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Republic of Iraq
Ministry of Higher Education and
Scientific Research
University of Baghdad
College of Education
Ibn-Al-Haitham
Insulin effect on
inflammatory response
Compared to sulfonylurea
in Diabetes Mellitus
patients.
A thesis
Submitted to the college of Education Ibn Al-Haitham,
University of Baghdad in Partial fulfillment of the
Requirement for the Degree of Master of Science in
Chemistry
By
Tamara Ala'a Hussein Al-Ubaidy
B.Sc. in chemistry(2005)University of Baghdad
Supervisors
Asst. Prof. Dr
Dr.
Zohair. I. Al-Mashhadani Nijoud. F. Al-Sarrag
2008 AD
1429 AH
‫بسم هللا الرحمن الرحيم‬
‫اقرأ باسم ربك الذي خلق‬
‫‪‬خلق اإلنسان من علق ‪‬‬
‫اقرأ وربك األكرم ‪ ‬الذي‬
‫علم بالقلم‪ ‬علم اإلنسان ما لم‬
‫يعلم‪‬‬
‫صدق هللا العظيم‬
‫سورة العلق‪ /‬اآلية ‪5-1‬‬
Certification of Supervisor
We certify that this thesis was performed under our
supervision at the department of chemistry, College of
Education Ibn AL – Haitham-University-of Baghdad in partial
fulfillment of the requirements for the degree of Master of
Science in chemistry.
Supervisor
Signature:
Supervisor
Signature:
Asst. Prof. Dr.
Zohair. I. Al-Mashhadni
Department of chemistry,
College of Education
(Ibn-Al-Haitham)
University of Baghdad
Dr. Nijoud. F.Al-Sarrag
Department of chemistry,
College of Education
(Ibn-Al-Haitham)
University of Baghdad
In view of the available recommendation I forward
this thesis for debate by the examining committee.
Signature:
Asst. Prof.
EMAD TAKI ALI
Head of Chemistry Department
College of Education
(Ibn-Al-Haitham)
Baghdad University
/ /2008
Certification
We, the examining, committee, after reading this
thesis “ Insulin effect on inflammatory response
compared to sulfonylurea in Diabetes mellitus
patients” and examining the student “ Tamara Alaà
Hussein” in its content, find that it is qualified for
pursuing the degree
of master of science in
chemistry with grade of (Excellent) on (21/ 12/
2008).
Signature
Name: Asst- prof. Dr. Wafa. F. AL- Taie
Chairmen
Date :
/
/ 2009
Signature:
Name: Asst. prof. Dr.
Sanad. B. Al-Arrji
Signature:
Name:. Dr. Enam. M. A.
Member
Date:
/
/ 2009
Signature:
Name: Asst. prof. Dr.
Zahair. I. AlMashhadani
Member
Date:
/
/ 2009
Signature:
Name: Dr. Nijoud F- ALSrrage
Member (Supervisor)
Date:
/
/ 2009
Member (Supervisor)
Date:
/
/ 2009
Approved by the dean of the college of education
( Ibn – Al – Haitham)
Signature:
Prof. Dr. Abdul Jabar A. Mukhlis
Address: Dean of college of Education
( Ibn – Al – Haitham) university of Baghdad
Date:
/
/ 2009
Dedications
To…
My parents
opportunities.
who
made
the
To…
My husband for his encouragement
patience continuous support and care.
To…
My brothers, sisters, baby and my Husband's
family.
To…
Every one helped me in this thesis.
Tamara
Acknowledgement
Firstly. I thanks the grace of God that has seen me
during the completion of this thesis.
I would like to express my sincere thanks and my
appreciation to my supervisor, assistant professor Dr. Zohair.
I. Al-Mashhadani and Dr. Nijoud. F. Al-Sarrag for their
endless help, guidance, advice and support through their
supervision of this work.
I would like to thank the head of chemistry department
Assistant professor Emad Taki and all members of staff in
college of Education Ibn-Al-Haitham specially assistant
Professor (Dr. Wafa Al-Taie) for their help. Also I would like to
thank all staff of Al-Khadhimyah teaching hospital specially
(Dr. Ala'a) for help and support. My gratitude and thanks to
my friend "Faeza; Ban; Enas; Sahar; Reem; Miss Defaf;
Hana'a, Ahmed; Naser" in Ibn-Al-Haitham College Education
of Baghdad.
Tamara Ala'a Al-Ubaidy
List of Abbreviations
A sample
A standard
ACEI
ATP
BCG
BMI
CRP
Cu+2
Da.
dL
DM
DNA
EDTA
ESR
FBS
g
GDM
GOD
HbA1c
h-chains
HpLc
IgA
IgD
IgE
IgG
IgM
IgS
IDDM
IL-6
JDA
L
L-chains
L
MCP
mg
Absorbance of sample
Absorbance of standard
Angiotension converting enzyme inhibitor
Adenosen tri phosphate
Bromo cresol green
Body mass index
C-Reactive protein
Cupric ion
Dalton
Deciliter
Diabetes mellitus
Deoxy ribonucleic acid
Ethylene diamine tetra acetic acid
Erythrocytes sedimentation rate
Fasting Blood sugar
gram
Gestational diabetes mellitus
Glucose oxidase
Glycosylated haemoglobin
Heavy chains
High pressure liquid chromatography
Immuno globulin A
Immuno globulin D
Immuno globulin E
Immuno globulin G
Immuno globulin M
Immuno globulins
Insulin dependent diabetes mellitus
Inter leukin 6
Japan diabetes association
Liter
Light chains
Micro liter
Monocyte chemoattractant protein
Milligram
mL
min
mmol
MMP
MODY
N.V.
NIDDM
nm
POD
SD
TNF
TP
Milliliter
minutes
Milli mole
Matrix metalloproteinases
Maturity onset diabetes of youth
Normal value
Non insulin dependent diabetes mellitus
Nano Meter
Peroxidase
Standard deviation
Tumor necrosis factor
Total protein
Summary
This study was designed to state the ground stone of the role of
the insulin as anti-inflammatory agent in different inflammation
processes.
Fasting venous blood samples were taken from 150
subjects of which 50 patients with type 1 diabetes, 50 patients
with type 2 diabetes, and 50 healthy individuals. All the blood
samples were analyzed for F.B.S, HbA1c %, TP, albumin, ESR,
CRP, alpha1-antitrypsine, immunoglobulin IgG, IgM and IgA.
Results of this study detected an increase in F.B.S, and
HbA1c % in sera of all patients of type 1 and type 2 compared
with control.
Total protein levels showed no alteration in sera of both
patients groups compared to control
Decrease in albumin level was detected in sera of patient
with type 2 group compared to patients with type 1 and control
groups.
The factors for diagnosis of any type of inflammatory
process ESR, CRP, alpha1- antitrypsin were raised in patients
with type 2 groups compared for patients with type 1 and
control groups.
Immunoglobulines, IgG, IgA levels were elevated in sera
of patients with type 2 group compared to patients with type 1
and control groups, while, IgM levels was decrease in sera of
patients with type 2 group compared for patients with type 1 and
control groups.
Figures index
No.
1.1
1.2
1.3
1.4
1.5
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Figures
Pancreas
Intimate relationship both insulin and glucagon
Structure of proinsulin, indicating the cleavage
sites at which insulin and C-peptide are roduced
Effect of insulin on glucose uptake and
metabolism
Structure of IgG
F.BS level in sera of three studied groups
HbA1c % in sera of three studied groups
Tp and albumin levels in sera of three studied
groups
ESR level in three studied groups
CRP level in sera of three studied groups
Alpha1-antitrypsin level in sera of three studied
groups
IgG, IgM, IgA levels in sera of three studied
groups
Pages
2
10
13
15
24
41
42
44
47
48
52
53
Tables Index
No.
1.1
3.1
3.2
3.3
3.4
3.5
Tables
Laboratory findings in hyperglycemia
F.B.S level in sera of three studied groups
HbA1c % in sera of three studied groups
Tp and albumin levels in sera of three studied
groups
ESR, CRP, Alpha1-antitrypsin levels in sera of
three studied groups
IgG, IgM, IgA levels in sera of three studied
groups
Pages
5
41
42
44
47
53
Contents Index
No.
