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Islamic University Gaza

Deanship of Graduate Studies


Faculty of Science
Biological Sciences Master Program
Medical Technology

Cystatin C and Other Markers of Nephropathy


Among Type 2 Diabetic Patients in Gaza Strip

By
Raffat M. El Telbani
Supervisor
Prof. Dr. Baker M. Zabut

A thesis Submitted in Partial Fulfillment of Requirement for the Degree of


Master of Biological Science /Medical Technology

2013

Cystatin C and Other Markers of Nephropathy Among Type


2 Diabetic Patients in Gaza Strip
Abstract

Background: Nephropathy is a significant cause of morbidity and mortality in patients


with diabetes mellitus (DM). The condition is characterized by persistent albuminuria
and may be decline in the glomerular filtration rate (GFR). Serum cystatin C has been
proposed as a simple, accurate, and rapid endogenous marker of GFR.
Aim of the study: To assess serum levels of cystatin C and some biochemical
parameters among type 2 diabetes mellitus (T2DM) patients in Gaza Strip and whether
these levels vary with stages of diabetic nephropathy (DNP).
Methods: In this study, 95 patients and 95 controls were enrolled. Body mass index
(BMI) and blood pressure were measured after conducting face to face interview for
each participant. Morning fasting blood and urine samples were obtained for
measurement of serum cystatin C, creatinine, urea, glucose, lipid profile, and urine
albumin and creatinine. The albumin to creatinine ratio (ACR) was calculated. Serum
cystatin C and urine albumin were measured by particle enhanced immunoturbidimetric
assay. Serum and urinary creatinine, serum urea, glucose and lipid profile were
measured using a specific enzymatic assay. The patients were divided into those with
normo-, micro-, and macroalbuminuria.
Results: About 51.6 % of diabetic patients had at least one diabetes complications.
Frequencies of diabetes complications were increased with increase the duration of
diabetes. Diabetes was found to be associated with family history and BMI (all P<0.05).
About half of patients were diabetics since 5 years or less. Serum cystatin C levels were
non-significantly changed in diabetic patients compared to controls (P>0.05). Serum
urea and creatinine were lower in diabetics (all P<0.05). Cholesterol, triglycerides and
low-density lipoprotein (LDL) were significantly higher in diabetics than controls (all
P<0.05). In contrast, high-density lipoprotein (HDL) was significantly lower in
diabetics (P<0.05). Diabetic patients showed higher levels of ACR (P<0.05). In
contrast, urine creatinine level was lower in patients (P<0.05). The results of the study

III

showed that 28.4% of the diabetic patients had microalbuminuria and 16.8% had
macroalbuminuria. The mean levels of serum cystatin C in macroalbuminuria were
significantly higher than those in normoalbuminuria or microalbuminuria (all P<0.05).
The mean levels of serum urea in microalbuminuria and macroalbuminuria were
significantly higher than those in normoalbuminuria (all P<0.05). However, the mean
levels of serum creatinine in macroalbuminuria were significantly higher than those in
normoalbuminuria or microalbuminuria (all P<0.05). The mean levels of ACR in
macroalbuminuria were significantly higher than those in normoalbuminuria or
microalbuminuria (all P<0.05). In addition, the mean levels of ACR in
microalbuminuria were significantly higher than in normoalbuminuria (P<0.05). For
diabetic patients there were positive significant correlation between serum cystatin C
and age (r=0.440, P=0.000), duration (r=0.372, P=0.042), serum urea (r=0.873,
P=0.000), serum creatinine (r=0.892, P=0.000), cholesterol (r=0.283, P=0.005), LDL
(r=0.416, P=0.000) and urinary albumin (r=0.579, P=0.000), In contrast, cystatin C
was negatively correlated with HDL (r=-0.645, P=0.000) and urinary creatinine (r=0.656, P=0.000). Receiver operating characteristic (ROC) plots demonstrated that with
a cutoff value of 30 mg/g, the area under the curve (AUC) was 0.719 for cystatin C and
0.624 for creatinine. With a cutoff value of 300 mg/g, the AUC was 0.907 for cystatin C
and 0.882 for creatinine.
Conclusion: The results of this study suggest that cystatin C measurement in serum is a
useful, practical tool for the evaluation of renal involvement in the course of diabetes,
especially in patients with DNP.
Key words: Type 2 diabetes mellitus, diabetic nephropathy, cystatin C, Gaza Strip.

IV




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VI

Declaration
I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains material neither previously published or written by
another person nor material which to a substantial extent has been accepted for the
award of any other degree of the university of other institute, except where due a
acknowledgment has been made in the next.

Signature

Raffat

Name

Date

Raffat M. El Telbani

30/04/2013

Copy right.
All Rights Reserved: No part of this work can be copied, translated or stored in a
retrieval system, without prior permission of the authors. E-mail: raffatmt@hotmail.com

VII

Dedication

With all the love and heartfelt gratitude these simple words can
convey, I dedicate this work to:
My father and my mother who taught me how to give.
My wife who supported me on the way of success.
My daughters Nour, Nada and Aeaa who change my life.
My brothers and sisters who encourages me in my study.
All my friends who spare no effort to help.

VIII

Acknowledgments

This research would not have been possible without the time and support of many
individuals. My sincere appreciation and deepest gratitude to all those who helped
accomplish this dissertation. Specifically:
My supervisor Prof. Dr. Baker M. Zabut, professor of biochemistry and nutrition,
faculty of science, for his initiation and planning of this study, keen supervision and the
great valuable scientific help that leads to achieve this work.
All the academic and administrative staff of the biological science master program for
their guidance and support.
My friends and colleagues, for their persistent support and for always being there for me
offering a helping hand.
Administration and laboratory staff of Shohada' El-Aqsa hospital laboratory for their
assistance and support.
My best friend Mr. Bassem Hassan, the head of chemistry department in Shohada' ElAqsa hospital laboratory for continuous support and encouragement particularly his
numerous suggestions and recommendations.
My father, mother, brothers, sisters and daughters for their unwavering support, love,
and encouragement all over the period of study and my wife who has tolerated during
the study and for her cooperation made this work possible.

IX

Table of contents
Contents

Page

Abstract ..

III

Arabic abstract ..

Declaration .....

VII

Dedication ...

VIII

Acknowledgements ....

IX

Table of contents ....

List of tables ...

XIV

List of figures .....

XVI

Abbreviations .

XVII

Introduction ..

1.1 Overview ...

1.2 General objective .......

1.3 Specific objectives .

1.4 Significant of the study ..

Chapter 2

literature review ...

2.1 Diabetes mellitus ...

2.1.1 Type 1 diabetes ....

2.1.2 Type 2 diabetes ....

2.1.3 Gestational diabetes mellitus .......

2.1.4 Other specific types of diabetes .......

2.2 Prediabetes .........

10

2.3 Prevalence of diabetes mellitus ...

10

2.4 Complications ....

12

2.4.1 Microvascular complications ...

13

2.4. 1.1 Diabetic neuropathy .

14

2.4.1.2 Diabetic retinopathy .....

15

2.5 Diabetic nephropathy .........

16

Chapter 1

2.5.1 Definition, epidemiology and clinical stages ..

16

2.5.2 Urinary albumin excretion ...

17

2.5.3 Risk factors for development of diabetic nephropathy

19

2.5.3.1 Hyperglycemia .

19

2.5.3.2 Dyslipidemia .....

19

2.5.3.3 Smoking ....

20

2.5.3.4 Arterial hyper tension ...

20

2.5.3.5 Genetic susceptibility ...

21

2.5.4 Screening and diagnosis of diabetic nephropathy .......

21

2.6 Cystatin C ..

24

2.6.1 Cystatin C and diabetic nephropathy ...

27

Materials and methods .

31

3.1 Study design ..

32

3.2 Study population ....

32

3.3 Sample size ....

32

3.4 Exclusion criteria ...

32

3.5 Ethical considerations ....

33

3.6 Data collection ...

33

3.6.1 Questionnaire ...

33

3.6.2 Measurement of blood pressure ...

33

3.6.3 Measurement of body mass index ...

34

3.6.4 Specimen collection .....

34

3.7 Biochemical analysis .....

35

3.7.1 Determination of serum cystatin C ..

36

3.7.2 Determination of serum glucose ..

37

3.7.3 Determination of serum urea ...

38

3.7.4 Determination of serum creatinine ..

39

3.7.5 Determination of serum cholesterol ....

40

3.7.6 Determination of serum triglycerides ..

41

3.7.7 Determination of high-density lipoprotein ..

42

Chapter 3

XI

3.7.8 Calculation of serum low-density lipoprotein ......

43

3.7.9 Determination of urine albumin ......

44

3.7.10 Calculated measurements ..

45

3.8 Statistical analysis .....

45

Chapter 4

Results .......

46

4.1 General characteristics of the study population .....

47

4.1.1 Distribution of the study population by governorates of the Gaza Strip ...

47

4.1.2 Distribution of study population by gender .

47

4.1.3 Age of the study population

48

4.1.4 Self-reported complications among the study population .....

49

4.1.5 Smoking and family history among the study population .

50

4.1.6 Body mass index of the study population ....

50

4.2 Diabetes mellitus ...

51

4.2.1 Age at diagnosis and duration of the disease ...

51

4.2.2 Diabetes duration and self-reported complication ...

53

4.2.3 Diabetes mellitus management ....

53

4.2.4 Blood glucose check ................................................................................

54

4.3 Biochemical analysis among the study population ...

54

4.3.1 Serum cystatin C among controls and diabetic patients ..

54

4.3.2 Serum glucose among controls and diabetic patients ..

55

4.3.3 Serum urea and creatinine among controls and diabetic patients

55

4.3.4 Lipid profile of controls and diabetic patients .....

56

4.3.5 Urine albumin, urine creatinine and eGFR among controls and diabetic
.. --patients ...

56

4.4 Comparison of serum cystatin C, urea and creatinine among males and
females in patients group .....

57

4.5 Occurrence of microalbuminuria and macroalbuminuria among Control and


...diabetic patients ....

58

4.6 Diabetic nephropathy .....

59

4.6.1 Gender and diabetic groups .

59

XII

4.6.2 Age, duration of diabetes and BMI of diabetic groups

60

4.6.3 Smoking and family history among diabetic groups ...

60

4.7 Biochemical analysis among diabetic groups ....

61

4.7.1 Serum cystatin C among diabetic groups ....

61

4.7.2 Serum glucose among diabetic groups ....

62

4.7.3 Serum urea and creatinine among diabetic groups ......

62

4.7.4 Lipid profile of diabetic groups ...

63

4.7.5 Urine albumin, urine creatinine and eGFR among diabetic groups ..

64

4.8 Association of cystatin C with some parameters in diabetic patients .....

66

4.8.1 Serum cystatin C in relation to age, duration of diabetes and BMI .....

66

4.8.2 Serum cystatin C in relation to serum glucose, urea and creatinine .....

67

4.8.3 Serum cystatin C in relation to lipid profile

68

4.8.4 Serum cystatin C in relation to urine albumin, creatinine, and GFR ..

69

4.9 Receiver operating characteristic curve analysis for the diagnostic accuracy
..of cystatin C and creatinine ...

70

Chapter 5

Discussion ..

72

Chapter 6

Conclusions & Recommendations ......

80

6.1 Conclusions ...

81

6.2 Recommendations .....

82

6.3 Limitation of the study ..

83

Chapter 7

References .

84

Appendices .....

103

XIII

List of tables
Table

Title

Page

Table 2-1

Other specific type of diabetes mellitus

Table 2-2

Definitions of abnormalities in albumin excretion

22

Table 2-3

Stages of chronic kidney disease

23

Table 2-4

Glomerular filtration rate estimating formulae based upon serum


cystatin C

26

Table 4-1

Frequencies and percentages of main self-reported complications


among controls and diabetic patients

49

Table 4-2

Frequencies and percentages of smoking and family history


among control and diabetic patients

50

Table 4-3

Weight classification by BMI of controls and diabetic patients

51

Table 4-4

The main self-reported complications and their relation to


duration of diabetes

53

Table 4-5

Serum cystatin C of controls and diabetic patients

54

Table 4-6

Serum glucose of controls and diabetic patients

55

Table 4-7

Serum urea and creatinine of controls and diabetic patients

55

Table 4-8

Lipid profile of controls and diabetic patients

56

Table 4-9

Urine albumin, urine creatinine, ACR and GFR of controls and


diabetic patients

57

Table 4-10

Serum cystatin C, urea and creatinine among males and females


in patients group

58

Table 4-11

Normoalbuminuria, microalbuminuria and macroalbuminuria


among controls and diabetic patients

59

Table 4-12

Association between gender and diabetic groups

59

XIV

Table 4-13

Mean age, duration and BMI of diabetic groups

60

Table 4-14

Frequencies and percentages of smoking and family history


among diabetic groups

61

Table 4-15

Mean values of serum cystatin C among diabetic groups

62

Table 4-16

Mean values of serum glucose among diabetic groups

62

Table 4-17

Mean values of serum urea and creatinine among diabetic groups

63

Table 4-18

Mean values of lipid profile among diabetic groups

64

Table 4-19

Mean values of urine albumin, urine creatinine, ACR and eGFR


among diabetic groups

65

Table 4-20

Correlation of serum cystatin C with age, duration of diabetes


and BMI

66

Table 4-21

Correlation of serum cystatin C with serum glucose, urea and


creatinine

67

Table 4-22

Correlation of serum cystatin C with lipid profile

68

Table 4-23

Correlation of serum cystatin C with serum urine albumin, ACR,


urine creatinine, and eGFR

69

XV

List of figures
Figure

Title

Page

Figure 2-1

Prevalence (%) of diabetes in (20-79 years), 2011

11

Figure 2-2

Natural history of diabetic nephropathy

17

Figure 2-3

Strategy for screening of microalbuminuria

23

Figure 2-4

Crystal structure of human cystatin C

25

Figure 4-1

47

Figure 4-2

Distribution of the study population by governorates of the Gaza


Strip
Distribution of study population by gender

Figure 4-3

Mean age of the study population

48

Figure 4-4

49

Figure 4-5

Distribution of self-reported complications among the study


population
Mean BMI of controls and diabetic patients

Figure 4-6

Mean age at diagnosis and mean duration of the disease

52

Figure 4-7

Distribution of diabetic patients by duration of the disease

52

Figure 4-8

Distribution of diabetic patients by type of diabetes management

53

Figure 4-9

Distribution of diabetic patients by glucose checking

54

Figure 4-10

Normoalbuminuria, microalbuminuria and macroalbuminuria


among controls and diabetic patients
Correlation of serum cystatin C with (a) age (b) duration of
diabetes
Correlation of serum cystatin C with (a) serum urea (b) serum
creatinine
Correlation of serum cystatin C with (a) serum cholesterol (b)
LDL (c) HDL
Correlation of serum cystatin C with (a) urine albumin (b) ACR
(c) urine creatinine (d) eGFR
Nonparametric receiver operating characteristic (ROC) curves
of cystatin c and creatinine for assessment of diabetic
nephropathy (a) cut-off value of ACR at 30 mg/g (b) cut-off
value of ACR at 300 mg/g

