osteoporosis in cholelithiasis: biochemical markers 2014/phd thesis... · pharmacy for offering the...

Republic of Iraq

Ministry of Higher Education and

Scientific Research

University of Baghdad

College of Pharmacy

OSTEOPOROSIS IN CHOLELITHIASIS: BIOCHEMICAL

MARKERS

A THESIS

SUBMITTED TO THE DEPARTMENT OF CLINICAL LABORATORY SCINCES AND THE

COMMITTEE OF GRADUATE STUDIES, IN THE COLLEGE OF PHARMACY UNIVERSITY

OF BAGHDAD IN PARTIAL FULFILLMENT FOR THE REQUIREMENTS OF THE DEGREE

OF DOCTOR OF PHILOSOPHY IN PHARMACY “CLINICAL LABORATORY SCINCES”

BY

Falah Hassan Shari AL-Maliki

B.Sc. Pharmacy 2002

M.Sc.Pharmacy 2008

Supervised By

Ass. Prof.Dr. Mohammed A. Taher

Dr.Salah K.AL-Ahmedi

2013 A.D 1434 A.H

بسن للاه الرهحمه الرهحين

أوتىا الهذيه وقال

لقد واليمان العلن

إلى للاه كتاب في لبثتن

يىم فهذا البعث يىم

ل كنتن ولكنهكن البعث

تعلمىن

65 - اآلية - الروم -

ألعظينصدق أهلل العلي

2

I dedicate this work to:

Everyone helped me.

To my family.

To my parents.

To my faithful wife & sons.

Dedication

3

I am sincerely grateful to my supervisor Ass.Prof.. Mohammed A.Taher for his

unlimited support, patience, fruitful encouragement and academic approach during the

working period.

I would like to express my deepest appreciation to my Co-Supervisor Dr. Salah K.AL-

Ahmmdi for his precise clinical touch and thoroughly insighting discussion during the work

period.

My genuine thanks to Prof. Dr. Alaa A. Abdulrasool, the dean of College of Pharmacy

for his support.

I am grateful to Ass. Prof. Dr. Mohammed Hassan, Assistant Dean for Postgraduate

Studies for his support.

I am also grateful to the College of Pharmacy, University of Baghdad College of

pharmacy for offering the opportunity to continue my graduate study.

My thanks go to the manager and the staff of Al- Basra general hospital Dept.of

surgeryfor permitting me to use their tools and places.

I thank College of Pharmacy – University of Basra for facilitating the work.

My deepest thanks for Prof. Dr. Nazar AL-Mahfood, Dr.Sarmed Manah,Dr.Hashim AL-

Khyat, for his assistance in . providing the patients.

I would like to express my deepest appreciation and gratitude to

Dr. Shatha Hussain Ali, for her kind and helps. And Dr. Eman Se'diy -Head of the Department of clinical

lab. Sciences- at College of Pharmacy, Baghdad Univer falah2013

Acknowledgements

4

Content Page

Dedication I

Acknowledgements II

Contents III

List of Tables VI

List of Figures VII

List of Abbreviations IX

Abstract XII

Chapter One: Introduction

1.1 Definition of Gallstone 1

1.2 Epidemiology of Gallstone 2

1.3 Etiology and risk factors 3

1.3.1 etnicity 3

1.3.2 Family history & genetics 3

1.3.3 Age 4

1.3.4 Geneder 4

1.3.5Obesity/metabolic,syndrome/diabetes,mellitus/dyslipidemia

and rapid weight loss.

4

1.3.6 Pregnancy, parity and female sex hormones 5

1.3.7 total parental nutrition (TPN) and Diet 6

1.3.8 Reduced physical activity 6

1.3.9 Underlying disease: cirrhosis, Crohn’s disease:cirrhosis 7

1.3.10 Drugs 7

1.4 Classification of Gallstones 8

1.4.1Cholesterol gallstones 8

1.4.2 Pigement stones 8

1.4.3 Mixed stones 8

1.5 Pathophysiology of gallstones 9

1.6 Investigations of gallstones 10

1.6.1 Ultrasonography 11

1.6.2 Computerize Tomography scans 11

1.6.3 Cholescintigraphy (HIDA scan) 11

1.7 Osteoporosis 11

5

1.8 Bone modeling and remodeling 14

1.8.1 Normal bone remodeling 14

1.9 Pathophysiology of Osteoporosis and Bone Fragility 15

1.10 Epidemiology of osteoporosis 21

1.11 Diagnosis and investigation of osteoporosis 23

1.11.1 measurement of bone mineral density 23

1.11.2 Assessment of clinical risk factors and fracture risk 24

1.11.3 Complet blood count and Chemistry Profile 27

1.11.4 Bone-Turnover Biomarkers 27

1.11.4.1 Bone resorption markers 27

1.11.4.2 Bone formation markers 28

1.12 Treatment of osteoporosis 29

1.14.1 Non-pharmacological intervention 29

1.12.2 Pharmacologic therapies for osteoporosis 30

1.13 Proinflammatory cytokines increase bone resorption 35

1.14 Aims of study 36

Chapter Two: Subjects, Materials & Methods

2.1 Subjects 37

2.1.1 Patients and Subjects 37

2.1.2 Inclusion criteria 38

2.1.3 Exclusion criteria 38

2.2 Chemicals 38

2.3 Instruments 39

2.4 Blood specimens 41

2.5 Biochemical assessment 41

2.5.1 Estimation of high sensitivity C-reactive protein 41

2.5.2 Estimation of Tumor Necrosis Factor Alpha 42

2.5.3 Estimation of Interleukine-1 42

2.5.4 Estimation of Interleukine-6 43

2.5.5 Estimation of serum free testosterone 44

2.5.6 Estimation of serum estradiol 44

2.5.7 Estimation of serum 25(OH) Vit. D 45

2.5.8 Estimation of Parathyroid Hormone 46

2.5.9 Estimation of Serum Total Calcium 47

2.5.10 Estimation of serum inorganic phosphate 47

2.5.11 Estimation of Serum Human C terminal Telopeptides of 48

6

types I Collagen

2.5.12 Estimation of Serum Human carboxyterminalpropeptide

of type I procollagen 48

2.6. Statistical analysis 49

Chapter Three: Results

3.1 Demographic characteristics of Cholelithiatic patients

&controls

50

3.2 Age 51

3.3 Body mass index 52

3.4 Waist to Hip Ratio 53

3.5 Biochemical parameters 56

3.5.1 Serum Tumor Necrosis Factor Alpha 56

3.5.2 Serum Interleukine-1 57

3.5.3 Serum Interleukine-6 58

3.5.4 Serum High sensitivity C-reactive protein 59

3.5.5 Serum 25(OH) Vitamin D 60

3.5.6 Serum Parathyroid hormone 61

3.5.7 Serum Inorganic phosphate 62

3.5.8 Serum total calcium 63

3.5.9 Carboxy terminal propeptide type I procollagen 65

3.5.10 Carboxy terminal telopeptide type I procollagen 66

3.5.11 Serum estradiol 68

3.5.12 Serum free testosterone 69

3.6 Correlation studies 71

Chapter Four: Discussion

4.1 Demographic facts and risk factor 76

4.1.1 Female gender & its related factors 76

4.1.2 Family History, nephrolithiasis and physical activity. 78

4.2 Obesity. 80

4.3 Age. 81

4.4 Serum concentrations of TNF-alpha, IL1, IL6 and HCRP. 81

4.5 Serum concentrationsof of Vitamin D, Parathyroid hormons,

inorganic phosphate and total serum calcium. 86

4.6 Serum concentrationsof of Carboxy terminal propeptide type

I procollagen(PICP) and Carboxy terminal telopeptide type I

procollagen(CTXI).

88

4.7 Serum concentrations of of estradiole and free teststeron 89

4.8 Correlation study 91

7

4.9 Conclusions 93

4.10 Recommendations for further work 94

Patient information sheet 95

List of normal values 96

References 97

No. Table Page

1.1 Secondary causes of Osteoporosis 13

1.2 show the World Health Organization Classification

of T Score

24

1.3 Differential diagnosis includes a thorough medical

history, physical examination and a range of

investigative tests which may be case-dependent

25

1.4 Laboratory evaluations for secondary causes of

osteoporosis

26

2.1 diagnostic kits and chemicals with their suppliers.

39

2.2 Instruments with their suppliers 40

3.1

Demographic characteristics of Cholelithiatic

patients &controls

50

3.2

Age in years, Body mass index and waist –hip ratios

for control and patient groups

51

serum concentrationsof TNF-alpha, IL1, IL6 and

HCRP in control and patients groups

55

8

3.3

3.4

serum concentrations of Vitamin D, Parathyroid

hormons, inorganic phosphate and total serum

calcium in control and patients groups

60

3.5

serum concentrations of C- terminal propeptide&

C- Terminal telopeptide , in ng/ ml in control and

patients groups

65

3.6

serum concentrations of estrogen in (pg/ml) &

free testeronein(mg/l) in control and patients

groups

68

3.7

Regression factors and P value for measured

parameters

72

Page Figure No.

8 The figure 2 show the shap of this stone 1.1

9

shape of mixed stones

1.2

10

Pathogenesis of stone formation is different for

cholesterol stones, soft brown stones, and pigment

stones

1.3

15 bone remodeling process 1.4

18 Osteoclasts, Immune Cells, and RBCs are Derived

from Marrow Hematopoietic Stem Cells

1.5

19

Osteoblasts, Cartilage, and Adipocytes are Derived

from Marrow Mesenchymal Stem Cells

1.6

20 RANK/RANKL/OPG Osteoimmunological System of

Bone Homeostasis

1.7

01

21 Chronic Immune Activation Leads to Bone Loss 1.8

30 Mechanisms of action of anti osteoporosis treatments 1.9

52 Histogram shows difference in Age, for both control

and study groups.

3.1

53

Histogram shows difference in BMI, for both control

and study groups.

3.2

54

Histogram shows difference in waist to hip ratio, for

both control and study groups

3.3

56 Histogram show serum level of TNF –α measured in

pg/ml, for both control and study group

3.4

57

Histogram shows serum level of interleukin -1

measured in Pg/ml, for both control and study groups.

3.5

58

Histogram shows serum level of interleukin -6

measured in Pg/ml, for both control and study groups.

3.6

59

Histogram shows serum level of HCRP measured in

mg/Liter, for both control and study groups.

3.7

61

Histogram shows difference in serum level of Vitamin

D measured in ng/ml , in both control and study

groups

3.8

62

Histogram shows difference in serum level of

parathyroid hormone measured in pg/ml, in both

control and study groups

3.9

63


inorganic phosphate measured in mg/dl, in both


3.10

00

64


calcium measured in mg/dl, in both control and study

groups

3.11

66

Histogram shows difference in serum level of C

terminal propeptide measured in ng/ml, in both control

and study groups

3.12

67

Histogram shows difference in serum level of C

terminal telopeptide measured in ng/ml, in both


3.13

69

Histogram shows difference in serum level of estradiol

measured in pg/ ml, in both control and study

groups; for female only

3.14

70

Histogram shows difference in serum level of free

testerone measured in mg/liter, in both control and

study groups.

3.15

73

Correlation between serum IL6 (pg/ml) and

serumC-terminal propeptide (ng/ml)

3.16

73

Correlation between serum IL-6(pg/ml) and

serumC-terminal telopeptide (ng/ml)

3.17

74

. Correlation between serum IL 1 (pg/ml) and

serumC-terminal propeptide (ng/ml)

3.18

74

Correlation between serum IL 1 (pg/ml) and


3.19

75 Correlation between serum TNF-α(pg/ml) and


3.20

Word Abbreviation

Acquired immune deficiency syndrome AIDS

Alkaline phosphatase ALP

Body mass index BMI

01

Bone mineral density BMD

Bone turnover markers BTM

C – reactive protein CRP

Calcium sensing receptors CasR

Cardiovascular CV

Chronic a calculous cholecystitis CAC

Chronic calculous cholecystitis CCC

Common bile duct CBD

Complete blood count CBC

Computerized tomography CT

C-terminal cross-linking telopeptide of type I collagen CTX

C-terminal cross-linking telopeptide of type I collagen

generated by matrix metalloproteinases

CTX-MMP

Deoxypyridinoline DPD

Dual x-ray absorpiometry DXA

Endoscopic retrograde cholagiopancreatography ERCP

Endoscopic sphincterotomy ES

Endoscopic ultrasoungraphy EUS

Enzyme Linked Immunosorbent Assay ELISA

Estradiol E2

Estrogen Receptor type one ESR1

Estrogen Receptor type two ESR2

Extracorporeal shock wave lithotripsy ESWL

Ezetemibe EZT

Family history FH

Farnasyl pyro phosphate FPP

farnesoid X receptor FXR

Follicle stimulating hormone FSH

Fracture risk assessment tool FRA

Gallbladder GB

Gallbladder cancer GBC

Gallstone GS

Gallstone disease GD

Gallstone pancreatitis GSP

Gaunosin tri phosphate GTP

high density lipoprotein HDL

Highly sensitive C-reactive protein Hs CRP

02

Horseradish peroxidase HRP

Hounsfield unit HU

Insulin like growth factor type one IGF-1

Interferon gamma IFN γ

Interleukin IL

Interleukin-type one IL-1

Interleukin-type six IL-6

Isoform 5b of tartrate resistant acid phosphatase TRACP 5b

Lipopolysaccharides LPS

Luteinizing hormone LH

Macrophage colony stimulating factor MCSF

Magnetic resonance cholangiopancreatography MRCP

Methyl tetrabutylether MTBE

Mirizzi syndrome MS

Mitogen active protein kinase MAPK-ERK1/2

Mixed stone Ms

Monoclonal antibody MAb

National Institute of Health NIH

Niemann-Pick C1-Like 1 NPC1L1

Non steroidal anti inflammatory drugs NSAIDS

N-terminal cross-linking telopeptide of type I collagen NTX

carboxyterminal procollagen type I propeptide PICP

N-terminal procollagen type I propeptide PINP

Nuclear factor kappa B NFκB

Nuclear factors of activated T-cells Wnt/NFA Tc

Nuclear receptors NRs

Osteocalcine OC

Osteoporosis OP

Osteoprotegrine OPG

Oxysterol X Receptor LXR

Parathyroid hormone PTH

Percutaneous transhepatic cholangiography PTC

Peroxisom proliferators activated receptor gamma PPARγ

Post menopausal osteoporosis PMO

Pyridinolin PYD

Receptor activator of nuclear factor kappa RANK

Receptor activator of nuclear factor kappa legand RANKL

03

Red blood cell RBC

Salmon calcitonin Sct

Selective estrogen receptors modulators SERMs

Serum C-terminal cross-linking telopeptide SCTX

Signal tranducer and activator of transcription STAT

Slandered deviation SD

Standard error SE

Sympathetic nervous system SNS

Taurou ursodeoxycholic acid TUDCA

Tetramethylbenzidine TMB

total parental nutrition TPN

Transforming growth factor –beta TGF-B

T-regulatory cell T-reg

Tumor necrosis factor alpha TNF – α

Tumor necrosis factor receptor association factor-6 TRAF-6

Ultra soungraphy US

Urinary C- terminal telopeptide UCTX

Urinary N-terminal cross-linking telopeptide UNTX

Ursodeoxycholic acid UDCA

Weight WT

World health Organization WHO

β-hydroxyl-β-methylglutaryl coenzyme A reductase HMG CoA

reductase

*

04

Background:

Gallstone disease (cholelithiasis) is one of the most common and most

costly digestive diseases that require hospitalization in world wide. Cytokines play

a pivotal role in the pathogenesis of cholelithiasis by driving the subsequent

inflammatory response which leads to tissue damage and organ dysfunction or

failure, in more severe cases. Local recruitment and activation of inflammatory

cells in cholelithiasis may lead to the production of proinflammatory cytokines,

such as interleukin [IL] 6, IL-1and tumour necrosis factor alpha [TNF-alpha].

Osteoporosis (OP) is the condition in which a low bone mass and altered

microarchitecture of the bone leads to increased risk of fracture. The major role

cytokines play in bone remodeling is demonstrated by the fact that receptors for

the proinflammatory cytokines [IL-1, IL-6, TNF-α] are present on both osteoclast

precursor cells and mature osteoclasts. Thus an elevation in these cytokines in

cholelithiatic patients act on their receptors in bone cells and induce bone

remodeling.In addition the decreased level of vitamin (D) in choleliyhiatic patients

due to malabsorption also leads to osteoporosis by several cascades.

Objective:

The aim of this study is to evaluate the proinflammatory cytokines effects on

cholilethiatic patients and evaluate levels of bone formation and bone resorption

markers. Also to study the biochemical changes in some parameters[ (IL-1 and IL-

6), TNF-α, highly sensitive C-reactive protein (hs – CRP), Vit.D,parathyroid

hormone (PTH),calcium( Ca++

), inorganic phosphate, C-terminal telopeptide of

type I collagen(CTX), Carboxyterminal propeptide of type I procollagen(PICP),

Abstract

05

free teststeron and estradiol]. in Cholelithiatic patients compared to controls, each

parameter may play a role in the pathogenesis of gallstones .also to study some risk

factors for gallstone formation and to correlate between demographic data and its

effect on gallstone formation.

Subjects and methods:

One hundred patients were participated in this study [90 females &10

males]; their age range of was (18-38) years. Only patients with cholelithiasis (non

complicated cases) selected for surgery were included in the present study.