1.1
1.1.1
1.1.1.1
1.1.1.2
1.2
1.3
1.3.1
1.4
1.4.1
1.5
1.5.1
1.5.1.1
1.6
1.7
1.8
1.9
1.9.1
1.9.2
1.10
2.1
2.2
Subject
List of abbreviations
Summary
Figures index
Tables index
Contents index
Chapter One
Introduction and Review of literature
Pancreas
Function
Endocrine
Exocrine
diabetes mellitus
Blood sugar
Normal regulation of blood glucose
Insulin
Effects of insulin
Oral medications
Daonil
Pharmacology and Mechanism of action
Glycosylated hemoglobin
The plasma proteins
Albumin
Acute phase proteins
C-Reactive protein (CRP)
1-Anti trypsin
Immunoglobulins (IgS)
Aim of study
Chapter Two
Subjects and Methods
Material and subjects
Instrument and Manufacturers
Pa
ges
I
III
IV
V
VI
1
1
1
3
3
7
8
11
13
16
16
16
17
18
19
19
20
21
21
25
26
26
2.3
2.4
2.5
2.5.1
2.5.2
2.5.3
2.5.4
2.6
2.6.1
2.6.2
2.6.3
2.6.3.1
2.6.3.2
2.6.3.3
2.6.4
2.7
2.7.1
2.7.2
2.7.3
2.7.4
2.8
2.8.1
2.8.2
2.8.3
2.8.4
2.9
2.9.1
2.9.2
2.10
2.10.1
2.10.2
2.10.3
2.10.4
2.10.5
Sampling
Collection of blood
Determination of fasting blood sugar (FBS)
Principle
Reagent concentration
Procedure
Calculation
Determination of Glycated haemoglobin
(HbA1c)
Principle
Reagents
Procedure
Preparation of HbA1c calibrator
Preparation of haemoglobin primer
Preparation of lyphochek control
Calculation
Determination of Total protein (TP)
Principle
Reagents
Procedure
Calculation
Determination of serum albumin
Principle
Reagents
Procedure
Calculation
Determination of Erythrocytes sedimentation
rate (ESR)
Principle
Procedure
Determination of C-Reactive protein (CRP)
Principle
Procedure
Interpretation of results of qualitative test
Positive reaction
Negative reaction
27
28
28
28
28
29
29
30
30
31
32
32
32
32
33
33
33
34
34
34
35
35
35
35
36
36
36
37
37
37
38
38
39
39
2.11
2.11.1
2.11.2
2.11.3
Determination of immunoglobulines (IgM,
IgG, IgA, 1-antitrypsin) in serum
Principle
Procedure
Statistical analysis
Chapter three
Results and Discussion
Conclusion
Future Work
Appendix
References
39
39
40
40
41
56
60
Chapter One
Introduction and Literature
Review
1.1. Pancreas:
The pancreas is a gland organ in the digestive and
endocrine system of vertebrates. It is both exocrine (secreting
pancreatic juice containing digestive enzyme) and endocrine
(producing several important hormones, including insulin,
glucagon and somatostatin)(1), Under
a microscope, stained
sections of the pancreas reveal two different types of
parenchymal tissue. Lightly staining clusters of cells are called
islets of langerhans, which produce hormones that underlie the
endocrine functions of the pancreas. Darker staining cells form
acini connected to ducts.
Acinar cells belong to the exocrine pancreas and secrete
digestive enzymes into the gut via a system of ducts(2).
1.1.1. Function:
The pancreas is a dual-function gland, having features of
both endocrine and exocrine glands.
1.1.1.1 Endocrine:
The part of the pancreas with endocrine function is made
up of a million(3) cell clusters called islets of langerhans(4). There
are four main cell types in the islets. They can be classified by
their secretion:  cells secrete glucagon,  cells secrete insulin,
 cells secrete somatostatin and gastrin, and pp cells secrete
pancreatic polypeptide(4, 5).
The islets are a compact collection of endocrine cells
arranged in clusters and cords and are crisscrossed by a dense
network of capillaries. The capillaries of the islets are lined by
layers of endocrine cells in direct contact with vessels, and most
endocrine cells are indirect contact with blood vessels, by either
cytoplasmic processes or by direct apposition. According to the
volume of the body, The islets are basely manufacturing their
hormone and generally disregarding the pancreatic cells all
around them, although they were located in some completely
different part of the body (6).
Figure (1-1): Pancrease (2)
1.1.1.2. Exocrine:
In contrast to the endocrine pancreas, which secretes
hormones into the blood, the exocrine pancreas produces
digestive enzymes and an alkaline fluid, into the small intestine
through a system of exocrine ducts. Digestive enzymes include
trypsin, chymotrypsin, pancreatic lipase, and
pancreatic
amylase, and are produced and secreted by acinar cells of the
exocrine pancreas. Specific cells that line the pancreatic ducts,
called centroacinar cells, secrete abicarbonate- and salt- rich
solution into the small intestine(7).
1.2. Diabetes Mellitus":
Diabetes mellitus is actually a group of metabolic diseases
characterized by hyperglycemia resulting from defects in insulin
secretion, insulin action, or both(8).
In 1979, the National Diabetes Data Group developed a
classification and diagnosis scheme for diabetes mellitus. This
scheme included dividing diabetes into two broad categories:(9)
type 1, insulin-dependent diabetes mellitus (IDDM); and
type 2, non-insulin-dependent diabetes mellitus (NIDDM).
According to classification proposed by the Japan Diabetes
Association (JDA) in 1999, type 1 diabetes was sub classified as
type-1A (autoimmune bases) and type-1B (idiopathic).
Type 1A is a result of cellular – mediated autoimmune
destruction of the  cells of the pancreas causing an absolute
deficiency of insulin secretion. Upper limit of 110mg/dL on the
fasting plasma glucose is designated as the upper limit of normal
blood glucose.
Type -1- constitutes only 10-20% of all diabetes and
commonly occurs in childhood and adolescence(11).
This disease is usually initiated by an environmental factor
of infection (usually a virus) in individuals with a genetic
predisposition and causes the immune destruction of the  cells
of the pancreas and, therefore, a decreased production of
insulincharacteristics of type 1 diabetes include abrupt onset,
insulin dependence, and ketosis tendency(12).
Signs and symptoms include polydipsia (excessive thirst),
polyphagia (increased food intake), polyuria (excessive urine
production), rapid weight loss, hyperventilation, mental
confusion, and possible loss of consciousness (due to increased
glucose
to
brain).
Complications
include
microvascular
problems such as nephropathy, neuropathy, and retinopathy.
Increased heart disease is also found in patients with diabetes.
Table 1.1 lists the laboratory findings in hyperglycemia(13).
Table (1.1): Laboratory findings in hyperglycemia(13)
Increased glucose in plasma and urine
Increased urine specific gravity
Increased serum and urine osmolality
Ketones in serum and urine (ketonemia and keton uria)
Decreased blood and urine pH (acidosis)
Electrolyte imbalance
Idiopathic type 1B diabetes is a form of type 1 diabetes that has
unknown etiology, is strongly inherited, and does not have cell autoimmunity. Individuals with this form of diabetes have
episodic requirements for insulin replacement.
Type- 1B diabetes is common form of diabetes most
commonly seen in obese African American individuals living in
large urban areas. This type of diabetes usually presents with
typical signs and symptoms of type 1 diabetes such as diabetic
ketoacidosis, but its subsequent clinical course often resembles
type 2 diabetes(14).
NIDDM type 2 is the most common form of diabetes,
accounting for 85-90% of the diabetic population(1), it is
characterized by two pathogenic defect, impaired insulin
secretion and insulin resistance(15).
Type -2 diabetes is most commonly associated with
obesity in middle-aged individuals. It is due to reduction in the
number or affinity of insulin receptors on the plasma membrane
of cells in target tissues, or an abnormal binding of insulin to the
receptors(16).The
resultant
hyperglycemia
is
largely
the
consequence of excessive release of endogenous glucose due to
increased gluconeogenesis.
Nevertheless, clinical experience has demonstrated that
therapies directed at improving beta cell function (sulfonylurea
such as Glibenclamid (Daonil)) and at Improving hepatic
(metformin) and muscle (thiazolidinediones) insulin sensitivity
are effective treatment for the condition(17).
Most patients in this type are obese or have an increased
percentage of body fat distribution in the abdominal region.
In obese individuals, persistent dietary excess may cause
excessive secretion of insulin, resulting in hyperinsulinemia
which leads to reduction in number of insulin receptors(16).
