 3.15 1.63 0.36

Control T4 5.30 13.30 8.32 1.83

TSH 1.06 4.69 2.59 1.01

Comparison of Thyroid Profile

50 45 40 35 30 25 20 15 10 5 Mean Thyroid Profile values Profile ThyroidMean 0 T3 T4 TSH Cases 1.15 6.29 49.73 Control 1.63 8.32 2.59

ASSOCIATION BETWEEN THYROID PROFILE AND GROUPS

Thyroid Group Mean SD P value Profile

Cases 1.15 .49 T3 <0.001 Control 1.63 .36

Cases 6.29 5.03 T4 0.009 Control 8.32 1.83

Cases 49.73 30.01 TSH <0.001 Control 2.59 1.01

TABLE 6 – GLUCOSE LEVEL IN CASES & CONTROLS

Std. GLUCOSE Minimum Maximum Mean P value Deviation

Cases 69.3 141.0 103.01 16.24 0.835 Control 88.0 115.0 102.48 6.56

Mean Glucose level

103.1 103 102.9 102.8 102.7 102.6 102.5

Mean Glucose Level GlucoseMean 102.4 102.3 102.2 Cases Control Mean Glucose 103.01 102.48

TABLE 7 - COMPARISION OF RENAL FUNCTION TESTS IN

CASES & CONTROLS

Std. Renal Function Test Minimum Maximum Mean Deviation

UREA 9.4 40.0 20.65 6.41 Cases CREATININE .70 1.81 .98 .21

UREA 10.5 15.0 13.28 1.13 Control CREATININE .56 .90 .75 .11

Renal

Function Group Mean SD P value

Test

Cases 20.65 6.41 Urea <0.001 Control 13.28 1.13

Cases .98 .21 Creatinine <0.001 Control .75 .11

Renal Function Test

10 Mean Level Mean 5

0 Cases Control Urea 20.65 0.98 Creatinine 13.28 0.75

TABLE 8 - COMPARISION OF LIPID PROFILE IN CASES &

CONTROLS

Std. Lipid Profile Minimum Maximum Mean Deviation

CHO 94.0 345.0 205.30 53.82

Cases TGL 70.0 441.0 195.41 85.74

HDL 35.0 96.6 53.98 11.39

CHO 95.0 165.0 123.53 15.52

Control TGL 123.0 180.0 149.64 12.70

HDL 42.0 55.0 47.40 3.31

Lipid Profile Group Mean SD P value

Cases 205.30 53.82 Cholesterol <0.001 Control 123.53 15.52

Cases 195.41 85.74 Triglycerides <0.001 Control 149.64 12.70

Cases 53.98 11.39 HDL <0.001 Control 47.40 3.31

Comparison of Lipid Profile Cases vs Control

250

200

150 ,l 100

0 Cholestrol Triglycerides HDL Cases 205.3 195.41 53.98 Control 123.53 149.64 47.4

TABLE 9 – COMPARISION OF LDH IN CASES & CONTROLS

Std. LDH Minimum Maximum Mean P value Deviation

Cases 236 986 515.52 153.85 0.002 Control 378 543 445.64 35.67

Mean LDH

520 500 480 460

Mean LDH Mean 440 420 400 Cases Control Mean LDH 515.52 445.64

TABLE 10 - COMPARISION OF LIVER FUNCTION TESTS IN

CASES & CONTROLS

Liver Function Group Mean SD P value Test Total Cases .51 .26 0.91 Bilirubin Control .49 .22 Direct Cases .21 .12 0.24 Bilirubin Control .19 .09 Cases 23.86 9.10 AST 0.45 Control 22.70 5.99 Cases 24.61 10.93 ALT 0.002 Control 31.04 9.49 Cases 92.18 30.82 ALP <0.001 Control 66.72 15.91 Cases 8.33 .99 Protein <0.001 Control 7.33 .56 Cases 4.42 .44 Albumin 0.090 Control 4.25 .52 Cases 3.92 .86 Globulin <0.001 Control 3.12 .44

Liver Function Test Cases vs Control

Globulin

Albumin

Protein

ALP

ALT

AST

Direct Bilirubin

Total Bilirubin

0 20 40 60 80 100

Total Direct AST ALT ALP Protein Albumin Globulin Bilirubin Bilirubin Control 0.49 0.19 22.7 31.04 66.72 7.33 4.25 3.12 Cases 0.51 0.21 23.86 24.61 92.18 8.33 4.42 3.92