Complicated gallstone cases, liver disorders, cardiovascular complications,

hypertension, asthma, diabetes mellitus,chronic renal failure, Cushing syndrome

and Drugs induced osteoporosis,cefatrixone, somatostatin , antioxidant were

excluded from this study.Apparently healthy persons were selected to participate as

control group with age range (19-40) years The diagnosis was made by a specialist

surgeon, depends on the presence of typical symptoms and the demonstration of

stones in gallbladder on diagnostic imaging. An abdominal ultrasonography is the

standard diagnostic test for gallstone detection. After 12 hours fasting, venous

blood samples, about 10 ml were collected from patients without use of tourniquet

before laparoscopic cholecystectomy and from healthy volunteers and placed in

plain tubes. After allowing the blood to clot at room temperature for 30 min, blood

samples were centrifuged at 3000 rpm for 15 min. stored serum at -20Cº was used

for the assessing the plasma levels of [(IL-1 and IL-6), TNF-α, hs - CRP,

25(OH)vit D, PTH, Ca++

, inorganic phosphate, C-terminal telopeptide of type I

collagen, Carboxyterminal propeptide of type I procollagen, free teststeron and

estradiol) by utilizing ELISA Kits. The present work was carried out at Al- Basra

General Hospital from January 2012 until May2012.

06

Results:

The present work show that female gender, over weight , obesity, oral

contraceptive usage, multiparity, renal stones and positive family history

were all associated with gallstones formation. whereas, aging did not have a

significant effect.also There was a significant increase (P<0.001) in serum

concentrations of TNF-alpha, IL1, IL6,hs- CRP,PTH, total Ca, C- terminal

propeptide& C- Terminal telopeptide and estradiol of patients with

gallstone compared to healthy controls.also There was a significant decrease

(P<0.007) in serum concentrations of 25(OH) vit D and inorganic

phosphate of patients with gallstone compared to healthy controls.

Meanwhile, serum concentration of free testosterone was significantly

reduced (P<0.05) in patients with gallstone compared to healthy controls.In

the current study results shows that there is a positive correlation between

interleukine-1[IL-1], interleukine-6[IL-6]andC-terminal propeptide

[r=0.4437,P,<0.05] and [r=0.4156, P<0.05] respectively. Also current study

showed that the postive correlations between interleukins 1 ,6 and TNF-α

with carboxyterminal telopeptide [r=0.3552, P<0.05], [r=0.3942, P<0.05],

[r=0.1529, P<0.05] ,respectively.

Conclusion:

From this study can conclude that Cholelithiatic patients have systemic

elevation of proinflammatory markers represented by IL-1,IL-6,TNF-α and

hs-CRP. Also gender, obesity, parity, contraceptive usage ,low physical

activity,positive family history and renal stones are associated significantly

with gallstones. Moreover, Cholelithiasis is considered as a risk factor for

osteoporosis as shown by elevation of bone turnover markers. High

association between cholelithiasis,obesity and osteoporosis.

Chapter One : Introduction 19

08

Chapter One

Introduction

1.1 Definition of Gallstone (GS):

Gallstone disease known as cholelithiasis is one

of the most common digestive surgical disorder. Cholelithiasis is common

with the incidence ranging from 10% to 20% of the world population, [1].

Gallstone disease is newly diagnosed in more than 1 million people annually in

the United States, and cholecystectomy is performed in 700,000 cases[2].

Cholelithiasis is one of the most common disorders among patients presenting

to emergency rooms with abdominal discomfort–e.g., epigastric pain, nausea,

vomiting, and loss of appetite [3]. Gallstone pathogenesis results from

complicated interactions between genetic factors and high-carbohydrate, high-

cholesterol, or Low-fiber diets [4]. Gallstone disease and cardiovascular

disease are both common conditions and share a number of risk factors,

particularly age, obesity, diabetes, and components of the metabolic

syndrome[5]. Approximately 80 percent of gallstones contain cholesterol (as

cholesterol monohydrate crystals). The remaining 20 percent are pigment

stones, which consist mainly of calcium bilirubinate. Cholesterol-containing

gallstones are divided into two subtypes: cholesterol stones [which contain 90-

to 100-percent cholesterol) and mixed stones (which contain 50- to 90-percent

cholesterol. Each subtype may also contain varying amounts of calcium salts,

bile acids, and other components of bile [6]. Cholelithiasis (gallstone

formation) results from a combination of several factors, including

supersaturation of bile with cholesterol, accelerated nucleation of cholesterol

monohydrate in bile, and bile stasis or delayed gallbladder emptying due to


11

impaired gallbladder motility. Cholesterol supersaturation can result from an

excessive concentration of cholesterol in bile, a deficiency of substances that

keep cholesterol in solution [i.e., bile salts and phospholipids], or a

combination of these factors. Accelerated nucleation of cholesterol is a

phenomenon not well understood [7]. Gallbladder hypomotility may occur

during pregnancy, with the use of oral contraceptives, after surgery, and in

patients with diabetes [8]. However, in many cases, the cause is not clear.

While most gallstones are asymptomatic, some patients experience biliary

colic, which is characterized by sudden and severe right-upper-quadrant pain

[often accompanied by nausea and vomiting], occurring postprandially and

lasting one to four hours Acute or chronic cholecystitis may also occur in

association with gallstones.

1.2 Epidemiology:

Gallstones disease is a common disorder all over the world [9]. The prevalence of

gallstone varies widely by region. In Iraq, the prevalence of gallstone disease

reportedly ranges from approximately 2.2%in men to 4.09% in women [10].. In

Asians, Cholelithiasis is also on the rise and becoming a major health problem in

Taiwan , with some estimates indicating an increase in prevalence from 4.3% in

1989 to as high as 10.7% in 1995 [11].

In the West, 80-90% of gallstones are cholesterol stones [12] whereas, the majority

of gallstones found in Asian populations are pigment stones [high-residue pigment,

low-residue pigment, and muddy pigment types], derived largely from salts or

polymers of bilirubinate [13] Ranges from 4.21% to 11% in China [14]. The

prevalence of gallstone is higher in Iran, the prevalence in men and women in the

age group of 31-40 years was very low [0.3% in men and 1.8% in women], but it


10

increased sharply by more than 10-fold in men older than 60 years and women

over 50 years [12.5% and 24.6% in males and females aged 71-80 years,

respectively][15].

1.3 Etiology and Risk Factors: Important risk factors have been identified

as being associated with gallstones.

1.3.1 Ethnicity

Ethnicity plays an important role in the prevalence of gallstone disease and also the

type of stone that forms: cholesterol gallstones highly prevalence in the developed

countries of the Western world; brown pigment stones in the bile ducts are more

common in Asia [9]. The highest prevalence of gallstone disease has been

described in North American Indians: 64.1% of women and 29.5% of men have

gallstones. This apparent epidemic reaches a high of 73% in Pima Indian women

over age 30 [16] . Intermediate prevalence rates occur in Asian Populations and

Black Americans (13.9% of women; 5.3% Of men [17]. The lowest frequencies

occur in sub-Saharan Black Africans (<5%) [18]. The majority of gallstones in the

developed countries Consist predominantly of cholesterol (>85%), whereas the

remainder constitutes black pigment stones (i.e. composed of calcium bilirubinate)

[3].

1.3.2 Family History & Genetics

Genetic susceptibility is a key factor in gallstone formation [19]. Familial studies

reveal an increased frequency: a nearly 5 times elevated risk in the relatives of


11

gallstone patients. These rates are even higher in monozygotic twins at 12% and

dizygotic twins at 6 % [20].genetic factors play a major role in the pathogenesis of

familial gallstone disease, characterized by autosomal dominant inheritance [21].

1.3.3 Age

Gallstone detection rates increase with age, which makes it possible to consider it

one of the risk factors for gallstone [22]. Gallstone disease is rare in neonates and

young children. Once thought only related to pigment stones developing in the

setting of hemolysis, cholesterol stones are becoming increasingly more common

in children [23].

1.3.4 Gender

One of the most important risk factors is female gender. Rates of gallstones are

two to three times higher among women than men [24]. Women predominate,

particularly when young [20-30 years of age], with a range of female-to-male ratio

from 10:1 in Pima Indians to 2-3:1 in Europeans women; this ratio declines after

the fifth decade.[25] The difference between the sexes narrows with increasing

age, particularly after the menopause. Estrogen is considered to be the obvious

reason for the gender difference. The liver has estrogen receptors, and the presence

of endogenous estrogens causes cholesterol saturation in the bile, inhibition of

chenodeoxycholic acid secretion, and an increase in cholic acid level [26].

1.3.5Obesity/Metabolic Syndrome/Diabetes Mellitus/Dyslipidemia

and Rapid Weight Loss.


12

Gallbladder (GB) stone formation is a type of metabolic syndrome due to

association of GB stone disease with metabolic abnormalities such as diabetes,

dyslipidemia, obesity, and hyperinsulinemia[27]. Hyperinsulinemia and insulin

resistance are common factors linking cholesterol GB stones, diabetes, and obesity

[28]. Insulin may increase GB stone formation through a mechanism in which

insulin increases the activity of hydroxyl- 3-methyglutaryl-coenzyme A reductase

and stimulates bile acid-independent flow of bile into perfused rat liver [29]

.Obesity is a well-established risk factor for GB stone formation because this

condition increases hepatic secretion of cholesterol . The risk is particularly high

in women and increases linearly with increasing obesity [30]. Recent studies

suggest that abdominal adiposity poses even greater risk to the development of

obesity-related complications including cholesterol gallstone disease than total

adiposity [31]. Rapid weight loss on low caloric diets or following bariatric

surgery, paradoxically, is also a major risk factor for cholesterol gallstone

formation.[32] Weight loss-associated gallstones are typically asymptomatic; Only

7% to 16% develop symptoms, best predicted by a postoperative weight loss

exceeding 25% of the body weight [33].

1.3.6 Pregnancy, Parity and Female Sex Hormones

The Pregnancy and parity are two important risk factors for the formation of

cholesterol gallstones. Female sex hormones are the obvious basis for this gender

difference. It is therefore not surprising to find that parity is a risk factor [34].

During pregnancy, biliary sludge (consisting of cholesterol crystals, calcium

bilirubinate and mucin) develops in up to 30%, while gallstones form in 1-3%. The

link may be biliary sludge, a potential precursor to cholesterol gallstone

formation[35].the Underlying mechanism is female sex hormones; parity, oral


13

contraceptive use and estrogen replacement therapy are stablished risk factors for

cholesterol gallstone formation [36]. Female sex hormones adversely influence

hepatic bile secretion and gallbladder function. Estrogens increase cholesterol

secretion and diminish bile salt secretion, while progestins act by reducing bile salt

secretion and impairing gallbladder emptying leading to stasis[37] . Among several

studies investigating the number of pregnancies in general populations, increase in

gallstone prevalence was found in women who have multiple pregnancies [38].

1.3.7 Total Parentral Nutrition (TPN) and Diet

Total parental nutrition (TPN) is a well-known risk factor for developing biliary

sludge and gallstone disease, along with a calculous cholecystitis in critically ill

patients . A possible explanation for this relates to loss of the enteric stimulation of

the gallbladder in the absence of eating, leading to gallbladder stasis . Long-term

parenteral nutrition promotes gallbladder dilatation and hypokinesia and gives rise

to gallstones [39].

The diet also may play significant role in gallstone formation. A high intake of

cholesterol increases its bile level [40]. in Spain,Italian patients with gallstones

were shown to have higher intake of saturated fatty acids than controls , dietary

intake of saturated fats was positively associated with gallstones . A low-fiber diet

slows transit of the intestinal contents, which promotes the increased formation and

absorption of secondary bile acids and the enhanced lithogenic properties of bile

[41]. Refined carbohydrates intake such as sucrose and fructose is associated with

a higher frequency of gallstones [42].. Humans have found that the prevalence of

gallbladder disease (asymptomatic gallstones or history of cholecystectomy) was

significantly lower in female vegetarians than female omnivores [43].

1.3.8 Reduced physical activity


14

Several studies indicate an inverse relationship between physical activity and risk

of gastrointestinal related diseases such as cholelithiasis[44] While the prevalence

of these diseases is relatively high and increases with age, participation in physical

activity is relatively low and decreases with age[45].

Physical activity may decrease risk for symptomatic gallstone disease through

several metabolic pathways both independently and as a result of its role in

maintaining body weight [46]. Serum high density lipoprotein (HDL) cholesterol

levels, which are increased in persons who exercise regularly, are inversely

associated with gallstone prevalence [47].

1.3.9 Underlying Diseases: Cirrhosis, Crohn’s disease:

Cirrhosis

Liver Cirrhosis is a well known risk factor for gallstones particularly in the among

population with advanced age [48]. Most stones in cirrhosis are of the black

pigment type. The mechanism of stone formatiom related to altered pigment

secretion, abnormal gallbladder motility, or increased estrogen levels [49].

Crohn‘s disease increased risk of developing gallstones. Although once believed

due to bile acid malabsorption and depletion leading to cholesterol gallstones [50].

Recent studies have found an increased frequency of pigment stones. Unabsorbed

bile acids that escape into the colon function as a biologic detergent to solubilize

bilirubin and thus increase its absorption and enterohepatic cycling. The resultant

increased pigment in bile then promotes stone formation [51].

1.3.10 Drugs


15

The drugs that cause cholelithiasis are : Estrogens, prednisolone, cyclosporine,

azathioprine, sandostatin[52], clofibrate, nicotinic acid and a number of other long-

term drugs increase the risk for GD[53].

Long-term therapy with each of these agents enhances cholesterol excretion into

bile and results in its supersaturation with cholesterol through competitive

inhibition of bile acid synthesis from cholesterol on cytochrome Р450 [54].

1.4 Classification of gallstones:

1.4.1 Cholesterol stones

Cholesterol stones are single, spheroidal an Coarsely nodular and they have a

translucent bluish white color. The figure1.1 shows the shape of this stone [55].

Figure 1.1 shapes cholesterol stones[103].

1.4.2 Pigment stones


16

Pigment gallstones contain large amounts of pigment material and little

cholesterol. They can be divided into black and brown stones according to their

colors: Brown stones are brownish yellow, soft and show alternate dark

And light layers in cross-section whereas black stones are black in color, hard in

consistency and show an amorphous appearance on cross section [56].

1.4.3 Mixed stones

Mixed gallstones (Ms) typically contain 30–70%, of cholesterol according to the

Japanese classification system]. Other common constituents are calcium carbonate,

palmitate phosphate, bilirubin, and other bile pigments .the figure1.2 show the

shape of this stone [57].

Figure 1.2 shapes of mixed stones [107].

1.5 Pathophysiology of gallstone

http://en.wikipedia.org/wiki/Calcium_carbonate

http://en.wikipedia.org/wiki/Palmitate

http://en.wikipedia.org/wiki/Bilin_(biochemistry)


17

The pathogenesis of gall stone disease is suggested to be multifactorial and

probably develops from complex interactions between many genetic and

environmental factors. The pathophysiology of GS formation involves three steps:

saturation, crystallization and growth. Bile cholesterol supersaturation is an

obligatory, but not the only, factor that contributes to GS formation.as the show in

the figure 1-3 [58-60].

Figure 1.3 Pathogenesis of stone formation is different for cholesterol stones,

soft brown stones, and pigment stones [58-60].

1.6 Investigations of gallstones:

Patients with suspected gallstones are usually investigated with A blood test for

liver biochemistry , including the hepatic aminotransferases, alanine

aminotransferase, and aspartate aminotransferase, which are elevated early in the

course of biliary obstruction[61]. Later, development of a more cholestatic picture

brings about an elevation of serum bilirubin, alkaline phosphatase, and


18

GammaGlutammyl trasferase (GGT). Bogue et al. reported abnormal hepatic

aminotransferases in 51% and an elevated GGT in 45% of patients with

symptomatic disease [62].

Although changes in serum transaminases or alkaline phosphatase are relatively

non-specific, they may be the only sign of bile duct stones. Serum bilirubin is less

reliable than changes in enzyme concentration. Other techeniqe used for diagnosis

which include:

1.6.1 Ultrasonography (US):

Transabdominal ultrasonography is the initial investigation of choice because it is

simple and readily available. Gallstones within the gallbladder demonstrate a

typical echogenic acoustic shadow[63].

1.6.2 Computerize Tomography Scans (CT Scans)

More than 80% of gallstones are radiolucent, and abdominal X-ray and CT scans

play a limited role in their diagnosis. Al- though CT is known to have a limited

sensitivity of 25%–88% for direct depiction of gallstones [64].

When compared with ultrasound, CT scans are more expensive, have substantial

radiation exposure and lower sensitivity, specificity, positive, and negative

predictive values for patients with gallstone disease [65].

1.6.3 Cholescintigraphy

The patient is injected with a small amount of nonharmful radioactive material

that is absorbed by the gallbladder, which is then stimulated to contract.The test is


21

used to diagnose abnormal contraction of the gallbladder or obstruction of the bile

ducts [66].

1.7 Osteoporosis (OP):

Osteoporosis is a systemic disease of the skeleton characterized by low bone mass

and microarchitectural deterioration of bone tissue leading to an increase in

skeletal fragility and fracture risk [67]. Bone mineral density (BMD) Assessment

measured by Dual X-ray Absorptiometry (DXA) is currently the ―gold standard‖

for the diagnostic of osteoporosis, based on the World Health Organisation (WHO)

criteria, as osteoporosis defined by a T-score [i.e. standard deviation compared to

the Mean value of BMD in a young adult women population] of −2, 5 or below

[68]. However, BMD measurement does not assess all risk factors for fracture.