This type of diabetes often goes undiagnosed for many
years and is associated with a strong genetic predisposition, with
patients at increased risk with an increase in age, obesity, and
lack of physical exercise. Characteristics usually include adult
onset of the diseases and milder symptoms than in type 1, with
ketoacidosis seldom occurring. However, these patients are
more likely to go into a hyperosmolar coma and are at an
increased
risk
microvascular(13).
of
developing
macrovascular
and
Other specific types of diabetes are associated with certain
conditions (secondary), including genetic defects of -cell
function or insulin action, pancreatic disease, diseases of
endocrine origin(18), drug or chemical induced insulin receptor
abnormalities(19)
and
certain
genetic
syndromes(20).
The
characteristics and prognosis of this form of diabetes depends on
the primary disorder. Maturity onset diabetes of youth (MODY)
is a rare form of diabetes that is inherited in an autosomal
dominant fashion(21,22).
Gestational diabetes mellitus (GDM) is any degree of
glucose intolerance with onset or first recognition during
pregnancy, Causes of GDM include metabolic and hormonal
changes(20).
Patients with (GDM) frequently return to normal
postpartum. However, this disease is associated with increased
perinatal complications and an increased risk for development of
diabetes in later years. Infants born to mothers with diabetes are
at increased risk for respiratory distress syndrome, hypocalcemia, and hyperbilirubinemia. Fetal insulin secretion is
stimulated in the neonate of a mother with diabetes. However,
when the infant is born and the umbilical cord is severed, the
infant's oversupply of glucose is abruptly terminated, causing
severe hypoglycemia(23).
1.3. Blood sugar:
Blood sugar is the amount of glucose in the blood.
Glucose, transported via the blood stream, is the primary source
of energy for the body's cells. Blood sugar concentration, or
glucose level, is tightly regulated in the human body. Normally,
the blood glucose level is maintained between about 4 and 6
mmol/L. Normal blood glucose level (homoeostasis) is about
90mg/100 ml (5mmol/L). The total measurement of glucose in
the circulating blood is therefore about 3.3 to 7g (assuming an
ordinary adult blood volume of 5 liters). Glucose levels rise
after meals and are usually lowest in the morning, before the
first meal of the day(24).
Failure to maintain blood glucose in the normal range
leads to conditions of persistently high (hyperglycemia) or low
(hypoglycemia) blood sugar.
Diabetes mellitus (characterized by persistent hyperglycemia of
several causes) is the most prominent disease related to failure
of blood sugar regulation.
Although it is called "blood sugar" sugars besides glucose
are found in the blood, such as fructose and galactose. Only
glucose levels are regulated via insulin and glucagon (25).
1.3.1. Normal regulation of blood glucose:
The human body requires blood glucose (blood sugar)
maintained in a very narrow range. The homeostatic effect that
keeps the blood value of glucose in a remarkably narrow range
is the result of many factors, of which hormone regulation is the
most important.
There are two types of mutually antagonistic metabolic
hormones affecting blood glucose levels:
 catabolic hormones such as glucagon which increase blood
glucose.
 and one anabolic hormone (insulin), which decreases
blood glucose(26).
The figure (1-2) shows the intimate relationship both insulin
and glucagon have to each other. The pancreas serves as the
central player in the scheme it is the production of insulin and
glucagon by the pancreas which ultimately determines if a
patient has diabetes, hypoglycemia or some other sugar
problem.
Figure (1-2): Intimate relationship both insulin and
glucagon (26).
1.4. Insulin:
Insulin is a peptide hormone composed of 51 amino acid
residues and has a molecular weight of 5808 Da. It is produced
in the islets of langerhans in the pancreas. The name comes from
the latin insula for "island".In mammals, insulin is synthesized
in the pancreas within the beta cells (-cells) of the islets of
langerhans. One million to three million islets of langerhans
(pancreatic islets) from the endocrine part of the pancreas,
which is primarily an exocrine gland. The endocrine portion
only accounts for 20% of the total mass of the pancreas. Within
the islets of langerhans, beta cells constitute 60-80% of all the
cells.In beta cells, insulin is synthesized from the proinsulin
precursor molecule by the action of proteolytic enzymes, known
as prohormone convertases (PC1 and PC2), as well as the
exoprotease carboxy peptidase E. These modifications of
proinsulin remove the center portion of the molecule (i.e., Cpeptide), from the C- and N- terminal ends of proinsulin. The
remaining polypeptides fifty one amino acids in total), the Band A- chains, are bound together by disulfide bonds/ disulphide
bonds. Confusingly, the primary sequence of proinsulin goes in
the order "B - C –A", since B and A chains were identified on
the basis of mass, and the C peptide was discovered after the
others(27) shown fig (1-3)(28).
Insulin is produced in the pancreas, and released when any
of several stimuli are detected. These include protein ingestion,
and glucose in the blood (from food which produces glucose
when digested characteristically this is carbohydrate, though not
all types produce glucose and so an increase in blood glucose
levels). Insulin causes most of the body's cells to take up glucose
from the blood (including liver, muscle, and fat tissue cells),
storing it as glycogen in the liver and muscle, and stops use of
fat as an energy source. When insulin is absent (or low), glucose
is not taken up by most body cells and the body begins to use fat
as an energy source (i.e., transfer of lipids from adipose tissue to
the liver for mobilization as an energy source). As its level is a
central metabolic control mechanism, its status is also used as a
control signal to other body systems (such as amino acid uptake
by body cells)(29).
It has several other anabolic effects throughout the body.
When control of insulin levels fail, diabetes mellitus results,
insulin is used medically to treat some forms of diabetes
mellitus. Patients with type 1 diabetes mellitus depend on
external insulin (most commonly injected subcutaneously) for
their survival because the hormone is no longer produced
internally. Patients with type 2 diabetes mellitus are insulin
resistant, have relatively low insulin production, or both, some
patients with type 2 diabetes may eventually require insulin
when other medications fail to control blood glucose levels
adequately(29.30).
‫النهاية الكاربوكسيلة‬
‫البداية االمينية‬
Figure (1-3): Structure of proinsulin, indicating the cleavage
sites at which insulin and C-peptide are produced(28).
1.4.1. Effects of insulin(31) :
The actions of insulin on the global human metabolism
level include:
 control of cellular intake of certain substances, most
prominently glucose in muscle and adipose tissue (about
2/3 of body cells).
 increase of DNA replication and protein synthesis via
control of amino acid uptake.
 modification
of
the
activity
of
numerous
enzymes(i.e,lipase) .
The actions of insulin on cells include:
 Increased glycogen synthesis- insulin forces storage of
glucose in liver (and muscle) cells in the form of glycogen;
lowered levels of insulin cause liver cells to convert
glycogen to glucose and excrete it into the blood. This is
the clinical action of insulin which is directly useful in
reducing high blood glucose levels as in diabetes.
 Increased fatty acid synthesis- insulin forces fat cells to
take in blood lipids which are converted to triglycerides.
 Increased esterification of fatty acids – forces adipose
tissue to make fats (i.e., triglycerides) from fatty acid
esters.
 Decreased proteinolysis- decreasing the breakdown of
protein.
 Decreased lipolysis- forces reduction in conversion of fat
cell lipid stores. Into blood fatty acids.
 Decreased gluconeogensis decreases production of glucose
from non- sugar substrates, primarily in the liver
(remember, the vast majority of endogenous insulin
arriving at the liver never leaves the liver); lack of insulin
causes glucose production from assorted substrates in the
liver and else where.
 Increased amino acid uptake- forces cells to absorb
circulating amino acids; lack of insulin inhibits absorption.
 Increased potassium uptake- forces cells to absorb serum
potassium; lack of insulin inhibits absorption.
 Arterial muscle tone- forces arterial wall muscle to relax,
increasing blood flow, especially in micro arteries; lack of
insulin reduces flow by allowing these muscles to contract
Figure (1-4): Effect of insulin on glucose uptake and metabolism. (28)
(Marc, et al,
(32)
found insulin to exert an anti inflammatory
effect on cellular mediators and the hepatic acute-phaseresponse.
1.5. Oral medications:
1.5.1. Daonil:
1.5.1.1. Pharmacology and Mechanism of action(33):
The Drug ( Daonil) is used as a second- generation
sulfonylurea antidiabetic agent, appears to lower the blood
glucose acutely by stimulating the release of insulin from the
pancreas, an effect dependent upon functioning beta cells in the
pancreatic islets. With chronic administration in type II diabetic
patients, the blood glucose lowering effect persists despite a
gradual decline in the insulin secretory response to the drug.