TABLE 11 – COMPARISION OF CALCIUM & PHOSPHOROUS

LEVEL

Serum Level Group Mean SD P value

Cases 8.65 1.62 Calcium 0.235 Control 8.33 .99

Cases 4.13 .98 Phosphorus <0.001 Control 3.07 .72

Mean Calcium and Phosphorus level

9 8 7 6 5 4

Mean Level Mean 3 2 1 0 Calcium Phosphorus Cases 8.65 4.13 Control 8.33 3.07

TABLE 12 – COMPARISION OF BMI CATEGORY WITH

GHRELIN LEVELS

Cases Control BMI P value Category Number Percentage Number Percentage

Normal (BMI – 18.5 11 22.0 32 64.0 to 24.9)

Overweight <0.001 (BMI – 25 to 39 78.0 18 36.0 29.9)

Total 50 100.0 50 100.0

Comparison of BMI Category Cases vs Control

40 35 30 25 20 15 Frequency 10 5 0 Cases Control Normal BMI 11 32 Overweight 39 18

TABLE 12 A:

Comparison Std. P of Sr.Ghrelin Group N Min Max Mean Deviation values with BMI

Normal Cases 11 2.08 9.86 5.93 2.73 (BMI – 18.5 <0.001 Control 32 8.99 11.03 10.08 .57 to 24.9)

Overweight Cases 39 1.38 8.77 3.87 1.75 (BMI – 25 to <0.001 Control 18 8.98 10.81 9.89 .46 29.9)

RESULTS

The Sex, Body Mass Index, serum ghrelin levels, of cases & controls are listed in Table 1, 2, 3.

The mean values of the studied parameters of the patients were compared with that of the controls using student’s independent t-test. P value <0.05 was taken as significant.

Mean BMI for cases with hypothyroid (26.14 +/- 1.72) is significantly higher than the mean BMI for controls (24.18 +/- 1.06). ( p

< 0.001) Table 3.

Mean Serum Ghrelin level for cases (4.32 +/- 2.15) is significantly lower than Mean value of controls (10.01 +/- 0.53), (p < 0.001) Table 4.

Mean T3, T4 values of cases is significantly higher than compared to controls Table 5.

Mean TSH value of cases (49.73+/-30.02) also significantly higher than controls (2.59+/-1.01), p < 0.001 Table 5.

Mean Glucose level increased in cases compared to controls, but not statistically significant Table 6.

Renal Function Test are statistically significant in cases compared to controls Table 7.

Lipid profile test also statistical significant in cases compared to controls Table 8.

In liver function tests total proteins, ALP, ALT, and LDH are significant in cases, all other parameters are not significant Table 9 & 10.

Mean calcium & phosphorous level, phosphorous level is significantly higher in cases. Calcium level not significant Table 11.

In BMI, further subdivided into Normal BMI, Overweight BMI.

This subdivision is compared with Serum Ghrelin levels are listed in

Table 12A.

Cases have a normal BMI shows low ghrelin level(5.93+/-2.73) & over weight BMI shows further low ghrelin levels ( 3.87 +/- 1.75) compared to controls. The relation between serum ghrelin with Normal BMI & overweight BMI is significant p<0.001.

DISCUSSION

Decreased production of thyroid hormones will lead to

Hypothyroidism. Hypothyroidism associated with changes in the body weight, appetite & energy expenditure(1).

Ghrelin is a secretogogue; it is also associated with feeding regulation, body weight, energy metabolism(44). We assessed the association between hypothyroidism and Serum Ghrelin level & BMI. We expected low level of ghrelin in hypothyroid state as in other positive energy balance states like obesity(31).

In this study, Serum Ghrelin levels are decreased in Hypothyroid patients. We observed in this positive correlation between Serum Ghrelin level with thyroid profile especially T3 & TSH values(45).

Giemenez-palop et al(46), shows no changes in circulating ghrelin levels to hypothyroid. But our study shows difference in Serum Ghrelin levels in hypothyroid patients compared to control group, That means decreased level of ghrelin levels in cases (4.32+/- 2.15) which is significant correlation with thyroid profile( p< 0.001).

Another study showed that hypothyroidism results in increase in

Ghrelin levels in rat, so they concluded hypothyroidism produce a state of resistance to orexigenic property of Ghrelin, but did not show relation between thyroid hormone levels with Ghrelin level. Also it was not clear there is increased level of Ghrelin in hypothyroid rat(47).

In study of kamegai et al, increased level of pituitary ghrelin was identified in hypothyroid rats(48). Hyperthyroidism is associated with decreased level of ghrelin reported in another study(49). Caminos et al, showed decreased level of ghrelin in hyperthyroid rats(50).

The reason for above results of Ghrelin levels in thyroid disease due to,

1. Duration of the thyroid illness.

2. Different forms of Ghrelin level measured (acylated &

desacylated).