58

Figure 4-11
Figure 4-12
Figure 4-13
Figure 4-14
Figure 4-15

XVI

48

50

66
67
69
70
71

Abbreviations
ACR

: Albumin to creatinine ratio

ADA

: American diabetes association

AER

: Albumin excretion rate

AGEs

: Advanced glycation end products

Ang II

: Angiotensin II

ANOVA : Analysis of variance


AUC

: Areas under curve

BMI

: Body mass index

CBVD

: Cerebrovascular disease

CHE

: Cholesterol esterase

CHOD

: Cholesterol oxidase

CSF

: Cerebrospinal fluid

CVD

: Cardiovascular disease

DKA

: Diabetic ketoacidosis

DM

: Diabetes mellitus

DN

: Diabetic neuropathy

DNP

: Diabetic nephropathy

DR

: Diabetic retinopathy

eGFR

: Estimated glomerular filtration rate

ESRD

: End stage renal disease

ET-1

: Endothelin-1

G3P

: Glycerol-3-phosphate

GDM

: Gestational diabetes mellitus

GFR

: Glomerular filtration rate

GK

: Glycerol kinase

GLDH

: Glutamate dehydrogenase

GOD

: Glucose oxidase

GPO

: Glycerol-3- phosphateoxidase

HbA1c

: Glycated hemoglobin A 1 c

HDL

: High density lipoprotein

HHS

: Hyperosmolar hyperglycemic state

XVII

HNF

: Hepatic nuclear factor

IDDM

: Insulin dependent diabetes mellitus

IDF

: International Diabetes Federation

IFG

: Impaired fasting glycemia

IGT

: Impaired glucose tolerance

IPF

: Insulin promoter factor

LA

: Lactic acidosis

LDL

: Low density lipoprotein

LPL

: Lipoprotein lipase

MDRD

: Modification of diet in renal disease

MODY

: Maturity onset diabetes of the young

MOH

: Ministry of health

NIDDM

: Non insulin dependent diabetes mellitus

NKF

: National Kidney Foundation

NO

: Nitric oxide

NPDR

: Nonproliferative diabetic retinopathy

NuroD1

: Neurogenic differentiation 1

OHA

: Oral hypoglycemic agents

PDR

: Proliferative diabetic retinopathy

PEG

: Polyethylenglycol

PKC

: Protein kinase C

POD

: Peroxidase

PVD

: Peripheral vascular disease

ROC

: Receiver operating characteristic

ROS

: Reactive oxygen species

T1DM

: Type 1 diabetes mellitus

T2DM

: Type 2 diabetes mellitus

UAE

: Urinary albumin excretion

UK

: United Kingdom

US

: United states

UTI

: Urinary tract infection

VLDL

: Very low density lipoproteins

WHO

: World health organization

XVIII

Chapter 1
Introduction

Chapter 1

Introduction

1.1 Overview
Diabetes mellitus (DM) is a serious disease and a cause for a growing public
health concern in both developed and developing countries [1]. It is a group of diseases
marked by high levels of blood glucose and characterized by disturbances of
carbohydrate, fat, and protein metabolism. It is resulting from absolute or relative
deficiency in the secretion and/or action of insulin [2].
Depending on the etiology, DM can be divided into two principal forms, type 1
diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) [3]. T1DM occurs in
childhood and is due primarily to autoimmune mediated destruction of pancreatic -cell
islets, resulting in absolute insulin deficiency [4]. People with T1DM must take
exogenous insulin for survival to prevent the development of ketoacidosis [5]. T2DM is
characterized by insulin resistance and/or abnormal insulin secretion. It is more
prevalent in adulthood, though it is becoming more common in children and
adolescents. Individuals with T2DM are not dependent on exogenous insulin, but may
require it for control of blood glucose levels if this is not achieved with diet alone or
with oral hypoglycemic agents (OHA) [6].
The chronic hyperglycemia and other metabolic disturbances of DM lead to
tissue and organ damage as well as dysfunction involving the eyes, kidneys, nervous
and vascular systems [7]. The injurious effects of hyperglycemia on the vascular system
are traditionally divided into microvascular and macrovascular complications.
Microvascular complications include diabetic retinopathy (DR), neuropathy (DN), and
nephropathy (DNP), whereas macrovascular complications include cardiovascular
disease (CVD), peripheral vascular disease (PVD), and cerebrovascular disease (CBVD)
[8].
Diabetic nephropathy is a serious, long-term complication of diabetes and the
leading cause of chronic renal disease throughout the world [9] and is responsible for
end stage renal disease (ESRD) in about one third of patients who undergo dialysis [10].
It is associated with increased cardiovascular mortality [11]. DNP has been classically
defined as increased protein excretion in urine. Early stage is characterized by a small

increase in urinary albumin excretion (UAE), also called microalbuminuria or incipient


DNP. More advanced disease is defined by the presence of macroalbuminuria or
proteinuria. The latter is classically named overt DNP [12].
Glomerular filtration rate (GFR) provides an excellent measure of the filtering
capacity of the kidneys. A low or decreasing GFR is a good index of kidney disease
[13]. The ideal marker of GFR should be an endogenous molecule, which being
produced at a constant rate, is cleared solely by the kidneys via free glomerular
filtration, with being neither secreted by tubular cells, nor reabsorbed into pertubular
circulation [14].
Cystatin C is a protein inhibitor of cysteine proteinases that is synthesized at a stable
rate by all nucleated cells. Because of its low molecular weight and high isoelectric
point, it can be eliminated almost exclusively by glomerular filtration [15]. Cystatin C
concentration is not influenced by age, sex, or protein ingestion, and it is sensitive to
small changes in glomerular filtration [16]. Because of these characteristics, cystatin C
concentration is considered among the best markers of glomerular filtration status [17].

1.2 General objective


The general objective of the current study is to evaluate serum levels of cystatin
C and other markers of DNP among a group of T2DM patients in Gaza Strip.

1.3 Specific objectives

To determine serum cystatin C and other biochemical parameters including urea,


creatinine, cholesterol, triglycerides, high-density lipoprotein (HDL) and lowdensity lipoprotein (LDL) in diabetic patients and healthy controls.

To determine urinary albumin, urinary creatinine, and GFR in diabetic patients


and healthy controls.

To investigate the relationship between these biochemical parameters and DNP


among T2DM patients.

To find out the relationship between serum cystatin C and the other studied
parameters.

To asses the significance of serum cystatin C as a sensitive marker for early


assessment of nephropathy in T2DM.

1.4 Significant of the study


Diabetic nephropathy represents one of the major problems developed in T2DM
patients, which is a common cause for renal failure. In Egypt, Afifi et al., [18] reported
that the prevalence of DNP among ESRD patients was 14.5%. However, Udayaraj et
al., [19] reported that in United kingdom (UK), diabetes mellitus was seen in 28.9% of
patients on renal replacement therapy while Malekmakan et al., [20] reported that
diabetes mellitus constitutes 30.1% of the causes of chronic renal failure in Iranian
hemodialysis patients. In Qatar, diabetic nephropathy was the commonest cause of
ESRD (48%) [21].
The ability to assess renal function in diabetic patients rapidly and early is of
major importance for the possibility of preventing the development of nephropathy.
Therefore, it is worth to discover a more sensitive or specific indicator for detecting
early renal impairment in diabetic patients. So, corrective measures could be adopted to
prevent the progression of kidney function impairment towards frank nephropathy.
There are some studies conducted in Gaza to detect some urinary enzymes,
transferrin and leptin as early markers for DNP among T2DM patients, but this study is
the first one to be executed in a group of patients in our area and dealing with cystatin
C.
Although numerous studies were carried out for long time to compare using of
cystatin C as nephropathy marker with other traditional ones for changing in GFR
among T2DM patients and considerable progress has been made according to these
studies, the assessment of cystatin C as sensitive marker of DNP is still controversial.

Chapter 2
Literature Review

Chapter 2

Literature Review

2.1 Diabetes mellitus


Diabetes mellitus is a clinically and genetically heterogeneous group of
disorders characterized by abnormally high levels of glucose in the blood
(hyperglycemia). The hyperglycemia is due to deficiency of insulin secretion or to
resistance of the body's cells to the action of insulin, or to a combination of these. Often
there are also disturbances of carbohydrate, fat, and protein metabolism [22]. World
health organization (WHO) has defined DM as having fasting plasma glucose 126
mg/dl or random plasma glucose > 200 mg/dl [23].
Diabetes mellitus may present with characteristic symptoms such as polydipsia,
polyuria, blurring of vision, sometimes with polyphagia and weight loss. Impairment of
growth and susceptibility to certain infections may also accompany chronic
hyperglycemia. In its most severe forms, diabetic ketoacidosis (DKA) or hyperosmolar
hyperglycemic state (HHS) may develop and lead to stupor, coma and in absence of
effective treatment, death [6]. The long-term effects of DM include progressive
development of the specific complications, retinopathy with potential blindness,
nephropathy that may lead to renal failure, and/or neuropathy with risk of foot ulcers,
amputation, Charcots (Neuropathic) joints, and features of autonomic dysfunction,
including sexual dysfunction. People with diabetes are at increased risk of brain, heart,
and blood vessels disease [3].

2.1.1 Type 1 diabetes


It was formerly termed insulin dependent diabetes mellitus (IDDM) or juvenile onset
diabetes, and it is usually occurs in children or young adults, but it can occur at any age,
it is accounts for 5-10% of diabetes [24]. It is characterized by severe insulin deficiency
as manifested by low or undetectable levels of plasma C-peptide. It is further divided
into type 1A, which is immune mediated, and type 1B, which has no defined etiology
for the insulin deficiency (Idiopathic) [22].
Immune mediated form of disease results from a cellular-mediated autoimmune
destruction of the -cells of the pancreas [6]. This autoimmune process is due to genetic
6

and environmental factors [24]. Markers of the immune destruction of the -cell include
islet cell autoantibodies, insulin autoantibodies, glutamic acid decarboxylase
autoantibodies, and autoantibodies to insulinoma antigen IA-2 and IA-2. One and
usually more of these autoantibodies are present in 85-90% of individuals when fasting
hyperglycemia is initially detected [6]. There is ample evidence suggesting that
autoimmune diseases, such as Addisons disease, pernicious anemia, and autoimmune
thyroid disease are involved in the etiology of T1DM [25].
Idiopathic diabetes has no known etiologies and a minority of patients with
T1DM falls into this category. Some of these patients have permanent insulinopenia and
are prone to ketoacidosis, but have no evidence of autoimmunity. This form of diabetes
is strongly inherited [6].

2.1.2 Type 2 diabetes


This type of diabetes previously referred to as noninsulin dependent diabetes
mellitus (NIDDM), or adult onset diabetes [3]. It comprises approximately 90-95% of
all patients with diabetes [24]. It is characterized by insulin resistance in muscle, liver,
and adipose tissue and usually individuals have relative (rather than absolute) insulin
deficiency (i.e. patients secrete insulin, but not enough to overcome the insulin
resistance) [26]. Whereas patients with this form of diabetes may have insulin levels
that appear normal or elevated, the high blood glucose levels in these diabetic patients
would be expected to result in even higher insulin values had their -cell function been
normal [6]. On the other hand, some individuals have essentially normal insulin action,
but markedly impaired insulin secretion [3].
In contrast to T1DM, patients with T2DM do not depend on exogenous insulin
to survive. However, they may require insulin for correction of fasting hyperglycemia if
this cannot be achieved with the use of diet or OHA [22]. Ketoacidosis seldom occurs
spontaneously in this type of diabetes; when seen, it usually arises in association with
the stress of another illness such as infection [6].
In most patients with T2DM diagnosis made in adult years, the disease also
occurs in young people who do not require insulin, not ketotic, and hence could not be
considered to have T1DM. In addition, the average age at diagnosis of T2DM is much
7

earlier in very high prevalence groups, and somewhat earlier in medium prevalence
groups [22].
The disease tends to develop slowly, and most patients of this condition are
undiagnosed for many years because the hyperglycemia is often not severe enough to
provoke noticeable symptoms of diabetes. Nevertheless, such patients are at increased
risk of developing macrovascular and microvascular complications for a long period of
time before diabetes is detected [3].
Type 2 DM is strongly associated with genetic predisposition, more so than is
the autoimmune form of T1DM [6]. However, the etiology of disease is heterogeneous
because a variety of lifestyle and environmental factors has been identified as being risk
factors for the condition [22]. The risk of developing this form of diabetes increases
with age, obesity, and lack of physical activity [3].
Many investigators have reported a strong correlation between obesity and
T2DM. In a study of the pattern of diabetes in Kuwait it was shown that 57.7% of the
diabetic women were obese and 30.2% were overweight [27]. A cross-sectional study in
Bahrain showed that 28% of diabetics were obese [28].

2.1.3 Gestational diabetes mellitus


Gestational diabetes mellitus (GDM) defined as glucose intolerance that begins
during pregnancy or first recognized during pregnancy. The definition applies
irrespective of whether management is limited to diet or requires insulin. It does not
exclude the possibility that the abnormal glucose tolerance may have predated the
pregnancy. The prevalence may range from 1-14% of all pregnancies, depending on the
population studied and the diagnostic tests employed [29, 30].
Women with GDM are at increased risk for development of diabetes. After
pregnancy, 5-10% of women with GDM are found to have T2DM. Women who have
had GDM have 35-60% chance of developing diabetes later in life [31].

2.1.4 Other specific types of diabetes


These groups of DM include various etiologies in which the cause is established
or at least partially known. The causes include known genetic defects of -cell function
8

or insulin action, diseases of the exocrine pancreas, drug or chemical induced pancreatic
changes, infections, genetic syndromes and other endocrinopathies (Table 2-1) [6].
Such types of diabetes account for 1-5% of all diagnosed cases [31].
Table 2-1 Other specific type of diabetes mellitus [6]
Genetic defects of beta cell function

Endocrinopathies

1.
2.
3.
4.
5.
6.
7.

Chromosome 12, HNF-1 - (MODY3)


Chromosome 7, glucokinase (MODY2)
Chromosome 20, HNF-4 - (MODY1)
Chromosome 17, HNF-1 - (MODY5)
Chromosome 2, NeuroD1 - (MODY6)
Mitochondrial DNA

1.
2.
3.
4.
5.
6.
7.

8.

Others

8.

Chromosome 13, insulin promoter factor-1 (IPF-1; MODY4)

Acromegaly
Cushings syndrome
Glucagonoma
Pheochromocytoma
Hyperthyroidism
Somatostatinoma
Aldosteronoma
Others

Genetic defects in insulin action

Drug or chemical induced

1.
2.
3.
4.
5.

Type A insulin resistance


Leprechaunism
Rabson-Mendenhall syndrome
Lipoatrophic diabetes
Others

1.
2.
3.
4.
5.

Vacor
Pentamidine
Nicotinic acid
Glucocorticoids
Thyroid hormone

Diseases of the exocrine pancreas

6.

Diazoxide

1.
2.
3.

Pancreatitis
Trauma/pancreatectomy
Neoplasia

7.
8.
9.

4.

Cystic fibrosis

10.

-adrenergic agonists
Thiazides
Dilantin
-Interferon

5.

Hemochromatosis

11.

Others

6.
7.

Fibrocalculous pancreatopathy
Others

Other genetic syndromes sometimes


associated with diabetes

Infections

1.

Down syndrome

1.
2.
3.

2.
3.
4.

Klinefelter syndrome
Turner syndrome
Wolfram syndrome

Uncommon forms of immune-mediated diabetes

5.

Friedreich ataxia

1.
2.
3.

6.
7.
8.

Huntington chorea
Laurence-Moon-Biedl syndrome
Myotonic dystrophy

9.

Porphyria

10.
11.

Prader-Willi syndrome
Others

Congenital rubella
Cytomegalovirus
Others
Stiff-man syndrome
Anti-insulin receptor antibodies
Others

MODY maturity onset diabetes of the young, HNF hepatic nuclear factor, IPF insulin promoter factor,
NuroD1 neurogenic differentiation 1

2.2 Prediabetes
Prediabetes represents intermediate states of abnormal glucose regulation that
exist between normal glucose homeostasis and diabetes [32]. It is a condition in which
individuals have blood glucose levels higher than normal but not high enough to be
classified as diabetes. People with prediabetes have an increased risk of developing
T2DM, heart disease, and stroke [31].
In general, people who have fasting plasma blood glucose in the 100-125 mg/dL
range are defined as having impaired fasting glycemia (IFG). Impaired glucose
tolerance (IGT) defined as two-hour oral glucose tolerance test values of 140-199
mg/dL [33].

2.3 Prevalence of diabetes mellitus


Diabetes mellitus can now be found in almost every population in the world and
epidemiological evidence suggests that, without effective prevention and control
programs, diabetes will likely continue to increase globally. The number of people with
diabetes is increasing due to population growth, aging, urbanization, and increasing
prevalence of obesity and physical inactivity. Quantifying the prevalence of diabetes
and the number of people affected by diabetes, now and in the future, is important to
allow rational planning and allocation of resources [34].
The global burden of diabetes has been estimated several times [35, 36]. In
1997, Amos et al., estimated the global burden of diabetes to be 124 million people, and
projected that this would increase to 221 million people by the year 2010 [35]. King et
al., also produced a report using epidemiological information and estimated the global
burden at 135 million in 1995, with the number reaching 300 million by the year 2025
[36].
The International Diabetes Federation (IDF) has produced other estimates [3740]. In the 2011, fifth edition of the Diabetes Atlas estimated that approximately 366
million people worldwide, or 8.3% of adults, are estimated to have diabetes in 2011.
About 80% live in low- and middle-income countries. If these trends continue, by 2030,
some 552 million people, or one adult in 10, will have diabetes. This equates to

10

approximately 10 million per year. The largest increases will take place in the regions
dominated by developing economies [40].
The estimates for both 2011 and 2030 showed little gender difference in the
number of people with diabetes. There are about four million more men than women
with diabetes (185 million men vs. 181 million women) in 2011. However, this
difference is expected to decrease to two million (277 million men vs. 275 million
women) by 2030 [40].
Separate estimates for urban and rural populations were undertaken for low and
middle income countries, and in 2011 the expected number of people with diabetes in
urban areas will be 172 million, compared to 119 million in rural areas. By 2030, it is
expected that this discrepancy will increase to 314 million urban and 143 million rural
people with diabetes (Figure 2-1) [40].

Figure 2-1 Prevalence (%) of diabetes in (20-79 years), 2011[40]

11

In Palestine, there is no or weak national data available on the overall incidence


and prevalence of DM. The current system counts mainly the visits of the patients to
primary health care centers, which does not reflect the real prevalence and incidence.
According to study conducted in cooperation with Al Quads University and Ministry of
Health (MOH) indicated that the prevalence of DM in Palestine was about 9% in 2000
[41].
In rural Palestinian population (Kober village), Husseini and his colleagues
investigated the prevalence of diabetes in which the prevalence was 9.6% and 10.0% in
females and males respectively [42]. Abdul-Rahim and his colleagues studied another
cross sectional survey of urban Palestinian population (old Rammallah city). Diabetes
was found in 12.0% of the survey population (including 9.4% previously diagnosed)
[43].
Diabetes mellitus in many countries is a leading cause of death, disability and a
significant contributor for rising health care cost. In USA, diabetes was the seventh
leading cause of death in 2007 [44]. In Australia, data indicated that diabetes was the
associated cause of death in 24% of CVD deaths and 8% of stroke deaths [45]. Diabetes
was not found to be among the ten leading causes of death in Palestine, it caused only
3.1% of total population deaths in 2005 with mortality rate of 8.5 per 100,000 [41].