Extrapancreatic effects may be involved in the mechanism
of action of oral sulfonyl-urea hypoglycemia drugs. The
combination of glibenclamide and metformin may have a
synergistic effect, scince both agents act to improve glucose
tolerance by different but complementary mechanisms. In
addition to its blood glucose lowering actions, glibenclamide
produces a mild diuresis by enhancement of renal free water
clearance.
Daonil is twice as potent as the related second generation
agent glipizide.
Sulfonylurea such as glibenclamide likely bind to ATPsensitive potassium- channel receptors on the pancreatic cell
surface,
reducing
potassium
conductance
and
causing
depolarization of the membrane. Depolarization stimulates
calcium ion influx through voltage – sensitive calcium channels,
raising intracellular concentrations of calcium ions, which
induces the secretion, or exocytosis, of insulin.
1.6. Glycosylated Hemoglobin:
Glycosylated hemoglobin is the term used to describe the
formation of a hemoglobin compound formed when glucose (a
reducing sugar) reacts with the amino group of hemoglobin (a
protein). The glucose molecule attaches nonenzymatically to the
hemoglobin molecule in a keto amine structure to form a
ketoamine(34). The rate of formation is directly proportional to
the plasma glucose concentration. Because the average red
blood cell lives approximately 120 days, the glycosylated
hemoglobin level at any one time reflects the average blood
glucose level over the previous 2-3 months. Therefore,
measuring the glycosylated hemoglobin provides the clinician
with a time- averaged picture of the patient's blood glucose
concentration
over
the
past
3
months(35).
Hemoglobin
A1c(HbA1c), the most commonly detected glycosylated
hemoglobin, is aglucose molecule attached to one or both Nterminus valines of the -polypeptide chains of normal adult
hemoglobin.
HbA1c is a reliable method of monitoring long-term
diabetes control rather than random plasma glucose (FBS)(36).
Normal values range from 4.5% to 8.0% using alinear
regression model (Rohlfing et al(37) determined that for every
1% change in HbA1c value, there is 35mg/dL (2 mmol/L)
change in the mean plasma glucose(37).
1.7. The plasma proteins(38,39):
The total protein of the plasma is about 7.0-7.5 g/dL. Thus,
the plasma proteins comprise the major part of the solids of the
plasma.
The proteins of the plasma are actually a very complex
mixture which includes not only simple proteins but also mixed
or conjugated proteins such as glycoproteins and various type of
lipoproteins in normal human plasma, six distinct moving
boundaries have been identified.
These are disignated in order of decreasing mobility as
albumin, alpha 1 and alpha 2 globulins, beta globulin,
fibrinogen, and gamma globulin.
The distribution of electrophoretic components of normal
human serum is as follows albumin 52-65% of total plasma
protein globulin 29.5-54.0% (3.2-5.6 g/dL).
1 2.5-5% (0.1-0.4 g/dL).
2 7-13% (0.4-1.2 g/dL).

8-14% (0.5-1.1 g/dL).

12-22% (0.5-1.6 g/dL).
Fibrinogen 6.5%.
1.8. Albumin:
Albumin, with a molecular weight of about 65000Da., is
synthesized by the liver. It has a normal plasma biological halflife of about 20 days.
About 60 percent in the extracellular fluid is in the
interstitial compartment. However, the concentration of albumin
in the smaller intravascular compartment is much higher
because of the relative impermeability of the blood vessel wall.
This concentration gradient across the capillary membrane is
important in maintaining plasma volume(28).
Albumin has two well-known functions. One is the
contribution of albumin to the colloid osmotic pressure of the
intravascular fluid. Because of its high concentration, albumin is
responsible for nearly 80% of this pressure, which maintains the
appropriate fluid in the tissue.The other prime function is its
propensity to bind various substances in the blood. For example,
albumin binds bilirubin, salicylic acid, fatty acids, calcium and
magnesium ions, cortisol, and some drugs.This characteristic is
also exhibited with certain dyes, providing a method for the
quantitation of albumin(13).
1.9. Acute phase proteins(40):
The levels of certain proteins in plasma increase during
acute inflammatory states or secondary to certain types of tissue
damage are called "acute phase proteins" or "reactants" and
include
C-Reactive
protein
(C.R.P).,
1-antitrypsin,
haptoglobin, 1-acid glycoprotein, and Fibrinogen.
1.9.1. C-Reactive protein(CRP):
CRP is one of the first acute phase proteins to rise in
response to inflammatory disease.
It is significantly elevated in acute rheumatic fever,
bacterial infections, myocardial infarcts, rheumatoid arthritis,
carcinomatosis, gout, and viral infections.
C-Reactive protein (CRP) is synthesized in the liver and
appears in the blood of patients with diverse inflammatory
diseases(41).
CRP was so named because it precipitates with the C
substance, a polysaccharide of pneumococci. However, it was
found that CRP rises sharply whenever, there is tissue necrosis,
whether the damage originates from apneumococcal infection or
some other source. This led to the discovery that CRP
recognizes and binds to molecular groups found on a wide
variety of bacteria and fungi. CRP bound to bacteria promotes
the binding of complement, which facilitates their uptake by
phagocytes. This process of protein coating to enhance
phagocytois is known as opsonization (42,43).
1.9.2. 1-Antitrypsin:
1-Antitrypsin is an acute-phase reactant. Its main
function is to neutralize trypsin-like enzymes (i.e., elastase) that
can cause hydrolytic damage to structural protein.
1-Antitrypsin is a major component (approximately 90%) of
the fraction of serum proteins that migrates electrophortically
immediately following albumin.
A deficiency of 1-antitrypsin is associated with severe,
degenerative, emphysematous pulmonary disease.
The lung disease is attributed to the unchecked proteolytic
activity of proteases from leukocytes in the lung during periods
of inflammation.
Juvenile hepatic cirrhosis is also a correlative disease in
1-antitrypsin deficiency.
The protein is synthesized but not released from the
hepatocyte(44).
Increased levels of 1-antitrypsin are seen in inflammatory
reactions, pregnancy, and contraceptive use(13).
1.10. Immunoglobulins (Igs)(45):
There are five major groups of immunoglobulins in the
serum: IgA, IgG; IgM, IgD, and IgE.
They are synthesized in plasma cells. Their synthesis is
stimulated by an immune response to foreign particles and
micro organisms.
The immunoglobulins are not synthesized to any extent by
the neonate. IgG crosses the placenta; the IgG present in the
newborn's serum is synthesized by the mother. IgM does not
cross the placenta but rather is the only immunoglobulins
synthesized by the neonate.
The concentration of IgM initially is 0.21 g/L, but this
increases rapidly to adult levels by about age 6 months. IgA is
virtually lacking at birth (0.003 g/L), increases slowly to reach
adult values at puberty, and continues to increase during the life
time.The immunoglobulins comprise two long polypeptide
chains (heavy, or H, chains) and two short polypeptides (light,
or L, chains), joined by disulfide bonds. An individual is
capable of producing 1 million different immunoglobulin
molecules.The differences among these molecules are found in a
region of the molecule called the variable region.This variable
region is located on the end of the molecule that contains both
the light and heavy chains and is the site at which the
immunoglobulin (antibody) combines with the antigen.The
differences in the heavy chains (H) are called idiotype and are
designated IgG, IgA; IgM, IgD, and IgE.
The heavy chains are called , , , , and , respectively.The
light chains (L) for all the immunoglobulin classes are of two
kinds, either K or . Each immunoglobulin or antibody
molecule has two identical H chains and two identical L chains.
For example, IgG has two  type H chains and two identical L
chains (either  or ) .
The basic immunoglobulin IgG is a Y-shaped molecule
depicted schematically in fig(1-5)(40).
Figure (1-5): Structure of IgG(40).
Aim of study :Evaluate the effect of insulin on some biochemical parameters
related to acute phase proteins and immunoglobulin in DM
patients.
Chapter two
Subjects and Methods
2.1. Material and Subjects:
Table (2.1): chemicals used their suppliers:
Chemicals
Suppliers
1.
Glucose MR-Kit
Segma Co. Germany
2.
Tri Sodium citrate
Segma Co. Germany
Na3C6H5O7. 2H2O
3.
Total protein- Kit
Linear. Es (Spain)
4.
Albumin- Kit
Linear. Es (Spain)
5.
C- Reactive protein- Kit
Randox- United Kingdom
6.
IgM- Kit
The Binding site. Co. USA
7.
IgG- Kit
The Binding site. Co. USA
8.
IgA- Kit
The Binding site. Co. USA
9.