The role of insulin secretion & ghrelin levels are inversely related

in human studies. Reduced ghrelin levels are reported in insulin

resistance conditions like obesity & Type 2 DM. Haqq et al, report that

serum ghrelin levels are inversely related with BMI, Age, insulin

concentrations(51). Recent studies showed that ghrelin is regulated by

glucose. In Schaller et al reported serum ghrelin levels are not regulate

by glucose as studied on healthy subjects(52).

In our study, ghrelin level are not associated with glucose concentration.

Hypothyroidism is a known cause of lipid abnormalities in patients.

Ghrelin has to increase liver TG content & induce tissue specific alteration in mitochondrial gene expression. Ghrelin also binds to a species of HDL cholesterol, this binding is important for relation of ghrelin to lipid transport & metabolism(34). In our study, Total cholesterol &

Triglycerides levels increased in hypothyroid patients compared to controls.

In this study, serum phosphorus level is increased in cases compared to controls and negatively correlate with serum ghrelin levels.

In comparison of BMI with serum ghrelin, cases of overweight

BMI have decreased ghrelin level compared to Cases of normal BMI. In

Euthyroid overweight BMI have decreased ghrelin levels are seen compared to normal BMI(51).

In Hashimoto’s thyroiditis(53) patients autoantibodies are produced against Thyroid Peroxidase & Thyroglobulin, both of this TPO

Ab is more important. The relationship between serum ghrelin & thyroid antibodies was not analyse in previous studies.

Altinova et al, studied thyroid antibodies compared with ghrelin levels, this study showed significant decreased ghrelin level seen in patients with high titre of TPO Ab compared to low level of TPO Ab titre(49).

Ghrelin was found mainly in stomach, but recent studies shows ghrelin cells present wide variety of tissues including thyroid cell. In thyroid tissues shows mRNA expression of both GHS-R Type 1a and

GHS-R Type 1b.

Recent studies shows, GHS-R is important for Ghrelin induced feeding in gene knockout mice was confirmed(43).

Genetic experiments also confirmed that the combination of

NPY/AgRP in hypothalamus is important for Ghrelin induced feeding behaviour(35). Ghrelin stimulates NPY/AgRP neurons in vivo, by following mechanisms(13).

1. Ghrelin is a hormone, major part its secretion is stomach into

the blood stream, through BBB Ghrelin enter into

NPY/AgRP neurons in hypothalamus.

2. Ghrelin is neurocrinepeptide, it increase food intake through

Afferent Vagal Nerve stimulation in foregut, this signals

indirectly goes to hypothalamus.

3. Ghrelin acts as a neuropeptide, release from hypothalamic

neurons. These neurons synapse with NPY/AgRP neuron

cells which are participated in energy homeostasis.

Raghay et al, study showed that serum ghrelin have a paracrine action in stimulation of thyroid follicular cell function(54).

Some autoimmune diseases shows ghrelin level alteration, probably due to B lymphocytes secreting immunoglobulins express

GHS-R type 1b and increase ghrelin mRNA expression(39).

CONCLUSION

CONCLUSIONS

The following conclusions are obtained from our study.

1. Serum ghrelin levels are decreased in positive energy balance

conditions like obesity, hypothyroid etc.

2. Thyroid hormones and antibodies are alter the circulating levels

of ghrelin in hypothyroid patients.

3. There is significant decrease in serum ghrelin levels in

overweight BMI hypothyroid patients, but there is no relation

between ghrelin level with blood glucose levels.

LIMITATIONS OF STUDY

LIMITATIONS OF THE STUDY

1. Small sample size.

2. Hypothyroidism is not categorised as per the causes.

3. In this study total Serum Ghrelin levels are assessed in hypothyroid

patients, To be more precise, it would be better if acyl ghrelin is

quantified as it is the active form of the hormone.

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ANNEXURES

PROFORMA

NAME: OP NO:

AGE/SEX:

ADDRESS: OCCUPATION:

DATE:

PRESENT HISORY: 1) The duration of illness

2) Any prior similar episodes or other diseases.

PAST HISTORY: History of any gastric diseases.

History of liver/kidney disease.

PERSONAL HOISORY:

 H/O Smoking. Alcohol intake.

 H/O DM, HT, CAD, bone diseases.

 H/O inflammatory bowel disease.

 H/O cancer.

 H/O drug intake for past 4 weeks.

FAMILY HISTORY:

 H/O Thyroid disorders

 H/O any autoimmune diseases.

PATIENT CONSENT FORM

STUDY TITLE: SERUM GHRELIN LEVELS IN HYPOTHYROID PATIENTS

Department of Biochemistry, CMCH,COIMBATORE.

Participant Name: Age/sex:

I.P. No:

I confirm that i have understood the purpose of above study. I have the opportunity to ask the question and all my questions and doubts have been answered to my satisfaction.

I understand that my participation in the study is voluntary and that I am free to withdraw at any time without giving any reason.