Skeletal turnover can be easily and non-invasively assessed by the measurement of

serum and urinary biochemical bone turnover markers (BTMs). These include the

serum bone formation markers, total osteocalcin (OC), the bone isoenzyme of

alkaline phosphatase (Bone ALP) and the C and N-propeptide of type I collagen

(PICP and PINP respectively). Bone resorption indices are either enzymes of the

osteoclastic cells such as the 5b isoenzyme of tartrate resistant acide phosphatase

(TRACP 5b) or the proteolytic fragments of bone collagen matrix including the

type I collagen crosslinks (pyridinoline, PYD and deoxypyridinoline, DPD) and

the related telopeptides [N-terminal crosslinking telopeptides of type I collagen,

NTX and C-terminal crosslinking telopeptides of type I collagen, CTX) which can

be measured in urine [u] or serum [s]. CTX peptide exists as two isomeric forms,

the native (α CTX) and the age-related (β CTX) forms. In addition to these markers

which have been widely evaluated, the resorption marker C-terminal crosslinking


20

telopeptide of type I collagen generated by matrix-metalloproteases (CTX-MMP or

ICTP) is also available [69].

Over 25 years ago, Riggs and Melton established a classification of osteoporosis

into primary and secondary forms [70]. Primary osteoporosis was further divided

into Type 1, which occurred in patients between 50 and 70 years old, and Type 2,

for patients over 70. The former type was mostly detected women with

postmenopausal osteoporosis, clearly related to the loss of estrogen with

menopause. In Type 1 osteoporosis, trabecular bone wasmore affected than cortical

bone, leading to fractures in regions high in trabecular bone content, the spine and

distal forearm. Type 2 primary osteoporosis affects both trabecular and cortical

bone, leading to hip fractures in addition to spine, forearm, rib, and humerus

Fractures [71].

Secondary osteoporosis occurs due to several causes mentioned in table 1.1

[72].Osteoporosis is correlated with aging and develops in a more gradual manner

in men.As the population continues to live longer, the effects of osteoporosis may

become more evident in older male patients [73]. Osteoporosis and particularly hip

fractures have a large economic impact; the direct costs of osteoporotic fractures in

the USA in 2005 were estimated to be $19 billion [74].

Table 1.1 Secondary Causes of Osteoporosis [72]

A-Pharmacologic or toxic treatment:

Glucocorticoids, Antiepileptics ,Aromatase inhibitors,Heparin, Alcohol, LHRH

analogues.

B-Endocrine diseases: Hypogonadism, Hyperthyroidism, Cushing‘s

disease,Growth hormone deficiency, Panhypopituitarism and


21

Hyperparathyroidism

C-Malnutrition or malabsorption: Anorexia nervosa

D-Inflammatory intestinal disease: Celiac disease, Intestinal resection

E-Chronic inflammatory disease (rheumatoid arthritis, SLE, etc.)

F-Liver disease. G- Osteogenesis imperfecta H- Transplant patients

(solid organs and bone marrow) I- HIV infection,Hemochromatosis

J-Idiopathic osteoporosis K- Osteoporosis associated to pregnancy

1.8 Bone Modeling and Remodeling

1.8.1. Normal Bone Remodeling

Being a primary structural framework of the body, bone undergoes dynamic micro

structural remodeling to accommodate mechanical stress and calcium demand [75].

Bone remodeling is a coupled process of bone resorption and formation, and

requires coordination of all three types of bone cells, namely osteocytes, steoblasts

and osteoclasts. Osteocytes play a role, forming an interconnected cellular network

that provides a basis for communication and transport between cells within the

bone matrix. Increasingly recognised additional key players in tissue turnover and

repair, including that of bone, are endothelial cells and associated pericytes of the

capillary vascular beds [76].


22

Osteoblasts later stimulate osteoclast differentiation and subsequent bone

resorption. Normally, osteoblastmediated bone formation takes place at the same

site to fill up the resorption pit with new bone [77]. Osteoclastic bone resorption

occurs in areas of structurally weak bone caused by mechanical stress or disuse. At

the cellular and molecular level, osteoclastmediated bone resorption commences

by osteoblasts initiating proliferation of osteoclast precursors and their differen

tiation into mature osteoclasts by secreting a cytokine called macrophage colony

stimulating factor (MCSF) [78]. Osteoblasts also secrete the key mediator for

osteoclastogenesis, receptor activator of nuclear factorκB (RANK) ligand

(RANKL), which binds to its receptor (RANK) on the plasma membrane of

osteoclast precursors, there by stimulating differentiation of preosteoclasts into

mature osteoclasts [79]. RANKL and MCSF are differentially upregulated by

various osteoclastogenic factors, such as parathyroid hormone (PTH), PTHrelated

peptide and prolactin [80] . Osteoprotegerin (OPG), a secreted member of the TNF

receptor superfamily, synthesize and secrete by osteoblasts acts as a natural

antagonist of RANKL , inhibit interaction with receptors and in turn prevent

osteoclastogenesis [81].

By presence of activated osteoclasts, bone resorption begins with dissolution of

inorganic and organic components by hydrochloric acid, cathepsin K and

lysosomal protease from osteoclasts [82] following bone resorption,

osteoblastmediated bone formation takes place to fill the resorption pits with

newly mineralized bone.

1.9 Pathophysiology of Osteoporosis and Bone Fragility

Bone health is maintained by a balanced remodeling process which play pivotal

role for bone health maintenance as it repairs areas of microdamage. This is a


23

cellular process as shown in (figure 1.4) involving coordinated actions of

osteoclasts [bone resorbing cells] and osteoblasts [bone forming cells], which form

the bone multicellular unit [83].

Failur the uncoupling of remodeling can result in bone fragility. The Role of

receptor activator of nuclear factorκB (RANK) ligand (RANKL), which binds to

its receptor (RANK) and osteoprotegerin RANK/RANKL/OPG and T Cells in

Bone Remodeling [84]. This osteoimmunological system determines the success

or failure of bone homeostasis. The common origin of bone and immune stem cell

is the key to understanding this system and the physiology of bone loss. It is also

the key to applying effective nutritional therapy for the inflammatory, catabolic-

based increase in bone fragility. Bone-resorbing cells (osteoclasts) and cells of the

immune system both originate in the bone marrow from hematopoietic cells.

Osteoclasts develop from Precursors of the mononuclear monocyte-macrophage

cell line after stimulation by macrophage colony-stimulating factor (M-CSF) and

receptor for activated nuclear-Factor kappa B (RANK) ligand (RANKL)

(Figure1.5)[85]. Bone-forming cells (osteoblasts) are of mesenchymal origin and

share a common precursor cell with adipocytes. During normal bone remodeling,

marrow stromal cells and osteoblasts produce RANKL, which binds to the

transmembrane receptor RANK on osteoclast precursors and induces

differentiation and activation (Figure1.6) [86]. This occurs through the

transcription factor, nuclear-factor kappa B (NFkB), which is responsible not only

for activating osteoclastogenesis but also the body‘s inflammatory response. Both

osteoclast differentiation and the inflammatory process occur via regulation of

interleukin-6 (IL-6). The major role cytokines play in bone remodeling is

demonstrated by the fact that receptors for the proinflammatory cytokines

interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α) are present on


24

both osteoclast precursor cells and mature osteoclasts. Estrogen exhibits its

nuclear regulatory effects by inhibiting IL-6 activation of NFkB during bone

remodeling [87]. Osteoblasts also produce osteoprotegerin ([OPG), a soluble decoy

receptor that blocks RANKL and maintains control of the remodeling process.

OPG is vital to the success of the RANK/RANKL/OPG system of bone

homeostasis [88]. RANKL is also produced by activated T cells. With reduced

estrogen levels and/or chronic or recurrent immune activation from either systemic

or gastrointestinal origin, there may be a reduction in the body‘s natural ability to

limit the production of RANKL [89]. This results in increased osteoclast

activation through a ―switch-like‖ diversion of osteoprogenitor-cell differentiation

away from monocyte-macrophage-cell development and toward

osteoclastogenesis. Osteoclastic activity, induced by proinflammatory cytokines

and activated T cell-induced RANKL, is thought to be modu- lated by the action

of interferon gamma (IFN-γ) on tumor necrosis factor receptor-associated factor 6

(TRAF-6) [90]. TRAF-6 is a RANK adaptor protein that mediates NFkB

activation [Figure 1.7] [91]. Activation of T cells is necessary for osteoclast di-

erentiation as the show in the [figure 1.8]. [92] RANKL activation of NFKB

through the RANK adaptor protein, TRAF6, increases osteoclastogenesis from

progenitor cells. IFN-G can either increase or limit bone resorption through

modulation of this cascade. This "fail safe" mechanism, under normal

circumstances, limits bone resorption. But with chronic T-cell activation and a

Predominate Th1 response, IFN-G no longer limits osteoclast activation and bone

resorption increases. Estrogen increases vitamin D receptor activation and

calcitonin release. It also increases osteoblast release of TGF-B, IGF-1 and OPG,

which limits M-CSF and RANKL and increases osteoclast apoptosis. With reduced

estrogen levels, TGF-B decreases and antigen presentation to T cells increases the


25

release of RANKL and TNF-α diverting progenitor cell dierentiation toward

osteoclastogenesis [93]. Vitamin D and normal gut -ora help preserve tolerogenic

dendritic cells and reduce activation of RANKL-induced osteoclastogenesis.

Figure 1.4 Bone Remodeling Process [83].


26

Figure 1.5 Osteoclasts, Immune Cells, and RBCs are Derived from Marrow

Hematopoietic Stem Cells [85].


27

Figure 1.6 Osteoblasts, Cartilage, and Adipocytes are Derived from Marrow

Mesenchymal Stem Cells [86].


28

Figure 1.7 RANK/RANKL/OPG Osteoimmunological System of Bone

Homeostasis [91].


31

Figure 1.8 Immune Activation Leads to Bone Loss [92].

1.10 Epidemiology of osteoporosis

Currently, it is estimated that over 200 million people worldwide have

osteoporosis [94]. And 44 million of these are in the United States [95].

Worldwide, osteoporosis causes more than 9 million fractures annually. These

fractures include more than 1.6 million hip fractures, 1.7 million forearm

fractures and 1.4 million clinically symptomatic vertebral fractures [96]. It is

projected that by the year 2050, the worldwide incidence of hip fractures


30

will increase by 240% in women and 310% in men [97]. The worldwide

health and economic burden of osteoporosis is likely to increase in the future, as

improvements in life expectancy will lead to a growing population of elderly

people with a high risk of fracture [98]. In the EU, the population of people aged

more than 65 years in 15member states is predicted to increase from 58million in

1995 to 108 million in 2040 [99]. In Canada, the incidence rate of hip fractures in

2005was 54.7 per 100,000 person-years for 55–64- year-old women, but was of

2636.6 per 100,000 person-years for women over 85 years [100]. A hip fracture,

the leading fracture in the elderly, has an incidence of 80,000 new cases per year in

France. This is a worldwide problem related to the aging of the population, and its

incidence is predicted to double if not triple in the coming 40 years [101].

Fracture rates are lower in Asian and black population [102].the number of hip

fractures seen in Asian population is rising; it has been estimated that by year

2050, more than half of all hip fractures worldwide will occur in Asia [103].the

projected number worldwide will be 6.3 million, with 3.2million in Asia.the

incidence of fractures in New zealand and south Africa are equal to or greater than

in men than women [104].

There is a variation in hip fracture incidence, with rates being much higher in

Caucasians living in Northern Europe and North America than in Asian and

Negroid populations. In the 1960s, the age-adjusted incidence of hip fracture in

Hong Kong Chinese was approximately 13% to 30% of that observed in

Caucasians [105]. However, with socio-economic development in many Asian

countries; the incidence of hip fractures has risen considerably. For instance, the

incidence of hip fractures in Hong Kong Chinese increased by more than 2-fold in

the last 2 decades [106]. In Singapore, the incidence of hip fracture increased from

7 per 10,000 women who were 60 years of age and older in 1957 to 15 per


31

10,000 in 1985 [107]. The results of the Asian Osteoporosis Study, which is the

first multi-centre epidemiological study conducted in Asia, confirmed that the hip

fracture incidence rates in Hong Kong and Singapore were approaching those

observed in American Caucasians [108]. Although the rates in Malaysia and

Thailand were much lower, these are likely to increase with urbanization and

ageing. According to projections by the World Health Organization, there will be a

total of 900 million men and women who are 65 years of age and older in Asia by

the year 2050. As a result, while only 30% of all hip fractures in the world

occurred in Asia in 1990, more than 50% of all hip fractures will occur in this

continent by the year 2050. By then, the total number of subjects with hip fracture

in Asia will be approximately 3.2 million per year [103].

1.11 Diagnosis and Investigation Of Osteoporosis

A major problem of osteoporosis management is that majority of those at high

fracture risk are not diagnosed or treated, despite availability of safe and effective

diagnostic tools and therapies [109]. Diagnosis of osteoporosis is based on:

1.11.1 Measurement of Bone Mineral Density (BMD):

Measurement of bone mineral density (BMD), using dual X-ray absorptiometry

(DXA), which is considered the gold standard. However, a personal history of

fragility fracture is consistent with a clinical diagnosis of osteoporosis,

regardless of BMD, in the absence of other causes of skeletal fragility. An

individual BMD value compared with the mean of a healthy young population

in terms of the number of standard deviations [SD] is termed the T-score. The

World Health Organization (WHO) has defined osteoporosis as a T-score of

less than −2.5 SD as in the table [1-3] [110]. BMD and age are not the only

factors that affect the frequency of fractures; other indicators, usually used in


32

combination with BMD assessments, include biochemical indices of bone

resorption and clinical factors such as age, previous fragility fracture, premature

menopause, family history, and use of oral corticosteroids [111]. Table 1.2

shows the World Health Organization Classification of T Score [112].

Table 1.2 the World Health Organization Classification of T Score [112].

1.11.2Assessment of Clinical risk Factors and Fracture Risk

Fracture is the outcome of multiple risk factors, and this multiplicity should be

taken into account in assessment of fracture risk for an individual. Several

important clinical risks Factors have been identified through epidemiological

studies, including age, falls and history of fragility fractures (table1.3)[113]. In

addition, several disorders and drugs can lead to increased bone loss and are

important secondary causes of osteoporosis.

Differential diagnosis includes a thorough medical history, physical examination

and a range of investigative tests which may be case-dependent, all these show in

the table 1.4.[114].


33

[Table 1.3] Clinical Risk Factors for Osteoporosis and ractures [113]

Age

Sex

Ethnicity

Femoral neck bone mineral density

Body weight

History of prior fractures

Height

Parental history of hip fracture

Current smoking

Glucocorticoid therapy

Alcohol consumption [3 or more units per day]

Rheumatoid arthritis

History of falls

Secondary osteoporosis (eg, hyperthyroidism, hypogonadism


34

or premature menopause, malabsorption, chronic liver

disease, inflammatory bowel disease)

Medications (eg, immunosuppressants, antiseizure medica-

tions, heparin, chemotherapy)

Table 1.4 Laboratory Evaluations for Secondary Causes ofOsteoporosis[114]

[114]

Initial laboratory tests

Renal profile (Urea, Creatinine) , Calcium, phosphorus, magnesium

Liver function tests

Full blood count

25 Hydroxyvitamin D, Thyroid stimulating hormone,Parathyroid hormone

Additional laboratory tests if indicated

Sex hormones (Testosterone, Oestradiol, LH, FSH)

Coeliac serology, Serum/Urine protein electrophoresis, Erythrocyte

sedimentation rate

24-h Urine calcium/Creatinine


35

Bone turnover markers, 24-h Urine free cortisol, Prolactin, Iron studies

Serum tryptase and histamine levels, Homocysteine, Rheumatoid factor

Skin biopsy for connective tissue disorders

LH, luteinising hormone; FSH, follicular stimulating hormone

In order to assist clinicians in their clinical management process, the WHO task

force has developed and introduced a country-specific Fracture Risk Assessment

Tool (FRA), based upon data collected from large international cohorts in which

clinical risk factors, BMD and fractures were evaluated. The tool combines BMD

measurement and clinical risk factors to derive a 10-year probability of hip fracture

or major osteoporotic fracture [combined hip, spine, humerus or wrist], thus

allowing identification of individuals at high fracture risk [115].

1.11.3 Complete Blood Count and Chemistry Profile:

A complete blood count (CBC) and chemistry profile provide the clinician with a

general survey of multiple organ systems. For example, a mild decrease in albumin

coupled with hypocalciuria may indicate malabsorption [116]. a low red blood cell

[RBC] count may be secondary to the effects of elevated proinflammatory

cytokines or the reduced hematopoietic capability of the osteoporotic patient‘s

fat-infiltrated bone marrow [117].

Alkaline phosphatase (ALP) is an enzyme found in bone, liver, intestine, kidneys,

and placenta. Although it is an indicator of osteoblastic activity, ALP is not


36

specific to bone tissue and is therefore not typically used in the management of

osteoporosis. ALP may be normal or increased in postmenopausal women [118]

and may be reduced in celiac disease, hypothyroidism, pernicious anemia, or zinc

deficiency [119]. Elevated ALP levels may also be an indication of cancer

metastasis to the liver or bone.

1.11.4 Bone-Turnover Biomarkers

1.11.4.1Bone Resorption Markers

Serum biochemical bone turnover markers [sBTM] are used in the management of

bone diseases including postmenopausal osteoporosis [PMO]. They allow a

dynamic assessment of bone remodeling, as they reflect bone cell activity. A high

bone remodeling, as reflected by a high sBTMlevel, is associated with accelerated

bone loss and thus can be associated with bone fragility in some patients [120].

Among bone resorption markers, serum C-telopeptide cross-link of type 1 collagen

([sCTX) is a highly sensitive indicator of bone resorption. SerumCTX is released

fromcathepsin K-mediated proteolytic degradation of α1- chain C telopeptide of

type 1 collagen during osteoclastic resorption and is assessed by ELISA. sBTM

assessment has several advantages over urinary markers of type I collagen

degradation such as uCTX, uNTX [N-telopeptide of type I collagen], or

deoxypyridinoline. First, it does not require urinary creatinine measurement.

