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
Histogram shows difference in serum level of
inorganic phosphate measured in mg/dl, in both
control and study groups
3.10
00
64
Histogram shows difference in serum level of
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
control and study groups
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
serumC-terminal telopeptide (ng/ml)
3.19
75 Correlation between serum TNF-α(pg/ml) and
serumC-terminal telopeptide (ng/ml)
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.
07
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
Chapter One : Introduction 20
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
Chapter One : Introduction 21
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
Chapter One : Introduction 22
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.
Chapter One : Introduction 23
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
Chapter One : Introduction 24
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
Chapter One : Introduction 25
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
Chapter One : Introduction 26
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
Chapter One : Introduction 27
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
Chapter One : Introduction 28
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
Chapter One : Introduction 29
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
Chapter One : Introduction 30
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
Chapter One : Introduction 31
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
Chapter One : Introduction 32
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].
Chapter One : Introduction 33
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
Chapter One : Introduction 34
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 mes- enchymal 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
Chapter One : Introduction 35
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
recep- tor 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
Chapter One : Introduction 36
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].
Chapter One : Introduction 37
26
Figure 1.5 Osteoclasts, Immune Cells, and RBCs are Derived from Marrow
Hematopoietic Stem Cells [85].
Chapter One : Introduction 38
27
Figure 1.6 Osteoblasts, Cartilage, and Adipocytes are Derived from Marrow
Mesenchymal Stem Cells [86].
Chapter One : Introduction 39
28
Figure 1.7 RANK/RANKL/OPG Osteoimmunological System of Bone
Homeostasis [91].
Chapter One : Introduction 40
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
Chapter One : Introduction 41
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
Chapter One : Introduction 42
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
Chapter One : Introduction 43
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].
Chapter One : Introduction 44
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
Chapter One : Introduction 45
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
Chapter One : Introduction 46
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
Chapter One : Introduction 47
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-
Chapter One : Introduction 48
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
Chapter One : Introduction 49
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].
Chapter One : Introduction 50
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
Chapter One : Introduction 51
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
Chapter One : Introduction 52
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 post- menopausal 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].
Chapter One : Introduction 53
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].
Chapter One : Introduction 54
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
Chapter One : Introduction 55
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
Chapter One : Introduction 56
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.
Chapter One : Introduction 57
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.
Chapter Two : Materials and Methods 59
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:
Chapter Two : Materials and Methods 60
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)
Chapter Two : Materials and Methods 61
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.
Chapter Two : Materials and Methods 62
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
Chapter Two : Materials and Methods 63
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
Chapter Two : Materials and Methods 64
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-
Chapter Two : Materials and Methods 65
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].
Chapter Two : Materials and Methods 66
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
Chapter Two : Materials and Methods 67
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
Chapter Two : Materials and Methods 68
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
Chapter Two : Materials and Methods 69
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
Chapter Two : Materials and Methods 70
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:
Chapter Two : Materials and Methods 71
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
Chapter three : Results 27
*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
Chapter three : Results 27
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
Chapter three : Results 27
Figure 3.2 Histogram show difference in BMI, for both control and study
groups.
*Significant if p value <0.05
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
Control group Study group
BM
I kgs
/m2
BMI
BMI
Chapter three : Results 27
.
Figure 3.3 Histogram show difference in waist to Hip Ratio, for both control
and study groups.
*Significant if p value <0.05
1.001
1.035
0.97
0.98
0.99
1.00
1.01
1.02
1.03
1.04
1.05
Control group Study group
Wei
st t
o h
ip r
atio
Waist to Hip Ratio
Weist to hip ratio
Chapter three : Results 22
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
Chapter three : Results 27
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
Control group Study group
Seru
m T
NF
alp
ha
(pg/
ml)
Serum TNF alpha
Serum TNF alpha
Chapter three : Results 27
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.
*Significant if p value <0.05.
46.9
190.8 *
0
50
100
150
200
250
Control group Study group
Seru
m in
terl
eu
kin
1 (
pg/
ml)
Serum IL 1
Serum IL 1
Chapter three : Results 78
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.
*Significant if p value <0.05.