I understand that investigator, regulatory authorities and the ethics committee will not need my permission to look at my health records both in respect to the current study and any further research that may be conducted in relation to it, even if i withdraw from the study. I understand that my identity will not be revealed in any information released to third parties or published, unless as required under the law. I agree not to restrict the use of any data or results that arise from the study.

I here by consent to participate in this study status.

Time: signature/thumb impression of patient

Date: Patient s name: ......

Place:

Signature of the investigator ......

Name of the investigator ......

ஒப்ꯁதல் ப羿வம்

ந ோயோளியின் பபயர்:

போலினம் : வய鏁:

பபற்ந ோர் பபயர் :

믁கவரி :

அர毁 நகோவவ ம쏁த்鏁வக் கல்쯂ரியில்உயி쏍வேதியிய쯍 鏁வ யில் பட்ட நமற்ப羿ப்ꯁ பயி쯁ம் மோணவி ம쏁. ப ொ.毁மதி அவர்கள் நமற்பகோள்쿁ம் ஹைப்வ ொஹதரொ뿍翁 வ ொயொளிகளின் ரத்தத்தி쯍 ஜிவரலின் அளஹே, பரிநெோதவன பற் ிய ஆய்வில் பெய்믁வ மற்쟁ம் அவனத்鏁 விளக்கங்கவள뿁ம் நகட்翁க் பகோண்翁சந்வதக柍கஹள என鏁 பதரிퟁப翁த்திக் பகோண்நடன் என்பவத பதரிவித்鏁க் பகோள்கிந ன்.

இந்த ஆய்வில் ோன் 믁폁 ெம்மதத்鏁டꟁம், 毁யெிந்தவன뿁டꟁம் கலந்鏁 பகோள்ள ெம்மதிக்கிந ன்.

இந்த ஆய்வில் என்வனப் பற் ிய அவனத்鏁 விவரங்கள் போ鏁கோக்கப்ப翁வ鏁டன் இதன் 믁羿ퟁகள் ஆய்விதழில் பவளியிடப்ப翁வதில் ஆட்நெபவன இல்வல என்பவத பதரிவித்鏁க்பகோள்கிந ன்.எந்த ந ரத்தி쯁ம் இந்த ஆய்விலி쏁ந்鏁 ோன் விலகிக் பகோள்ள எனக்埁 உரிவம உண்翁 என்பவத뿁ம் அ ிநவன்.

இடம் :