2.4 Complications
Diabetes as a chronic condition requires careful control. Without proper control,
management and follow-up it can lead to various complications. These complications
may be divided to short- and long-term complications. Short-term complications refer to
acute metabolic complications of diabetes, and it consists of DKA, HHS, lactic acidosis
(LA), and hypoglycemia. DKA and HHS are related to insulin deficiency.
Hypoglycemia results from the treatment of diabetes, with either oral agents or insulin.
LA is usually associated with other factors that may be related to diabetes, such as CVD
associated with hypoxia and excess lactic acid production [46].
Mainly direct and indirect effects of diabetes on human vascular system cause
long-term complications. Generally, the injurious effects of hyperglycemia are separated

12

into macrovascular complications (CVD, PAD, and CBVD) and microvascular


complications (DN, DR and DNP) [47].

2.4.1 Microvascular complications


Several theories have emerged regarding how hyperglycemia induces the
pathologic changes leading to microvascular complications. An increase in intracellular
glucose will lead to an increased flux through the polyol pathway, excessive formation
of advanced glycation end products (AGEs), over activity of the hexosamine pathway,
and activation of protein kinase C (PKC) isoforms. Each of these pathways can generate
toxic and reactive metabolites that promote cellular dysfunction and damage [48].
The polyol pathway converts excess glucose to sorbitol and fructose by aldose
reductase and sorbitol dehydrogenase, which is accompanied by increased oxidation of
NADPH to NADP+ and increased reduction of NAD+ to NADH. This pathway may
impair endothelial function through three mechanisms. First, increased sorbitol
accumulation will increase osmotic stress. Secondly, the increase in the cytosolic
NADH/ NAD+ ratio results in a redox imbalance, which termed hyperglycemic
pseudohypoxia. Thirdly, the redox imbalance favors the accumulation of triose
phosphates that increases the formation of methylglyoxal and AGEs and enhances
oxidative stress [49].
When hyperglycemia is present, proteins undergo nonenzymatic glycosylation
generating AGEs, intracellularly and extracellularly. Intracellular AGEs damages target
cells by three general mechanisms. First, intracellular proteins modified by AGEs have
altered function. Second, extracellular matrix components modified by AGEs precursors
interact abnormally with other matrix components and with the receptors for matrix
proteins (integrins) on cells. Third, plasma proteins modified by AGEs precursors bind
to AGEs receptors on endothelial cells, mesangial cells and macrophages, inducing
receptor-mediated production of reactive oxygen species (ROS) [50].
The hexosamine pathway becomes activated when glucose levels are high in
cells. Fructose 6-phosphate is diverted from glycolysis to provide substrate for the ratelimiting enzyme of this pathway. The product of the pathway, N-acetyl-glucosamine
causing a permanent modification of proteins and transcription factors [51].
13

Protein kinase C is a family of serine-threonine kinases that plays an important


role in signal transduction mechanisms. High levels of intracellular glucose activate the
enzyme PKC via diacylglycerol, which are synthesized directly from glycolytic
intermediates such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.
When activated, for example, PKC affects the activation of a number of growth factors
and changes the function of vasoactive factors. These vasoactive factors include
vasodilators such as nitric oxide (NO) as well as vasoconstrictors such as angiotensin II
(Ang II) and endothelin-1 (ET-1) [48].

2.4. 1.1 Diabetic Neuropathy


An internationally agreed simple definition of DN is the presence of symptoms
and/or signs of peripheral nerve dysfunction in people with diabetes after the exclusion
of other causes. However, the diagnosis cannot be made without a careful clinical
examination and absence of symptoms must never be equated with absence of
neuropathy [52].
Diabetic neuropathy is a common complication of DM, it is generally considered
to be related to the magnitude and duration of hyperglycemia. Usually more than 50%
of patients with duration of diabetes of 25 years or more are affected, making it as one
of the most common disease of the nervous system [53].
Diabetic

neuropathy

can

be

classified

as

generalized

symmetrical

polyneuropathies or focal and multifocal neuropathies. Each affects different parts of


the body in various ways. Generalized symmetrical polyneuropathies include acute
sensory, chronic sensorimotor and autonomic neuropathy [54]. The most common forms
of DN are chronic sensorimotor polyneuropathy, which is often accompanied by
autonomic dysfunction. Late phase of neuropathy includes insensate foot ulceration,
Charcots joints, and occasionally even amputation [55].
Symptoms of the disease depend on the type of neuropathy and affected nerves.
Some people with nerve damage have no symptoms at all. For others, the first symptom
is often numbness, tingling, or pain in the feet. Symptoms are often minor at first, and
because most nerve damage occurs over several years, mild cases may go unnoticed for
a long time [56].
14

A large American study estimated that 47% of patients with diabetes have some
peripheral neuropathy [57]. Cross-sectional study of diabetic patients in the UK found
the prevalence of diabetic neuropathy to be 28.5% [58]. In another study, Peripheral
neuropathy was found in 41.6 % of diabetic patients [59]. In Spain, the prevalence of
diabetic neuropathy was 22.7% in the whole sample, 12.9% among patients with T1DM
and 24.1% among patients with T2DM [60].
Development of DN depends on many risk factors, such as poor glycemic
control, advanced age, hypertension, long duration of DM, dyslipidemia and smoking
[61].

2.4.1.2 Diabetic Retinopathy


Diabetic retinopathy may be defined as the presence of characteristic evolution
of typical retinal microvascular lesions in an individual with diabetes [62]. It is the most
frequent cause of new cases of blindness among adults aged 20-74 years. During the
first two decades of disease, nearly all patients with T1DM and > 60% of patients with
T2DM have some degree of retinopathy [63].
Diabetic retinopathy is a progressive disease predominantly affecting the
integrity of the microscopic vessels found in the retina. It can be classified into two
clinical stages: nonproliferative diabetic retinopathy (NPDR) and the more advanced,
proliferative diabetic retinopathy (PDR) [64].
The prevalence rates for DR are widely varied in the literature. According to the
Eye Disease Prevalence Research Group, 40.3% of the adult diabetic populations of the
United States (US) have DR, and 8.2% have vision-threatening retinopathy [65]. In
cross-sectional study conducted on diabetic patients, in 20052008, the estimated crude
prevalence of DR and vision threatening was 28.5% and 4.4% among US adults with
diabetes, respectively [66]. A cross-sectional study of patients with T1DM in the Brazil
found the DR was present in 44.4% of subjects [67]. Another study was done on T2DM,
28.9% had evidence of retinopathy [68]. At diagnosis, the prevalence of any retinopathy
in T1DM is low, ranging from 0-3%, while in T2DM the range is between 6.7-30.2%,
this suggests that many people with T2DM go undiagnosed for years [64].

15

2.5 Diabetic nephropathy


2.5.1 Definition, epidemiology and clinical stages
Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. It
is also known as Kimmelstiel Wilson syndrome and it was discovered in 1936 by
Clifford Wilson and Paul Kimmelstiel [69]. The classical definition of DNP is a
progressive rise in UAE, coupled with increasing blood pressure, when, untreated, there
is a progressive decline in glomerular filtration, until ESRD is reached. Patients
generally have DR [70].
Patients with T1DM more frequently develop nephropathy as a complication
with approximately 20-40% of persons affected. This compares to 10-20% of those with
T2DM [71]. Worldwide, DNP has become the leading cause of ESRD. In the US, DNP
accounts for about 40% of new cases of ESRD [72]. In many countries, including the
Middle East the majority of diabetic patients starting kidney replacement therapy now
have T2DM rather than T1DM [73].
Many studies have confirmed the close links between DNP and CVD,
cardiovascular risk increasing in parallel with albuminuria. In T1DM the relative risk of
CVD is 1.2 fold in microalbuminuric than that of normoalbuminuric patients [74], and
10 fold higher in proteinuric than normoalbuminuric patients [75]. In T2DM, with
microalbuminuria, the risk is increased 2-3 fold [76] and with proteinuria 10 fold
compared with normoalbuminuric patients [77].
Clinically, the natural history of kidney involvement in T1DM is described to
consist of five stages as suggested by Mogensen and his colleagues (Figure 2-2) [78].
Stage 1 is characterized by hyperfiltration and hypertrophy of the glomerulus, leading to
elevated GFR and renal enlargement. Stage 2 is clinically similar to stage 1, but
morphological lesions (glomerular basement membrane thickening and mesangial
expansion) are present in the kidney on biopsy. Stages 1 and 2 are generally clinically
silent. By stage 3, microalbuminuria (incipient DNP) is present with mild to moderate
decrease in GFR. It is usually occurring at least 5-10 years after the onset of diabetes.
Without treatment, however, patients may progress to stage 4, with overt DNP (also
referred to as macroalbuminuria or proteinuria). Stage 4 is associated with moderate to
16

severe decrease in GFR. Subsequently, some patients evolve to stage 5 which defined as
ESRD a stage in which patients require dialysis or kidney transplantation [79].

Figure 2-2 Natural history of diabetic nephropathy [80]

In the general clinic population, the time of onset of T2DM is often unclear and
may be delayed by many years. Hence, this group of patients may present with
advanced stages of DNP at the time of initial diabetes diagnosis. Additionally, death
from CVD often precedes progression to advanced renal dysfunction or renal failure in
patients with T2DM [81].

2.5.2 Urinary albumin excretion


Albumin is generally the most prevalent protein circulating in the blood. The
kidney normally excretes very little albumin into the urine, as the glomerular filtration
barrier prevents passage of the majority of albumin into the urinary space. Normal UAE
has been defined as less than 30 mg daily [79].
The earliest manifestation of DNP is the appearance of low, yet abnormal, levels
of albumin in the urine, referred to as microalbuminuria. Microalbuminuria refers to
albumin excretion between 30 and 300 mg daily. These patients are categorized as
having incipient nephropathy. More advanced disease is defined by the presence of

17

macroalbuminuria or proteinuria; albumin excretion greater than 300 mg daily. The


latter is classically named overt nephropathy [82].
In adults with T1DM, the prevalence of microalbuminuria in clinic-based studies
is 10-20% [83] and 15-30% in patients with T2DM [76, 83, 84]. The prevalence of
macroalbuminuria is about 10-20% in T1DM but varies in T2DM from 5-50% in
different populations [84-87]. A higher proportion of persons with T2DM are found to
have microalbuminuria and overt nephropathy at the time they are diagnosed as having
diabetes. This is likely due to their diabetes being present for many years prior to an
actual diagnosis being made [88].
While microalbuminuria is considered a risk factor for the development of
macroalbuminuria, not all patients progress to this stage, and some may regress to
normoalbuminuria [89,90]. The initial studies suggested that about 80% of T1DM
patients with microalbuminuria would progress to proteinuria over a period of 6-14
years [91-93]. More recent studies suggest that only 30-45% of microalbuminuric
patients will progress to proteinuria over 10 years of follow-up [89].
There is accumulating evidence suggesting that the risk for developing DNP
starts when UAE values are still within the normoalbuminuric range. In patients with
T2DM, the progression to micro- or macroalbuminuria is more frequent in individuals
whose baseline UAE was normal but above 2.5 mg/day [94]. Furthermore, in another
study after 10 years of follow-up, patients with T2DM and UAE values above 10
g/min were at 29 times higher risk of developing DNP [95]. Similar results were
observed in patients with T1DM [96].
In the microalbuminuric stage, no decline in GFR is expected. Once the subject
has developed macroalbuminuria, the expected GFR decline is 1.2 ml/min/month in
T1DM [97]. In T2DM, the rate of GFR decline is less predictable. A mean decline of
approximately 0.5 ml/min/month has been described [98], but in some patients, GFR
may remain stable for long periods of time [99]. However, it is clear today that some
subjects could have DNP without increased UAE [100,101]. About 10% of subjects
with T2DM will have low GFR without micro or macroalbuminuria [102]. This was
also observed among patients with T1DM [103].

18

2.5.3 Risk factors for development of diabetic nephropathy


A study in patients who have or do not have DNP identified a number of factors
has being associated with increased risk of renal involvement.

2.5.3.1 Hyperglycemia
Hyperglycemia is a prerequisite for the development of DNP, and it is a
significant risk factor for the development of microalbuminuria in T1DM [96] and
T2DM [104]. Microalbuminuria is closely associated with glycated hemoglobin
(HbA1c) levels over 8.0% in T1DM [105] and in T2DM patients [106]. In an
observational, clinic-based study in T1DM and T2DM patients, the percent rate of
increase in albumin excretion rate (AER) was closely related to mean HbA1c levels
[107]. Observational studies in T1DM patients with overt nephropathy have shown that
the rate of decline of GFR is related to glycaemic control [108]. In patients with existing
microalbuminuria, the Diabetes Control and Complications Trial [109] and a metaanalysis study in T1DM patients [110] showed that intensive glycaemic control reduced
the progression to overt DNP. In T2DM patients, tight glycaemic control reduced the
development of overt DNP [111].
On other hand, in patients with T2DM and microalbuminuria, intensive
glycaemic control is associated with a decreased rate of progression of AER, but not
renal function as measured by creatinine clearance [112].

2.5.3.2 Dyslipidemia
Type 2 DM is one component of the metabolic syndrome, which includes
impaired glucose tolerance, insulin resistance, central obesity, hypertension, combined
dyslipidemia, impaired fibrinolysis and hyperuricemia [113]. Microalbuminuria has also
been linked to this pattern of metabolic disturbances [114]. A cross-sectional study of
T2DM

patients

showed

that

microalbuminuria

was

associated

with

hypertriglyceridaemia and low HDL [115]. In an Israeli study of T2DM patients, total
cholesterol predicted the risk of development of microalbuminuria and total cholesterol
also predicted a decline in renal function [116]. In T1DM patients increased serum
triglycerides, cholesterol and LDL were associated with micro- and macroalbuminuria
19

[117]. High serum cholesterol also seems to be a risk factor for GFR loss in
macroalbuminuric T1DM subjects [118]. In longitudinal studies, the determinants of
progression of microalbuminuria to overt DNP include serum cholesterol in T1DM
[119] and serum triglycerides in T2DM [120]. By contrast, serum cholesterol and
triglycerides were not related to the development of microalbuminuria in a UK study
[121].

2.5.3.3 Smoking
Several lines of evidence have shown that smoking increases the risk and
progression of DNP [94, 122-124]. In a study of patients with T2DM, 61% of the
patients were smokers, 26% had microalbuminuria, and 14% had overt nephropathy.
Analysis of a number of risk factors showed a 1.6-fold increased risk of nephropathy
among smokers [124]. Chase et al., [125] found more progression of albuminuria in
smokers and noted that albuminuria decreased significantly when subjects stopped
smoking. In cross-sectional studies of patients with newly diagnosed T2DM, smoking
was associated with a higher UAE [126]. Sawicki et al., [122] examined T1DM patients
with long duration and DNP. They were well controlled with respect to glycemia and
blood pressure. Progression occurred in 53 % of smokers compared to 11 % of nonsmokers and 33 % of ex-smokers.
While some cross-sectional analyses failed to confirm an association between
smoking and nephropathy [104, 120, 127]. In a long-term prospective observational
cohort study on progression of DNP in T1DM patients, there is no association between
smoking status and rate of decline in GFR [127].

2.5.3.4 Arterial hypertension


Hypertension (defined as a blood pressure 140/90 mmHg) is an extremely
common comorbid condition in diabetes, affecting ~2060% of patients with diabetes,
depending on obesity, ethnicity, and age [128]. In US, from 2005-2008, there is 67% of
diabetic patients aged 20 years or older had blood pressure greater than or equal to
140/90 (mmHg) or used prescription medications for hypertension [44]. In people with

20

T2DM, the prevalence of hypertension is 50% at the time of diagnosis, increasing to


80% in the presence of microalbuminuria and to > 90% with macroalbuminuria [129].
Hypertension substantially increases the risk of both macrovascular and
microvascular complications [128]. Analysis of UK study showed that every 10 mmHg
reduction in systolic blood pressure is associated with a 13% reduction in the risk of
microvascular complications [130]. The studies in elderly patients with T2DM have
suggested that systolic blood pressure relates to the rate of progression of albuminuria
[131]. Some studies show that an increase in blood pressure follows the onset of
microalbuminuria in patients with T1DM, [132,133] while others suggest either a close
association or that a rise in blood pressure precedes the onset of microalbuminuria
[134]. By contrast, in elderly patients with T2DM, arise in blood pressure usually
precedes the onset of microalbuminuria [84].
In accordance with the American Diabetes Association (ADA), the primary goal
of therapy for diabetic patients is to decrease blood pressure to and maintain it at ~130
mmHg systolic and ~80 mmHg diastolic [135].

2.5.3.5 Genetic susceptibility


In addition to the risks of hyperglycemia and hypertension, a subset of patients
may be at greater risk for nephropathy has long been interpreted as evidence that there is
a genetic susceptibility to the development of nephropathy [136]. Several studies have
shown that the likelihood of developing DNP is markedly increased in patients with a
diabetic sibling or parent who has DNP, these observations have been made in both
T1DM and T2DM [137, 138]. In a study of Brazilian families with two or more diabetic
members, the presence of DNP was significantly associated with a 3.7-fold increased
risk of DNP in the diabetic siblings [138].