1.2
14.7 *
0
2
4
6
8
10
12
14
16
18
Control group Study group
Seru
m in
terl
eu
kin
6(p
g/m
l)
Serum IL 6
Serum IL 6
Chapter three : Results 78
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.
*Significant if p value <0.05
12.5
35.5 *
0
5
10
15
20
25
30
35
40
Control group Study group
se
rum
HC
RP
(mg/
L)
Serum H CRP
Serum H CRP
Chapter three : Results 77
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
Chapter three : Results 77
Figure 3.8 Histogram show difference in serum level of Vitamin D measured
in ng/ml , in both control and study groups.
*Significant if p value <0.05
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
Control group Study group
Seru
m V
it D
(n
g/m
l)
Vitamin D
Vitamin D
Chapter three : Results 77
Figure 3.9 Histogram show difference in serum level of parathyroid hormone
measured in pg/ml, in both control and study groups.
*Significant if p value <0.05
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
Control group Study group
Seru
m P
arat
hyr
oid
ho
rmo
ne
(pg/
ml)
Serum PTH
Serum PTH
Chapter three : Results 77
Figure 3.10 Histogram show difference in serum level of inorganic phosphate
measured in mg/dl, in both control and study groups.
*Significant if p value <0.05.
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
Control group Study group
Seru
m p
ho
sph
ate
(m
g/d
l)
Serum Inorganic phosphate
Serum phosphate
Chapter three : Results 77
Figure 3.11 Histogram show difference in serum level of calcium measured in
mg/dl, in both control and study groups.
*Significant if p value <0.05
8.6
9.7 *
7.5
8.0
8.5
9.0
9.5
10.0
10.5
Control group Study group
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.
Chapter three : Results 72
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)
*Significant at p<0.05 as compared with values of control group
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).
Chapter three : Results 77
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.
*Significant if p value <0.05
0.9
2.0 *
0.0
0.5
1.0
1.5
2.0
2.5
Control group Study group
seru
m C
-te
rmin
al p
rop
epti
de(
ng/
ml)
Serum C-Terminal Propeptide
Chapter three : Results 77
Figure 3.13 Histogram show difference in serum level of C terminal
telopeptide measured in ng/ml, in both control and study groups.
*Significant if p value <0.05
2.7
5.6 *
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Control group Study group
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
Chapter three : Results 78
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)
*Significant at p<0.05 as compared with values of control group
Chapter three : Results 78
Figure 3.14 Histogram show difference in serum level of estradiol measured in
pg/ ml, in both control and study groups; for female only.
*Significant if p value <0.05
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
Control group Study group
Seru
m E
stra
dio
l (p
g/m
l)
Serum Estradiol
Serum estrogen
Chapter three : Results 77
Figure 3.15 Histogram show difference in serum level of free testerone
measured in mg/liter, in both control and study groups.
*Significant if p value <0.05
3.1
2.2 *
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Control group Study group
Seru
m f
reet
est
ste
ron
(m
g/lit
er)
Serum free testerone
Serum free testerone
Chapter three : Results 77
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).
Chapter three : Results 77
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
*Significant if p value <0.05
Chapter three : Results 77
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
Chapter three : Results 77
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
Chapter three : Results 72
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
Chapter three : Results 77
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 three : Results 77
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].
Chapter Four: Discussion 101
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
Chapter Four: Discussion 102
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].
Chapter Four: Discussion 103
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].
Chapter Four: Discussion 104
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
Chapter Four: Discussion 105
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
Chapter Four: Discussion 106
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
Chapter Four: Discussion 107
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-
Chapter Four: Discussion 108
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
Chapter Four: Discussion 109
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
Chapter Four: Discussion 110
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:
Chapter Four: Discussion 111
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
Chapter Four: Discussion 112
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
Chapter Four: Discussion 113
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
Chapter Four: Discussion 114
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:
Chapter Four: Discussion 115
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,
Chapter Four: Discussion 116
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
Chapter Four: Discussion 117
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
Chapter Four: Discussion 118
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.
Chapter Four: Discussion 119
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.
Chapter Four: Discussion 120
Patient information list
Patient name: Age: Sex:
Occupation: Living place:
Chapter Four: Discussion 121
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]
Chapter Four: Discussion 122
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
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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
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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
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،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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