நததி : வகபயோப்பம் / நரவக

MASTERCHART CASES CONTROLS

S.NO SEX AGE SERUM_GHRELINT3 T4 TSH BMI GLUCOSE UREA CREATININECHO TGL HDL T.BILIRUBIND.BILIRUBINAST ALT ALP URIC ACID CALCIUM LDH PHOSPHORUSPROTEIN ALBUMIN GLOBIN 1 2 28 10.11 1.38 8.3 1.55 25 98 13 0.9 102 180 55 0.24 0.1 11 15 34 2.9 6.3 402 2.2 7.2 3 4.2 2 2 40 9.99 1.68 9.07 2.27 24 100 15 0.8 115 160 49 0.71 0.21 15 24 44 3.1 7.5 422 3.1 8 4.2 3.8 3 2 26 10.2 2.55 9.06 3.04 25 101 15 0.8 122 176 51 0.6 0.22 23 33 56 3.9 8.1 434 4 7.5 4 3.5 4 2 44 10.72 2.33 11.01 1.88 24 110 14 0.7 111 150 47 0.71 0.4 31 24 45 5.9 10.4 407 2.3 8.1 4 4.1 5 2 40 10.55 1.48 5.86 3.15 23 104 13.5 0.6 98 146 55 0.53 0.22 30 18 56 3.3 7.6 423 3.1 7.8 3.8 4 6 2 50 9.78 1.84 10.54 1.49 24 110 12.9 0.81 122 152 48 0.13 0.05 23 24 67 2.9 7.3 450 2.4 8 5.1 2.9 7 2 55 10.12 1.39 9.97 2.42 24 115 14.3 0.9 122 160 50 0.6 0.21 22 44 76 3.5 7.1 432 3.3 7.8 4.6 3.2 8 2 26 11.01 3.15 13.3 1.58 23 98 10.5 0.6 127 150 55 0.55 0.22 26 37 78 3.2 7.6 403 3.4 7.6 4.4 3.2 9 2 31 11 1.22 11.41 2.88 23 98.9 11.3 0.7 110 155 49 0.48 0.15 28 36 66 4.5 8.6 409 4 8 4.5 3.5 10 1 53 10.76 1.48 8.64 1.77 22 96.5 12.3 0.8 121 165 46 0.55 0.24 17 38 65 4.3 9 423 5 7.7 4.5 3.2 11 2 34 10.23 1.57 9.31 4.69 23 100 13.4 0.71 111 154 45 0.72 0.21 19 39 69 3.2 7.7 456 2.2 7.8 4 3.8 12 2 57 9.97 1.8 9.5 2.72 24 115 12 0.8 110 145 43 0.45 0.2 20 40 76 3.4 7.9 523 3.1 7.9 4.9 3 13 2 59 9.87 2.06 6.91 2.3 25 102 13 0.7 115 156 47 0.54 0.28 21 41 77 3.1 7.6 514 3 6.5 3.5 3 14 2 49 10.3 1.67 7.76 2.98 23 105 14 0.81 98 139 48 0.31 0.1 30 42 98 3.7 7.7 453 3.4 6.7 3.4 3.3 15 2 43 11.03 1.86 8.34 1.9 24 101 13 0.9 99.5 143 45 0.3 0.21 32 44 78 3.9 8.2 465 2.2 7 4.8 2.2 16 2 43 10.64 1.36 8.11 1.16 25 100 12.7 0.8 95 154 43 0.78 0.32 15 34 87 3.6 8.8 476 3.4 8.1 5.1 3 17 2 42 9.57 1.61 5.61 3.59 23 98 13.4 0.71 101 145 45 0.27 0.11 17 36 54 4 8.4 484 2.2 7.2 4.2 3 18 2 36 9.87 1.53 8.89 2.81 25 101 13.5 0.6 142 135 51 0.22 0.05 18 37 43 4.3 9.5 456 2.1 7 4.3 3.7 19 2 40 10.09 1.55 5.73 3.81 23 98 12.4 0.8 134 145 48 0.22 0.09 23 42 76 4 9.5 432 2.4 8 4.7 3.3 20 2 45 9.86 1.33 5.3 4.56 26 112 13.4 0.9 143 142 52 0.57 0.17 25 22 75 3.3 8.7 411 3.8 6.7 3.7 3 21 2 55 9.54 1.44 5.79 2.89 25 98 12.5 0.6 132 145 48 0.14 0.05 30 24 73 4.2 10.2 422 3.5 6.8 3.5 3.3 22 2 41 9.95 1.29 6.48 2.21 24 99 15 0.7 122 135 51 0.28 0.15 27 32 81 3.4 9.6 432 2.8 7.6 4.6 3 23 2 56 9.89 2.01 9.61 1.31 26 110 14.3 0.8 132 145 50 0.71 0.32 31 31 83 3.5 7.7 483 2.9 8 4.8 3.2 24 2 58 9.54 1.14 9.65 3.25 25 109 12 0.9 122 156 49 0.43 0.11 12 34 65 4.7 8.7 432 3.9 7.6 4.2 3.4 25 2 25 10.01 1.96 9.6 2.76 23 110 13.6 0.8 132 154 52 0.54 0.25 16 14 35 3.5 7.3 456 2.2 6.7 4.5 2.2 26 1 43 10.81 1.57 7.77 2.62 26 99 12.6 0.7 122 155 51 0.28 0.15 19 17 75 2.6 7.5 431 3.1 7.2 4.2 3 27 2 25 9.54 1.54 9.51 2.73 24 105 13.4 0.8 132 165 45 0.51 0.2 23 18 86 3.5 8.2 422 4 7.6 4.6 3 28 2 60 8.98 1.68 8.13 4.65 25 107 13 0.9 134 156 51 0.32 0.17 27 22 53 4.2 8.4 451 2.3 6.8 4.4 3.4 29 2 55 9.78 1.44 9.22 2.09 23 111 14 0.7 135 125 47 0.52 0.2 29 18 65 4.5 8.7 444 3.1 7 4.2 2.8 30 2 60 10 1.98 5.43 4.31 26 107 15 0.8 122 140 46 0.41 0.2 30 34 53 7.1 11.1 403 2.4 7.8 4.5 3.3 31 2 56 9.65 1.21 7.7 1.12 24 101 13 0.6 112 135 47 0.77 0.31 30 28 53 3.6 8.2 433 3.3 7.4 4.4 3 32 2 56 8.99 1.16 10.1 1.5 23 97 14 0.8 102 132 43 0.64 0.32 32 27 57 3.4 7.7 502 3.4 6.8 3.5 3.3 33 2 37 9.32 1.61 7.05 3.24 24 98 15 0.9 111 130 45 0.4 0.16 21 43 43 4.1 8.2 481 4 6 3.2 2.8 34 2 30 9.34 1.53 7.75 1.87 23 99 12 0.6 101 123 43 0.6 0.25 22 29 46 3.2 8.6 473 5 6.8 3.8 3 35 2 57 10.08 1.53 10.32 3.52 24 88 11 0.7 120 146 43 0.42 0.11 32 38 54 5.5 10.4 444 2.2 7 4.3 2.7 36 2 43 11.02 1.34 9.51 1.75 24 97 13 0.6 124 155 48 0.6 0.24 30 50 65 5.5 10.4 453 3.1 8 4.5 3.5 37 2 50 10.09 1.43 8.51 2.26 25 99 14 0.8 125 134 44 0.21 0.05 16 33 87 3.9 8.3 423 3 6.4 4.4 2 38 2 26 9.99 1.96 9.04 3.03 26 89 11 0.7 122 165 43 0.24 0.07 17 32 77 2.8 8 430 3.4 7.2 4.2 3 39 2 46 9.52 1.82 6.9 3.81 24 90 12 0.9 132 135 44 1.11 0.5 19 42 76 3.5 8.7 465 2.2 7.9 4.7 3.2 40 2 27 9.01 1.79 9.67 1.38 25 107 14.3 0.6 142 162 47 0.72 0.32 23 16 56 2.6 6.8 433 3.4 8 5.3 2.7 41 2 50 10.54 1.75 9.1 2.19 23 111 13 0.7 132 142 50 0.32 0.11 21 19 65 3.7 7.3 504 2.2 7.8 4.3 3.5 MASTERCHART CASES CONTROLS