Second, it is available for standard clinical laboratory as either a manual ELISA

[121] or an immunochemiluminescent assay adapted on fully automated platforms,

and is more convenient to use than manual assays, with improved analytical

performances and higher throughput [122]. Third, reference ranges established in

large and well-documented populations of healthy premenopausal women are

available [123,124]. Fourth, sCTX has both short-term and long-term within-


37

subject variability, which is approxi mately twice lower than urinarymarkers

[around 10-15% compared with around 20-30%] [125].

Elevated levels of resorption markers indicate increased osteoclastic activity and

a higher risk for osteoporotic hip fracture, independent of BMD [126,127]. Even

when BMD is not in the osteoporotic range, increases in urine N-Tx [cross-links of

N-terminal telopeptide of type-1 collagen] and/or Dpd indicate increased

osteoclastic-bone resorption and risk for fracture [116]. C-telopeptide (C-Tx) is a

serum marker for C-terminal telopeptide of type-1 collagen used predominately in

Europe.

1.11.4.2 Bone Formation Markers

Among bone formation markers, N-terminal and C-terminal propeptides of type I

procollagen (PINP and PICP) are cleaved during procollagen extracellular

metabolism and are released in the blood. The intact form of PINP presents the

advantage of not being affected by glomerular filtration rate [128]. Bone ALP and

P1NP are considered early markers of formation, while osteocalcin, which is

greatly influenced by genetics [129] is a later marker of osteoblastic activity;

osteocalcin, although related to fracture risk,[130] is a less responsive indicator.

Serum concentration of P1NP is directly proportional to the amount of new

collagen produced by osteoblasts [131]. P1NP is useful for assessing bone turnover

in postmenopausal women and is the best marker for monitoring patients on

teriparatide (recombinant human PTH) therapy [132].

1.12 Treatment Of Osteoporosis

1.12.1 Non-pharmacological Intervention


38

This includes modification of general lifestyle factors, such as balanced diet

containing calcium and vitamin D, smoking cessation and avoidance of heavy

alcohol use. A regular exercise routine should be encouraged, including weight-

bearing and muscle-strengthening exercises [133]. The role of calcium and vitamin

D supplementation in reducing falls and fractures are not clear. A reduction in

calcium intake or absorption and/or vitamin D deficiency/insufficiency leads to

secondary hyperparathyroidism, which contributes to accelerated bone loss in the

elderly [134]. the greatest effect being with a daily dose of 1200 mg calcium and

800 IU vitamin D . It has been shown that an adequate calcium intake (1000–

1500 mg/day) reduces bone loss in adults,[135] while vitamin D is useful

for calcium [136] intestinal absorption, so calcium and vitamin D are essential

in maintaining bone homeostasis [137]. It has been shown that vitamin D

deficiency is more evident in the elderly population [138].

1.12.2 Pharmacologic Therapies For Osteoporosis

The available antiosteoporosis treatments include bisphosphonates, selective

estrogen receptor modulators (SERMs), calcitonin, strontium ranelate, and agents

derived from parathyroid hormone (PTH). Fig.1.10 illustrates the mechanism of

action of these antiosteoporosis medications [139].


41

Figure 1.9 Mechanisms of action of antiosteoporosis treatments [139].

1.12.2.1Anti-Resorptive Therapy

I-Bisphosphonates

Bisphosphonates are the most widely used antiresorptive agents for treatment of

osteoporosis. Bisphosphonates are synthetic analogues of naturally occurring


40

inorganic pyrophosphate, Bisphosphonates bind strongly to mineral especially at

sites of active bone remodelling, where they are then taken up by boneresorbing

osteoclasts [139]. The most potent bisphosphonates are nitrogen-containing

(amino-) bisphosphonates (alendronate,risedronate, ibandronate, pamidronate and

zoledronate), which inhibit the farnesyl pyrophosphate (FPP) synthase, an enzyme

in the mevalonate pathway, and prevent prenylation of small guanosine

triphosphate (GTP)-binding proteins (GTPases), which are essential for osteoclast

function and survival [140]. Non- nitrogen containing

bisphosphonates(etidronate, tiludronate) are metabolized to cytotoxic ATP

analogues,which induce osteoclast apoptosis. Thus bisphosphonates reduce bone

turnover allowing for more complete mineralization of existing [but reduced] bone

tissue mass, which is reflected in an increase in BMD observed in clinical trials,

however they do notincrease bone tissue mass per se [in contrast to anabolic

agents] [140].

II-Selective Estrogen Receptor Modulators (SERMs)

Selective oestrogen receptor modulators (SERMs) are compounds which in general

act as oestrogen agonists on bone and oestrogen antagonists on breast and brain

tissue. The effect on the uterus can be neutral, as seen with raloxifene, or

antagonistic in the presence of oestrogen, as seen with bazedoxifene.Other

available drug is raloxifene, which is somewhat less effective than

bisphosphonates, though direct comparison trialsare lacking [141]. Raloxifene is

associated with an increased risk of venous thromboembolic events, particularly

during the first 4 months of treatment, and it can cause or exacerbate vasomotor

symptoms associated with menopause. While there aredata to suggest raloxifene

may be effective in men, it is not currently recommended for use in men . In the


41

UK, raloxifene is not recommended for primary prevention of postmenopausal

osteoporosis but can be used in secondary prevention as alternative treatment to

patients intolerant to or who have contraindications to bisphosphonate.

III-Strontium Ranelate

Strontium ranelate (2 g/day) is now considered as an effective Anti-osteoporotic

drug developed for the treatment of postmenopausal osteoporosis [142]. In vitro

and in vivo experimental studies [1–9] have demonstrated that strontium

ranelate has a dissociating effect on bone remodeling by maintaining bone

formation and decreasing bone resorption, resulting in an increase in bone

mass and strength [143]. Current pharmacological studies showed that strontium

activates multiple signaling pathways in bone cells to achieve its pharmacological

actions. Notably, activation of the calcium-sensing receptor by strontium in

osteoclasts or osteoblasts leads to activation of phospholipase Cβ, inositol 1,4,5-

triphosphate,release of intracellular Ca2+, and activation of Extracellular-signal-

regulated kinase ½—Mitogen-activated protein kinase (MAPK ERK1/2)and

Nuclear factors of activated T-cells; Wnt/NFATc signaling [144,145]. Strontium-

mediated activation of these pathways results in the modulation of key molecules

such as RANKL and OPG that control bone resorption, and to the regulation of

genes promoting osteoblastic cell replication, differentiation and survival [146].


42

IV-Cathepsin K inhibitors

Cathepsin K inhibitors blocking a 24 kDa cysteine protease of the papain

superfamily, which is expressed by osteoclasts reduce collagen type I, collagen

type II [cartilage] and bone matrix protein degradation [147]. Nevertheless,

cathepsin K expression has been observed in many tissues, including osteoclasts,

ovary, colon and other sites [148].

Odanacatib has been claimed to offer a better specificity to cathepsin K as

compared to other inhibitors and, therefore, a better toxicity profile [149].

Cathepsin K inhibition appears to suppress osteoclastic function but preserves the

cells viability.

V-Calcitonin

calcitonin is produced by the parafollicular cells (C cells) of the thyroid gland and

this polypeptide hormone is secreted in response to elevated calcium serum levels

[88]. sCT therapy inhibits or slows osteoclast-mediated resorptive bone loss .

Calcitonin antiresorptive activity is particularly mediated through an inhibition of

osteoclastic bone resporption [150] Calcitonin has been reported to reduce the

activity of active osteoclast and an algesic effects have lately been described as

well as efficacy against cartilage erosion in ovariectomized rats [151]. A targeting

to osteoclasts would intuitively appear reasonable. calcitonin is well tolerated, such

that targeting for toxicological reasons lacks an immediate rationale. A conjugate

system with a Bisphosphonates has been successfully developed [152].


43

VI-Denosumab

Denosumab is a monoclonal antibody binding to receptor activator of NF-κB

ligand (RANKL) and, thereby, preventing the binding of RANKL to RANK, a cell

surface receptor on osteoclasts and preosteoclasts [153]. Denosumab – like

RANKL's natural decoy receptor osteoprotegerin (OPG) – interferes with these

processes. RANKL is secreted by osteoblasts, T, and B lymphocytes and stromal

bone marrow cells and blockade by denosumab reduced bone resorption to what

was observed for the alendronate and fracture rate[154].

1.12.2.2 Anabolic Therapy

Parathyroid hormone (PTH) is an 84-amino acid peptide hormone and is

synthesized and secreted by parathyroid glands. PTH regulates the calcium-

phosphate metabolism in response to the low serumcalcium (Ca2+

) levels and

elevates phosphate levels sensing through the calcium-sensing receptors (CaSR)

[155]. In response to the low Ca2+ levels in blood PTH is secreted by the chief

cells of parathyroids glands to increase the Ca2+ levels by increasing the

reabsorption of Ca2+from the kidneys and intestine and mobilizing Ca2+ from the

skeleton. PTH promotes the synthesis of 1, 25(OH) vitamin D3 thus enabling the

increase absorption of Ca2+ from the intestine. Therefore PTH through its receptor

PTH1R plays a very crucial role in maintenance of Ca2+ homeostasis.

Hypersecretion of PTH as occurs in primary hyperparathyroidism cause

hypercalcemia and osteopenia in bone showing the catabolic effect of PTH. On the

other hand intermittent subcutaneous administration of low doses of PTH leads to

bone formation and increase bone mass in osteporotic patients through its action on


44

osteoblasts showing its anabolic role. Therefore PTH is considered an endogenous

peptide hormone having both catabolic and anabolic effects on bone [156].

1.13 Proinflammatory Cytokines Increase Bone Resorption

Bone is a dynamic organ that continuously undergoes significant turnover, a

process called modeling/remodeling involving bone resorption by osteoclasts and

bone formation by osteoblasts . Therefore, bone mass at any particular time reflects

the balance between bone formation and resorption [157]. At the cellular level,

osteoblast number and activity decrease while osteoclast number and activity

increase with aging [158]. It is now established that osteoblasts regulate the

recruitment and activity of osteoclasts through the expression of the receptor

activator of NF-B ligand (RANKL) and osteoprotegerin (OPG) . RANKL is

expressed on the osteoblast/ stromal cell surface and binds to its receptor RANK,

on the surface of hematopoietic precursor cells to stimulate osteoclast

differentiation and maturation in the presence of macrophage colony stimulation

factor (M-CSF) [159]. OPG, a decoy receptor secreted by osteoblasts, binds

RANKL to prevent the activation of RANK and, therefore, to prevent osteoclast

differentiation and activation [160]. It has been demonstrated that increased

steoclastic activity and increased bone resorption in postmenopausal women is

positively correlated with the upregulation of RANKL[161].

Proinflammatory cytokines including TNF- , IL-1, and IL-6 are key mediators in

the process of osteoclast dif ferentiation and bone resorption. Experimental studies

in animals have provided substantial evidence suggesting that certain inflammatory


45

ctokines, including IL-1, IL-6, and TNF-, play an important role in the

pathogenesis of osteoporosis [162]. However, evidence that these inflammatory

cytokines play a similar role in human studies is limited [163]. Recently, growing

understanding of the bone remodelling process suggests that factors involved in

inflammation are linked with those critical for bone physiology and remodelling,

supporting the theory that inflammation significantly contributes to the

aetiopathogenesis of osteoporosis [164,165]. It has also been established that

upregulated proinflammatory cytokines are primary mediators of osteoporosis. The

accelerated bone loss at menopause is linked to increased production of

proinflammatory cytokines including TNF- , IL-1, and IL-6 [166]. These

proinflammatory cytokines are capable of stimulating osteoclast activity through

the regulation of the RANKL/RANK/ OPG pathway [167].

1.14 Aims of Study

1- To investigate possible association between gallstone and developing the

osteoporosis.

2- To study the biochemical changes in some parameters [ Interleukines1 and 6

(IL-1 and IL-6), TNF-α, hs - CRP, Vit.D, PTH, Ca++

, inorganic phosphate, C-

terminal telopeptide of type I collagen, Carboxyterminal propeptide of type I

procollagen, free teststeron and estradiol.) in symptomatic gallstone patients

compared to controls, each parameter may play a role in the pathogenesis of

gallstones.

3- To confirm some risk factors for devloping symptomatic gallstone

disease(gender,age,pregnancy,ethinicity,gentic,diabetes,metabolic

syndrome,diet,drugs like cefatrixone,liver disease like cirrhosis and total

parenteral nutrition.


46

4- To find possible correlation between demographic data and its effect on

gallstone formation.

Chapter Two : Materials and Methods 58

Chapter Two

Subjects and Methods

Subjects and Methods

2.1 Subjects:

2.1.1 Patients and Subjects:

The present work was carried out at Al- Basra General Hospital from January 2012

until May2012. One hundred patients were participated in present study including

(90 females&10males). The age range was (18-38) years. Apparently healthy

subjects were selected to participate as a normal group for comparison (control)

with age group and sex (90female &10male) matching of patients. The age range

of these subjects was (19-40). Diagnosis was made by a specialist surgeon.

Depends on the presence of typical symptoms and the demonstration of stones in

gallbladder on diagnostic imaging. An abdominal ultrasonography is the standard

diagnostic test for gallstone detection.


2.1.2 Inclusion Critria:Only patients with cholelithiasis (non complicated

cases) were selected for surgerywere included in the present study.

2.1.3 Exclusion Criteria:

1. Complicated gallstone diseases like (acute cholecystitis, pancreatitis).

2. Patients with chronic disease like liver disorders, and cardiovascular

complications.

3. Patients with a history of alcohol consumption.

4. Subjects suffered from diseases (hypertension, asthma and diabetes

mellitus,chronic renal failure, Cushing syndrom.

5. A history of ceftriaxone Drugs induced osteoporosis (cortecosteroides,loop

diuretics,ect).

6. Drugs used in prevention osteoperosis like calcium and vit.D supplements.

7. Total parenteral nutrition (TPN),somatostatin and antioxidant intake that

may interfere with the data obtained were also excluded.

8. A questionnaire was designed to obtain the information from the patients

and control groups. It contained the name, age,

weight,genederheight,waist,hipe occupation, family history,drug history,

renal stones, parity, and contraceptive use (last two only for females).

Ultrasound for patients with cholelithiasis and urolithiasis were checked to

confirm the presence of calculi. All the laboratory investigations were done

in Laboratory, Biochemistry and Immunity Departments, in AL-Basra

general hospital.

2.2 Chemicals:

The following diagnostic kits and chemicals were used in the study are listed

with their suppliers in table (2.1) below:


Table (2.1) Diagnostic Kits and Chemicals with Their Suppliers.

Supplier

kit or chemical

Diagnostic Kits

Myobiosource (Denmark) Carboxyterminal propeptide of type I

procollagen

Myobiosource (Denmark) C-terminal telopeptide of type I collagen

Biocheck ESRTADIOL(E2)

DIA METRA(Italy) Free teststeron

Monobind Inc. USA hs C-reactive protein ELISA Kit

BIOLABO(France) Inorganic phosphate

ANOGEN Interleukine 1(IL-1)

ANOGEN Interleukine 6(IL-6)

EUROIMMUN(U.K) 25-OH Vitamin D Highly sensitive C-

reactive protein

DRG(U.S.A) Parathyroid hormone intact

Randox (U.K) Serum calcium

ANOGEN Tumor Necrosis Factor Alpha (TNF- α)


2.3. Instruments:

The instruments that are utilized throughout the study are listed in table (2.2)

with their suppliers:

Table (2.2) Instruments with Their Suppliers

Suppliers

Instruments

Hettich 210,Germany Centrifuge

Bio -Tek instruments, INC. USA ELx 800 universal micro plate reader

Bio -Tek instruments, INC. USA ELx 800 universal micro plate washer

Fisher scientific, Germany Isotemp incubator

Teknolabo A.S.S.I.S.R.I. Italy Kahn-shaker

Eppendorf, Germany Micropipette 5-50µL

Brand, transferpette, Germany Micropipette 10-100µL

Slamedautoclvable, Germany Micropipette 100-1000µL

Apel PD 303,Japan Spectrophotometer

K&K scientific supplier, Korea Water bath.


2.4. Blood Specimens:

Ten milliliters of venous blood sample were drawn from each patient after

12 hours fasting. Blood specimens were collected from patients and healthy

persons. The sample was transferred into a clean plain tube, left at room

temperature for at least 30 minutes for clotting, centrifuged, then serum was

separated to be used for measuring the following variables: IL-1, IL-6, TNF-α, hs -

CRP,Vit.D,PTH,Ca++

,inorganic phosphate,C-terminal telopeptide of type

Icollagen, Carboxy terminal propeptide of type I procollagen,free teststeron and

estradiol.

2.5. Biochemical Assessment Method

2.5.1. Estimation of High sensitivity C-reactive protein (hs-CRP):

Principle of Assay

The hs-CRP ELISA Kit is based on the principle of a solid phase enzyme –

linked immunosorbent assay [168].

The assay system utilizes a unique monoclonal antibody directed against a

distinct antigenic determinant on the CRP molecule. This mouse monoclonal anti –

CRP antibody is used for solid phase immunization (on the micro titer wells). A


goat anti – CRP antibody is used in the antibody – enzyme (horse radish

peroxidase) conjugate solution. The test sample is allowed to react simultaneously

with the two antibodies, resulting in the CRP molecule being sandwiched between

the solid phase and enzyme – linked antibodies. After 45 minute incubation at

room temperature, the wells are washed with water to remove the unbound labeled

antibodies. A tetramethylbenzidine (TMB) reagent is added and incubated for 20

minutes, resulting in the development of a blue color. The color development is

stopped with the addition of 1N HCL changing the color to yellow. The

concentration of CRP is directly proportional to color intensity of the test sample.