2.5.4 Screening and diagnosis of diabetic nephropathy


Screening for DNP must be performed at the time of diagnosis in patients with
T2DM, since these individuals may have had a silent form of DM for some time
already. For patients with T1DM, it is recommended that screening be performed

21

beginning in the fifth year after DM diagnosis or earlier if the DM is chronically poorly
compensated, or if the patient is an adolescent [139].
Screening for microalbuminuria can be performed by three methods: first by
measurement of the albumin in a random spot collection, second by twenty four hour
collection with creatinine, allowing the simultaneous measurement of creatinine
clearance, and third by timed (e.g., 4-h or overnight) collection [139].The first step in
screening for DNP is to measure albumin in an isolated urine sample [135]. The results
of albuminuria in an isolated sample can be expressed as albumin concentration (mg/l)
or as albumin to creatinine ratio (ACR) (mg/g) [140]. Recent reviews and guidelines
define a normal ACR as less than 30 mg/g, microalbuminuria as 30 mg/g to 299 mg/g,
and overt albuminuria as 300 mg/g or greater (Table 2-2) [82,141,142].

Table 2-2 Definitions of abnormalities in albumin excretion [82]


Spot collection
24-h collection
Category
(mg/g creatinine)
(mg/24 h)
Normal
Microalbuminuria
Macroalbuminuria

30
30-299
300

Timed collection
(g/min)

30
30-299
300

20
20-199
200

In situations in which UAE measurement is not available, semiquantitative


dipstick measurements of albuminuria can be used, although these tests are less accurate
and all positive tests should be confirmed by specific methods [143].
Screening should not be performed in the presence of conditions that increase
UAE, such as urinary tract infection (UTI), hematuria, acute febrile illness, vigorous
exercise, short-term pronounced hyperglycemia, uncontrolled hypertension, and heart
failure. Bacteriuria had also been considered a factor that could interfere in the measure
of urinary albumin [144], but in a recent study, this finding was not confirmed,
suggesting that it is not necessary to exclude bacteriuria to measure albuminuria [145].
Due to the known day-to-day variability in UAE, all abnormal tests must be
confirmed in two out of three samples collected over a three to six month period before

22

designating a patient as having microalbuminuria [139]. Strategy for microalbuminuria


screening is given in (Figure 2-3).

Figure 2-3 Strategy for screening of microalbuminuria [82]


Although the measurement of UAE is essential to diagnose DNP, some patients
with either T1DM or T2DM have decreased GFR; have normal UAE values
[100,101,146]. Based on this, the classification of the National Kidney Foundation
(NKF) can also be used to stage chronic kidney disease in these patients (Table 2-3)
[135]. Therefore, GFR should be routinely estimated for appropriate screening of DNP.
Table 2-3 Stages of chronic kidney disease [135]
Stage

Description

GFR (ml/min/1.73m2)

Kidney damage* with normal or increased GFR

90

Kidney damage* with mildly decreased GFR

60-80

Moderately decreased GFR

30-59

Severely decreased GFR

15-29

Kidney failure

15 or dialysis

*Kidney damage defined as abnormalities on pathologic, urine, blood, or imaging tests.

23

The GFR is generally considered to be the best index of renal function in health
and disease. Rigorous assessment of GFR requires the measurement of an ideal
filtration marker, defined as a substance that is freely filtered by the kidney, not bound
to plasma proteins, non-toxic and does not undergo metabolism, tubular secretion or
absorption [147]. The clearance of endogenous creatinine is commonly used, despite its
limitations [148]. However, in clinical practice, GFR is estimated by equations that take
into account serum creatinine concentration and some or all of the following variables:
age, sex, body weight and race. The equation recommended by the NKF is that of the
study on Modification of Diet in Renal Disease (MDRD):

Estimated glomerular filtration rate (eGFR) (ml/min/l.73m2) = 186 [(serum


creatinine (mg/dl)

-1.154

age (years)

-0.203

(0.742 if a woman) (1.21 if African-

American)] [149].

2.6 Cystatin C
Cysteine proteinases comprise a group of proteolytic enzymes that cleave
peptide bonds by use of a reactive cysteine residue at the catalytic site. In general,
cysteine proteinases are involved in the intracellular catabolism of peptides and
proteins, processing of proenzymes and prohormones, breakdown of collagen, and bone
resorption. The activities of the cysteine proteinases are controlled by naturally
occurring inhibitory proteins such as 2-macroglobulin and cystatins [150].
Cystatins are single chain proteins that reversibly inhibit cysteine proteinases
belonging to the papain and legumain families [151]. The human cystatins form three
groups based on molecular organization. Family 1 cystatins are found primarily
intracellularly, without disulfide bonds and no carbohydrate side chains. There are two
human representatives, cystatin A and cystatin B. Family 2 cystatins are mainly
extracellular, contain two disulfide bridges. In humans, eight members of the cystatin
family have been identified: cystatin C, D, E/M, F, G, S, SN and SA. Family 3 cystatins
are multidomain proteins. These proteins are of quite high molecular mass, contain
disulfide bonds and are glycosylated. The human representatives of this group are the
kininogens [152].

24

Cystatin C, formerly known as -trace or post-- globulin, is a cysteine


proteinase inhibitor that belongs to family 2 of the cystatin superfamily [16], and is a
potent inhibitor of lysosomal cathepsins B, H, and L [151]. The cystatin C protein has a
molecular mass of 13.343 kDa and consists of 120 amino acid residues in a single
polypeptide chain with two disulfide bonds [16]. The crystal structure of human cystatin
C composed of five-stranded antiparallel -sheets partially wrapped around a central helix (Figure 2-4) [153].

Figure 2-4 Crystal structure of human cystatin C [154]


Determination of the structure of the human cystatin C gene and its promoter has
demonstrated that the gene is of the housekeeping type, which indicates a stable
production rate of protein by most nucleated cell types. The presence of a hydrophobic
leader sequence in pre-cystatin C precursor strongly indicates that the protein is
normally secreted. Cystatin C is widely distributed in body fluids such as cerebrospinal
fluid (CSF), seminal fluid, saliva, blood plasma, and urine. It is also present in tissues,
including brain, kidney, liver, placenta, and seminal vesicles [16].
The knowledge that most human tissues produce cystatin C and that it, being a
low molecular mass protein, is removed from plasma by glomerular filtration, suggested
that its plasma or serum level might be a potentially good marker for GFR. Cystatin C
was first suggested as a new marker for GFR in 1979, when it was observed that the

25

plasma level of cystatin C was up to 13 times higher in patients on hemodialysis than in


healthy persons [155].
Several recent studies have compared the use of serum cystatin C and creatinine
as markers for GFR as determined by gold standard procedures based upon
determinations of the plasma clearance of injected low molecular mass substances like
chromium-51

ethylenediamine

tetraacetic

acid

(51Cr-EDTA),

technetium-99m

diethylene triamine pentaacetic acid (99mTc-DTPA) and iohexol. These studies have
indicated that serum cystatin C is either had a significantly better diagnostic
performance than serum creatinine, particularly for individuals with small to moderate
decreases in GFR, or that the two parameters are of equal value as GFR indicators.
[156-162]. The main advantage of cystatin C compared to creatinine as a GFR marker is
that cystatin C is less dependent upon the body composition of a patient than creatinine.
Whereas the muscle mass strongly influences creatinine, it does not, or only marginally,
affects cystatin C [163-165]. The results for adults have generally shown that there are
no sex differences for any age group and that the well-known decrease in GFR with age
is mirrored by an increase in the cystatin C level with age [166]. the results for children
have demonstrated that the cystatin C level, In contrast to the creatinine level, does not
significantly influence by age and it was constant for children beyond the first year and
with no difference between the sexes. Therefore, cystatin C is a more suitable GFR
marker in pediatric populations [167-169].
Recently, several formulae for estimation of GFR from cystatin C values have
been proposed. A summary of the eGFR formulae based on serum cystatin C is shown
in (Table 2-4).

Table 2-4 Glomerular filtration rate estimating formulae based upon serum cystatin C
Reference

Proposed formula

Unit

Patient characteristics
2

Tan et al. [170]

(87.1 / cystatin C) 6.87

ml/min/1.73 m

Diabetic patients

Filler et al. [171]

91.62 s-cystatin C1.123

ml/min/1.73 m2

Children

Hoek et al. [172]


MacIsaac et al. [173]

(80.35 / cystatin C) 4.32


(86.7 / cystatin C) 4.2

ml/min/1.73 m2
ml/min/1.73 m2

Adults with renal disease


Diabetic patients

Rule et al. [174]

66.8 / cystatin C1.30

ml/min/1.73 m2

26

Several studies show that cystatin C is stable for at least 7 days at room
temperature, for several weeks in a refrigerator and for several months frozen at -20 C
or at -80C. In addition, its level will be unchanged after seven freeze/thaw cycles and
storage of unseparated blood for 24 hours will not affect the cystatin C level [175, 176].

2.6.1 Cystatin C and diabetic nephropathy


Many studies on the cystatin C and creatinine as markers of GFR in diabetes
indicated that cystatin C is the best marker in this case, but sometimes studies have not
show superiority for cystatin C
Piwowar et al., (1999) evaluated the clinical usefulness of measuring plasma
cystatin C compared with other parameters related to kidney dysfunction in T2DM. The
concentration of cystatin C was increased in diabetic patients about 2.5 times, and
creatinine was elevated only about 1.1 with respect to the controls. Plasma cystatin C
concentration increased significantly in patients with microalbuminuria in comparison
to patients with normoalbuminuria. Additionally, the mean levels of cystatin C was
significantly higher in the patients with macroalbuminuria in compared to patients with
normoalbuminuria and microalbuminuria. Plasma concentration of creatinine was
significantly higher only in the group with macroalbuminuria in compared to patients
with normoalbuminuria. Calculations of sensitivity of the plasma cystatin C and
creatinine showed that the sensitivity of cystatin C was 82 % and that of creatinine was
48% [177].
Mojiminiyi et al., (2000) assessed whether serum concentration of cystatin C
can be used as markers of nephropathy in patients with T2DM. Serum cystatin C and
creatinine were significantly higher in patients with DNP than in normoalbuminuric
patients. In normoalbuminuric patients, one of seven patients with calculated creatinine
clearance less than 80 mL/min had elevated cystatin C and none of these patients had
elevated serum creatinine. In the patients with DNP, two of nine patients with calculated
creatinine clearance less than 80 mL/min had increased serum creatinine compared with
four of nine patients with elevated serum cystatin C [178].
Oddoze et al., (2001) evaluated serum cystatin C as a potential new marker of
GFR in diabetic patients with early renal impairment. The mean levels of serum

27

creatinine in group 2 (GFR 60 to 80 mL/min) and group 3 (GFR 60 mL/min) were


significantly higher than those in group 1 (GFR 80 mL/min). The mean levels of
serum creatinine in group 2 were also significantly higher than those in group 3.
However, the mean levels of serum cystatin C in group 3 were significantly higher than
those in group 1 and group 2.There were no significant changes in the levels of serum
cystatin C between group 1 and group 2. The areas under curve (AUC) with a cutoff
value of 60 mL/min were 0.925 for cystatin C, and 0.916 for serum creatinine. With a
cutoff value of 80 mL/min, the AUC were 0.780 for cystatin C, and 0.905 for serum
creatinine. For the two parameters, areas under the curve did not differ statistically
between the two definitions of renal failure [179].
Mussap et al., (2002) compared traditional markers of changes in GFR with
cystatin C in patients with T2DM, and determined whether diagnostic accuracy of
serum cystatin C is better than of serum for the early assessment of DNP. In the group
with normal GFR (GFR 80 ml/min/1.73m2), serum creatinine values significantly
differed between males and females, while no difference was observed for cystatin C.
Likewise, in the group with reduced GFR (GFR 80 ml/min/1.73m2), the serum
creatinine values in males significantly differed from those in females, while serum
cystatin C values were not significantly different. In patients 61-70 years old, serum
creatinine was significantly higher than in patients 51-60 years old with normal GFR,
while serum cystatin C did not differ between patients of these two decades. Non
parametric receiver operating characteristic (ROC) plots for serum cystatin C and serum
creatinine demonstrated the AUC of cystatin C was significantly greater than that of
serum creatinine in distinguishing between the T2DM patients with a normal and
reduced GFR. The maximum diagnostic accuracy of serum cystatin C (90%) was
significantly better than that of serum creatinine (77%) in distinguishing between the
T2DM patients with a normal and reduced GFR [180].
Shimizu et al., (2003) determined the relationship between levels of serum
cystatin C or serum creatinine and prognostic stages of diabetic nephropathy in T2DM
patients. The mean levels of serum cystatin C in macroalbuminuric patients without
renal dysfunction were significantly higher than those in normoalbuminuric patients or
microalbuminuric patients. The mean levels of serum cystatin C in macroalbuminuric

28

patients with renal dysfunction and patients with renal failure were also significantly
higher than those in normoalbuminuric patients. There were no significant changes in
the

levels

of

serum

cystatin

between

normoalbuminuric

patients

and

microalbuminuric patients. However, the mean levels of serum creatinine in


macroalbuminuric patients with renal dysfunction were not significantly higher than
those in normoalbuminuric patients or microalbuminuric patients. The levels of serum
creatinine in macroalbuminuric patients with renal dysfunction and patients with renal
failure were significantly higher than those in normoalbuminuric patients. There were
no significant changes in the levels of serum creatinine between normoalbuminuric
patients and microalbuminuric patients. ROC plots demonstrated that the AUC of
cystatin C was greater than that of creatinine. To distinguishing between
microalbuminuric patients and macroalbuminuric patients without renal dysfunction,
sensitivity and specificity of serum cystatin C were better than those of serum creatinine
[181].
Xia et al., (2004) determine whether cystatin C can replace serum creatinine as
the screening marker for reduced GFR in T2DM patients. Mean Serum cystatin C was
significantly higher in patients with reduced GFR (GFR 68 ml/min) than in normal
group (GFR 68 ml/min). Mean Serum creatinine was significantly higher in patients
with reduced GFR than in normal group. Mean creatinine clearance was significantly
lower in patients with reduced GFR than in normal group. In the normal group, serum
creatinine was significantly different between males and females, but no difference was
found in serum cystatin C between males and females. In the reduced GFR group,
however, no differences between males and females were found in both serum
creatinine and serum cystatin C. The AUC was statistically different between serum
cystatin C and creatinine clearance, but not between serum cystatin C and serum
creatinine [182].

Yang et al., (2007) compared serum cystatin C and serum creatinine as markers
for early decline of GFR in T2DM, and explored the relationship of urine albumin,
GFR, the serum creatinine and cystatin C concentrations. The mean value of the serum
creatinine significantly differed between females and males, while no difference was
observed for cystatin C values. Analysis of the data on the basis of age, the mean value

29

of the serum creatinine increase with increase of age. While no difference was noted for
cystatin C. Serum cystatin C concentration increased significantly in patients from
normoalbuminuria to macroalbuminuria and microalbuminuria to macroalbuminuria.
The serum creatinine also revealed significance, but only in microalbuminuria to
macroalbuminuria group. ROC curve analysis for all patients included in the study
showed that serum cystatin C had significantly higher diagnostic accuracy than serum
creatinine. [183].
El-Shafey et al., (2009) assessed serum cystatin C as a marker of GFR for
detection of early renal impairment in patients with T2DM. Diabetic patients with
macroalbuminuria and renal dysfunction showed significantly higher concentrations of
serum cystatin C and creatinine compared to normoalbuminuric patients with normal
renal function, microalbuminuric patients with normal renal function microalbuminuric
patients with normal renal function, and macroalbuminuric patients with normal renal
function. In the early diabetic nephropathy group (microalbuminuric patients with
normal renal function), the AUC for cystatin C was greater than that of creatinine
clearance and creatinine as well. Therefore, sensitivity and diagnostic accuracy of
cystatin C was higher than creatinine clearance, and both were better than serum
creatinine [184].

30

Chapter 3
Materials and Methods

31

Chapter 3

Materials and Methods

3.1 Study design


The present study is an observational retrospective case control design.

3.2 Study population


The target population of this study comprises adult males and females who have
T2DM for different period of time and aged between 30-60 years from outpatient
diabetic clinics in Gaza Strip. Another group of apparently healthy individuals
represented the control group.

3.3 Sample size


The sample size was 95 patients which previously diagnosed as having T2DM
(52 males and 43 females) as cases and 95 healthy individuals (52 males and 43
females) as controls. Controls and patients were matched with age, sex and residence.

3.4 Exclusion criteria


A. Cases

Diabetics with UTI.

Diabetics suffering from renal or liver disease.

Diabetics who have high blood pressure: more than 130/80 mmHg.

Females who are pregnant.

B. Controls

Subjects with fasting blood glucose 125mg/dl.

Subjects with UTI.

Subjects suffering from renal or liver disease.

Subjects who have high blood pressure: more than 130/80 mmHg.

Females who are pregnant.

32

3.5 Ethical considerations


The study was approved by:

Local Helsinki committee in Gaza Strip (Appendix A).

Palestinian ministry of health to conduct the study (Appendix B, C).

Each volunteers involved in the study, which gave them a clear explanation
about the purpose of this research and to ensure the confidentiality of
information and that such participation completely optional (Appendix D).