42 2 55 9.43 1.42 7.57 1.36 24 108 14 0.9 165 143 49 0.24 0.14 27 26 77 3.6 7.8 398 2.1 7.9 5.3 2.6 43 2 55 9.67 1.53 12.3 1.06 25 109 15 0.8 153 153 45 0.23 0.1 29 23 97 4.2 9 378 2.4 8 5 3 44 2 32 9.43 1.78 6.15 2.79 24 100 12 0.9 146 162 42 0.35 0.12 14 34 92 3.1 7.7 388 3.8 6.9 3.9 3 45 2 30 9.87 1.47 6.12 2.62 23 99 13 0.6 134 165 47 0.63 0.1 17 43 52 3.4 7.9 433 3.5 7.4 4.2 3.2 46 2 30 10.24 1.5 7.56 3.94 26 104 14 0.7 142 164 45 0.8 0.3 19 28 52 3.1 7.6 453 2.8 7 4.2 2.8 47 2 44 10.9 1.49 6.02 1.76 24 103 15 0.56 144 134 45 0.65 0.23 21 39 62 3.6 7.7 463 2.9 6.7 4.2 2.5 48 2 52 9.78 1.77 6.29 4.28 26 113 14 0.6 134 154 47 0.3 0.12 21 41 73 3.9 8.2 431 3.8 6.5 3.5 3 49 1 54 10.3 1.08 6.61 1.31 24 104 13 0.7 120 150 45 0.79 0.21 13 33 71 3.6 8.8 503 3.1 7 4 3 50 2 33 9.85 1.91 8.38 3.65 23 90 14 0.9 132 170 46 1.1 0.23 21 14 92 4.1 8.4 543 3.3 6.5 3.5 3 MASTERCHART CASES CONTROLS