1-anti trypsin- Kit
The Binding site. Co. USA
2.2. Instrument and Manufacturers:
Table (2.2): The instruments and their manufacturers:
Instruments
Manufactures
Centrifuge
Universal 16A, (Germany)
Variant
Bio-Red, (USA)
Incubator
Fisher Scientific, (USA)
Auto Vortex
Stuart Scientific, (USA)
Oven 50oC
Memert, (Germany)
UV.vis Spectrophoto Meter
Milton Roy Co., (USA)
2.3. Sampling:
The
samples
were
collected
from
"Al-Kadhimyah
Teaching Hospital". They have been classified into three groups
as the following:1) Control group:- include (30) healthy individual from both
sexes, with age range (20-70) years and no previous
disease which may interfer with the parameters analyzed
in this study.
2) Type-1- (Insulin Dependent Diabetes Mellitus) IDDM
group: include (50) patients from both sexes, with age
range (20-60) years.
3) Type-2- (non-Insulin Dependent Diabetes Mellitus)
NIDDM group: include (50) patients from both sexes,
with age range (30-70) years.
Excluding criteria:Male patients suffer from infection of the renal tubules or
fungus of the renal system.
Female patients were suffering from acute reproductive system
infections.
All patients were not taking any non steroidal anti inflammatory,
aspirin and statin drugs, also not taking Angiotension
Converting Enzyme Inhibitor (ACEI) and anti-diabetic drug
Thiazolidinediones (Glitazones).
2.4. Collection of Blood:
10 ml vienous blood was taken from the above groups,
place in a plane tube (no anti coagulant) left for (15min) at room
temperature, then centrifuged (at 2500 rpm from 10min). to get
the serum, which is stored at (-20oc) unless used immediately.
Whole blood was used for ESR and HbA1c determination.
2.5. Determination of Fasting Blood Sugar
(FBS):
2.5.1. Principle:
The determination of serum glucose was done for each
blood specimen using glucose enzymatic colorimetric test
(GOD-PAP).
The enzymatic color test was done on basis of trinder
reaction(47,48).
Glucose + O2 + H2O
Glucose
Oxidase
Gluconic acid + H2O2
2H2O2+ Phenol + 4-aminoantipyrine peroxidase red chinonimin + 4H2O
2.5.2. Reagent concentration:
(a) Reagent 1. (Buffer solution):
- pipes pH. 7.5
150 mmol/L
- P. chlorophenol
7.5 mmol/L
(b) Reagent 2. (Substrate):
- GOD
12000 U/I
- POD
660 U/I
-4-amino anti pyrine
0.40 mmol/L
(c) Reagent 3. (Standard):
Standard
100mg/dI
(d) Preparation and stability:
Dissolve the contents of one bottle of R2 with one bottle
buffer Reagent R1. This working reagent is stable for 2 weeks at
20-25oC or 2 months at 2-8 oC.
2.5.3. Procedure:
1) Tenl of serum/ standard was added to 1ml of working
reagent.
2) Were mixed and left for 30 minutes at room temperature.
3) Then the absorbance of sample was read against the
reagent blank "which has been adjucted to zero" at 500nm
with in 60 mints.
2.5.4. Calculation:
A sample
Glucose concentration (mg/dl) = A standard
x C standard
Reference values : Serum. Plasma 70 – 115 mg/dl
2.6. Determination of Glycated Haemoglobin
(HbA1c):
2.6.1. Principle)(49):
The levels of HbA1c were determined utilizing the variant
haemoglobin A1c program. The principles of determination was
based on ion exchange high performance liquid chromatography
(HPLC) for the automatic and accurate separation of HbA1c. The
separation of HbA1c is performed rapidly and precisely,with out
interference from Schiff
base, lipemia, or temperature
fluctuations. The variant's two dual- piston pumps deliver a
programmed buffer gradient of increasing ionic strength to the
system. Prepared samples are automatically injected into the
analytical flow path and applied to the cation exchange column,
where the haemoglobin is separated, based upon the attraction of
haemoglobin to the column material.
The separated haemoglobin then passes through the flow cell of
the filter photometer where changes in the absorbance (415 nm)
are measured. A graph of the changes in the absorbance is
plotted versus the retention time(49).
2.6.2. Reagents:
1.
2.
3.
4.
5.
Reagent type
Reagent (1)
Buffer (1)
Reagent (2)
Buffer (2)
Reagent (3) wash
Solution
Reagent (4) hemolysis
Reagent
Reagent (5) hemoglobin
A1c Calibrator.
6.
Reagent (6)
Calibrator diluent
7.
Reagent (7) hemoglobin
Primer
8.
Reagent (8) lyphochek
Control
Material and concentration
Sodiumphosphate buffer,
pH 5.9
Sodiumphosphate buffer,
pH 5.6
Deionized water, pH 6.6
Citrate solution, pH 5.0
Lyophilized human red blood cell
hemolysate containing
gentamicin.Tobramycin. and EDTA
as preservative
Deionized water, plus EDTA and
potassium cyanide as preservatives,
pH 7.2
Lyophilized human red blood cell
hemolysate containing gentamicin,
tobramycin, and EDTA as
preservatives.
Lyophilized.
2.6.3. Procedure:
2.6.3.1. Preparation of HbA1c Calibrator:
The lyophilized HbA1c Calibrator (R5) was reconstituted
with 10 ml of cold calibrator diluent (R6), allowed the calibrator
to stand for 5-10 min, swirled gently to dissolve.
2.6.3.2. Preparation of haemoglobin primer:
The haemoglobin primer (R7) was reconstituted with 1ml
of deionized water (R3), swirled gently to dissolve after standing
for 10 min at 15-30oC.
2.6.3.3. Preparation of lyphochek control:
The lyphochek control (R8) was reconstituted with 0.5 ml
deionized water (R3), then let to stand for 2 to 3 min., swirled
gently to dissolve.
1) The sample test tubes of anticoagulated whole blood
placed on a plate for mixing until samples are
homogenous.
2) A laboratory marker was used to label two 1.5 ml sample
vials for HbA1c calibrator and put it into the sample tray
wells 1 and 2.
3) 1.0 ml of reconstituted calibrator was added into the
properly labeled vial. No dilution is required for the
calibrator.
4) 1.0ml of haemolysis reagent was added to each of the
control and patient sample vials.
5) Five L of whole blood patient sample or reconstituted
control were removed and displaced into the bottom of the
properly labeled sample vial.
6) Then the samples stand at 18-28oC for at least 15 min. to
allow Schiff base removal. The sample tray and cover
were placed into the sample compartment.
7) The key was pressed to begin the system flush.
* Fresh aliquots of hemoglobin primer were used at the
beginning of each run to condition the cartridge for analysis.
2.6.4. Calculation:
The value of HbA1c was given directly by the instrument(3).
(N-V) = 4.1 – 6.5%).
2.7. Determination of total proteins (TP).
Total proteins were determined "according to biuret methods. (50)
2.7.1. Principle:
This method depends on the reaction of peptide bond of
the protein with cupric ion (Cu+2) in alkaline medium to form
colored products whose absorbance is measured at 540nm(50,51).
Cu+2 + serum protein
pH >12
(25-37)oC
copper-protein complex
2.7.2. Reagents:
1) Biuret reagent (100mmol/L sodium hydroxide. NaOH,
16mmol/L Na-K-tartrate, 15 mmol/L potassium iodide,
and 6mmol/L cupric sulphate).
2) Blank reagent: (100 mmol/L NaOH and 16 mmol/L NaK- tartrate).
3) Standard protein solution (6 gm/dl).
2.7.3. Procedure:
Into a curvett the following solutions were pipetted:
Reagent
Distilled water
Standard
Serum sample
Biuret reagent
Standard
0.02 ml
1 ml
Sample
0.02 ml
1 ml
Blank
0.02 ml
1 ml
The a bove solutions were mixed, incubated for 10 min. at 2537oC.
The absorbance of sample were measured (A sample) and
of the standard (A standard) against the reagent blank.at540 nm
2.7.4. Calculation:
Total protein concentration(gm/dl) =
Asample
Astandard
x standard
concentration of protein
Standard concentration of protein = 6.0 gm/dl.
2.8. Determination of serum albumin(52):
2.8.1. Principle:
The measurement of serum albumin was based on its
quantitative binding to the indicator 3.3`, 5.5`- tetra bromo-m
cresol. Sulphonphthalein (bromo cresol green, BCG).