3.6 Data collection


3.6.1 Questionnaire
A meeting interview was used for filling the questionnaire from both cases and
controls. The questionnaire (Appendix E, F) was based on diabetic clinic questions at
Al Rimal Medical Center with some modifications. All interviews were conducted face
to face by the researcher through using a clear Arabic language. Personal data was
entered to the questionnaire using English language. The questionnaire includes issues
about personal information like name, sex, age, weight, height, time of diagnosis (for
patients only), smoking and family history of diabetes in first-degree relatives (father,
mother, sister and brother). In addition, it includes other information like type of
medication and complication of diabetes (retinopathy, CVD and neuropathy). Most
questions were yes or no question that offers a dichotomous choice.

3.6.2 Measurement of blood pressure


Blood pressure (systolic blood pressure and diastolic blood pressure) were
obtained twice (before and after meeting interview), from the individuals in the sitting
position using calibrated mercury sphygmomanometer in order to exclude cases that
have blood pressure more than 130/80 mmHg.

33

3.6.3 Measurement of body mass index


The body weight of each individual dressed in light clothing without shoes was
measured using a carefully calibrated electrical balance (Camry EF711H, China). The
height of each individual was measured with shoes removed using height measuring
tape (Seca 206, USA). Body mass index (BMI) was calculated as person's weight in
kilograms divided by height in meters squared (BMI=kg/m2). People with BMI=18.524.9 were considered to have normal weight, people with BMI=25.0-29.9 were
classified overweight, people with BMI=30.0-34.9 were considered obese of type I, and
those with BMI=35.0-39.9 were obese of type II, and people with BMI 40 were
classified as obese of type III [185].

3.6.4 Specimen collection


Convenient sampling method was used for selection of the study population, in
order that every individual has to meet the criteria of being included in the sample. For
both members of the case and control group, blood and urine samples were collected
under quality control and safety procedure.

A. Blood sampling and processing


Fasting overnight venous blood sample (about 5 ml) was drawn from each
control and diabetic individual. The blood was collected in plain vacutainer tubes and
left for a while to allow blood to clot. Then clear serum sample was obtained by
centrifugation at room temperature at 3500 rpm for 10 minutes and collected into two
plastic tubes, then stored at -20C for no more than one month until the time of analysis.

B. Urine sampling and processing


Urine sample from both patients and controls were collected in a plastic
container. The urine samples were immediately centrifuged at 2000 rpm for 10 minutes.
Routine urine test were performed to each sample in order to exclude cases that have
UTI. 5 ml from each sample supernatant was distributed equally into two plastic tubes
and then stored at -20C for no more than one month until the time of chemical assay.

34

3.7 Biochemical analysis


Serum cystatin C, glucose, urea, creatinine, cholesterol, triglycerides and urine
albumin were analyzed using chemistry automated analyzer (Hitachi 902; Roche,
Manheim, Germany). A part of urine was diluted 1/50 (20l urine+ 980l distilled
water) and then analyzed by automated analyzer to determine urine creatinine. HDL was
determined manually using spectrophotometer (Biosystems BTS-310, Barcelona, Spain)
and then LDL was calculated.
For serum cystatin C, two levels of human-based control serum were analyzed
with each run. For other serum parameters (glucose, urea, creatinine, cholesterol and
triglycerides) two levels of lyophilized multi-control sera; normal and abnormal levels
were analyzed with each run. For urine albumin, two levels of aqueous control serum
were analyzed with each run.
The concentration of enzymatic tests for glucose, urea, creatinine, cholesterol
and triglycerides which performed by the automated analyzer calculated according to
beer's law after calibration and adjustment of the photometers against water blank using
a specific program of every test inserted to the instrument.

Beers law: Concentration = (A) Sample x Concentration of Calibrator / (A)


Calibrator

(A) = Absorbance

The cystatin C and urine albumin concentration of test samples was derived
from the calibration curve using an appropriate mathematical model (spline). The
calibration curve is obtained with five calibrators at different levels and distilled water
for determination of the zero value.

35

3.7.1 Determination of serum cystatin C


Serum cystatin C was determined by particle enhanced immunoturbidimetric
assay [186] using a commercially available kits (Diasys, Halzheim, Germany).

Principle
Cystatin C in the sample binds to the specific anti-cystatin C antibody, which is
coated on latex particles, and causes agglutination. The degree of the turbidity caused by
agglutination is measured optically and is proportional to the amount of cystatin C in the
sample.

Reagents
Reagent

Component

Concentration

TRIS pH 7.4

100 mmol/L

NaCl

200 mmol/L

Reagent 1
Polyethylenglycol (PEG)
Detergents, Stabilizers
Borate
Reagent 2

7.5 mmol/L

Polyclonal Antibodies (goat) against human cystatin C


bound to carboxylated polystyrene particles, Stabilizers

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:

Parameter

Value

Parameter

Assay code

2 Point Sample volume (l)

Value

Parameter

Value

Calibrator 2 (mg/l)

0.50

Reaction time (m)

10

Reagent 1 volume (l)

180

Calibrator 3 (mg/l)

1.50

Wave length (nm)

505

Reagent 2 volume (l)

60

Calibrator 4 (mg/l)

3.00

Assay point 1

22

Calibrator type

spline

Calibrator 5 (mg/l)

5.50

Assay point 2

35

Calibrator 1 (mg/l)

0.0

Calibrator 6 (mg/l)

8.00

36

3.7.2 Determination of serum glucose


Serum glucose was determined by enzymatic photometric test (glucose oxidase
(GOD)) [187] using a commercially available kits (Diasys, Halzheim, Germany).

Principle
Under the catalytic action of GOD glucose oxidized to gluconic acid and
hydrogen peroxide (H2O2), which in the presence of peroxidase (POD) it is reacts with
4-aminoantipyrine and phenol to form a red complex (quinoneimine).

GOD

Glucose + O2

2 H2O2 + 4-Aminoantipyrine + Phenol

Gluconic acid + H2O2

POD

Quinoneimine + 4 H2O

The intensity of the color is proportional to glucose concentration in the sample.

Reagents
Reagent

Component

Concentration

Phosphate buffer pH 7.4

250 mmol/L

Phenol
Reagent

5 mmol/L

4-Aminoantipyrine

0.5 mmol/L

GOD

15 KU/L

POD

1 KU/L

Standard

100 mg/dL

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:

Parameter

Value

Parameter

Assay code

1 Point Wave length (nm)

Value

Parameter

Value

505

Calibrator type

linear

Reaction time (m)

10

Sample volume (l)

2.5

Calibrator 1 (mg/dl)

0.0

Assay point 1

35

Reagent volume (l)

250

Calibrator 2 (mg/dl)

100

37

3.7.3 Determination of serum urea


Serum urea was determined by enzymatic test (urease/glutamate dehydrogenase
(GLDH)) [188] using a commercially available kits (Diasys, Halzheim, Germany).

Principle
Urea hydrolyzed enzymatically by urease into ammonia (NH4+) and carbonate
ions. Ammonia ions reacts with 2-oxoglutarate in a reaction catalyzed by GLDH with
simultaneous oxidation of NADH to NAD+.
Urea + 2 H2O

Urease

2-Oxoglutarate + NH4+ + NADH

2 NH4+ + 2 HCO3-

GLDH

L-Glutamate + NAD+ + H2O

The decrease in concentration of NADH at 340 nm is proportional to the urea


concentration in the sample.

Reagents
Reagent

Component
TRIS PH 7.8
2-Oxoglutarate

Reagent 1

Reagent 2

ADP

Concentration
150 mmol/L
9 mmol/L
0.75 mmol/L

Urease

7 KU/L

GLDH

1 KU/L

NADH

1.3 mmol/L

Standard

50 mg/dL

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:
Parameter

Value

Parameter

Assay code

Rate A Wave length (nm)

Value

Parameter

Value

340

Calibrator type

linear

Reaction time (m)

10

Sample volume (l)

2.5

Calibrator 1 (mg/dl)

0.0

Assay point 1

21

Reagent 1 volume (l)

200

Calibrator 2 (mg/dl)

50

Assay point 2

24

Reagent 2 volume (l)

50

38

3.7.4 Determination of serum creatinine


Serum creatinine was determined by kinetic test without deproteinization
according to the Jaff method [189] using a commercially available kits (Diasys,
Halzheim, Germany).

Principle
Creatinine forms a colored orange-red complex in an alkaline picrate solution.
The difference in absorbance at fixed times during conversion is proportional to the
concentration of creatinine in the sample.
Creatinine + Picric acid

Creatinine picrate complex

Reagents
Reagent

Component

Concentration

Reagent 1

Sodium hydroxide

0.2 mol/L

Reagent 2

Picric acid

20 mmol/L

Standard

2 mg/dL

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:
Parameter

Value

Parameter

Assay code

Rate A Assay point 4

Value

Parameter

Value

15

Calibrator type

linear

Reaction time (m)

10

Wave length (nm)

505

Calibrator 1 (mg/dl)

0.0

Assay point 1

20

Sample volume (l)

15

Calibrator 2 (mg/dl)

Assay point 2

26

Reagent 1 volume (l)

200

Assay point 3

10

Reagent 3 volume (l)

50

39

3.7.5 Determination of serum cholesterol


Serum cholesterol was determined by enzymatic photometric test (cholesterol
oxidase (CHOD)) [190] using a commercially available kits from (Diasys, Halzheim,
Germany).

Principle
The cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase
(CHE). The free cholesterol is then oxidized by CHOD with the simultaneous
production of H2O2. The hydrogen peroxide produced couples with 4-aminoantipyrine
and phenol, in the presence of POD, to yield a chromogen.

CHE

Cholesterol ester + H2O


Cholesterol + O2

CHOD

Cholesterol + Fatty acid

Cholesterol-3-one + H2O2

POD

2 H2O2 + 4-Aminoantipyrine + Phenol


Quinoneimine + 4 H2O
The intensity of the color produced is directly proportional to the concentration of total
cholesterol in the sample.

Reagents
Reagent

Component

Concentration

Good's buffer pH 6.7 50 mmol/L

Reagent

Phenol

5 mmol/L

4-Aminoantipyrine

0.3 mmol/L

CHE

200 KU/L

CHOD

50 KU/L

POD

3 KU/L

Standard

200 mg/dL

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:
Parameter

Value

Parameter

Assay code

1 Point Wave length (nm)

Value

Parameter

Value

505

Calibrator type

linear

Reaction time (m)

10

Sample volume (l)

2.5

Calibrator 1 (mg/dl)

0.0

Assay point 1

35

Reagent volume (l)

250

Calibrator 2 (mg/dl)

200

40

3.7.6 Determination of serum triglycerides


Serum triglycerides was determined by colorimetric enzymatic test (glycerol-3phosphateoxidase (GPO)) [191] using a commercially available kits (Diasys, Halzheim,
Germany).

Principle
The triglycerides are hydrolyzed by lipoprotein lipase (LPL) and the liberated
glycerol is converted to glycerol-3-phosphate (G3P) by the glycerol kinase (GK). The
G3P will be oxidized by GPO and generating quantitatively hydrogen peroxide. The
hydrogen peroxide produced couples with 4-aminoantipyrine and 4-Chlorophenol, in
the presence of POD, to form a red color.
Triglycerides
Glycerol + ATP
Glycerol-3-phosphate + 02

LPL
GK

Glycerol + fatty acid


Glycerol-3-phosphate + ADP

GPO

Dihydroxyaceton phosphate + H2O2

2 H2O2 + 4-Aminoantipyrine + 4-Chlorophenol

POD

Quinoneimine + HCl + 4 H

The absorbance measured is proportional to the concentration of triglycerides


and free glycerol in the sample.

Reagents
Reagent

Reagent

Component

Concentration

Good's buffer pH 7.2

50 mmol/L

4-Chlorophenol

4 mmol/L

ATP

2 mmol/L

Mg2+

15 mmol/L

GK

0.4 KU/L

POD

2 KU/L

LPL

4 KU/L

4-Aminoantipyrine

0.5 mmol/L

GPO

1.5 KU/L

standard

200 mg/dL

41

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:

Parameter

Value

Parameter

Assay code

1 Point Wave length (nm)

Value

Parameter

Value

505

Calibrator type

linear

Reaction time (m)

10

Sample volume (l)

2.5

Calibrator 1 (mg/dl)

0.0

Assay point 1

35

Reagent volume (l)

250

Calibrator 2 (mg/dl)

200

3.7.7 Determination of high-density lipoprotein


High-density lipoprotein was determined by precipitation of LDL, very lowdensity lipoprotein (VLDL) and chylomicrons method [192] using a commercially
available kits (Diasys, Halzheim, Germany).

Principle
Chylomicrons, VLDL and LDL are precipitated by adding phosphotungstic acid
and magnesium ions to the sample. After centrifugation only HDL leaves in the
supernatant, their cholesterol content is determined enzymatically using cholesterol
reagent.

Reagents
Reagent
Reagent

Component

Concentration

Magnesium chloride

25 mmol/L

phosphotungstic acid

0.55 mmol/L

Assay Procedure
Precipitation
Sample / Stander

200 L

Precipitation reagent

500 L

Mix well; allow standing for 10 minute at room temperature, and centrifuging for 10 minute at
4000 rpm. After centrifugation, separate the clear supernatant from the precipitate within 1 hour.

42

Cholesterol determination
Wavelength

500 nm

Optical path

1 cm

Temperature

37 oC

Measurement
Blank
Distil water

Sample or Stander

100L
100 L

Sample / Stander
Cholesterol Reagent

1000L

1000 L

Mix, incubate for 5 minute at 37 C, then read absorbance within 60 minute against reagent
blank.

Calculation:
HDL = (A) Sample / (A) Standard X Concentration (Standard)
(A) = Absorbance
Concentration of standard = (50 mg/dL)

3.7.8 Calculation of serum low-density lipoprotein


Low-density lipoprotein can be calculated using the empirical relationship of
Friedewald [193].

Principle
The ultra-centrifugal measurement of LDL is time consuming and expensive and
requires special equipment. For this reason, LDL is most commonly estimated from
quantitative measurements of cholesterol, HDL and plasma triglycerides using the
empirical relationship of Friedewald.
LDL = Total Cholesterol - HDL - Triglycerides/5
The Friedewald equation should not be used when chylomicrons are present, and when
plasma triglycerides concentration exceeds 400 mg/dl.

43

3.7.9 Determination of urine albumin


Urine albumin was determined by immunoturbidimetric assay [194] using a
commercially available kits (Diasys, Halzheim, Germany).

Principle
Anti-albumin antibodies react with the antigen in the sample to form
antigen/antibody complexes, which causes agglutination. The degree of the turbidity
caused by agglutination is measured optically and is proportional to the amount of
albumin in the sample.

Reagents
Reagent

Reagent 1

Component

Concentration

TRIS pH 7.5

100 mmol/L

NaCl

50 mmol/L

PEG
Detergents, Stabilizers
TRIS pH 8.0

83 mmol/L

NaCl
165 mmol/L
Polyclonal antibodies (goat) against human albumin
bound to carboxylated polystyrene particles, Stabilizers

Reagent 2

Assay Procedure
About 0.5 ml of serum was transferred to the Hitachi 902 chemistry
autoanalyzer, to perform the test according to these parameters:

Parameter

Value

Parameter

Assay code

2 Point Sample volume (l)

Value

Parameter

Value

12

Calibrator 2 (mg/l)

11.4

Reaction time (m)

10

Reagent 1 volume (l)

250

Calibrator 3 (mg/l)

20.9

Wave length (nm)

415

Reagent 2 volume (l)

50

Calibrator 4 (mg/l)

44.5

Assay point 1

17

Calibrator type

line

Calibrator 5 (mg/l)

151

Assay point 2

35

Calibrator 1 (mg/l)

0.0

Calibrator 6 (mg/l)

310

44

3.7.10 Calculated measurements


A. Glomerular filtration rate
Glomerular filtration rate was estimated according to the listed formulae:

MDRD formula: eGFR (ml/min/1.73 m2) = 186 (Serum creatinine in mg/dL)1.154

age-0.203 [149]. A correction factor of 0.742 was used for women.

Hoek formula: eGFR (ml/min/1.73 m2) = (80.35 / Cystatin C in mg/L) 4.32


[172].

B. Albumin/Creatinine ratio
The urine creatinine value was divided by 100 to convert mg/dL to g/L and then
divide the urine albumin value by the urine creatinine value to express ACR as (mg
albumin/g creatinine).
ACR (mg/g) = Urine albumin (mg/L) x100 /Creatinine in urine (mg/dl)

3.8 Statistical analysis


Data were computer analyzed using Statistical Packages for Social Science (SPSS 17.0
USA). The relationships between some qualitative categories were identified
statistically by using chi-square test. Independent T test was applied to compare means
of two quantitative variables. The one-way analysis of variance (ANOVA) is used to
determine whether there are any significant differences between the means of groups.
To assess the correlation between biochemical parameters, Pearsons correlations
coefficient (r) was applied. ROC analysis was employed to calculate the AUC. All
results were considered significant if P < 0.05.

45

Chapter 4
Results

46

Chapter 4

Results

4.1 General characteristics of the study population


The present study is a case control that included 190 individuals (95 patients and
95 controls).