Absorbance is measured spectrophotometrically at 450nm by comparison to

standards of hs-CRP. The values were expressed as (mg/l).

2.5.2. Estimation of Tumor Necrosis Factor Alpha (TNF-α)

Principle of Assay

TNF-α -ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay

performed on microtiterplate. Theassay uses monoclonal antibodies (MAbs)

directed against distinct epitopes of TNF-α. Standards and samples react withthe

capture monoclonal antibody (MAb 1) coated on microtiter well and with a

monoclonal antibody (MAb 2) labeled with horseradish peroxidase (HRP). after an

incubation period allowing the formation of a sandwich: coated MAb 1 –human

TNF-α – MAb 2 – HRP, the microtiterplate is washed to remove unbound enzyme

labelled antibody.Boundenzyme-labelled antibody is measured through a

chromogenic reaction. Chromogenic solution (TMB) is added andincubated. The

reaction is stopped with the addition of stop solution and the microtiterplate is then

read at theappropriate wavelength. The enzyme-substrate reaction is terminated by

the addition of a sulphuric acid solution and the color change is measured


spectrophotometrically at a wavelength of 450 nm ± 2 nm.A calibration curve is

plotted and TNF-α concentration in samples is determined by interpolation from

the calibrationcurve. The use of the EASIA reader (linearity up to 3 OD units) and

a sophisticated data reduction method(polychromatic data reduction) result in a

high sensitivity in the low range and in an extended calibration range Values are

expressed a pg/l[169].

2.5.3. Estimation of Interleukine-1(IL-1):

Principle of Assay

The microtiter plate provided in this kit has been pre-coated with

anAntibodyspecificto IL-1α. Standards or samples are then added toThe

appropriate microtiter plate wells with a biotin-conjugatedAntibody preparation

specific for IL-1α and Avidin conjugated toHorseradish Peroxidase (HRP) is added

to each microplate wellAndincubated. Then a TMB (3, 3', 5,5'tetramethyl-

benzidine)Substrate solution is added to each well. Only those wells thatContain

IL-1α, biotin-conjugated antibody and enzyme-conjugatedAvidin will exhibit a

change in color. The enzyme-substrate reactionis terminated by the addition of a

sulphuric acid solution and thecolor change is measured spectrophotometrically at

a wave length of 450 nm ± 2 nm. The concentration of IL-1α in the samples isthen

determined by comparing the O.D. of the samples to thestandard curve.The values

were expressed asa pg/l[170].

2.5.4. Estimation of Interleukine-6(IL-6):

Principle of Assay

The micro titer plate provided in this kit has been pre-coated with anantibody

specific to IL-6. Standards or samples are then added to theappropriate micro titer

plate wells with a biotin-conjugated polyclonalantibody preparation specific for IL-


6 and Avidin conjugated to HorseradishPeroxidase (HRP) is added to each micro

plate well and incubated. Then aTMB (3, 3', 5, 5' tetramethyl-benzidine) substrate

solution is added to eachwell. Only those wells that contain IL-6, biotin-conjugated

antibody andenzyme-conjugated Avidin will exhibit a change in color.

Theenzyme-substrate reaction is terminated by the addition of a sulphuric

acidsolution and the color change is measured spectrophotometrically at

awavelength of 450 nm ± 2 nm. The concentration of IL-6 in the samples isthen

determined by comparing the O.D. of the samples to the standardcurve.The values

were expressed asa pg/l [171].

2.5.5. Estimation of Serum Free Testosterone:

Principle of assay

Free Testosterone (antigen) in the sample compets with horse radish

peroxidasetestosterone(enzyme labeled antigen) for binding on to the limited

number of anti-testosterone (antibody) sites on the microplates (solid phase).After

incubation the bound/free separation is performed by a simple solid-phase

washing.The enzyme substrate (H2O2) and the TMB-Substrate(TMB) are added.

After an appropriate time has elapsed for maximum color development, the

enzyme reaction is stopped and the absorbance is determinated. Free Testosterone

concentration in the sample is calculated based on a series of standard.The color

intensity is inversely proportional to the Free Testosterone concentration in the

sample(Read the absorbance (E) at 450 nm against Blank).Testosterone in the

blood is bound to SHBG (60 %) and in lower quantity to other protein. Only the

measurement of Free Testosterone (< 1% of Total Testosterone) permits the

estimating of the hormone biologically active. The values were expressed as

apg/ml [172].


2.5.6. Estimation of Serum Esradiol:

Principle of assay

The estradiol (E2) Enzyme Immunoassay( E2 EIA) is based on the principle of

competitive binding betweenE2 in the test specimen and E2-HRP conjugate for a

constant amount of rabbit anti-estradiol. In the incubation, goat anti-rabbitIgG-

coated wells are incubated with 25 µL E2 standards, controls,patient samples, and

100µL estradiol-HRP Conjugate Reagent and 50µLrabbitanti-Estradiolreagentat

room temperature (18-25oC) for90 minutes. During the incubation, a fixed amount

of HRP-labeledE2 competes with the endogenous E2 in the standard, sample, or

quality control serum for a fixed number of binding sites of the specific E2

antibody. Thus, the amount of E2 peroxidase conjugates immunologically bound to

the well progressively decreases as the concentration of E2 in the specimen

increases. Unbound E2peroxidase conjugate is then removed and the wells washed.

Next,a solution of TMB Reagent is added and incubated at room temperature for

20 minutes, resulting in the development of blue color. The color development is

stopped with the addition of 1NHCl, and the absorbance is measured

spectrophotometrically at 450nm. The intensity of the color formed is proportional

to the amount of enzyme present and is inversely related to the amount of

unlabeled E2 in the sample. A standard curve is obtained by plotting the

concentration of the standard versus the absorbance. The E2 concentration of the

specimens and controls run concurrently with the standards can be calculated from

the standardcurve. The values were expressed aspg/ml [173].

2.5.7. Estimation of Serum 25(OH)Vit D:

Principle of assay


The assay utilizes of a competitive ELISA technique with a selected

monoclonal antibody recognizing 25(OH)-vitamin D. For a reliable

determination of 25(OH)-vitamin D, it is necessary to release it from the

25(OH)-vitamin D-DBP-complex. Standards, controls and patient samples

which are assayed for 25(OH)-vitamin D are incubated with the releasing

reagent. The pre-incubated solutions are then transferred to the microplate

coated with 25(OH)-vitamin D, and an anti-25(OH)-vitamin D antibody is

added. During an overnight incubation step, 25(OH)-vitamin D in the sample

and a fixed amount of 25(OH)-vitamin D bound to the microtiter well compete

for the binding of the antibody. Then aperoxidase-conjugated antibody is added

into each microplate well. A complex of 25(OH)-vitamin D - anti-25(OH)-

vitamin D antibody – peroxidase conjugate is formed. Tetra methyl benzidine

(TMB) is used as a peroxidase substrate. Finally, an acidic stop solution is

added to terminate the reaction, whereby the color changes from blue to yellow.

The intensity of the yellow color is inversely proportional to the concentration

of 25(OH)-vitamin D. A dose response curve of the absorbance unit (optical

density, OD at 450 nm) vs. concentration is generated using the values obtained

from the standard. 25(OH)-vitamin D in the samples is determined from this

curve. The values were expressed as ng/ml [174].

2.5.8. Estimation of Parathyroid Hormone:

The DRG intact PTH immunoassay is a two-site ELISA for the

measurement of the biologically intact 84 amino acid chain of PTH [175]. Two

different goat polyclonal antibodies to human PTH have been purified by affinity

chromatography to be specific for well defined regions on the PTH molecule. One

antibody is prepared to bind only the mid regions and c-terminal PTH 39-84 and


this antibody is biotinylated. The other antibody is prepared to bind only the N-

terminal PTH 1-34 and this antibody is labeled with horseradish peroxidase [HRP]

for detection. Streptavidin well biotinylated Anti-PTH(39-84)-intact PTH-HRP

conjugated Anti-PTH(1-34) although mid region and c-terminal fragments are

bound by the biotinylated anti-PTH(39-84), only the intact PTH 1-84 forms the

sandwich complex necessary for detection. The capacity of the biotinylated

antibody and the streptavidin coated microwell both have been adjusted to exhibit

negligible interference by inactive fragments, even at very elevated levels. In this

assay, calibrators, controls, or patients samples are simulataneously incubated with

enzyme labeled antibody and biotin coupled antibody in a streptavidin-coated

microplate well.At the end of the assay incubation, the microwell is washed to

remove unbound component and the enzyme bound to the solid phase is incubated

with substrate, tetramethylbenzidine [TMB]. An acidic stopping solution is then

added to stop the reaction and converts the color to yellow. The intensity of the

yellow color is directly proportional to the concentration of the intact PTH in the

sample. Absorbance was measured spectrophotometrically at 450nm. And the

results were expressed as pg/ml.

2.5.9. Estimation of Serum Total Calcium:

The method is based on the specific binding of o-cresolphtaleine

complexone(o-cc) a metallo chromic indicator,and calcium at alkalin PH with the

resulting cromophore was determined at 570nm [176].The intensity of the

cromophore formed is proptional to the concentration of total calcium in the

sample .

O-cc+ calcium PH 10.7

OCC –calicum complex


The results expressed as mg/dl.

2.5.10. Estimation of Serum Inorganic Phosphate

Inorganic phosphate can react with ammonium molybdate in the presence of

sulfuric acid to form a phosphmolybdate complex [177] .The resultant colour of

molybdenum blue is proportional to the phosphorus concentration present in the

sample . And read at 680 nm, the result expressed as mg/dl.

PO4 +H+ + (NH4)6MO7 O24 Phosphomolybdic complex

Phosphomolybdic complex PH10

molybdenum blue

Reductant

2.5.11. Estimation of Serum Human C terminal Telopeptides of

types I Collagen:

Principle of assay:

ThisCTX1 enzyme linked immunosorbent assay applies a technique called a

quantitative sandwich immunoassay [178]. The microtiterplate provided in this kit

has been pre-coated with a monoclonal antibody specific forCTX1. Standards or

samples are then added to the microtiter plate wells andCTX1 if present, will bind

to the antibody pre-coated wells. In order to quantitatively determine the amount

ofCTX1 present in the sample, a standardized preparation of horseradish

peroxidase (HRP)-conjugated polyclonalantibody, specific forCTX1 are added to

each well to ―sandwich‖ theCTX1 immobilized on the plate. The microtiter plate


undergoes incubation, and then the wells are thoroughly washed to remove all

unbound components. Next, A and B substrate solution is added to each well.

The enzyme (HRP) and substrate are allowed to react over a short incubation

period. Only those wells that containCTX1 and enzyme-conjugated antibody will

exhibit a change in color. The enzyme-substrate reaction is terminated by the

addition of a sulphuric acid solution and the color change is measured

spectrophotometrically at a wavelength of450 nm. And the results were expressed

as ng/ml.

2.5.12. Estimation of Serum Human carboxy terminal propeptide of

type I procollagen(PICP):

Principle of assay:

The microtiter plate provided in this kit has been pre-coated with an antibody

specific to PICP. Standards or samples are then added to the appropriate microtiter

plate wells with abiotin-conjugated antibody preparation specific for PICP and

Avidin conjugated to Horseradish Peroxidase (HRP) is added to

each microplate well and incubated. Then a TMB (3, 3', 5, 5'tetramethyl-benzidine)

substrate solution is added to each well.Only those wells that contain PICP, biotin-

conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color.

The enzyme-substrate reaction is terminated by the addition of a sulphuric acid

solution and the color change is measured spectrophotometrically at a wavelength

of 450 nm. The concentration of PICP in the samples is then determined by

comparing the O.D. of the samples to the standard curve [179]. The results in the

present study were expressed as ng/ml.

2.6. Statistical Analysis:


The statistical analysis was done by using Microsoft excels 2010; our results

included:

1- Mean ± Standard error of the mean.

2- The student's t test was used to determine the significant difference in

means of each two groups.

3- Pearson correlation (r).The results of analysis with (P) values <0.05 was

considered significant [180].

Chapter three : Results 27

Chapter three

Results

3.1. Demographic Characteristics of Cholelithiatic Patients & Controls:

Demographic presentation of 100cholelithiatic-patients & 100 healthy

controls was elucidated in table (3-1).

Table 3.1: Demographic Data of Cholelithiatic Patients & Controls.

Characters

Groups

Patients

N=100(%)

Controls

N=100(%)

Gender

Female 90 (90%)

90 (90%)

Male 10 (10%) 10 (10%)

Female/male 9/1 9/1

Number of children*

No. ≥ 4

No. < 4

58(64.44%)**

32(35.55%)

55(61.11%) 35(38.88%)

Contraceptive usage*

User

Non-user

55(61.11%)**

35(53.03%)

39(43.33%)

51(56.66%)

Family history // Positive

Negative

35(38.88%) * 65(72.22%)

20(20%) 80(80%)

Occupation// Employed

Unemployed

36(36%)

64(64%)

27(27%)

73(73%)

Renal stone

Present

Absent

25(25%)*

75 (75%)

8 (8%)

92(92%)

Body mass index(BMI) 34.4±0.9* 28.7±0.5

Waist/hip ratio(WHR)\ 1.035±0.01* 1.001±0.01


*significantly different as compared to control (p<0.05)

** significantly different at (p<0.05) when compare Patients and control are

females.

3.2. Age

In the present study,as summarized in table(3.2) and figure (3.1), the Results of

the study showed no significant difference (P > 0.05) regarding the mean age

between patients (34.5 ± 1.1) and controls (35.4 ± 1.1).

Table 3.2 Show Age in years, Body mass index and waist –hip ratios

for control and patient groups; data are expressed as mean + standard

error of mean.

P value patients

(N=100) control

(N=100)

Groups

P=0.28 34.5 ± 1.1 35.4 ± 1.1 AGE(years)

p<0.001 34.4 ±0.9* 28.7± 0.5 Body mass

index (BMI)

p<0.001 1.035 ±0.01* 1.001 ± 0.01

Waist to hip

ratio

(WHR)

*Significantly different( p<0.05) compared to control group


Figure 3.1 histogram show average Age (in years) , for both control and study

groups.

*Significant if p value <0.05

3.3. Body Mass Index (BMI):

Table (3-2) and figure (3-2) showed that body mass index (BMI) was

significantly higher P<0.05 in patients (mean ±SEM34.4 ±0.9) than the controls

(28.7± 0.5), it also showed that the highest percentage of patients are obese while

most of the controls are with normal weight.

35.4

34.5

32

32

33

33

34

34

35

35

36

36

37

37

Control group Study group

Age

in y

ears

Age

Age


Figure 3.2 Histogram show difference in BMI, for both control and study

groups.


3.4. Waist to Hip Ratio (WHR):

Table (3-2) and figure (3-3) showed that waist to hip ratio was significantly

higher (P<0.001)in patients (1.035 ±0.01) than controls (1.001 ± 0.01), it also

showed that the highest percentage of patients are obese while most of the controls

are with innormal weight.

28.7 34.4 *

0

5

10

15

20

25

30

35

40


BM

I kgs

/m2

BMI

BMI


.

Figure 3.3 Histogram show difference in waist to Hip Ratio, for both control

and study groups.


1.001

1.035

0.97

0.98

0.99

1.00

1.01

1.02

1.03

1.04

1.05


Wei

st t

o h

ip r

atio

Waist to Hip Ratio

Weist to hip ratio


3.5.

Bioc

hemi

cal

Para

mete

rs:

3.5.1. Serum Tumor Necrosis Factor Alpha (TNF-α)

Table (3-3) and figure (3-4) showed thatTumor Necrosis Factor Alpha

(TNF-α) was significantly higher (P<0.001) in patients (mean ±SEM49.5 ±2.75)

than controls (39 ± 2.08).

Table 3.3 Show serum concentrations of TNF-alpha, IL1, IL6 and hs-

CRP in control and patients groups; data expressed as mean +

standard error of mean.

P value patients

(N =100)

Control

(N=100)

Groups

p<0.001 49.5 ±2.75* 39 ± 2.08 TNF-α

(pg/ml)

p<0.001 190.8 ±10.62* 46.9 ± 1.99

IL-1(pg/ml)

p<0.001 14.7 ±1.82* 1.2 ± 0.06 IL-6(pg/ml)

p<0.001 35.5 ±1.6* 12.5 ± 0.54 hs-

CRP(mg/l)

*Significantly different (p<0.05) as compared with control group


Figure 3.4 Histogram show serum level of TNF –α measured in Pg/ml, for

both control and study groups.

*Significant if p value <0.05.

3.5.2. Serum Interleukine-1(IL-1):

39.0

49.5 *

0

10

20

30

40

50

60


Seru

m T

NF

alp

ha

(pg/

ml)

Serum TNF alpha

Serum TNF alpha


Table (3-3) and figure (3-5) showed that serum interleukine-1(IL-1) was

significantly higher (P<0.001) in patients (mean ±SEM190.8 ±10.62) than controls

(46.9 ± 1.99).

Figure 3.5 Histogram show serum level of interleukin -1 measured in Pg/ml,

for both control and study groups.


46.9

190.8 *

0

50

100

150

200

250


Seru

m in

terl

eu

kin

1 (

pg/

ml)

Serum IL 1

Serum IL 1


3.5.3. Serum Interleukine-6(IL-6):

Table (3-3) and figure (3-6) showed that serum interleukine-6(IL-6) was

significantly higher (P<0.001) in patients (mean ±SEM 14.7 ±1.82) than controls

(1.2 ± 0.06).