SERUM_GHRE GLUCOS CREATININ T.BILIRUBI D.BILIRUBI URIC CALCIU PHOSPHOR S.NO SEX AGE T3 T4 TSH BMI UREA CHO TGL HDL AST ALT ALP LDH PROTEIN ALBUMIN GLOBIN LIN E E N N ACID M US 1 2 28 6.408 1.45 9.8 98.78 26 83.6 9.4 0.79 184 355 71.8 0.34 0.2 13 13.2 123 8.3 6.3 601 2.82 6.3 3.4 2.9 2 2 58 5.026 1.16 6.82 88.16 24 110.5 21.7 0.96 321 241 50.8 0.81 0.34 31 41 102 7.2 6.6 463 3.89 7.5 4.4 3.1 3 2 55 2.674 0.78 4.29 63.03 24.2 120 25 1.3 192 222 35 0.72 0.42 35 47 109 7 7 532 3.1 8.1 4.2 3.9 4 2 55 9.862 1.38 4.54 72.62 23 104.5 25.3 0.89 179 91.6 96.6 0.71 0.4 25 19.2 110 7 7.2 796 4.67 10.4 4.5 5.9 5 2 57 5.548 0.98 5.75 18.57 26 138 20 0.94 145 186 43.4 0.62 0.38 35.2 30.3 95 6.5 6.9 708 5.83 7.6 4.3 3.3 6 2 30 4.346 1.19 7.63 29.08 25 107 16.4 1.07 186 177 48.9 0.13 0.5 15.7 16.6 96 6.3 8.3 365 3.81 7.3 4.4 2.9 7 2 55 4.481 0.98 7.04 20.75 26 129 35.2 1.22 197 160 46.1 0.6 0.11 10.3 30.2 95 7.1 8.7 639 7.22 7.1 3.8 3.3 8 2 55 3.151 0.95 1.89 100 27 106.4 20.6 1.01 233 202.1 72.8 0.88 0.33 30.1 20.2 66 7.3 8.9 599 4.8 7.6 4.4 3.2 9 2 37 5.007 1.34 5.18 26.24 24 130 22.6 1.16 180 219.7 58 0.48 0.19 40.1 23 101 5.8 8.5 425 4.3 8.6 4.2 4.4 10 2 24 5.066 1.41 5.13 23.85 26 90 13.9 0.96 168 96.7 55.7 0.45 0.24 13 17.4 91 4.8 8.2 365 4.8 9 4.7 4.3 11 2 28 1.584 0.8 4.71 79.06 28 82.5 24.4 1.05 187 88.8 77.1 0.82 0.31 25.2 17.9 39 7.3 8.2 986 4.4 7.7 4.6 3.1 12 2 26 2.431 1.63 7.43 20.27 27 85.6 26.2 1.31 161 94.3 51.1 0.45 0.19 36.1 65.1 65 3.4 6.6 499 3.99 7.9 4.5 3.4 13 2 39 5.685 1.51 6.91 33.38 26 92 19.3 1.07 187 175.9 53.1 0.5 0.18 30.8 27.3 100 6.7 7 569 4.35 7.6 4.5 3.1 14 2 39 3.095 1.51 6.98 28.07 25 86.4 19.3 0.97 238 266.4 50.7 0.31 0.11 40 18.7 142 4.3 7.3 471 3.69 7.7 4.1 3.6 15 2 33 2.722 0.9 5.4 69.8 27 116.6 19.2 0.93 257 219.5 47.6 0.25 0.11 19.4 13.7 96 4.7 10.8 623 5.63 8.2 4.3 3.9 16 1 57 8.776 2.2 7.7 43.7 29 111.5 23.7 1.18 258 133.8 52.9 0.78 0.2 35 45.5 68 8.6 11.8 805 3.41 8.8 5.2 3.6 17 2 54 3.285 0.9 5.1 80.1 26 100.1 14.3 0.99 240 434 46.2 0.17 0.11 12.2 16.8 94 5.3 13.1 395 4.52 8.4 4.3 4.1 18 2 43 4.1515 1 6.1 20.7 27 110.7 23.5 1.02 255 261.6 46.2 0.11 0.05 16.1 12.5 72 6.5 11.1 347 4.72 9.5 4.2 5.3 19 2 55 2.556 0.7 5.8 42.2 27 120 31.5 1.1 214 277.6 42.4 0.22 0.09 37.2 40.5 178 6.9 11.7 566 4.31 9.5 4.7 4.8 20 2 55 3.828 0.9 38.4 88.4 26 125 34.6 1.81 327 440.6 43.3 0.47 0.17 12.8 13.1 134 7 9.2 880 4.52 8.7 5.4 3.3 21 2 40 2.367 1 5.9 23.7 28 111 17.9 1.08 231 111.2 66.1 0.12 0.07 42.2 26.2 103 3.9 10.5 598 4.57 10.2 5.3 4.9 22 2 37 2.0856 0.9 5.8 32.7 24 115 30 1.3 345 288.5 52.9 0.38 0.15 17.8 13.1 111 5.6 13.7 465 3.76 9.6 5.1 4.5 23 2 29 5.9651 1.4 6 27.8 26 82 19.5 0.83 176 290.2 47.4 0.91 0.32 14.8 15.6 89 6.2 8.3 459 4.17 7.7 3.8 3.9 24 2 20 4.66 1.1 4.1 97.7 25 103.4 22 0.91 247 330.1 40.7 0.33 0.11 13.6 11.1 106 5.7 8.5 609 4.32 8.7 4 4.7 25 2 