The albumin-BCG- complex absorbs maximally at 578nm.
2.8.2. Reagents:
Contents
Initial concentration of solutions
1- BCG concentrate
succinate buffer
75.00mmol/L, pH =4.2
Bromo cresol. Green 0.15 mmol/L
Brig 35
Preservative
2- Standard Human
45.00 gm/L
serum Albumin
2.8.3. Procedure:
Reagent blank
Distilled water 0.01 ml
Standard
Serum
BCG reagent 3.00 ml
Standard
0.01 ml
3.00 ml
Sample
0.01 ml
3.00 ml
Each of the above solutions (Blank; standard and sample)
were pipetted into test tubes, mixed, and incubated for 5 minutes
at +20 to +25oC. The absorbance of the sample and the standard
solutions were measured against the blank at 630nm.
2.8.4. Calculation:
The albumin concentration in the sample was calculated
from the following formula:
Albumin concentration (gm/L)=
standard.
Asample
Astandard
"Concentration of standard = 45.00 gm/L"
x Conc. of
2.9. Determination of Erythrocytes
Sedimentation Rate (ESR)(52):
2.9.1. Principle:
The procedure is an indirect method quantifying red cell
agglomeration or rouleaux formation. In normal anticoagulated
blood, red cell form loose agglomerations or aggregations and
since the sedimentation rate of the agglomerants increases as
their diameter increases, therefore; the time course of the red
cell sedimentation; if carefully observed, has a slow phase
followed by a more rapid phase. However, in disease cases, the
levels of variety of plasma proteins increase as a result for
increased red cell agglomeration leading to accelerated
sedimentation(53).
2.9.2. Procedure:
1) Two ml of blood sample, 0.5ml of ESR-solution sodium
citrate, were mixed in anticoagulant tube contain EDTA.
2) The pipette tube "Westergren tube" was inserted to the
bottom of the anticoagulant tube and the mixture with
drawn as well as its level was adjusted to the zero mark.
3) The blood was allowed to sediment. for 60 minutes, the
distance in mm was noted between the plasma meniscus
and the top of the red cell column.
2.10. Determination of C-reactive protein(CRP)(54,55):
2.10.1. Principle:
CRP was measured by rapid test for the qualitative and
semiquantitative
determination
of
CRP
in
serum
by
agglutination of latex particales on slide.
The latex reagent is a suspension of polystyrene latex
particles of uniform size with the IgG fraction of an anti-human
CRP specific serum.
Latex particles allow visual observation of antigenantibody, if the reaction takes place due to the presence of CRP
in the serum, the latex suspension changes its uniform
appearance and a clear agglutination becomes evident.
This change occur because the CRP present in the serum
reacts with IgG coated to the latex particles, starting the
formation of a web between them. When the latex reagent is
mixed with the serum contains approximately more than 6mg/L
of CRP a clear agglutination will appear.
2.10.2. Procedure:
1) The reagent and serum were kept at room temperature.
2) One drop (50l) of serum was added to the test circle on
the slide.
3) The latex reagent was shaked, then one drop of suspension
was added to the test circle.
4) The drops were mixed using adiposable stirrer, in which
the test circle was covered with the mixture.
5) Gently and evently; rock and rotate the test slide for 2
minutes whilst examining the test slide for agglutination.
2.10.3. Interpretation of results of qualitative test:
The presence of agglutination indicates a content of CRP
in the serum is equal to or greater than 6 mg/L. The absence of
agglutination indicates a content of CRP. in the serum is less
than 6mg/L.
2.10.4. Positive reaction:
+++ large clumping with clear back ground.
++ moderate clumping with fluid a paque in back ground.
+
small clumping with a paque fluid in back ground.
+
represent value 6 mg /L
++ represent value 12 mg /L
+++ represent value 18 mg /L
2.10.5. Negative reaction:
No visible clumping, uniform suspension.
2.11. Determination of Immunoglobulines (IgM;
IgG; IgA; 1-antitrypsin) in Serum(56,57):
2.11.1. Principle:
The procedure consists in animmuno precipitation in
a agarose between an antigen and its homologous antibody.
It is performed by incorporating one of the two immune
reactants (usually antibody) uniformly throughout a layer of
agarose gel, and then introducing the other reactants (usually
antigen) into well punched in the gel. Antigen diffuses radially
out of the well into the surrounding gel- antibody mixture, and
a visible ring of precipitation forms where the antigen and
antibody reacted.
2.11.2. Procedure:
Radial immuno diffusion (RID) plates were:
1) Opened for few minutes to allow evaporation of the
moisture.
2) Five l of serum was applied into walls on the plate.
3) The lid was closed firmly and the plate were incupated at
room temperature for three days.
4) The diameters were measured accurately to with in 0.1
mm with magnifying glass lens (Jeweller's eye piece)
capable of measuring with 0.1mm precision.
5) The results were evaluated using the table of reference
provided with the plates.
2.11.3. Statistical analysis:
Data presented were the means and standard deviations,
student -t- test was used to compare the significance of the
difference in the mean values of any two groups. (P0.05) was
considered statistically significant(58).
The overall predictive values for the results in all studied
groups were performed according to program of office XP 2002.
Chapter Three
Results and Discussion
3. Results and Discussion
Table (3-1) and (3-2) and figures (3-1) and (3-2) showed
the levels of F.B.S and HbA1c in sera of patients with type 1
(IDDM), type 2 (NIDDM) and control.
A marked increase in F.B.S (8.33.08, 13.31.970) and
HbA1c (70.62,101.416) levels in sera of type 1 and type 2
compared to control (4.230.93,5.270.726) respectively was
found. All elevated levels were significant between both patients
groups and control also between the groups themselves for
F.B.S and HbA1c.
A healthy person has around 20.000 insulin receptors sites
per cell, while people with insulin resistance can have as low as
5000 of these sites per cell, the result of this is that glucose can
not be efficiently transferred by insulin through these receptor
sites from the blood stream into the cell to be burned as energy
This causes elevated blood sugar level(59) Two factors determine
the glycosylated hemoglobin levels: the average glucose
concentration and the red blood cell life span if the red blood
cell life span is decreased because of another disease state such
as hemoglobinopathies, the hemoglobin will have less time to
become glycosylated and the glycosylated hemoglobin level will
be lower
(13)
because A1c based on hemoglobin both qualitative
and quantitative variations in hemoglobin can effect the A1c
value (60).
Some researches demonstrated the relationship between
iron and glucose metabolism, because iron modulates insulin
action in human(61-63).
Table (3-1): F.B.S level in sera of three studied groups
GROUPS
NO.
Control
F.B.S (mmol/L)
Mean ± SD
2.4 ± 0.93
50
Type 1
3.8 ±3.08
05
Type 2
P
50
13.3 ±1.97
P≤0.05
P≤0.05
*P≤0.05
F.B.S
mmol/L
13.3
14
12
10
8
6
4
2
0
8.3
4.2
control
type 1
type 2
Figure (3-1): F.B.S level in sera of three studied groups
P*
= P . value between Type1 and Type2
Table (3-2): HbA1C% in sera of three studied groups
NO.
GROUPS
Control
HbA1c%
Mean ± SD
50
5 ± 0.72
05
Type 1
Type 2
P
50
7 ± 0.62
P≤0.05
10 ±1.41
P≤0.05
*P≤0.05
HbA1C%
10%
10.00%
%
8.00%
7%
5.00%
6.00%
4.00%
2.00%
0.00%
control
type 1
type 2
Figure (3-2): HbA1C% in sera of three studied groups
Table (3-3) and figure (3-3) show the results of total
protein (TP) in (g/dL) and albumin in (g/dL) in sera of type 1
(IDDM), type 2 (NIDDM), and control group. Total protein and
albumin for control group is (6.880.77) (4.830.009), for type
1 group is (6.850.651) (4.7600.227) g/dL, and for type 2
group is (6.990.716) (3.430.177) g/dL respectively.
From the table (3-3) there was no significant difference in
total protein levels between group type1 and group of control,
with P value equal (0.856) which is high than (0.05) as (P0.05)
is considered significant, no significant difference between
control and type 2 group P value equal (0.462) also no
significant differences between both groups of patient type 1
and type 2 with P value equal (0.323).
A significant reduction of albumin level for type 2 group.
compared to control with P value (7.43x10-76) while no
alteration in albumin level between type 1 and control groups
was found. Also significant differences were found between
type 1 and type 2 with P value(2.77x10-54).