4.1.1 Distribution of the study population by governorates of the Gaza Strip


Figure 4-1 shows the distribution of the study population by governorates of the
Gaza Strip. The number of individuals from middle area (Deir Al-Balah Governorate)
was 37 (38.95%) patients and 37 (38.95%) controls. The number of individuals from the
north area (Gaza and North Gaza governorate) was 33 (34.74%) patients and 33
(34.74%) controls, while 25 (26.32%) patients and 25 (26.32%) controls were from the
south area (Khan yunis and Rafah governorate).

Figure 4-1 Distribution of the study population by governorates of the Gaza Strip

4.1.2 Distribution of the study population by gender


Figure 4-2 shows the distribution of the study population by gender. The males
represented 54.7% (52) of the controls and 54.7% (52) of the patients, whereas females
represented 45.3% (43) of the controls and 45.3% (43) of the patients. The case group
was matched by sex with the control group.

47

Figure 4-2 Distribution of study population by gender

4.1.3 Age of the study population


Figure 4-3 shows mean age of the study population. The mean age of control
males was 49.336.22 years and that of females was 47.267.02 years. For diabetic
patients the mean age of males was 49.336.22 years whereas that of females was
47.267.02 years. The control and case group was matched by age, were the mean age
of controls and patients was 48.386.64 and 48.386.64 years, respectively.

Figure 4-3 Mean age of the study population

48

4.1.4 Self-reported complications among the study population


Figure 4-4 shows the distribution of main self-reported complications among
the study population, whereas 51.6% of diabetic patients have at least one complication
compared to 3.2% of controls.

Figure 4-4 Distribution of self-reported complications among the study population


Table 4-1 illustrates frequencies and percentages of main self-reported
complications among controls and diabetic patients. The percentages of retinopathy,
neuropathy and CVD were higher in diabetic patients compared to the controls (28.4,
15.8 and 7.4% vs. 1.1, 2.1 and 0.0%, respectively). The results showed significant
association between all complications and diabetes (X2=28.316, P=0.000, X2=10.918,
P=0.001 and X2=7.268, P=0.007, respectively), indicating that diabetes mellitus is an
independent risk factor for vascular complications.

Table 4-1 Frequencies and percentages of main self-reported complications among


controls and diabetic patients
Controls

Diabetics

Statistics

Males
(N=52)
N
%

Females
(N=43)
N
%

Males
(N=52)
N
%

Females
(N=43)
N
%

Retinopathy

1.92

12

23.1

15

Neuropathy

1.92

2.32

13.5

CVD

5.8

Complication

X2

P-Value

34.9

28.316

0.000

18.6

10.918

0.001

9.3

7.268

0.007

P value by chi-square test, P < 0.05 is statistical significant. CVD, cardiovascular disease.

49

4.1.5 Smoking and family history among the study population


Table 4-2 illustrates frequencies and percentages of smoking and family history
of diabetes among controls and diabetic patients. There was no association between
smoking and diabetes, where smokers representing 23.2% (22) of controls and 15.8%
(15) of diabetic patients (X2=1.645, P=0.200). However, family history was found to be
statistically significant associated with diabetes, where 31.6% (30) of controls and
52.6% (50) of patients reported to have a family history of diabetes (X2=8.636,
P=0.003).
Table 4-2 Frequencies and percentages of smoking and family history among controls
and diabetic patients
Controls
(N=95)

Variable

Diabetics
(N=95)

Yes

22

23.2

15

15.8

No

73

76.8

80

84.2

Yes

30

31.6

50

52.6

No

65

68.4

45

47.4

Smoking

Family history

Statistics
X2

P-Value

1.645

0.200

8.636

0.003

P value by chi-square test, P < 0.05 is statistical significant

4.1.6 Body mass index of the study population


Figure 4-5 shows mean BMI of controls and diabetic patients, where the mean
BMI of controls was 27.594.94 kg/m2 and that of diabetic patients was 31.445.96
kg/m2. BMI of patients was significantly higher than that of controls (t=-4.839,
P=0.000).

Figure 4-5 Mean BMI of controls and diabetic patients

50

Table 4-3 illustrates weight classification by BMI of controls and diabetic


patients. About 32.6% (31) of controls were normal, 41.1% (39) were overweight,
14.7% (14) were obese I, 11.6% (11) were obese II and 0.0% (0) were obese III.
Whereas in patients they were 15.8% (15), 25.3% (24), 30.5% (29), 21.0% (20) and
7.4% (7), respectively. There was a statically significant higher frequency of obesity
among diabetic patients than controls (X2=23.982, P=0.000), indicating that obesity is a
risk factor of diabetes.

Table 4-3 Weight classification by BMI of controls and diabetic patients


Controls
Diabetics
Statistics
Weight
(N=95)
(N=95)
classification
N
%
N
%
X2
P-Value
Normal

31

32.6

15

15.8

Over weight

39

41.1

24

25.3

Obese I

14

14.7

29

30.5

Obese II

11

11.6

20

21.0

Obese III

0.0

7.4

23.982

0.000

P value by chi-square test, P < 0.05 is statistical significant. Weight classifications by BMI:
people with BMI=18.5-24.9 have normal weight, people with BMI=25.0-29.9 were classified
overweight, people with BMI=30.0-34.9 were considered obese of type I, and those with
BMI=35.0-39.9 were obese of type II, and people with BMI=40 were classified as obese of
type III [185].

4.2 Diabetes mellitus


4.2.1 Age at diagnosis and duration of the disease
Figure 4-6 shows mean age at diagnosis and mean disease duration of diabetic
patients. The mean age of diabetic males at diagnosis was 42.174.94 years while that
of females was 40.285.77 years (t=1.722, P=0.088). The mean duration of disease for
males was 7.154.90 years while for females it was 6.984.81 years (t=0.177,
P=0.860).

51

Figure 4-6 Mean age at diagnosis and mean duration of the disease

Figure 4-7 shows the distribution of diabetic patients by the duration of disease.
More than half of patients were found to be diabetics since 5 years or less; 27 (51.9%)
males and 22 (51.2%) females, whereas those with diabetes duration of 6-10 years
represented 27.4% of diabetic patients; 14 (26.9%) males and 12 (27.9%) females. The
rest of population was distributed over the intervals from 11-20 years and they represent
21.1% of diabetic patients; 11 (21.2%) males and 9 (20.9%) females.

Figure 4-7 Distribution of diabetic patients by duration of the disease

52

4.2.2 Diabetes duration and self-reported complication


Table 4-4 demonstrates main self-reported complications and their relation with
duration of diabetes. There was an increase in self-reported complication frequency with
increase the duration of diabetes. This positive relationship was statically significant for
retinopathy (X2=7.083, P=0.029) and neuropathy (X2=9.226, P=0.010). On the other
hand, such relationship was not statically significant for CVD (X2=1.641, P=0.440).
Table 4-4 The main self-reported complications and their relation to duration of
diabetes
Duration (years)
Complication

Statistics

5 (N=49)

6-10 (N=26)

>10 (N=20)

Retinopathy

18.4

30.8

10

Neuropathy

6.1

19.2

CVD

4.1

11.5

X2

P-value

50.0

7.083

0.029

35.0

9.226

0.010

10.0

1.641

0.440

P value by chi-square test, P < 0.05 is statistical significant. CVD, cardiovascular disease.

4.2.3 Diabetes mellitus management


Figure 4-8 illustrates the distribution of diabetic patients by type of diabetes
management. The majority of patients 48.4% (46) used OHA to manage diabetes and
25.3% (24) were on insulin therapy, whereas 15.8% (15) of patients bring together
between OHA and insulin, and 10.5% (10) depend on diet only.

Figure 4-8 Distribution of diabetic patients by type of diabetes management

53

4.2.4 Blood glucose check


Figure 4-9 demonstrates the distribution of diabetic patients by blood glucose
checking. About 81.1% (77) of patients check blood glucose regularly, whereas 18.9%
(18) of patients checking blood glucose sporadically.

Figure 4-9 Distribution of diabetic patients by glucose checking

4.3 Biochemical analysis among the study population


4.3.1 Serum cystatin C among controls and diabetic patients
Table 4-5 illustrates the comparison of the mean level of cystatin C between
controls and diabetic patients. The difference between controls and diabetic patients in
the mean values of cystatin C was non-significant (0.710.23 vs. 0.710.29 mg/l,
t=0.008, P=0.994).
Table 4-5 Serum cystatin C of controls and diabetic patients
Serum
parameter
Cystatin C
(mg/l)

Controls
Males
MeanSD
0.74
0.25

Females
MeanSD
0.69
0.22

Diabetics
Males
MeanSD
0.75
0.29

Females
MeanSD
0.67
0.29

P value by independent samples T test, P < 0.05 is statistical significant.

54

Statistics
t

PValue

-0.008

0.994

4.3.2 Serum glucose among controls and diabetic patients


Table 4-6 illustrates the comparison of the mean level of fasting blood glucose
between controls and diabetic patients. The results showed that the mean serum glucose
level in diabetic patients was significantly higher than that in controls (185.1566.12 vs.
87.8715.03 mg/dl, t=-13.983, P=0.000).

Table 4-6 Serum glucose of controls and diabetic patients


Serum
parameter
Glucose
(mg/dl)

Controls
Males
MeanSD
87.92
15.54

Females
MeanSD
87.81
14.57

Diabetics
Males
MeanSD
187.32
68.80

Statistics

Females
t
MeanSD
182.53
-13.983
63.44

PValue
0.000

P value by independent samples T test, P < 0.05 is statistical significant.

4.3.3 Serum urea and creatinine among controls and diabetic patients
Table 4-7 illustrates the comparison of the mean level of serum urea and
creatinine between controls and diabetic patients. The results showed that the mean
serum urea and creatinine levels were significantly decreased in diabetic patients
(23.359.88 and 0.660.26 mg/dl respectively) compared to controls (28.489.90 and
0.750.27 mg/dl, respectively) (t=3.571, P=0.000 and t=2.299, P=0.023, respectively).

Table 4-7 Serum urea and creatinine of controls and diabetic patients
Serum
parameter
Urea
(mg/dl)
Creatinine
(mg/dl)

Controls
Males
MeanSD
32.25
9.36
0.83
0.24

Females
MeanSD
23.93
8.60
0.65
0.27

Diabetics
Males
MeanSD
24.26
10.13
0.72
0.26

Females
MeanSD
22.25
9.57
0.58
0.23

P value by independent samples T test, P < 0.05 is statistical significant.

55

Statistics
t

PValue

3.571

0.000

2.299

0.023

4.3.4 Lipid profile of controls and diabetic patients


Table 4-8 illustrates the comparison of the mean level of lipid profile
(cholesterol, triglycerides, HDL and LDL) between controls and diabetic patients. The
results showed that the mean levels of cholesterol, triglycerides and LDL were higher in
diabetic patients (196.9449.37, 212.6889.62 and 109.9248.88 mg/dl, respectively)
than in controls (171.0444.35, 155.3569.86 and 85.6916.41 mg/dl, respectively)
with (t=-3.804, P=0.000, t=-4.917, P=0.000 and t=-4.579, P=0.000, respectively). In
contrast, HDL was significantly lower in diabetic patients than in controls (44.4812.06
vs. 54.2715.48 mg/dl, t=4.862, P=0.000).
Table 4-8 Lipid profile of controls and diabetic patients
Serum
parameter
Cholesterol
(mg/dl)
Triglycerides
(mg/dl)
HDL
(mg/dl)
LDL
(mg/dl)

Controls

Diabetics

Males
MeanSD

Females
MeanSD

Males
MeanSD

Females
MeanSD

170.50
42.70
162.51
68.44
53.76
15.79
84.22
13.92

171.69
46.78
146.69
71.38
54.88
15.25
87.47
19.02

193.34
47.90
208.86
86.89
44.11
12.55
107.45
50.80

201.30
50.33
217.30
93.64
44.93
11.56
112.91
46.88

Statistics
t

PValue

-3.804

0.000

-4.917

0.000

4.862

0.000

-4.579

0.000

P value by independent samples T test, P < 0.05 is statistical significant. HDL, high-density
lipoprotein. LDL, low-density lipoprotein.

4.3.5 Urine albumin, urine creatinine and eGFR among controls and
diabetic patients
Table 4-9 demonstrates the comparison of the mean level of urine albumin,
urine creatinine, calculated ACR and eGFR between controls and diabetic patients. The
results showed that the mean levels of urine albumin and ACR were markedly higher in
diabetic patients (144.34231.24 mg/l and 178.82309.16 mg/g, respectively) than in
controls (38.1665.79 mg/l and 37.8883.51 mg/g, respectively) with t=-4.304,
P=0.000 and t=-4.829, P=0.000, respectively). In contrast, urinary creatinine level were
significantly decreased in patients compared to controls (113.4060.89 vs.

56

145.3765.87 mg/dl, t=3.474 and P=0.000). On the other hand, both eGFR (eGFR
calculated by the MDRD equation and eGFR calculated by the Hoek equation)
increased in patients versus controls (147.8593.44 and 126.4452.60 vs. 128.1979.88
and 122.0849.15 ml/min/1.73m). This increment was not statically significant (t=1.558, P=0.121 and t=-0.585, P=0.559, respectively).

Table 4-9 Urine albumin, urine creatinine, ACR and eGFR of controls and diabetic
patients
Urine
parameter
Albumin
(mg/l)
Creatinine
(mg/dl)
ACR
(mg/g)
eGFR
calculated by the
MDRD equation
(ml/min/1.73m2)

Controls

Diabetics

PValue

-4.304

0.000

3.474

0.000

-4.829

0.000

152.93
117.08

-1.558

0.121

136.89
60.93

-0.585

0.559

Males
MeanSD

Females
MeanSD

Males
MeanSD

Females
MeanSD

37.55
73.77
158.48
67.47
37.21
92.34

38.90
55.47
129.53
60.95
38.69
72.49

137.27
227.69
122.13
65.56
161.85
292.92

152.89
237.87
102.83
53.58
199.34
330.05

120.75
58.19

138.25
100.69

143.65
69.03

125.71
46.58

117.79
45.14

eGFR
119.08
calculated by the
51.44
Hoek equation
(ml/min/1.73m2)
P value by independent samples
creatinine ratio. eGFR, estimated
Renal Disease.

Statistics

T test, P < 0.05 is statistical significant. ACR, albumin


glomerular filtration rate. MDRD, Modification of Diet in

4.4 Comparison of serum cystatin C, urea and creatinine among males


and females in patients group
Table 4-10 demonstrates the comparison of the mean level of serum cystatin C,
urea and creatinine between males and females in patients group. The results showed
that the difference between males and females in the mean levels of cystatin C
(0.750.29 vs. 0.670.29 mg/l) and urea (24.2610.13 vs. 22.259.57 mg/dl) nonsignificant (t=1.285, P=0.202 and t=0.988, P=0.326, respectively). In contrast, there
was statistically significant increase in the mean level of serum creatinine of males
0.720.26 compared to females 0.580.23, (t=2.699, P=0.008).

57

Table 4-10 Serum cystatin C, urea and creatinine among males and females in patients
group
Serum
parameter

Diabetics patients

Statistics

Males
MeanSD

Females
MeanSD

P-Value

Cystatin C (mg/l)

0.750.29

0.670.29

1.285

0.202

Urea (mg/dl)

24.2610.13

22.25 9.57

0.988

0.326

Creatinine (mg/dl)

0.720.26

0.580.23

2.699

0.008

P value by independent samples T test, P < 0.05 is statistical significant.

4.5 Occurrence of microalbuminuria and macroalbuminuria among


Controls and diabetic patients
Figure 4-10 and table 4-11 show the occurrence of normoalbuminuria,
microalbuminuria and macroalbuminuria among controls and diabetic patients. Were
87.4% (83) of controls and 54.7% (52) of diabetic patients was normoalbuminuric, 9.5%
(9) of controls and 28.4% (27) of diabetic patients have microalbuminuria and 3.2% (3)
of controls and 16.8% (16) of patients have macroalbuminuria. There was a statically
significant association between microalbuminuria and macroalbuminuria with DM
(X2=25.013, P=0.000), implying that diabetes is a risk factor for the development of
DNP.

Figure 4.10 Normoalbuminuria, microalbuminuria and macroalbuminuria


among controls and diabetic patients

58

Table 4.11 Normoalbuminuria, microalbuminuria and macroalbuminuria among


controls and diabetic patients
Controls

Diabetics

Statistics

Males
(N=52)
N
%

Females
(N=43)
N
%

Males
(N=52)
N
%

Females
(N=43)
N
%

Normoalbuminuria

45

86.6

38

88.4

31

59.6

21

48.8

Microalbuminuria

9.6

9.3

12

23.1

15

34.9

Macroalbuminuria

3.8

2.3

17.3

16.3

Item

X2

PValue

25.013 0.000

P value by chi-square test, P < 0.05 is statistical significant.

4.6 Diabetic nephropathy


Depending on their UAE patients were categorized into three diabetic groups:
normoalbuminuric group and diabetic nephropathy groups (microalbuminuric and
macroalbuminuric group).