41 5.05 1.2 7.2 39.7 27 141.3 20.8 0.85 194 103.2 56 0.61 0.25 18.2 12.4 52 2.4 7.9 632 3.96 7.3 3.8 3.5 26 2 57 5.6079 1.6 8.9 15.5 27 100.3 17 0.85 168 118.3 52 0.36 0.15 22.7 15.6 59 4.3 8.9 670 4.74 7.5 3.9 3.6 27 2 51 5.3906 0.5 2.3 100 26 87.6 24 0.76 154 140.3 48.3 0.5 0.23 14.2 17.6 135 6 8.7 660 3.85 8.2 3.7 4.5 28 2 34 9.2633 1.4 8.6 29.1 23 119.9 17.7 0.83 164 136.1 39.3 0.42 0.17 19.9 7.8 78 4.5 8.3 251 3.01 8.4 4.2 4.2 29 2 36 6.5979 1.1 5.6 34.4 26 86.1 40.3 0.75 244 213 52.2 0.52 0.18 17.6 14.3 100 4.2 9.9 236 4.73 8.7 4.2 4.5 30 2 35 3.4437 1.5 3.2 43.9 28 89.1 17.4 0.88 185 107.4 42.9 0.41 0.16 24.4 23.5 100 4.9 7.8 509 3.93 11.1 4 7.1 31 2 30 3.863 0.8 4.7 26.6 27 95.6 15.5 0.91 184 128.3 50.2 0.86 0.31 18.3 24.1 68 4.7 7.7 682 3.25 8.2 4.6 3.6 32 2 33 3.5045 0.9 4.6 30.1 27 102.4 15.8 0.76 166 69.9 69.6 0.64 0.68 18.7 15.2 45 3.9 8.1 451 3.63 7.7 4.3 3.4 33 2 45 2.525 1.6 5.1 46.9 28 84.3 19.3 0.97 176 154 55.2 0.39 0.16 17.2 22.2 99 4.7 9.5 325 3.12 8.2 4 4.2 34 2 55 5.7869 0.85 6.21 25.75 23 123 27.7 1.57 251 296.9 57.7 0.6 0.25 18.3 28.4 114 7.4 10.8 498 4.63 8.6 5.4 3.2 35 2 42 7.2767 2.02 6.31 44.89 24 120 21.1 1.1 246 172.4 70.5 0.21 0.11 41.7 29 144 5.6 8.8 385 4.4 10.4 4.9 5.5 36 2 49 5.535 1.32 4.52 18.24 25 102 23.4 1.1 190 157.5 61.7 0.53 0.24 20 31.6 161 7 9.4 466 6.3 10.4 4.9 5.5 37 2 43 3.621 1.71 6.62 26.2 23 77.5 27 0.96 151 140.8 45.8 0.21 0.1 13.8 23.4 69 8.2 9 387 3.75 8.3 4.3 4 38 2 42 5.105 1.54 7.25 27.06 24 69.3 24.2 0.74 233 103.2 67.2 0.24 0.1 28.7 29.9 77 3.8 9.2 301 3.48 8 5 3 39 2 35 6.9816 1.08 5.16 39.44 25 96.5 10.9 0.81 104 175 56.3 1.11 0.49 16.1 20 42 2.3 7.7 350 2.19 8.7 4.7 4 40 2 56 9.603 1.33 9.718 40.04 24 83.5 16.3 0.8 94 120 57.9 0.63 0.32 13.9 16.6 56 5.3 8 390 2.2 6.8 4.2 2.6 41 1 48 2.626 0.82 1.2 100 29 95 15.9 0.94 138 196 50 0.24 0.11 24.8 29.9 103 6.3 7.3 452 2.65 7.3 4.2 3.6 42 2 31 2.4634 0.746 5.3 100 28 90 13.3 0.88 169 112.6 41.6 0.18 0.14 26.7 35.1 52 5.2 8.2 450 2.32 7.8 4.2 3.6 43 1 45 3.006 1.05 8.68 13.04 27 95 12.3 0.7 110 175 56 0.12 0.1 38.1 33 66 3.3 9 420 4.8 9 4.7 4.3 44 2 52 2.2474 0.709 5.7 100 28 98 14 0.88 190 290 41 0.25 0.12 32 35.5 111 4.3 8.2 553 4.4 7.7 4.6 3.1 45 2 27 1.8061 1.377 6.47 20.14 29 102 21.3 0.91 230 190 47 0.75 0.1 19.8 28.8 134 5.5 7.3 432 3.99 7.9 4.5 3.4 46 2 33 1.3805 1.44 5.07 40.97 28 110 12.9 1 220 210 45 0.82 0.13 21.1 33 79 5.3 7.8 456 4.35 7.6 4.5 3.1 47 2 47 3.3449 1.091 5.41 34.19 25 97 17 0.86 234 221 55 0.69 0.32 24.5 27.7 80 4.5 7.4 467 3.69 7.7 4.1 3.6 48 2 20 2.399 0.431 1.002 100 28 121 13 0.79 197 189 57 0.29 0.22 22 29.8 76 5.2 6.5 478 5.63 8.2 4.3 3.9 49 2 45 1.5837 1.141 5.62 62.2 28 100 18.9 0.92 275 287 61 0.79 0.13 28 20.9 56 4.6 8.1 543 3.41 8.8 5.2 3.6 50 2 53 1.6485 0.28 0.42 100 28 103 20 0.88 290 200 67 1.1 0.33 31 30 68 3.7 8.8 567 4.52 8.4 4.3 4.1