Albumin normally makes the largest single contribution to
plasma total protein.
Total protein levels may be misleading, and may be
normal in the face of quite marked changes in the constituent
proteins.
For example: A fall in albumin may roughly be balanced
by arise in immunoglobulin levels. This is quite a common
combination. Most individual proteins, other than albumin,
make a relatively small contribution to total protein, quite a
large percentage change in the concentration of one of them may
not be detectable as a change in total protein (64).
Constituent proteins, only low albumin levels are of a
clinical importance
(65)
. A low plasma albumin level despite a
normal total body albumin may be due to dilution by an excess
of protein – free fluid, or to redistribution into the interstitial
fluid due to increased capillary permeability. There may be true
albumin deficiency due to a decreased rate of synthesis, or to an
increased rate of catabolism or loss from the body.
The slight fall in the albumin level found in even mild
acute illness may be due to a combination of the above two
factors(66).
Reduction in albumin concentration was reported in
inflammatory processes, including acute-phase response and
chronic inflammatory disorders and in neoplastic diseases(67).
Table (3-3) TP and Albumin levels in sera of three studied groups
TP(g/dl )
GROUPS
NO.
p
Albumin
Mean ± SD
P
(g/dl)
Mean ± SD
Control
50
6.88 ±0.77
4.83 ±0.009
Type 1
50
6.85 ±0.65
P>0.05
4.76 ±0.22
P>0.05
Type 2
50
6.99 ±0.71
P>0.05
3.43±7.43
P≤0.05
*P>0.05
7
(TP)
Albِumin
6
6.88
4.83
6.85
*P≤0.05
6.99
4.76
5
3.43
4
TP
Albumin
3
2
1
0
control
type 1
type 2
Figure (3-3) TP and Albumin levels in sera of three studied
groups
Table (3-4) and figure (3-4),(3-5),(3-6) showed the results
of ESR, CRP and 1-antitrypsin in sera of patients with type 1
type 2 DM and control groups.
A significant increase in level of ESR, CRP, 1-antitrypsin
levels in sera of patients with type 2 DM compared with
control(P0.05), while no significant alteration in ESR, CRP,
1-antitrypsin levels in sera of patients with type 1 DM
compared with control, also a significant differences was found
between both patients groups themselves.
Under physiologic conditions the liver synthesizes mainly
constitutive hepatic proteins, such as albumin, prealbumin, or
transferrin. After trauma the synthesis shifts from constitutivehepatic proteins to acute phase proteins, such as haptoglobin,
2-macroglobulin, 1-acidglycoprotein, and c-reactive protein
(CRP)(68).
This reaction of the liver is called the hepatic acute phaseresponse. The goal of the hepatic acute-phase response is to
restore homeostasis, however, a prolonged and exaggerated
response leads to the enhancement of hypermetabolism and
catabolism, thus to increase morbidity and mortality(69-73).
Mediators
of
the
acute-phase-response
are
pro-
inflammatory cytokines, such as interleukin-1 (IL-1B), inter
leukin-6 (IL-6), interlukin-8 (IL-8),tumor-necrosis factor (TNF),
or the anti-inflammatory cytokine interleukin -10 (IL-10) (68).
An inflammatory pattern indicating an inflammatory
condition is seen when there is a decrease in albumin and an
increase in the 1-globulins (1-acid glycoprotein, 1antitrypsin), 2-globulis (Ceruloplasmin and haptoglobin), and
-globulin blood (C-Reactive protein).
(13)
Although the main
physiological abnormalities are insulin resistance and impaired
insulin secretion(74,75), specific underlying determinates of these
metabolic defects remain uncertain. An accumulating body of
evidence suggests that inflammation may play a crucial
intermediary role in pathogenesis, thereby linking diabetes with
a number of commonly coexisting conditions thought to
originate through inflammatory mechanisms(76).
Inflammation as measured by c-reactive protein (CRP) has
been shown to be increased in people with type 1 and type 2
diabetes who have macrovascular complications(77-80).
Increased serum levels of inflammatory biomarkers of
arteriosclerosis, like c-reactive protein, cytokins, like tumor
necrosis factor-alpha or interleukin-6, as well as novel markers
like monocyte chemoattractant protein (MCP)-1, soluble CD40
ligand (sCD40L), and matrix metalloproteinases (MMP) have
been shown to predict cardiovascular risk and seen to reflect the
over all burden of vascular disease in patients(81).
Our results agree with studies claimed that some of these
markers are elevated in patients with type 2 diabetes and insulin
resistance, indicating a pivotal role of inflammation in this
metabolic disorder (82-84). However, The results of present study
agree with previous study found a positive correlation
between inflammatory markers and type 2 diabetes(85, 86).
The research data suggest that the release of inflammatory
mediators like tumor necrosis factor-alpha and interleukin -6
(IL-6) from the visceral adipose tissue as well as an activation of
vascular cells itself contribute to the inflammatory state in these
patients with metabolic syndrome (87, 88).
Adipose tissue (body fat) has been lately regarded as a
separate body organ which can produce a number of different
biologically active molecules-such as cytokine proteins that are
associated with inflammation, and the hormone resistance,
which is linked to insulin resistance and the development of type
two diabetes(89).
(Jerome, and Rotter(90)) showed that four specific gene
variations were significantly linked with high levels of insulin
resistance.
Among these inflammatory genes, IL4, IL 4R and C4 were
found to have significant variation despite the patients age, sex,
or body mass index (BMI), while variation in IL6 affected
insulin resistance only through excess body fat "Rotter said" in
other words, it appears that low grade inflammation causes
insulin resistance and is not just a consequence of insulin
resistance(90).
There is now an evidence that insulin improves hyper
metabolism by affecting pro-inflammatory cytokine production
and hepatic signal transcription factor expression(91).
In the present study we investigated the effects of insulin
hormone and daonil drug on the systemic inflammatory
response in patients with type 1 and type 2 DM respectively
both suffering from the same inflammatory diseases. Without
taking any non steroidal anti inflammatory, aspirin and statin
drugs, ACEI, and Glitazones. Showed that insulin is decreasing
pro-inflammatory hepatic acute-phase protein concentrations in
sera of patients with type 1 DM group compared with the effects
of daonil drug on the systemic inflammatory response in
patients with type 2 DM. Results given the fact that the insulin
treat
diabetes,
also
may
have
potential
treatment
for
inflammatory diseases.
These data suggest that insulin acts as an antiinflammatory molecule through direct cellular effects rather
than through indirect effects, which would be by modulating
glucose concentration.
Insulin at a dose that kept blood glucose below 110mg/dL
decreased mortality and prevented the incidence of multi-organ
failure in critically ill patients(92).
In an animal model, insulin had anti-inflammatory effects
by decreasing pro-inflammatory signal transcription factors and
pro-inflammatory cytokines, while increasing anti-inflammatory
cytokines. However, it is still unknown whether insulin exerts
its effects directly through modulating pro-inflammatory
mediators
or
indirectly
through
modulating
glucose
concentration(91).
Table (3-4) ESR, CRP, Alpha 1- antitrypsin levels in sera of three
studied groups
No.
GROUPS
ESR(mm/1hr)
P
Mean ± SD
CRP(mg/dl)
Alph 1 –
Mean ± SD
antitrypsin(mg/d
P
l) Mean ± SD
50
10.4 ±3.79
Type 1
50
12.5 ±3.78
Type 2
50
32.4±8.67
-
131.1± 45.3
P≤0.05
-
130.4±32.8
P>0.05
P≤0.05
9 ±0.302
194±4.6
P≤0.05
Control
*P≤0.05
*P≤0.05
mm/ hr.
ESR
32.4
40
30
20
10.4
12.5
10
0
control
type 1
type 2
Figure (3-4) ESR level in three studied groups
CRP
1.5
1.2
1
0.5
0
control
type 1
type 2
Figure (3-5) CRP level in sera of three studied groups
Alpha1-antitrypsin
194
200
131.1
130.4
150
100
50
0
control
type 1
type 2
Figure (3-6) Alpha 1- antitrypsin level in sera of three
studied groups
Table (3-5) and figure (3-4), (3-5), (3-4) showed the
results of IgG, IgM, IgA in sera of patients with type 1, type 2
DM and control groups.
A
marked
significant
increase
in
IgG
and
IgA
(1911281.2)(447.6172.2) levels respectively and a significant
decreased in IgM(26.97.3) level in sera of patients with type 2
DM compared to control, while no significant alteration in IgG,
IgM and IgA was found in sera of patients with type 1 DM
compared to control group. Also a significant differences in
IgG, IgM & IgA levels between both patients groups themselves
was found.