4.6.1 Gender and diabetic groups


Table 4-12 illustrates the association between gender and different diabetic
groups (normoalbuminuric, microalbuminuric and macroalbuminuric group). There
were non-significant association between sex and the three diabetic groups (X2=1.669,
P=0.434) where 59.6% of males and 40.4% of females were normoalbuminuric, 44.4%
of males and 55.6% of females were microalbuminuric and 56.3% of males and 43.7%
of females were macroalbuminuric.
Table 4-12 Association between gender and diabetic groups
Diabetic nephropathy
Gender

Normoalbuminuric
(N=52)

Microalbuminuric
(N=27)

Statistics

Macroalbuminuric
(N=16)

Males

31

59.6

12

44.4

56.3

Females

21

40.4

15

55.6

43.7

P value by chi-square test, P < 0.05 is statistical significant.

59

X2

P-Value

1.669

0.434

4.6.2 Age, duration of diabetes and BMI of diabetic groups


Table 4-13 illustrates the mean age, diabetes duration and BMI of diabetic
groups (normoalbuminuric, microalbuminuric and macroalbuminuric group). The means
of age were 47.197.17, 50.035.09 and 49.506.67 years and the means of BMI were
31.335.06, 31.096.79 and 32.367.41 kg/m2 for normoalbuminuric, microalbuminuric
and macroalbuminuric group, respectively. The ANOVA test showed non-significant
differences between the mean of age and BMI among different groups (F=1.937,
P=0.150 and F=0.241, P=0.787, respectively). In contrast the ANOVA test showed
significant differences between the means of diabetes duration among diabetic groups
(F=6.096 and P=0.003), where the means of diabetes duration in normoalbuminuric,
microalbuminuric and macroalbuminuric group were 5.614.72, 8.404.44 and
9.564.42 years, respectively. In Scheffe test diabetes duration increased significantly
from normoalbuminuric to microalbuminuric group and normoalbuminuric to
macroalbuminuric group (all P<0.05). Whereas there were non-significance difference
between microalbuminuric and macroalbuminuric group (P>0.050).
Table 4-13 Mean age, duration and BMI of diabetic groups
Diabetic nephropathy
Variables

Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

Age (years)

47.197.17

50.035.09

Duration
(years)

5.614.72

BMI (kg/m2)

31.335.06

Statistics
F

PValue

49.506.67

1.937

0.150

8.404.44u

9.564.42u

6.096

0.003

31.096.79

32.367.41

0.241

0.787

P value by one way ANOVA, P < 0.05 is statistical significant, u significance in relation to the
normoalbuminuric group,. BMI, body mass index.

4.6.3 Smoking and family history among diabetic groups


Table 4-14 illustrates the frequencies and percentages of smoking and family
history among different diabetic groups (normoalbuminuric, microalbuminuric and
macroalbuminuric group). There was no association between smoking and diabetic
groups

(X2=1.995, P=0.369) where 19.2% of normoalbuminuric, 7.4% of

60

microalbuminuric and 18.8% of macroalbuminuric patients are smokers. In addition


there was also no association between family history of diabetes and three diabetic
groups (X2=2.249, P=0.325) where 59.6% of normoalbuminuric, 44.4% of
microalbuminuric and 43.8% of macroalbuminuric patients have family history of
diabetes.
Table 4-14 Frequencies and percentages of smoking and family history among
diabetic groups
Diabetic nephropathy
Variable

Normoalbuminuria
(N=52)

Microalbuminuria
(N=27)

Statistics

Macroalbuminuria
(N=16)

Yes

10

19.2

7.4

18.8

No

42

80.8

25

92.6

13

81.2

Yes

31

59.6

12

44.4

43.8

No

21

40.4

15

55.6

56.2

Smoking

Family
History

X2

PValue

1.995

0.369

2.249

0.325

P value by chi-square test, P < 0.05 is statistical significant.

4.7 Biochemical analysis among diabetic groups


4.7.1 Serum cystatin C among diabetic groups
Table 4-15 illustrates the mean levels of serum cystatin C among diabetic
groups (normoalbuminuric, microalbuminuric and macroalbuminuric group). The
results showed stepwise increase in the level of cystatin C in diabetic groups. Where,
the mean levels of cystatin C in normoalbuminuric, microalbuminuric and
macroalbuminuric group were 0.600.16, 0.710.31 and 1.110.27 mg/l respectively.
The ANOVA test showed statistical significant difference in the mean levels of cystatin
C among different diabetic groups with F=29.406 and P=0.000. In Scheffe test serum
cystatin

concentration

increased

significantly

from

normoalbuminuric

to

macroalbuminuric group and microalbuminuric to macroalbuminuric group (all


P<0.05). Whereas there were non-significance difference between normoalbuminuric
and microalbuminuric group (P>0.05).

61

Table 4-15 Mean values of serum cystatin C among diabetic groups


Serum
parameter
Cystatin C
(mg/l)

Diabetic nephropathy

Statistics

Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

0.600.16

0.710.31

1.110.27uv

PValue

29.406

0.000

P value by one way ANOVA, P < 0.05 is statistical significant, u significance in relation to the
normoalbuminuric group, v significance in relation to the microalbuminuric group.

4.7.2 Serum glucose among diabetic groups


Table 4-16 illustrates the mean levels of serum glucose among diabetic groups
(normoalbuminuric, microalbuminuric and macroalbuminuric group). The results
showed progressively increase in the mean level of glucose in diabetic groups. Where,
the

mean

levels

of

glucose

in

normoalbuminuric,

microalbuminuric

and

macroalbuminuric group were 171.5762.81, 189.7771.85 and 221.5054.32 mg/dl


respectively. The ANOVA test showed statistical significant difference in the mean
level of glucose among different diabetic groups with F=3.792 and P=0.026. In Scheffe
test serum glucose concentration increased significantly from normoalbuminuric to
macroalbuminuric group (P<0.001). Whereas there were non-significance difference
between normoalbuminuric and microalbuminuric group and microalbuminuric and
macroalbuminuric group (all P>0.05).

Table 4-16 Mean values of serum glucose among diabetic groups


Serum
parameter
Glucose
(mg/dl)

Diabetic nephropathy
Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

171.57 62.81

189.77 71.85

221.50 54.32u

Statistics
F

PValue

3.792

0.026

P value by one way ANOVA, P < 0.05 is statistical significant, u significance in relation to the
normoalbuminuric group.

4.7.3 Serum urea and creatinine among diabetic groups


Table 4-17 illustrates the mean levels of serum urea and creatinine among
diabetic groups (normoalbuminuric, microalbuminuric and macroalbuminuric group).

62

Where, the mean levels of urea in normoalbuminuric , microalbuminuric and


macroalbuminuric group were 19.445.61, 26.6611.28 and 30.5012.54 mg/dl,
respectively and that of creatinine were 0.600.19, 0.600.23 and 0.970.26 mg/dl,
respectively. The ANOVA test showed statistical significant difference in the mean
level of both urea and creatinine among different diabetic groups with F=12.064, P=
0.000 and F=19.138, P=0.000, respectively. The Scheffe test revealed that there was
significantly increased in the mean level of serum urea from normoalbuminuric to
microalbuminuric group and normoalbuminuric to macroalbuminuric group (all
P<0.05), whereas, there were non-significance differences between microalbuminuric
and macroalbuminuric group (P>0.05). As for creatinine the concentration increased
significantly from normoalbuminuric to macroalbuminuric group and microalbuminuric
to macroalbuminuric group (all P<0.05) whereas, there were non-significance
differences between normoalbuminuric and microalbuminuric group (P>0.050).
Table 4-17 Mean values of serum urea and creatinine among diabetic groups
Serum
parameter

Diabetic nephropathy

Statistics

Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

19.445.61

26.6611.28u

0.600.19

0.600.23

Urea
(mg/dl)
Creatinine
(mg/dl)

PValue

30.5012.54u

12.064

0.000

0.970.26uv

19.138

0.000

P value by one way ANOVA, P < 0.005 is statistical significant, u significance in relation to
the normoalbuminuric group, v significance in relation to the microalbuminuric group

4.7.4 Lipid profile of diabetic groups


Table 4-18 illustrate the mean levels of lipid profile including cholesterol,
triglycerides,

HDL,

and

LDL

among

diabetic

groups

(normoalbuminuric,

microalbuminuric and macroalbuminuric group). The results showed increase in the


mean levels of cholesterol, triglycerides and LDL in diabetic groups. Where the values
of 193.6144.28, 195.8156.12 and 209.6854.21 mg/dl for cholesterol, 207.7596.42,
210.3784.85

and

232.6275.70

mg/dl

for

triglycerides

and

106.4544.42,

110.3354.06 120.53551.2 mg/dl for LDL in normoalbuminuric, microalbuminuric


and macroalbuminuric group respectively. In contrast the mean values for HDL showed

63

slightly decrease with values of 45.6114.21, 43.408.84 and 42.628.85 mg/dl in


normoalbuminuric, microalbuminuric and macroalbuminuric group respectively. There
were non-significance differences for all lipid profiles among diabetic groups by
ANOVA test (all P>0.05).
Table 4-18 Mean values of lipid profile among diabetic groups
Serum
parameter
Cholesterol
(mg/dl)
Triglycerides
(mg/dl)
HDL
(mg/dl)
LDL
(mg/dl)

Diabetic nephropathy
Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

193.6144.28

195.8156.12

207.7596.42

Statistics
F

PValue

209.6854.21

0.653

0.523

210.3784.85

232.6275.70

0.478

0.606

45.6114.21

43.408.84

42.628.85

0.521

0.596

106.4544.42

110.3354.06

120.5355.12

0.504

0.606

P value by one way ANOVA, P < 0.005 is statistical significant, HDL, high-density
lipoprotein; LDL, low-density lipoprotein.

4.7.5 Urine albumin, urine creatinine and eGFR among diabetic groups
Table 4-19 illustrates the mean levels of urine albumin, urine creatinine, ACR
and eGFR among diabetic groups (normoalbuminuric, microalbuminuric and
macroalbuminuric group). The results showed markedly increase in the mean levels of
urine albumin and ACR in diabetic groups. Where, the values of 12.801.42,
107.7720.23 and 633.55129.28 mg/l for urine albumin and 13.446.28, 138.5380.53
and 784.31314.32 mg/g for ACR in normoalbuminuric, microalbuminuric and
macroalbuminuric group respectively. The ANOVA test showed statistical significant
difference in the mean level of both urine albumin and ACR among different diabetic
groups with F=838.331, P=0.000 and F=204.073, P=0.000, respectively. The Scheffe
test revealed that there were significantly increased in the mean levels of urine albumin
and ACR from normoalbuminuric to microalbuminuric group and normoalbuminuric to
macroalbuminuric group (all P<0.05). In addition, significant differences were found
between microalbuminuric group and macroalbuminuric group (P<0.05). On the other
hand, the mean levels of both eGFR (eGFR calculated by the MDRD equation and
eGFR calculated by the Hoek equation) gradually decreased with values of

64

168.53112.81, 146.9652.95 and 82.1422.40 ml/min/1.73m2 for eGFR calculated by


the MDRD equation and 140.4945.33, 131.2961.92 and 72.5720.99 ml/min/1.73m2
for eGFR calculated by the Hoek equation in normoalbuminuric, microalbuminuric and
macroalbuminuric group, respectively. The differences in mean levels of eGFR
calculated by the MDRD equation and the Hoek equation were statically significant
(F=5.759, P=0.004 and F=12.491, P=0.000, respectively). Scheffe test showed
significant difference in eGFR calculated by the MDRD equation only between
normoalbuminuric group and macroalbuminuric group (P<0.05). The differences in
eGFR calculated by the Hoek equation were significant between normoalbuminuric
group and macroalbuminuric group and between microalbuminuric group and
macroalbuminuric group (all P<0.05). In contrast, the mean values for urine creatinine
showed decrease with values of 123.8268.80, 102.8549.61 and 97.3144.98 mg/dl in
normoalbuminuric, microalbuminuric and macroalbuminuric group respectively. By
ANOVA test there was non-significant difference between diabetic groups with regard
to urine creatinine (all P>0.05).
Table 4-19 Mean values of urine albumin, urine creatinine, ACR and eGFR among
diabetic groups
Urine
parameter
Albumin
(mg/l)
Creatinine
(mg/dl)
ACR
(mg/g)
eGFR
calculated by the
MDRD equation
(ml/min/1.73m2)

Diabetic nephropathy

Statistics

Normoalbuminuria

Microalbuminuria

Macroalbuminuria

MeanSD

MeanSD

MeanSD

12.801.42

107.7720.23u

123.8268.80

102.8549.61

13.446.28

138.5380.53u

168.53112.81

146.9652.95

82.1422.40u

5.759

0.004

140.4945.33

131.2961.92

72.5720.99uv

12.491

0.000

PValue

633.55129.28uv 838.331 0.000


97.3144.98

1.753

0.179

784.31314.32uv 204.073 0.000

eGFR
calculated by the
Hoek equation
(ml/min/1.73m2)

P value by one way ANOVA, P < 0.05 is statistical significant, u significance in relation to
the normoalbuminuric group, v significance in relation to the microalbuminuric group. ACR,
albumin creatinine ratio; eGFR, estimated glomerular filtration rate; MDRD, Modification of
Diet in Renal Disease.

65

4.8 Association of cystatin C with some parameters in diabetic patients


4.8.1 Serum cystatin C in relation to age, duration of diabetes and BMI.
Table 4-20 shows the relationship of serum cystatin C with age, duration of
diabetes and BMI of cases. Pearson's correlation test showed positive significant
correlation between cystatin C and age or duration of diabetes (r=0.440, P=0.000 and
r=0.372, P=0.000, respectively) (Figure 4-11 a and b). On the other hand, very week
positive correlation was found between cystatin C and BMI, the correlation was nonsignificant (r=0.041, P=0.693).

Table 4-20 Correlation of serum cystatin C with age, duration of diabetes and BMI
Cystatin C (mg/l)
Variable
Pearson's correlation (r)

P-value

Age (years)

0.440

0.000

Duration (years)

0.372

0.000

BMI (kg/m2)

0.041

0.693

(a)

(b)

Figure 4-11 Correlation of serum cystatin C with (a) age (b) duration of diabetes

66

4.8.2 Serum cystatin C in relation to serum glucose, urea and creatinine


Table 4-21 shows the relationship of serum cystatin C with serum glucose, urea
and creatinine of cases. Pearson's correlation test showed very week positive correlation
between cystatin C and glucose levels. This correlation was not significant (r=0.082,
P=0.428). On the other hand, strong positive significant correlation was found between
cystatin C and each of urea and creatinine (r=-0.873, P=0.000 and r=0.892, P=0.000,
respectively) (Figure 4-12 a and b).

Table 4-21 Correlation of serum cystatin C with serum glucose, urea and creatinine
Cystatin C (mg/l)

Serum
Parameters

Pearson's correlation (r)

P-value

Glucose (mg/dl)

0.082

0.428

Urea (mg/dl)

0.873

0.000

Creatinine (mg/dl)

0.892

0.000

(a)

(b)

Figure 4-12 Correlation of serum cystatin C with (a) serum urea (b) serum creatinine

67

4.8.3 Serum cystatin C in relation to lipid profile


Table 4-22 shows the relationship of serum cystatin C with each of lipid profile
(cholesterol, triglycerides, HDL and LDL) of cases. Pearson's correlation test revealed
significant positive correlation between cystatin C and each of cholesterol and LDL
levels (r=0.283, P=0.005 and r=416, P=0.000, respectively). (Figure 4-13 a and b). In
contrast, negative significant correlation between cystatin C and HDL was achieved
(r=-0.645, P=0.000) (Figure 4-13 c). On the other hand, very week positive correlation
but not statically significant was found between cystatin C and triglycerides (r=-0.079,
P=0.444).

Table 4-22 Correlation of serum cystatin C with lipid profile


Cystatin C (mg/l)

Lipid
Profile

Pearson's correlation (r)

P-value

Cholesterol (mg/dl)

0.283

0.005

Triglycerides (mg/dl)

0.079

0.444

HDL (mg/dl)

-0.645

0.000

LDL (mg/dl)

0.416

0.000

(a)

(b)

68

(c)
Figure 4-13 Correlation of serum cystatin C with (a) serum cholesterol (b) LDL (c)
HDL

4.8.4 Serum cystatin C in relation to urine albumin, ACR, creatinine, and


eGFR
Table 4-23 shows the relationship of serum cystatin C with urine albumin, ACR,
urine creatinine and eGFR of cases. Pearson's correlation test revealed relatively strong
positive correlation between cystatin C and urine albumin levels and ACR (r=0.579,
P=0.000 and r=0.769, P=0.000, respectively) (Figure 4-14 a and b). In contrast, strong
negative significant correlation between cystatin C and each of urine creatinine and
eGFR was achieved (r=-0.656, P=0.000 and r=0.595, P=0.000, respectively) (Figure
4-14 c and d).