Figure 3.6 Histogram show serum level of interleukin -6 measured in Pg/ml ,

for both control and study groups.


1.2

14.7 *

0

2

4

6

8

10

12

14

16

18


Seru

m in

terl

eu

kin

6(p

g/m

l)

Serum IL 6

Serum IL 6


3.5.4. Serum High sensitivity C-Reactive Protein (hs-CRP):

Table (3-3) and figure (3-7) showed that serum High sensitivity C-reactive

proteinwas significantly higher (P<0.001) in patients (mean ±SEM 35.5 ±1.6) than

controls (12.5 ± 0.54).

Figure 3.7 Histogram show serum level of HCRP measured in mg/Liter , for

both control and study groups.


12.5

35.5 *

0

5

10

15

20

25

30

35

40


se

rum

HC

RP

(mg/

L)

Serum H CRP

Serum H CRP


3.5.

5.

Ser

um

25(

OH

)

Vit

D

(VI

T

D):

T

able

(3-

4)

and figure (3-8) showed that serum 25(OH) Vit D was significantly lower

(P<0.007) in patients (mean ±SEM 21.3 ±1.15) than controls (25.3 ± 1.11).

Table 3.4 Show serum concentrations of Vitamin D, Parathyroid

hormons, inorganic phosphate and total serum calcium in control and

patients groups; data expressed as mean + standard error of mean.

P value patients

(N=100) control

(N=100)

Groups

P<0.007 21.3 ±1.15* 25.3 ± 1.11 VIT D(ng/ml)

p<0.001 32.5 ±3.19* 21.5 ± 0.77 PTH(pg/ml)

p<0.007 4.8 ±0.08* 5 ± 0.04

Inorganic

phosphate

(mg/dl)

p<0.001 9.7 ±0.16* 8.6 ± 0.04 Total

calcium(mg/dl)

*Significant at p<0.05 as compared with values of control group


Figure 3.8 Histogram show difference in serum level of Vitamin D measured

in ng/ml , in both control and study groups.


3.5.6. Serum Parathyroid Hormone (PTH):

Table (3-4) and figure (3-9) showed that serum Parathyroid hormone was

significantly higher (P<0.001) in patients (mean ±SEM 32.5 ±3.19) than controls

(21.5 ± 0.77).

25.3 21.3 *

0

5

10

15

20

25

30


Seru

m V

it D

(n

g/m

l)

Vitamin D

Vitamin D


Figure 3.9 Histogram show difference in serum level of parathyroid hormone

measured in pg/ml, in both control and study groups.


3.5.7. Serum Inorganic phosphate:

Table (3-4) and figure (3-10) showed that serum inorganic phosphatewas

significantly higher (P<0.007) in patients (mean ±SEM 4.8 ±0.08) than controls (5

± 0.04).

21.5

32.5 *

0

5

10

15

20

25

30

35

40


Seru

m P

arat

hyr

oid

ho

rmo

ne

(pg/

ml)

Serum PTH

Serum PTH


Figure 3.10 Histogram show difference in serum level of inorganic phosphate

measured in mg/dl, in both control and study groups.


3.5.8. Serum Total Calcium:

Table (3-4) and figure (3-11) showed that serum total calcium was

significantly higher (P<0.001) in patients (mean ±SEM9.7 ±0.16) than controls

(8.6 ± 0.04).

5.0

4.8 *

4.5

4.6

4.7

4.8

4.9

5.0

5.1


Seru

m p

ho

sph

ate

(m

g/d

l)

Serum Inorganic phosphate

Serum phosphate


Figure 3.11 Histogram show difference in serum level of calcium measured in

mg/dl, in both control and study groups.


8.6

9.7 *

7.5

8.0

8.5

9.0

9.5

10.0

10.5


Seru

mC

alci

um

(m

g/d

l)

Serum Total Calcium

Serum Calcium

Table 3.5 Show serum concentrations of C- terminal propeptide& C-

Terminal telopeptide , in ng/ ml in control and patients groups; data

expressed as mean + standard error of mean.


P value patients

(N=100) control (N=100)

Groups

p<0.001 2 ±0.09* 0.9 ± 0.05

C-terminal

propeptide

(ng/ml)

p<0.001 5.6±0,47* 2.7 ± 0.15

C-terminal

telopeptide

(ng/ml)


3.5.9.Carboxy Terminal Propeptide Type I Procollagen(PICP):

Table (3-5) and figure (3-12) showed that serum Carboxy terminal

propeptide type I procollagen(PICP)was significantly higher (P<0.001) in

patients (mean ±SEM 2 ±0.09) than controls (0.9 ± 0.05).


3.5.

10.

Car

box

y

Ter

min

al

Tel

ope

ptid

e

Typ

e I

pro

coll

age

n(CTXI):

Table (3-5) and figure (3-13) showed that serum Carboxy terminal

telopeptide type I procollagen(CTXI)was significantly higher (P<0.001) in patients

(mean ±SEM 5.6±0,47) than controls (2.7 ± 0.15).

Figure 3.12 Histogram show difference in serum level of C terminal

propeptide measured in ng/ml, in both control and study groups.


0.9

2.0 *

0.0

0.5

1.0

1.5

2.0

2.5


seru

m C

-te

rmin

al p

rop

epti

de(

ng/

ml)

Serum C-Terminal Propeptide


Figure 3.13 Histogram show difference in serum level of C terminal

telopeptide measured in ng/ml, in both control and study groups.


2.7

5.6 *

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0


Seru

m c

-te

lop

epti

de

(ng/

ml)

C -Terminal telopeptide

C telopeptide

Table 3.6 Show serum concentrations of estrogen in (pg/ml) & free

testeronein (mg/l) in control and patients groups; data expressed as


3.5.

11.

Ser

um

estr

adio

l:

T

able

(3-

6) and figure (3-14) showed that serum estradiolwas significantly elevated

(P<0.001) in patients (mean ±SEM 58.7 ±3.14) than controls (31.2 ± 1.52).

mean + standard error of mean.

P value patients

(N=100) control (N=100)

Groups

p<0.001 58.7 ±3.14* 31.2 ± 1.52 Estrogen

(pg/ml)

p<0.05 2.2 ± 0.21* 3.1 ± 0.35

Free

testosterone

(µg/L)



Figure 3.14 Histogram show difference in serum level of estradiol measured in

pg/ ml, in both control and study groups; for female only.


3.5.11. Serum free testosterone:

Table (3-6) and figure (3-15) showed that serum free teststerone was

significantly reduced (P<0.05) in patients (mean ±SEM 2.2 ± 0.21) than controls

(3.1 ± 0.35).

31.2

58.7 *

0

10

20

30

40

50

60

70


Seru

m E

stra

dio

l (p

g/m

l)

Serum Estradiol

Serum estrogen


Figure 3.15 Histogram show difference in serum level of free testerone

measured in mg/liter, in both control and study groups.


3.1

2.2 *

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0


Seru

m f

reet

est

ste

ron

(m

g/lit

er)

Serum free testerone

Serum free testerone


3.6. Correlation Studies:

In the present study, as the show in the table (3.7) There was a positive

Correlation between serum IL6 (pg/ml) and serumC-terminal propeptide (ng/ml)

(r=0.4156, P<0.05), serum IL-6(pg/ml) and serumC-terminal telopeptide (ng/ml)

(r=0.3942, P<0.05), serum IL 1 (pg/ml) and serumC-terminal propeptide (ng/ml)

(r=0.4437, P<0.05), serum IL 1 (pg/ml) and serumC-terminal telopeptide (ng/ml)

(r=0.3552, P<0.05)and serum TNF-α(pg/ml) and serumC-terminal telopeptide

(ng/ml) (r=0.1529, P<0.05).

Positive correlations between different parameters in this study are shown in

figures (3-16), (3-17), (3-18), (3-19) and (3-20).


Table 3.7 Show regression factors & p values for IL1, IL6,TNF α , C

terminal telopeptide and c terminal propeptide.

C-terminal

propeptide

C- terminal

telopeptide TNF α IL 6 IL 1

Groups

P

value

R

value

P

value

R

value

P

value

R

value

P

value

R

value

P

value

R

value

.014* 0.444 0.018* 0.355 0.029* 0.234 0.013* 0.43 0.000 1 IL 1

0.015* 0.415 0.015* 0.394 0.031* 0.217 000 1 0.013* 0.48 IL 6

0.09 0.078 0.045* 0.153 000 1 0.031* 0.217 0.029* 0.234 TNF α

0.024* 0.283 000 1 0.045* 0.153 0.016* 0.334 0.018* 0.355 C- terminal

telopeptide

000 1 0.024* 0.283 0.09 0.078 0.015* 0.416 0.014* 0.444 C-terminal

propeptide



Figure

(3.16): Correlation between serum IL6 (pg/ml) and serum C-terminal

propeptide (ng/ml) (r=0.4156, P<0.05)

0 10 20 30 40 50 60

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

IL6

C p

rop

epti

de

Y= 1.2347 + 0.02545 X

R2 = 0.1727

r=0.4156


Figure (3.17): Correlation between serum IL-6(pg/ml) and serum C-terminal

telopeptide (ng/ml) (r=0.3942, P<0.05).

0 5 10 15 20 25

0

10

20

30

40

50

60

C_telopeptide

IL6

y= 1.6383 + 1.5216 X

R2 = 0.1552

r=0.3942


Figure

(3.18): Correlation between serum IL 1 (pg/ml) and serumC-terminal

propeptide (ng/ml) (r=0.4437, P<0.05).

0 100 200 300 400 500

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

IL 1

C p

rop

ep

tid

e

Y =0.9902 + 0.003762 X

R2 =0.1969

Y =0.9902 + 0.003762 X

R2 =0.1969

r=0.4437

0 100 200 300 400 500

0

5

10

15

20

25

IL 1

C_te

lop

ep

tid

e

Y =77.7027 +9.9057 X

R2 = 0.1262

r=0.3552


Figure (3.19): Correlation between serum IL 1 (pg/ml) and serumC-terminal

telopeptide (ng/ml) (r=0.3552, P<0.05).

Figure (3.20): Correlation between serum TNF-α(pg/ml) and serumC-

terminal telopeptide (ng/ml) (r=0.1529, P<0.05).

0 5 10 15 20 25

0

20

40

60

80

100

120

C_telopeptide

TN

F a

lph

a

Y= 40.0312 + 1.0094 X

R2= 0.02338

r=0.1529

Chapter Four: Discussion 100

Chapter Four

Discussion

4.1 Demographic facts and risk factors:

4.1.1 Female gender & Its Related Factors:

Cholelithiasis represents one of the major sources of morbidity industrialized

countries. Advanced age and female gender are well-established risk factors for

gallstone disease and among the avoidable risk factors, food pattern, a sedentary

lifestyle, and being overweight have been found to be associated with the risk of

gallstone formation [181] . There remains some controversy about gender as a

risk factor for cholelithiasis. While the majority of studies conducted in the West

have concluded that females are more likely than males to develop cholelithiasis .

The present study reveals that females with symptomatic gallstone disease are

approximately 9 times (90%) more than males(10%) as showed in table (3-1) .this

study came in tune with study of Brown AC et al in 2009 they conclude that the

incidence of gallstone disease is two to three times higher in women than in men,

and female sex hormones, particularly estrogens, have been implicated as

contributory factors and suggest that estrogen interacts with cholesterol-rich rafts

in vesicles within bile to promote cholesterol nucleation and predispose females to

gallstone formation (182). Meanwhile other study showed the role of sex and

gender in development of cholelithiasis, Grigorieva et al. in 2007 assume that

age and sex are profoundly associated with the incidence of gallstone disease; the

metabolic risk factors for gallstone disease are different between men and women

[183].

http://www.ncbi.nlm.nih.gov/pubmed?term=Brown%20AC%5BAuthor%5D&cauthor=true&cauthor_uid=19898891


In the present work females with multiparity more susceptible for gallstone

disease than nulliparous females as stated in table [3-1]. Such results are supported

by study of Ko et al. (2006) which suggest that women with more pregnancies and

longer lengths of fertility periods appear to have a higher likelihood of developing

gallstones than those who remain nulliparous [184]. In other hand this study

revealed that women using contraceptive pills for at least 6 months have more

incidences for cholelithasis as compared with non user, an explanation for such

finding that estrogens increases biliary cholesterol secretion causing cholesterol

super saturation of bile and then precipitation leading to stone formation

[185].From this hormone replacement therapy in postmenopausal women has been

described to be associated with an increased risk for gallstone disease also there

are relation between oral contraceptive use and a high prevalence of gallstone

disease [186].

It has been found that estrogens increase cholesterol secretion and diminish bile

salt secretion; while progestins act by reducing bile salt secretion and impairing

gallbladder emptying leading to stasis so female sex hormones adversely influence

hepatic bile secretion and gallbladder function [187]. During pregnancy when

female sex hormones are endogenously raised, biliary sludge [particulate material

that is composed of cholesterol, calcium bilirubinate, and mucin] appears in 5% to

30% of women. Resolution frequently transpires during the post-partum period:

sludge disappears in two-thirds; small [<1 cm] gallstones (microlithiasis) vanish in

one-third, but definitive gallstones become established in ~5% [188]. Previous

studies described the influence of sex hormones on gallbladder emptying. A

significant delay in gallbladder emptying during the luteal phase of the menstrual

cycle and an increased incidence of cholelithiasis in women using sex hormones


have been reported [189]. Experimental investigations have shown the

sensitivity of the gallbladder to neurohormonal stimulation and the inhibitory

effect of sex hormones, mainly progesterone, on gallbladder contractility [190].

Impairment of gallbladder emptying is increasingly suspected to be a potential

pathophysiological factor in the development of Gallstones. The longer gallbladder

emptying delay in women compared to men and the coincidence of elements

related to decreased gallbladder emptying and the pathogenesis of gallstones

support this hypothesis [191].

4.1.2. Family History, Nephrolithiasis and physical activity:

Gallstones are likely to result from a complex interaction of the environment

and the effects of multiple undetermined genes [192]. As showed in table (3-1), the

percentage of symptomatic gallstone patients with a positive family history (FH)

was greater significantly than negative FH. These results may consider (in

concordance with other studies)such as Study in Swedish twins provided

conclusive evidence for the role of genetic factors for the development of

symptomatic gallstone disease in which , the genetic effects accounted for 25% of

the phenotypic variation among twins [193].

The genetic susptibility of gallstone disease has been estimateed in several studies,

like Katsika et al reported a heritability component of 25% in Swedish twins and

Nakeeb et al a 29% in the USA [194] . Familial studies indicating an increased

frequency in families, twins and a nearly 5 times elevated risk in the relatives of

gallstone patients [195].


The present study found that (25%) of patients and only ([8%) of controls had

renal stones. So there were close relation between cholelithiasis and renal stone. In

a study of Akoudad et al[2010] the ARIC cohort, individuals with a history of

gallstones were 54% more likely to report a history of nephrolithiasis after

adjusting for age, gender, body size and other factors they also reported that

ahistory of gallstones was associated with prevalent nephrolithiasis [196]. The

mechanisms underlying the association between gallstone disease and kidney

stones are unknown where it is difficult to speculate on the nature of a shared

metabolic defect that would predispose to the development of both diseases.

Insulin resistance is associated with an increased risk of gallstones and kidney

stones [197]. Factors affecting the intestinal handling of bile acids and oxalate may

account for the associations between gallstones and kidney stones. Lower biliary

concentrations of bile acids increase the risk for cholesterol gallstones, the most

common type of gallstone, and can result from impaired enterohepatic circulation

of bile salts [198]. Severe states of intestinal malabsorption can lead to reduced

intestinal bile acid reabsorption and simultaneous increases in urinary oxalate [via

reduced intestinal oxalate secretion or increased absorption of dietary oxalate]

[197].

In the present study as stated in table (3-1), suggest that lower physical activity

was associated with increased incidence of the symptomatic gallstone as compared

to normal physical activity healthy volunteers, the predominance of overweight

and obesity in the patients may confirm this suggestion. Reduced physical activity

heightens the risk of gallstone disease whereas increased physical activity helps

prevent cholelithiasis, independent of its role in weight loss [199].


Increased endurance exercise [to 30 minutes 5 times a week] may avert

symptomatic gallstones developing in men [200]. The present study is agreement

with other study, in which physical activity inversely proportional with gallstone

formation [201]. Several mechanisms were suggested to contribute to this

association, including a direct effect of colonic motility , and the effect of

reduction in insulin and insulin resistance [202].

4.2. Obesity:

The results in the present study indicate that over weight and higher

BMI persons more prone for cholelithiasis, this come in concordance with other

studies they considered obesity as a risk factor for cholelithiasis [203]. However,

obesity-related disease risk is also increased in individuals with normal weight and

BMI who have an increased waist circumference: a waist circumference of more

than 102 cm [40 inches] in men and more than 88 cm [35 inches] in women, poses

a significant risk.

Waist circumference is an indirect measurement of visceral adiposity, which is

metabolically active and responsible for the secretion of inflammatory cytokines

that are, in part, responsible for the pathogenesis of insulin resistance and the

metabolic syndrome [204].also the data in table (3-2) and figure (3-3) show that


the patients had significantly higher waist-hip ratio [mean ±SEM 1.035 ±0.01] than

control (1.001 ± 0.01).

Obesity is a major, well- established risk factor for gallstone disease, particularly

abdominal/centripetal obesity (eg, as measured by waist-to-hip ratio). The risk is

especially high in women and rises linearly with increasing obesity . Women with

extreme obesity have a 7- fold elevation in the development of gallstones

compared with non obese controls. The current more accepted pathogenic link

between insulin resistance and GS is the increase of cholesterol saturation in

gallbladder bile, a phenomenon related to increase of body cholesterol synthesis

and hypersecretion of biliary cholesterol as observed in obesity [205] .