Infections stimulate the immune system and may result in
increased immunoglobulin levels(93, 94).
It has been postulated that type 2 DM may represent a
disease of the innate immune system(95), a hypothesis of
particular interest because both of these
inflammatory
biomarkers also are known to predict the development of
cardiovascular disease in otherwise healthy populations(96-98).
Interleukin 6, a major proinflammatory cytokine, is
produced in a variety of tissues, including activated leukocytes,
adipocytes,
and endothelial cells. C-reactive protein is the
principal downstream mediator of the acute phase response and
is primarily derived via IL-6-dependent hepatic biosynthesis. In
rodent models of glucose metabolism, the in vivo infusion of
human
recombinant
IL-6
has
been
shown
to
induce
gluconeogenesis, subsequent hyperglycemia, and compensatory
hyperinsulinemia(100). Our results are in agreement with similar
metabolic responses have been observed in humans after
administration of subcutaneous recombinant IL-6
(101)
. Cross-
sectional investigations further support a role for inflammation
in the etiology of diabetes; several studies have demonstrated
elevated levels of IL-6 and CRP among individuals both with
features insulin resistance syndrome and clinically over type 2
DM(101-105).
Table (3-5) IgG, IgM, IgA levels in sera of three studied groups
No.
IgG(mg/dl)
P
IgM(mg/dl)
P
IgA(mg/dl)
P
Mean ± SD
Mean ± SD
Mean ± SD
50
919.8 ±307.5
141.9±85.7
158.1±92.3
50
919.3±368.36
P>0.05
133.2±62.5
P>0.05
159.5±81.3
P>0.05
50
1911.9±281.5
P≤0.05
26.9±7.36
P≤0.05
447.6±172.2
P≤0.05
GROUPS
Control
Type 1
Type 2
P≤0.05
P≤0.05
1911.9
2000
1500
919.3
919.8
1000
447.6
500
P≤0.05
141.9
158.1
133.2 159.5
IgG
IgM
IgA
26.9
0
control
type 1
type 2
Figure (3 - 7) IgG, IgM, IgA levels in sera of three studied
groups
Conclusions & Future Work
And Appendix
((Conclusion))
The data obtained from this study enable us to conclude the
following:1. A significant increase in F.B.S. and HbA1c level in sera of
patients with D.M. (type 1 &type2) compared to control.
2. There is no significant difference in total protein level in
sera of patients with D.M. (type 1 & type 2) compared
with control.
3. A significant reduction in albumin level in sera of patients
with type 2 DM compared to control, while there is no
alteration in albumin level in sera of patient with type 1
DM compared to control.
4. A significant increase in ESR, CRP, and
 1-antitrypsin
level in patients with type 2 DM compared with control,
while, there is no significant differences in ESR, CRP, and
 1-
antitrypsin levels in sera of patient with type-1-DM Compared
with control.
5. A significant increase in IgG & IgA levels and a
significant decrease in IgM level in sera of patients with
type 2 DM compared to control, while there is no
significant difference in IgG, IgM and IgA levels in sera of
patients with type 1 DM compared to control.
6.
Our results suggest that insulin could act as an anti –
inflammatory molecule through direct cellular effects
rather than through indirect effects by reduction glucose
concentration.
((Future Work))
1. To find a relation between insulin resistance ((IR)) and
HbAic in two types of DM.
2. To correlate Insulin resistance ((IR)) with oxidative stress
and proteins of Iron and copper transport.
Appendix
((Study protocol))
The role of insulin effect on inflammatory response
Compared to Daonil in DM Patients:-
Code No:
Data:
Hospital:
Name :
Age:
Drugs:
A- History:- Duration of Symptoms .
> 1 year
- Medical History .
- Surgical History.
- Habits.
B- Research Tests:- FBS =
mmol/L
-HbAic =
%
-Total Proteins =
g/dl
-Albumin =
g/dl
-ESR=
mm/ 1hr
< 1 year
- CRP=
g/dl
-IgM =
g/dl
-IgA=
g/dl
- IgG=
g/dl
-
anti trypsin =
g/dl
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‫الخالصة‬
‫صممت هذه الدر ِ‬
‫اسة لتكون الحجر االساس ِ‬
‫األنسولين كعامل مضاد‬
‫لدور‬
‫ُ ّ ْ‬
‫َ‬
‫ِ‬
‫ِ‬
‫المختلفة‪.‬‬
‫اإللتهاب‬
‫لاللتهاب في عمليات‬
‫عينات الدم الور ِ‬
‫ِ‬
‫يدية للصائمين ِم ْن ‪ 005‬فردا و التي منها ‪05‬‬
‫اخذت نماذج‬
‫ّ‬
‫مر ِ‬
‫يض بنوِع مرض السكر ‪ 05 ،0‬مريض بنوِع مرض السكر ‪ ، 4‬و‪ 05‬فرِد من‬
‫االصحاء‪.‬‬
‫حللتتت كتتل نمتتاذج التتدم لكيتتاس ستتكر دم الصتتائم و النستتية المئويتتة للهيمو لتتوبين‬
‫المستكر والبتتروتين الكلتتي و االلبتومين ومعتتدك ترستتيب كريتات التتدم الحمتراء وبتتروتين –‬
‫س‪-‬التفاعلي والفا ‪-0-‬مضاد للتربسين والكلوبيولينات المناعية ‪IgA, IgM ,IgG‬‬
‫بينتتت نتت ِ‬
‫تائ ُ الد ارست ِتة الحاليت ِتة يتتاد فتتي معتتدك ستتكر دم الصتتائم والنستتية المئويتتة‬
‫َ‬
‫للهيمو لوبين المسكر ‪ ،‬في ُك ّل من مرضى النوِع االوك والثاني ُمَك َارنة للسيطرِ ‪.‬‬
‫َكان ت ْتت مس تتتويات الب ت ِ‬
‫تروتين الكل تتي ب تتدون ت يي تتر ف تتي مص تتل دم كلتت تا مج تتاميع‬
‫ّ‬
‫المرضى مكارنة يالسيطر ‪.‬‬
‫وجت تتد نكصت تتان فت تتي مست تتتو االلبت تتومين فت تتي مصت تتل مرضت تتى النت تتوِع ‪ 4‬مكارنت تتة‬
‫لمجاميع مرضى النوع ‪0‬و السيطر ‪.‬‬
‫ِ‬
‫لتشخيص الي نوع من العملية االلتهابية معدك ترسيب كريتات‬
‫ارتفعت عوامل ا‬
‫الدم الحمراء وبروتين –س‪-‬التفاعلي والفا ‪ -0-‬مضاد للتربسين عند مرضى نتوِع ‪4‬‬
‫مكارنة لمجاميع مرضى نوِع ‪ 0‬والسيطر ‪.‬‬
‫ارتفعت تتت مست تتتويات الكلوبولينت تتات المناعيت تتة كلوبت تتولين جت تتي وكلوبت تتولين اي فت تتي‬
‫مصل دم مجموعتة مرضتى النتوع ‪ 4‬مكارنتة يمجتاميع مرضتى النتوع ‪ 0‬والستيطر بينمتا‬
‫قل مستو الكلوبيولين ام في مصل دم مرضى نوع ‪ 4‬مكارنة يمجتاميع مرضتى النتوع‬
‫‪ 0‬والسيطر ‪.‬‬
‫جمهورية العراق‬
‫وزارة التعليم العالي والبحث العلمي‬
‫جامعة بغداد – كلية التربية ابن الهيثم‬
‫تأثيـر األنسوليـن علـى االستجـابة االلتهابيـة‬
‫\‬
‫مقـارنـة بالسلفونيل يوريا في مـرضى داء‬
‫السكـري‬
‫رسالة‬
‫مقدمة إلى مجلس كلية التربية ‪ /‬ابن الهيثم – جامعة بغداد كجزء‬
‫من متطلبات نيل درجة ماجستير علوم في الكيمياء‬
‫من قبل‬
‫تمارا عالء حسين العبيدي‬
‫بكالوريوس كيمياء – جامعة بغداد – ‪5005‬‬
‫األستاذ المساعد الدكتور‬
‫زهير إبراهيم المشهداني‬
‫‪ 1459‬هـ‬
‫إشراف‬
‫المدرس الدكتورة‬
‫نجود فيصل السراج‬
‫‪ 5002‬م‬