Obesity is a well-established risk factor for gallbladder (GB) stone formation

because this condition increases hepatic secretion of cholesterol [206]. The risk is

particularly high in women and increases linearly with increasing obesity [207].

Thus, gallstone disease is closely related to abdominal obesity.

Numerous experimental, epidemiological, and clinical studies have established

that obesity is associated with a chronic inflammatory response, abnormal cytokine

production, increased acute-phase reactants, and activation of inflammatory

signaling pathways and that these processes are involved in and responsible for the

development of obesity-related diseases [208]. Adipose tissue also produces other

pro inflammatory factors including interlukin-6 (IL-6) and C-reactive protein

(CRP). Obesity has also been implicated in the development or progression of

musculoskeletal diseases such as osteoarthritis. [209].

4.3. Age

In the current study, there is increase risk of gallstone formation at younger age

group. This results in line with other studies [210] which found the higher


incidence of cholelithiasis among younger age group of 20-30 years. In contrast to

this idea western countries and other regions of Asia, showed that an older age is a

significant risk factor for gallstone disease [211]. Studies in Western children have

shown an increase in The prevalence of gallstones during the last three decades

[212]. The explanation for this phenomenon such as decreased gallbladder motility

and the decrease of cholesterol 7 -hydroxylase activity, which regulate de novo

bile salt synthesis [213].

4.4. Serum concentrations of TNF-alpha, IL1, IL6 and hs-CRP:

Table (3-3) and figures (3-4),(3-5),(3-6) and (3-7) showed that the levels of

tumor necrosis factor alpha (TNF-α), Interleukine 1(IL-1), Interleukine 6(IL-6) and

Highly sensitive C-reactive protein (Hcrp) significantly higher (p<0.001) as

compared with control group. An reasonable interpretation for such elevation may

be due to psrence of specific Bacteria in the bile of the gallbladder and bile ducts

responsible for increase production of such proinflammatory cytokines, the

presence of which may have relevance to clinical diseases of stone formation

[214]. Exposure of sub-epithelial tissue to lipopolysaccharid LPS from gram

negative bacteria causes release of cytokines by resident leukocytes and activation

of macrophages [215]. From this the biliary fluid of these patients has increased

concentrations of the inflammatory cytokines TNF-α, IL-6 and IL-1 into the bile

[216].

Biliary epithelium possess receptors for some of these cytokines and chemokines

[including tumor necrosis factor (TNF-α), indicating that these secreted cytokines

can act in both an autocrine and a paracrine manner [217]. Inflammation, and more

specifically the acute phase response, produces marked alterations in the

metabolism of a variety of proteins and lipids; these changes are extensive, such


acute inflammation does alter the hepatic metabolism of both cholesterol and bile

salts [218].

Some expermintal studies show that the administration of LPS proinflammatory

cytokines [I L-1, TNF] raises serum cholesterol levels and increases the production

of HMG-CoA reductase at both the transcription and translational levels [219].

This change augments de novo cholesterol synthesis. Also The gallbladder, like

most organs, when it becomes inflamed and damaged, it loses its ability to perform

concentrative, pH modification, absorptive, and contractive functions. Unlike most

other organs in the body, the gallbladder can be exposed to large concentrations of

free [unesterified] cholesterol and potentially cytotoxic detergent-like bile salts.

These molecules, as well as other proinflammatory molecules, can induce potent

inflammatory responses these in total support our study of elevation of

inflammatory cytokines [220,221].

IL-6 is related in part to promoting the production of C-reactive protein(CRP),

indeed, IL-6 and CRP also play a pathogenic role in a range of diseases associated

with disability (osteoporosis, arthritis, and congestive heart failure, among others)

[222]. This comes in agreement with our study in which levels of either

concentration were elevated. the Previous studies have oreported an early increase

in IL-6, which regulates the acute-phase response, such as CRP produced by

hepatocytes, and a significant difference in plasma levels between mild and severe

acute pancreatitis that induced by gallstone has been found [223].In other hand an

explaination for elevated levels of proinflammatory cytokines is the increase body

mass index (BMI) which lead to activation of inflammatory processes in

metabolically active sites such as liver, adipose tissue and immune cells. The

consequence of this response is an increase in circulating levels of pro-


inflammatory cytokines, adipokines and other inflammatory markers [224].

Another explanation for such finding is the invasion of inflammatory cells that

release cytokines that plays an important role in the pathogenesis of the disease,

although the exact mechanisms that trigger the inflammatory and necrotizing

process are not well understood [225].

The increased secretion of the proinflammatory cytokines from activated

monocytes and mononuclear phagocytes is pivotal to this process.

In present study increase levels of interleukins 1, 6 and tumor necrosis factor-

alpha and highly sensitive C-reactive protein has important role in pathogenesis of

osteoporosis. The obtained results show that certain proinflammatory cytokines,

and predominantly IL-6, IL-1 at higher concentrations are assumed to play a

crucial role in inducing idiopathic osteoporosis and osteopenia in the

developmental age [226]. Interleukine-1 (IL-1) exert its action on bone cells

through number of pathways, affecting mainly osteoblast receptors, also stimulates

resorption through enhancement of synthesis and collagenase release by

osteoblasts, inhibits osteo-calcin synthesis, and stimulates the production of an

osteoclast precursor recruitment-inducing factor by osteoblasts and their fusion

into polynuclear osteoclastic cells IL-1 also it directly stimulates osteoclasts

through receptor binding [227]. When acting on stromal cells TNF-α stimulates IL-

1 production which mediates a substantial component of TNF-α‘s osteoclastogenic

effect. [228]. IL-1 also stimulates osteoclast activity by increasing production of

M-CSF [229], but it enhances osteoclastogenesis only in the presence of

permissive concentrations of RANKL . Cohen-Solal et al.(1993) reported a

positive correlation between IL-1β concentration and the resorptive activity of

peripheral monocytes[230] . Ghazali et al.[231].Observed a relationship between


IL-β and the decreased bone mineralization in patients with idiopathic

hypercalciuria, similarly Salamone et al. [232]and Zheng et al. [233] found the

same results in postmenopausal and premenopausal women respectively.

Moreover (IL-1, IL-6andTNF- α) can influence osteoclasts directly via their

specific receptors located on osteoclasts or via modulation of the RANK/ RANK

ligand (RANKL)/osteoprotegerin (OPG) system [234]. IL-6 is a pleiotropic

cytokine and has important effects on osteoclast differentiation and function. Its

expression was upregulated in bone tissue from osteoporotic patients . IL-6 may

exert its inhibitory effect on bone formation directly through [glycoprotein 130

Signal transducer and activator of transcription (STAT)-3) or indirectly by

influencing balance between osteoprotegerin (OPG)and receptor activator of

nuclear factor B (RANK) and its ligand (RANKL) [235,236].The influence of IL-

6 on RANK-RANKL/OPG was suggested to be more powerful effect leading to

increased bone mass loss . this results suggest that IL-6 plays a key role in

stimulation of RANKL-RANK/OPG system and this effect is strongly enhanced

bone turnover which in turn induce bone resorption. This came inconcodence with

animal study Mysliwiec et al in 2011 [237]. Moreover Chung et al in 2003 and

Garnero et al in 2002 support our finding in relation to role of IL-6 and bone mass

in pre and postmenopausal women, respectively [238].

There are two mechanisms by which TNF-α act in osteoclasts, both marrow

stromal cells and osteoclast precursors express TNF-α receptors. The main process

occurs when stromal cells are exposed to TNF-α and produce RANKL, M-CSF,

and IL-1, which promote osteoclast formation and activation. TNF-α and RANKL

are synergistic, and minimal levels of one markedly enhances the osteoclastogenic

capacity of the other [239]. TNF-α also has potent antiapoptotic effects on


osteoclasts, prolonging their lifespan [240]. The second mechanism occurs when

the inflammatory process becomes more aggressive and TNF-α may promote

osteoclast formation by directly stimulating its precursors in the absence of stromal

cells responsive to the cytokine, perhaps through activation of transforming growth

factor [TGF]-β [241].The ability of TNF to increase the osteoclastogenic activity

of RANKL is due to synergistic interactions at the level of NFkB and AP-1

signaling [242]. Another target of TNF is the RANKL receptor RANK, whose

expression in osteoclasts precursors is synergistically upregulated by TNF and

RANKL [243]. The present study is agreement with other study that correlate

elevation of TNF-α and Osteoporosis. [244]

The explanation for C-reactive protein elevation that glycoprotein produced in the

liver and upregulated by IL-1, IL-6 and TNF-α, an is regarded as a sensitive

marker of systemic inflammation [245]. An association between circulating high

sensitive (hs) CRP level and bone mineral density has been observed in several

immune and inflammatory diseases, as well as in healthy individuals, suggesting a

relationship between subclinical systemic inflammation and osteoporosis .T he

present study is in concordance with other studies that confirm such correlation

Koh et al and Ganesan et al in 2005 [246,247]. Moreover, CRP has consistently

been found to be related to increased fracture risk in elderly women [248].

4.5. Serum concentrations of of Vitamin D, Parathyroid hormones,

Inorganic phosphate and Total serum Calcium:


In the present study as summarized in table [3.4] and figure [3.8] showed that the

level of (VIT D)in the cholelithiatic patients were significantly lower (p<0.007)

compared to healthy control. There are two explanations for such reduction in the.

One of these is malabsorption of VitD in patient with cholelithiasis and this

comes intune with our study [249].Accordingly the levels of parathyroid hormone

[PTH] rise [250]. Low circulating levels of 25-hydroxyvitamin D (25(OH)D) and

increased secretion of parathyroid hormone [PTH] increase bone loss through

increased bone resorption [251].

The Second explanation for vitamin D deficiency is Obesity is often accompanied

by low levels of 25-OH D and normal or high levels of 1,25(OH)2D(252). The

mechanism[s] for this inverse relationship between body weight and serum 25-

OHD is not well defined. Vitamin D is fat soluble and it is likely that with greater

fat mass more vitamin D is stored in adipocytes. But the precise mechanism

whereby vitamin D storage is enhanced by greater fat mass leading to lower

circulating levels of the 25-OHD metabolite has not been described. On the other

hand, 1, 25(OH) 2D, which is much more constant in the circulation even in obese

individuals, appears to be necessary for both insulin secretion and insulin action.

As such, several studies have suggested an association of vitamin D deficiency

with obesity and insulin resistance [253]. this will confirm the present study since

the obesity is one of risk factor for gallstone formation and because most of

cholelithiatic patients are obese .

In the present study as shown in the table (3.4) and figure [3.9] the level of

parathyroid hormone significantly higher (p<0.001) as compared to healthy

control. The explanation for these elevation is reported previously ,as Low

circulating levels of 25-hydroxyvitamin D (25(OH)D) and increased secretion of


parathyroid hormone (PTH).Moreover in the bone PTH acts on an osteoblast cell

membrane receptor, activating adenylate cyclase and increasing intracellular

cAMP, which increases the cell permeability to calcium. The increase in

cytosolic calcium activates a pump that drives calcium from the bone to the

extracellular fluids (ECF). The pump is enhanced by 1,25 (OH)2D3 [254].

Meanwhile in the Kidney, PTH acts on a renal tubule membrane receptor,

activating adenylate cyclase and increasing intracellular and urine cAMP which,

in turn, decreases Proximal renal tubule phosphat reabsorption. PTH also increases

distal nephron calcium reabsorption and stimulates the 1α -hydroxylase conversion

of 25 hydroxycholecalciferol [25 (OH)D3) to 1,25 (OH)2D3, thereby acting

indirectly on the gastrointestinal tract by increasing absorption of calcium [255].

In the present study as shown in the table [3.4] and figure (3.10) the level of

Inorganic phosphate were significantly lower (p<0.007)as compared to healthy

control .The explanation for these lowering is the increment of parathyroid

hormone in the bone this will increases bone turnover, resulting in a release of

calcium and phosphorus from bone. Meanwhile, PTH works directly on the kidney

to increase renal calcium reabsorption and decrease renal phosphorus reabsorption.

The net effect is a rise in serum calcium but no net change in serum phosphorus

[256].

In the present study as shown in the table (3.4) and figure (3.11) the level of total

calcium in the patients were significantly higher (p<0.001) compared to healthy

control. Such elevation in serum concentration of total calcium result from several

pathways :one of these In parathyroid glands, vitamin D helps to suppress the

gland by a negative feedback mechanism where


The parathyroid gland is hyperproliferates due to vitamin D deficiency, even in the

presence of normal serum calcium [257]. The other pathway at the kidney

parathyroid hormone increases the conversion of calcidiol to calcitriol by 1alpha-

hydroxylase, an enzyme that is activated by low phosphorus and high PTH levels.

Also, under the direction of PTH, renal distal tubule reabsorption of calcium, and

excretion of phosphorus, increases. Finally, via binding to cell surface PTH

receptors on the osteoblast, PTH stimulates a cascade that results in increased bone

turnover and calcium/phosphorus mobilization from bone [258].

4.6. Serum Concentrationsof of Carboxy Terminal Propeptide Type

I procollagen[PICP] and Carboxy Terminal Telopeptide Type I

Procollagen[CTXI]:

In the present study as they showed in the table (3.5)and figure (3.12) showed that

the level of Carboxy terminal propeptide type I procollagen(PICP) in the patients

were significantly higher (p<0.001) compared to healthy control. Serum

biochemical bone turnover markers (sBTM) are allow a dynamic assessment of

bone remodeling, as they reflect bone cell activity. A high bone remodeling, as

reflected by a high sBTM level, is associated with accelerated bone loss and thus

can be associated with bone fragility in some patients [259]. Bone metabolism can

be assessed using biochemical bone turnover markers (BTM). Bone formation can

be assessed by osteocalcin (OC), bone-specific alkaline phosphatase (bone-ALP)

and N-terminal and C-terminal propeptides of type I procollagen (PINP, PICP)

[260].Recently, an expert panel convened by the International Osteoporosis

Foundation and the International Federation of Clinical Chemistry and Laboratory


Medicine proposed that serum concentrations of PINP and CTX-I become the

reference markers of bone formation and resorption, respectively [261]. At present,

the most sensitive markers for bone formation are serum bone the procollagen type

I C-terminal propeptide (PICP) elevation is due to cleaved during procollagen

extracellular metabolism and are released in the blood [262] several human studies

confirm the present study that suggest elevation of bone turnover markers during

osteoporosis [263] .

In the present study as they showed in the table (3.5) and figure (3.13) showed that

the level of Carboxy terminal telopeptide type I procollagen(CTXI) in the patients

were significantly higher (p<0.001) compared to healthy control.CTX, a biomarker

of bone resorption, increased in cholelithiatic patients is suggestive that increased

bone resorption may have contributed to the increased fracture risk in the women

[259]. The majority of markers of bone resorption are degradation products of type

I collagen and include the carboxy-terminal cross-linked telopeptides of type I

collagen (CTX-I and ICTP), and pyridinolines [264].Biochemical markers of bone

turnover in osteoporosis have been suggested to be useful to assess fracture risk

together with BMD and clinical risk factors, although currently one of the most

promising roles is to assess efficacy of antiresorptive therapies. Indeed markers of

bone turnover decrease rapidly within a few months after initiation therapy and the

magnitudes of observed changes are associated with the reduction in risk of

fracture [265].as mentioned previously the elevation in these markers due to

extracellular metabolism of procollagene and released into blood [262].

4.7 Serum Concentrations of of Estradiole and Free Testosterone:


In the present study as they showed in the table (3.6) and figure (3.13) showed that

the level of estradiole in the patients were significantly higher (p<0.001) compared

to healthy control. An interpretation of these is because the gallstones are highely

prevalence in women than men at all ages as found by epidemiological studies.

Also long-term administration of oral contraceptive steroids and conjugated

estrogens markedly increases the risk of

Cholesterol gallstones, it is hypothesized that estrogen could enhance cholesterol

cholelithogenesis by augmenting functions of estrogen receptors in the liver and

gallbladder [266]. Such action of estradiole (E2) contribute to biliary cholesterol

hypersecretion and cholesterol supersaturation of bile, which significantly enhance

the formation of cholesterol gallstones. Furthermore, these findings show that

estradiol promotes gallstone formation by up-regulating hepatic expression of

ESR1 but not ESR2, several evidences has revealed that estrogen increases the risk

for the formation of cholesterol gallstones by promoting hepatic secretion of biliary

cholesterol that induces an increase in cholesterol saturation of bile in humans and

in several animal models of cholesterol gallstones [267]. high levels of estrogen

significantly enhance the activity of 3-hydroxy-3-methylglutaryl coenzyme A

(HMG-CoA) reductase, the rate-limiting enzyme in hepatic cholesterol

biosynthesis, even under high dietary cholesterol loads. These findings suggest that

there could be an increased delivery of cholesterol to bile from de novo synthesis

in the liver. In addition, estrogen could decrease plasma low-density lipopro-tein

(LDL) cholesterol and increase plasma high-density lipoprotein (HDL) cholesterol

because high doses of E2 amplify expression levels of HDL receptor SR-BI and

LDL receptor [268]. The decrease in plasma LDL is a result of increased hepatic

LDL receptor expression, which increases the clearance of plasma LDL. Therefore,


the increased uptake of LDL by the liver may result in increased secretion of

cholesterol into the bile. These alterations could induce an apparent increase in

hepatic output of biliary cholesterol derived from circulating lipoproteins such as

HDL and LDL, although LDL cholesterol could have a less effect on biliary

secretion. Moreover, estrogens could also act at the canalicular membrane by

increasing ABCG5/ABCG8 activity [269] .These results have led to a model in

which estrogen induce cholesterol gallstone formation by promoting cholesterol

biosynthesis through SREBP2 and hepatic biliary cholesterol secretion.

In the present study as they showed in the table (3.6) and figure (3.14)

demonstrated that the level of free teststeron in the patients were significantly

lower (p<0.05) compared to healthy control. An interpretation for these result since

the most patients participate in our study are females (90%), therefore the level of

free testosterone is within lower limit. The effects of low testosterone on bone in

men and women have been less definitive. As men age, there is a rise in the

proinflammatory cytokine marker soluble IL-6 receptor secondary to reduced sex

steroid levels [270]. This is an indication that androgens may play a role in both

the inflammatory changes and increased bone loss related to aging.

The relationship of testosterone to fracture risk has remained unclear. In a recent

study, Mellstrom et al in 2006 ,showed low-normal levels of serum free

testosterone were an independent predictor of BMD and increase in

fractures.[271].Meanwhile Lorentzon et al in 2005, showed the effects of sex

hormone-binding globulin may be independent of estrogen and testosterone and

that its effects on bone may be age dependent [272].In another study of 609 men

over the age of 60, low serum testosterone and high sex hormone binding globulin

(SHBG) levels were associated with increased risk of osteoporotic fractures


independent of BMD [273].Elevated SHBG in young men may be beneficial for

improved bone mass, whereas in elderly men it appears to be a negative predictor

of BMD [274].Testosterone replacement therapy in men with severe subnormal

serum testosterone levels has been shown to reduce bone resorption and reverse

deterioration of trabecular architecture[275].

4.8. Correlation Study: In the present study, as the show in the table (3.7) There

was a positive Correlation between serum IL6 (pg/ml) and serumC-terminal

propeptide (ng/ml) (r=0.4156, P<0.05), serum IL-6(pg/ml) and serumC-terminal

telopeptide (ng/ml) (r=0.3942, P<0.05), serum IL 1 (pg/ml) and serumC-terminal

propeptide (ng/ml) (r=0.4437, P<0.05), serum IL 1 (pg/ml) and serumC-terminal

telopeptide (ng/ml) (r=0.3552, P<0.05])and serum TNF-α(pg/ml) and serumC-

terminal telopeptide (ng/ml) (r=0.1529, P<0.05).

Positive correlations between different parameters in this study are shown in

figures (3-16), (3-17), (3-18), (3-19) and (3-20) respectively. An interpretation for

these results was agreement with Cohen-Sol al et al. in 2002. This reported a

positive correlation between IL-1 concentration and the resorptive activity of

peripheral monocytes. Also because these proinflammatory cytokines are capable

of stimulating osteoclast activity through the regulation of the RANKL/RANK/

OPG pathway[276].In present study as shown in the figures[3,4 and 5] the postive

correlations between interleukins 1 ,6 and TNF-α and carboxyterminal telopeptide

(r=0.3552, P>0.05]), (r=0.3942, P<0.05), (r=0.1529, P<0.05) respectively. As

levels of these proinflammatory cytokines increased, the level of bone resorption

marker elevated, this indicated the osteoclast activity increased which in turn lead


to bone resorption and osteoporosis. Thus many of the cytokines released during

inflammation also have stimulatory effects on osteoclast development and activity,

resulting in increased bone resorption; these include interleukin-6 ([IL-6), tumor

necrosis factor alpha(TNFα) and receptor activator of NFkB ligand (RANKL)

(277).

4.9. Conclusions

1. Cholelithiatic patients have systemic elevation of inflammatory markers

resemble by IL-1, IL-6, TNF-α and CRP.

2. From this it is show that geneder,obesity,pairty,contraceptive use,low

physical activity,positive family history and renal stones are associated with

gallstones.

3. Cholelithiasis is considered as a risk factor for osteoporosis shown by

elevation of bone turnover markers.

4. Highly significant association between cholelithiasis, obesity with

osteoporosis.


4.10. Recommendations for future work:

1. Study association between cholelithiasis and insulin resistance.

2. Study the osteoporosis induces cholelithiasis.

3. Use other markers for assessment osteoporosis.


Patient information list

Patient name: Age: Sex:

Occupation: Living place:


parity: contraceptive use: Date:

Telephone Nummber Height:

WT. Waist circumference: hip circumference: BMI:

WHR:

History of renal stone: family history:

Drug therapy in use:

Parameter

BMI Inorganic phosphate

WHR Estradiol

IL-1 C-terminal popeptide

IL-6 C-terminal telopeptide

TNF- α PTH

hs CRP Total Calcium

VIT.D

[278]


Parameters Values

1. Serum Tumor necrosis factor -α[pg/ml] 7.36

2. Serum Interleukine-1 [pg/ml ] 0-3.72

3. Serum Interleukine-6 [pg/ml ] 1.56-8.6

4. Serum Highly Sensitive CRP [mg/L ] ≤ 1.1

5. Serum 25 [OH] Vit. D [ng/ml] 30-74

6. Serum Parathyroid Hormone[pg/ml] 10-65

7. Serum calcium [mg/dl] 9-10.5

8. Serum Inorganic phosphate[mg/dl] 3-4.5

9.

Serum Estradiole [pg/ml]

Day 1-10 of Menstrual cycle

11-20 of Menstrual cycle

21-30 of Menstrual cycle

14-27

14-54

19-40

10. Serum Free testosterone s[ng/dl] for

female 20-75

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جمهورية العراق وزارة التعليم العالي والبحث العلمي

كلية الصيدلة - جامعة بغداد

هشاشت العظام لذى الورضي الوصابن بحصي

الورارة :الوؤشراث الحوت

اليو العلوم الوختبرت السررتهقذهت الي فرع رسالة

لجنت الذراساث العلا ف كلت الصذلت/ جاهعت بغذاد ف علوم ألذكتوراه فلسفتكجزء هن هتطلباث الحصول علي درجت

(العلوم الوختبرت السررت)الصذلت/

من قبل

فالح حسن شر الوالك

(2002بكالوريوس علوم الصيدلة )

2002 مختبرية سريريةماجستير علوم

بإشراف

هحوذ عباس طاهرالذكتور الوساعذاألستار

صالح كاظن االحوذالذكتور

1434 م2013 هـ

الخالصة :خلفية الدراسة

كلفددوروة دد ركثددصرةالرةضددصةىرة ازددعهرة ز ددض ررة شددعةعوروروةاددمةرضدد رىةضددصرةاليعددمرضددصىرالددارة ضددصةص ر اددمرةورة سددي وكياعدرموصةرضاوصيددعر دد ررة عوةضددلرة خلويددو لعدد ر.اضيددارةااددععرة عددع رمخولر لضس شددفار دد رة ددر طلدد

و د رادعالدرةكثدصر لد رةناسداوررةال زعبيدورة د ر د ملرة داةالسد اعبوررخدللرض ررة سب ر ر اصرة لفصةعلفصةولري ملرإ دارة اصرة ررة ضفعلورضو عيعرة خليعرةال زعبيورة .شم رة ار ع رةور شلرةازه رة اس

روررععضلراخصرة وص ر فع.ر1ورر 6إا عجرة عوةضلرة خلويورة ضضزم ر ل زع ر،رضثلرةا ص وكي ر

بدعرربايورة عظ رة ضازصيورضضعريهيمرخطدصرةاللدعبورةاخفعىرك لورة عظع رو غييصريامثهشعشورة عظع ر راع وررظددد ر ددد ركدددو ة عربادددععررعضليدددورإعدددعم ددد روي الدددارة دددموصرة صةيسددد ر لعوةضدددلرة خلويدددور ة سدددعي وكياعد رر. كسدددوص

ضوادوم رعلداركدلروععضلراخدصرة دوص ر فدعررر6ور1ضضزم رةال زع رضثلرةا ص وكي رة ضس قبلدرهذهرة سي وكياعدرهدذهرة سدي وكياعدرضسد و ررإص فدععادع ر.روبع دع راعق دورة عظد سدلة رة ض رة خليعراعق دورة عظد رة اع داورور

رربادععرة عظد رةعدعم لاثرعلار رخليعرة عظع ررة سعي وكياعدرعلارضس قبلدري ثصعامرضص ارالارة ضصةص رسددوعرةالض لددعصررا ياددورب الدد رة لددفصةعة ضلددعبي رة ضص دداررعاددمرD ي ددعضي رراقددصر ددع رة ددارذ دد رإ ددع و

ر.رسلسلورض رةالامةثهشعشورة عظع ربوةسطوررة اري ملر ي ع

رر:لهدفاضضزدددم رةال زددددع رعلدددارة ضص ددددارر ة سددددعي وكياعد ة زدددم رضددد رهددددذهرة مصةسدددورهددددور قيدددي ر ثددددعصرة عوةضدددلرة خلويور

مصةسددور ددلرعدد رة ضلددعبي رب الدد رة لددفصةعرو قيددي رضسدد ويعدررض شددصةدر كددوي رة عظددع روةص شددع رة عظددع ر.ر،ة بدصو ي رة فدععل رر،ععضلراخدصرة دوص رة فدعر6ورر1ةا ص وكي ر ة غيصةدرة كيضيعةيورة ايويور ربعىرة ض غيصةدر

C وض شددصةدرباددععرة عظدد ر،رة فوسددفعدرريددصرة ع ويو،ر،رة كع سدديو ي ددعضي رمل،ة زصضددو رة ضاددعوصر لمصقيددور،ررهصضدددو ررذورة كعصبوكسددديلرة طص يدددو،ررة كدددوالاي رة ادددوعرةنولرة بصوبيب عيدددمروض شدددصةدراقدددىرة عظددد رة يلوبيب عيدددم

رايددث،رضاضوعددورة سدديطص ضددارضقعصادورضلددعبي ربالددارة ضددصةص رة وةسدد صةميول. ر دد رة ضص دارة س سد يصو رة اددصرمصةسدوربعدىرعوةضدلرة خطدصرنضدصةىررالارة ضصةص .ض رةالهدمة رةي دعموصةر رة سب ر رركلرض غيصريلع

ض دععفعدررو دي رةخصة،وادمدر.وةخيصعلار كدوي رة الدوهرعبي رة بيعاعدرة ميضغصة يورور أثيصهرهرة صةبطرة الوروقددمر اصيدددرهددذهرة مصةسددور دد رضس شددفارة بلددص رة عددع رضدد رياددعيصرزشعشددورة عظددع ربر ضثددلاميددم ر الددو رة ضددصةص ر

2112ةريعصا ارر2112

:ىالطرق والمرضري دصةوحربدي ىة ضص دارهد العررضدم رةعضدعصر،كدع رذكوص[ر11إادعثرورر01]رضدصيىضعةدورر رهذهرة مصةسدورشعص م رضد رة الدد رة لدفصةولرة ضعدم ر قدطر ل دمةخلرة اصةاد روةمصةازددعرو د رةخ يدعصرة ادعالدرة غيدصرضعقدر. 33-13

رة قلبيدددوروة ض دددععفعدرة كبددم،روة دددطصةبعدرضعقدددم ،ة رالددو ة رادددعالد كذذذ تم اذذذع د ذذذا . دد رة مصةسدددورة اع يددوررة ضسدددببورمويدددوةنركوشددديا رض لهضدددورة ضدددهض ،رة كلدددولرة فشدددلرة سدددكصل،روة صبدددو،رة دددم ،ر دددغطروةص فدددععرة وععةيدددو،

و غدددصىر.ة مصةسدددورهدددذهرضددد ر ألكسدددم رة ض دددعم رةسددد بععمرورة سوضع وسددد ع ي ر،،رة سيف صيعكسدددو ة عظدددع رزشعشدددو افدد رة فةددورة عضصيددوروافدد رة اددا ررضدد ةخ يددعصرةشددخعصرةلدداععر لضشددعصكوركضاضوعددور]ة سدديطص [رر دد رة ضقعصاددو

ة شخيصرضد ررةاصةعر ررر.]1,1±ر35,4]ري صةوحربي ره العةالشخعصرضم رةعضعصذكوص[ركع ر11ةاعثرو01]الددددار دددد رة ضددددصةص رعلددددارواددددومرةنعددددصةىرة شددددخيصررايثريع ضددددمقبددددلرة طبيدددد رة ض خلددددصر دددد رة اصةاددددو.

ر عددمرة ضواددعدر ددوصرة لددو يور دد رة ددبط رة ة لددويصرة شخيلدد .ربوةسددطورلدداة اضوذايددو،روة ددم يلرعلددارة ارضد رعيادعدر رةخدذ،سدععور12ر ضدم ر..روبعدمرة لديع الدو رة ضدصةص لكش رع رررة شخيل رة قيعس رخ بعصةال

قبلرةس ةلعلرة ضصةص ربع ضاظعصروض رربمو رةس خمة رة ععلبوررضلرض رة ضص ار11ة م رة وصيمل،راضاراوة رر31 ددددد رمصادددددورادددددصةص رة غص دددددور ضدددددم رر دددددص رة دددددم ر يددددد الط.رو ددددد ر اعبيددددد رععميدددددروو دددددعدرض طدددددوعي ر لددددداعع

سد خم رةمقيقدو.رر15موص ر د رة مقيقدور ضدم رر3111وو عدرعياعدرة م ر رازدعهرة طدصمرة ضصكدهلربضعدملمقيقو،،ععضدددلراخدددصرة دددوص رة فدددعر،ة بدددصو ي رر6ورر1ةا ص دددوكي ر[ضددد رر كدددلررة ضلدددلرة ضادددعع ر قيدددي رضسددد ويعدرة بلهضدددع

،ر ي دعضي رمل،ة زصضدو رة ضادعوصر لمصقيدور،رة كع سديو ر،رة فوسدفعدرريدصرة ع ويو،وض شدصةدربادععرر Cة فدععل رشصةدراقىرة عظ رة يلوبيب عيمرة كوالاي رة اوعرةنولرذورة كعصبوكسيلرة طص يو،ررهصضو رة عظ رة بصوبيب عيمروض رر.[ELISA]ربوةسطور اصرةاهي رضص بطرة ضاعع ]س صةميولةالة س س يصو رة اصرور

: النتائجة ددوه روة سددضاو،رةسدد خمة ررهيددعم رر،ع اا رةالاثددولبددر ضثددلري رعوةضددلة ددارواددومرعلقددوربددرة عضددلرة اددع يشدديصر

ياعبيددورروة كلويددرة الدداوسددعةلرضاددارة اضددلرعدد رطصيددصرة فدد ،ر كددصصرة ددوالمةد،ر بددي رةضكعايددورورعددعةل رة ددعصي رة وة هاددع رهيددعم ررة كضددعرر. دد راددي ر دد ريكدد رهاع دد ر ععضددلرة قددم ر دد رة عضددصرةثددصريددذكصررالددار دد رة ضددصةص .راددموث

،رر C،ععضدلراخدصرة دوص رة فدعر،ة بدصو ي رة فدععل رر6ورر1ةا ص دوكي ر[ [ضلدلر صةكيهر ر د رP <0.001ضعاويور ،ة زصضو رة ضاعوصر لمصقيور،رة كع سيو ر،روض شصةدرباععرة عظد رة بصوبيب عيدمروض شدصةدراقدىرة عظد رة يلوبيب عيدمر

صةص رضدة رة ضص ارة ذي ريععاو رضد رالدو ر ] س صةميولرةالة كوالاي رة اوعرةنولرذورة كعصبوكسيلرة طص يو،ررور ي عضي ر ر ر صكيهةدرضللرP <0.007وكع رهاع رةاخفعىركبيصر بضاضوعورة سيطص رض رةاللاععررضقعصاو.ربضاضوعدورة سديطص رضد رةاللداععرضقعصادورة ضدصةص رالدو برة لعبي ة ضص ارر وة فوسفعدرريصرة ع ويوررمل

ة ضصةص ررالو برة ضلعبي راة ضص ر رP <0.05 س وس يصو رة اصر ة هصضو رركضعرامثرةاخفعىركبيصر ر1ةا ص وكي ررعلقورإياعبيوربي واومرة مصةسورة اع يوررهذةرو واظر ربضاضوعورة سيطص رض رةاللاعع.رضقعصاو

روكدددذ رعلقدددورةياعبيدددوربدددي ركدددلرضددد .روبدددي رض شدددصةدربادددععرة عظددد رة بصوبيب عيدددمرذورة كعصبوكسددديلرة طص يدددور6وررض شصةدراقىرة عظ رة بصوبيب عيمرذورة كعصبوكسيلرة طص يوورععضلراخصرة وص رة فعرورر6،ر1ةا ص وكي رر:ررراالستنتاجر ددد رض شدددصةدةص فدددععرازدددعهلرضددد رهدددذهرة مصةسدددور رة ضص دددارة ضلدددعبي ربالدددارة ضدددصةص ريكدددو ر دددميز رراسددد ا ر

،رة اددا ركددلرضدد رCرة فددعروة بددصو ي رة فددععل رة ددوص راخددصلروععضددر6و1ا ص وكي رعبددة ض ضثلددوررة ضوة يددور ل زعبددعدياددددعب رةإلعددددعةل رة ددددعصي رة ةسدددد خمة روسددددعةلرضاددددارة اضددددل،روة اشددددعطرة بددددما رة ضدددداخفى،ر،رر عممة اضلة سددددضاو،

صقدصر الد رة ضدصةص رععضدلرخطيدصرالار رة ضصةص .روعلو رعلارذ ريع بدصرراموثركلويورضاة اليعدرة ورالدارروراو،روهشعشدورة عظدع كبيصربدي رة سدضهاع رةص بعطرموصة رة عظع .رض شصةدة ذلر ظزصهرةص فععرورة عظع رر.ة ضصةص ر

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