Table 4-23 Correlation of serum cystatin C with serum urine albumin, ACR, urine
creatinine, and eGFR
Cystatin C (mg/l)

Urine
Parameters

Pearson's correlation (r)

P-value

Albumin (mg/l)

0.579

0.000

Creatinine (mg/dl)

-0.656

0.000

ACR (mg/g)

0.769

0.000

eGFR (ml/min/1.73m2)

-0.595

0.000

69

(a)

(b)

(c)

(d)

Figure 4-14 Correlation of serum cystatin C with (a) urine albumin (b) ACR (c) urine
creatinine (d) eGFR

4.9 Receiver operating characteristic curve analysis for the diagnostic


accuracy of cystatin C and creatinine
Figure 4-15 illustrates the ROC plots to assess the diagnostic efficiency of
serum cystatin C and serum creatinine. ROC curve analysis for all patients included in
the study showed that serum cystatin C had significantly higher diagnostic accuracy
than serum creatinine in diagnosis of DNP. With a cut-off value of ACR at 30 mg/g, the
AUC was 0.7190.056 with P<0.001 for serum cystatin C, and 0.6240.060 with
P<0.038 for serum creatinine (Figure 4-15 a). With a cut-off value of ACR at 300
mg/g, the AUC was 0.9070.034 with P<0.001 for serum cystatin C, and 0.8820.038
with P<0.001 for serum creatinine (Figure 4-15 b)

70

(a)

(b)
Figure 4-15 Nonparametric receiver operating characteristic (ROC) curves of cystatin c
and creatinine for assessment of diabetic nephropathy (a) cut-off value of ACR at 30
mg/g (b) cut-off value of ACR at 300 mg/g

71

Chapter 5
Discussion

72

Chapter 5

Discussion

Diabetes is one of the most common chronic diseases in the young, and is a
substantial cause of morbidity as well as mortality at all ages. It was estimated that the
global number of adults suffering from any form of diabetes would reach 552 million by
2030 [40]. Diabetes increases the risk of premature death mainly due to an increased
risk of cardiovascular events. In addition, people suffering from diabetes have a greater
risk of developing visual problems, nerve disease as well as renal disease.
There is widespread agreement that specific tests are necessary to monitor for
early signs of DNP. Few studies have been carried out on microalbuminuria and other
early markers for diabetic nephropathy among T2DM patients in the Gaza Strip [195197]. In another study, leptin was investigated in different stages of DNP among type 2
diabetic males [198]. In the present study, it is aimed to evaluate the serum cystatin C
levels in a small cohort of patients with T2DM patients by categorizing them into three
groups

depending

on

their

different

degrees

of

ACR

(normoalbuminuria,

microalbuminuria and macroalbuminuria).


Data presented in this study dealt with 95 T2DM patients (52 males and 43
females) as cases and 95 healthy individual (52 males and 43 females) as controls. The
mean age of diabetic males was 49.36.2 years and that for females was 47.27.0 years.
It was reported that T2DM usually develops after age 40 years [199]. This was
confirmed by the mean age at diagnosis; 42.14.9 years for males and 40.25.7 years
for females.
The data from the current study revealed that more than half of patients had
diabetes since 5 years or less and they can develop at least one form of diabetic
complications within this period. This confirmed the idea that T2DM has long
asymptomatic preclinical phase, which frequently goes undetected [200,201]. The
present study showed a significant association between prevalence of complications and
DM. The common complications among the diabetics were retinopathy (28.4%),
neuropathy (15.8%) and CVD (7.4%). A rising trend in the prevalence of diabetic
complications with advancing years of DM was found in this study. Several studies

73

observed a similar upsurge in trend in the prevalence of diabetic complications with


increasing duration of DM [202-204].
The present study showed that the prevalence of microalbuminuria and
macroalbuminuria among diabetic patients was 28.4 and 16.8% respectively. In
previous studies carried out in Gaza Strip on diabetic patients Altibi [195] demonstrated
that 22.2% of patients were microalbuminuric and 22.2% were macroalbuminuric, and
Abu Mustafa [198] showed that 32.6% of patients were microalbuminuric and 14.7%
were macroalbuminuric. Various epidemiological and cross sectional studies have
reported marked variation in the prevalence of microalbuminuria. Wu et al., [205] in
their study of Asian T2DM patients, found the prevalence of microalbuminuria to be
39.8% and a prevalence rate of 18.8% for macroalbuminuria. While Al Maskari et al.,
[206] reported that 61.2% had microalbuminuria and 12.5% had clinical proteinuria
among T2DM patients and Bruno et al., [207] reported the prevalence of
microalbuminuria and macroalbuminuria were 32.1% and 17.6%, respectively. This
variation in prevalence can be attributed to factors such as differences in populations,
diagnostic criteria, duration of disease, stage of the disease, and method of assessment
[208]. Microalbuminuria was more frequent in females (34.9 vs. 23.1%) as compared to
males, which have been observed in other study [209].
There is a strong association between obesity and T2DM, in both genders and all
ethnic groups and obesity is involved in the pathologic process that culminates in the
development of frank T2DM [210]. According to a survey by the Centers for Disease
Control and Prevention, the prevalence of overweight among persons diagnosed with
diabetes was 30.4%, and the prevalence of obesity was 54.8% [211]. In the current
study, 58.9% of patients were obese and 25.3% were overweight, and it showed a
significant association between BMI and DM when compared to controls. This confirms
the fact that obesity is a risk factor for diabetes. However, there were non-significance
differences between mean BMI among normoalbuminuric, microalbuminuric and
macroalbuminuric group. This means that the development of the DNP is not affected
by obesity. Similar conclusions were recorded in studies conducted by Altibi [195] and
Abu Mustafa [198].

74

The association between family history of diabetes and risk for the disease has
been well documented [212,213]. Harrison et al., [213] have indicated that, those who
have a family history of diabetes are two to six times more likely to have T2DM than
people without a family history. In agreement with these studies, the results of the
current study demonstrate that there was a significant association between diabetes and
family history of the disease. Whereas, 52.6% of diabetic patients had a positive family
history for T2DM and 31.6% of health controls had a family history of the disease.
However, there was no association between a history of diabetes and development of
DNP.
The results of the present study showed that serum glucose levels were significantly
higher in diabetic patients than controls. 48.4% of those patients used OHA to manage
diabetes and 25.3% of them were on insulin therapy, whereas 15.0% of patients used
both OHA and insulin and about 10.5% of patients depended on diet only. Palestinian
2005 reports showed that in West Bank (part of Palestinian territories) about 64.7% of
diabetics were managed by tablets and 28.6% were managed by insulin treatment.
Whereas 5.0% of patients were treated with a combined therapy (insulin and OHA), and
1.7% on diet control [41]. The majority of patients, about 81.1%, were checking blood
glucose regularly, and 18.9% of patients were checking blood sporadically. The findings
showed that the difference between the mean of the serum glucose levels among the
three diabetic groups were statistically significant. Whereas, serum glucose was
significantly higher in macroalbuminuric than in normoalbuminuric group, there were
non-significance differences between normoalbuminuric and microalbuminuric group,
and between microalbuminuric and macroalbuminuric group.
Data of the current study showed significant decreases in serum urea and
creatinine concentrations among diabetics compared to controls. These results may be
explained based on glomerular hyperfiltration that may develop at initial stages of the
DNP [214]. This is supported by the observed increase in GFR in diabetic patient
compared to controls. Nelson et al., [215] and Serri et al., [216] reported that GFR
increased in diabetes. Serum urea was significantly increased in micro- and
macroalbuminuria compared to normoalbuminuria. Serum creatinine significantly
increased only in macroalbuminuria when compared to normoalbuminuria or
microalbuminuria. This is a logic result as creatinine may be used as an indicator for the

75

late stages of renal diseases. The change in serum urea and creatinine may be related to
disturbance of kidney function toward the development of DNP. This funding could be
explained by impairment of kidney function and supported by the observed significant
decrease of GFR in micro- and macroalbuminuria.
Diabetes mellitus is often associated with cardiovascular morbidity and this may
partly be explained by the abnormal lipid profile, which is sometimes a feature of DM.
In the present study the total cholesterol, triglycerides, and LDL were significantly
higher in diabetic patients when compared to controls, while HDL was significantly
lower in diabetic patients than in controls. This is in agreement with Suryawanshi et al.,
[217], and Smith et al., [218], who reported that serum total cholesterol, LDL and
triglycerides were significantly raised, whereas the level of HDL was significantly
lower in diabetic subjects as compared to controls. Cross-sectional studies have
suggested that raised lipid levels are involved in the pathogenesis and progression of
renal diseases, and treatment of dyslipidemia can reduce albumin excretion [219]. The
present study revealed that microalbuminuria and macroalbuminuria group had high
cholesterol, triglycerides and LDL, and low HDL than normoalbuminuria group, with
the difference between the three diabetic groups were not statistically significant. This
finding is in concord with Altibi [195] who found no significant differences in lipid
profile between normoalbuminuria and microalbuminuria or macroalbuminuria group.
Jha P et al., [220] have reported significant difference in the mean triglycerides level
between the normoalbuminuric and macroalbuminuric group, but not in cholesterol,
HDL and LDL.
Urine analysis showed significant increase in ACR among diabetic patients
compared to controls. Abu Hilal [197], and Abu Mustafa [198] documented similar
finding. This may be attributed to impairment of kidney filtration efficiency in diabetic
patient. ACR showed marked elevation in micro and macroalbuminuria. On the other
hand, urinary creatinine was significantly decreased in patients compared to controls.
This finding was in agreement with that obtained by Altibi [195], and Abu Mustafa
[198]. The mean values of urinary creatinine showed gradually decrease among three
diabetic groups, but with non-significant difference. The decrease in urinary creatinine
in nephropathy is coincides with its increase in blood as previously mentioned.

76

The

routine

classical

evaluation

of

DNP

includes

appearance

of

microalbuminuria, decreased creatinine clearance and increased serum creatinine [221].


However, it has been reported that a decline in the renal function of patients with
diabetes was not always accompanied by an increased AER [222-227]. To overcome
these limitations, many clinicians additionally used creatinine in evaluating such
patients. However, serum creatinine also depends on creatinine production, extrarenal
elimination and tubular handling [228]. Therefore, other biomarkers for estimation of
renal function have been searched for, and one of them was cystatin C [229].
Cystatin C, a small cationic 13.3 kDa protein, is produced by all nucleated cells
at a constant rate and is freely filtered by the glomerulus. Subsequently it is not secreted
in the tubulus but mainly reabsorbed by tubular epithelial cells and catabolized
completely. Therefore, it is considered an excellent marker of GFR [230]. The results of
current study revealed that the mean serum cystatin C concentrations of diabetics were
not significantly changed compared to that of controls. In diabetic groups, the mean
value of the cystatin C was significantly higher in the macroalbuminuric group
compared to normoalbuminuric group, and it also significantly higher in the
macroalbuminuric group compared to microalbuminuric group. In agreement with these
results, Mojiminiyi et al., [178] found that cystatin C was significantly higher in patients
with DNP than in normoalbuminuric diabetic patients, which can be explained in that
there is already impaired renal function in DNP patients. In addition, Yang et al., [183]
reported that serum cystatin C concentration increased significantly in patients from
normo- to macro- and micro- to macroalbuminuria.
The production of cystatin C has been extensively reported to be independent of
and unaffected by sex, age, height, weight, and muscle mass [16]. However, there is
conflicting evidence regarding whether cystatin C levels vary by gender. Some
investigators found statistically significant differences between genders in adults [231233], with males having higher cystatin C levels than females, whereas others did not
[166,234,235]. In agreement with the majority of previous studies, the results of the
present study showed non-significant difference between males and females in the mean
values of cystatin C. In contrast, there was statistically significant increase in the mean
serum creatinine of males compared to females which may be due to the difference in

77

muscle mass of males and females. Thus, we can confirm that, cystatin C, unlike
creatinine was independent of gender.
In the present study, the significant positive correlation noticed between serum
cystatin C and age indicating that serum cystatin C increased with age. Earlier studies
have also shown positive correlation of serum cystatin C with age of the patients [236,
237]. Several authors noted an age-related rise in cystatin C levels after the 50 years
[166,232, 238], which presumably reflects a decline of kidney function with age. In
addition, cystatin C also had a significant positive correlation with duration of diabetes.
These results are consistent with Hosokawa et al., [237]. In disagreement with these
results, Shafey et al., [184] reported that no correlation was found between cystatin C
and duration of diabetes.

On the other hand, there was non-significant correlation

between cystatin C and BMI, and this is generally consistent with Galteau et al., [239]
who have reported a moderate but biologically insignificant correlation between BMI
and cystatin C. In contrast, Al Wakeel JS et al., [240] and Muntner P et al., [241] have
reported a significant correlation between serum cystatin C and BMI.
The results of the current study revealed significant positive correlation between
serum cystatin C and each of serum urea and creatinine suggesting that serum cystatin C
increased similar to serum urea and creatinine. Similar findings were observed in the
previous study done by Tian et al., [242]. There was non-significant correlation between
cystatin C and glucose level indicating that serum cystatin C levels are independent of
blood sugar level.
In the present study, cystatin C levels showed positive significant correlation
with cholesterol, LDL, and inversely correlated with HDL levels. These results are in
accordance with the study done by Krishna et al., [243]. There was non-significant
correlation between cystatin C and triglycerides.
The results of the current study demonstrated a strong positive statistical
correlation between cystatin C and ACR. In contrast, cystatin C showed a strong inverse
correlation with urine creatinine and eGFR.
Based on ROC plots the AUC for serum cystatin C (0.719) was significantly
greater than that for serum creatinine (0.624) at a cut-off value of ACR at 30 mg/g, and

78

the AUC for serum cystatin C (0.907) was significantly greater than that for serum
creatinine (0.882) at a cut-off value of ACR at 300 mg/g. Therefore, sensitivity and
diagnostic accuracy of cystatin C was better than serum creatinine to detect
nephropathy. Some previous studies on the role of cystatin C in detecting early renal
failure in diabetic patients were contradictory. Some authors showed that cystatin C was
more effective than creatinine in detecting initial reduction of GFR in T2DM as well as
in T1DM. Mussap et al., [180], Xia et al., [182] and Harmoinen et al., [244] showed
that serum cystatin C was more sensitive than serum creatinine for estimation of GFR in
T2DM patients and Tan et al., [170] showed the same in T1DM patients. In addition
Shimizu et al., [181] found that AUC of cystatin C was greater than that of creatinine
and they stated that, serum cystatin C was better than serum creatinine in terms of
sensitivity and specificity for an early prognostic marker of DNP. On the other hand,
Oddoze et al., [179] and Donadio et al., [245] found that sensitivity, specificity, and
positive predictive value for serum cystatin C were not superior to creatinine for
detecting early renal failure in diabetic patients. Keevil et al., [246] and ORiordan et
al., [247] studies findings did not reach a level of significance. Such discrepancies may
be attributable at least in part to intraassay variations for creatinine and cystatin C
measurements related to differences in assay techniques.

79

Chapter 6
Conclusions and
Recommendations

80

Chapter 6

Conclusions and Recommendations

6.1 Conclusions

There was significant association between DM and complications. The main


self-reported

complications

among diabetic

patients

were

retinopathy,

neuropathy and CVD. The longer the duration of diabetes, the higher the
percentage of complications.

Family history, obesity and overweight are risk factor for DM.

More than half of patients had diabetes since 5 years or less.

The majority of patients used OHA to manage diabetes and most of patients
check blood glucose regularly.

Serum urea and creatinine was found to be lower in diabetic patients than in
their non-diabetic counterparts.

People who have T2DM tend to have high levels of cholesterol, triglycerides,
LDL and low HDL levels.

Serum cystatin C and creatinine were significantly higher in macroalbuminuric


patients compared to norm- or microalbuminuric patients.

Lipid profile including, total cholesterol, triglyceride, HDL and LDL did not
affected by the presence of microalbuminuria or macroalbuminuria.

The serum level of cystatin C was positively correlated with age and diabetes
duration whereas sex and body mass index did not affect cystatin C level.

Serum cystatin C had significantly higher diagnostic accuracy in distinguishing


patients with nephropathy than serum creatinine.

81

6.2 Recommendations

As many diabetes complications start before the diagnosis of disease, it is


recommended to screening for diabetes in adults of any age who have risk factors
like obesity, dyslipidemia and first degree family history.

There is a need to improve the diabetic patients' and general populations' awareness
of diabetic complications, risk factors and the importance of lifestyle modifications
in order to early protect themselves from the complications of this disease. As a
result, they will not face future adverse consequences.

Screening for diabetic nephropathy must be initiated at the time of diagnosis in


patients with T2DM and yearly thereafter.

Frequent monitoring of microalbuminuria and urinary ACR to avoid the future


development of diabetic nephropathy.

Serum cystatin C and other biochemical parameters should be measured annually in


patients with diabetes to help for detect the early stages of diabetic nephropathy
patient.

Notification of health authorities on the results of this study, to investigate the


introduction of new laboratory tests for early diagnosis of DNP.

Further research is required to investigate other factors affecting cystatin C across a


broader range of populations and to define the use of both creatinine and cystatin C
in GFR estimation.

82

6.3 Limitation of the study

The study included only outpatients diabetics registered in MOH diabetes clinics,
diabetic patients treated in private sector or in UNRWA are not included, so the
results may not be generalizable to the overall diabetic patients.

Part of the questionnaire was based on self-report, so there was the potential of
recall bias.

Cases and controls may not free from other undiagnosed diseases.

Not probability sample.

83

Chapter 7
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84

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102

Appendices

103

Annex A

104

Annex B

105

Annex C

106

Annex D

................................................:
:

) (

)
(
. .

) (

107

Annex E
Diabetic patient questionnaire.
Questionnaire No

Personal data
Name:

Phone No:

Age:
Weight:

Years
Kg

Sex: Male

Height:

cm

Female
Kg/m2

BMI:

Family history of diabetes

Yes

No

Smoking

Yes

No

Duration of DM:

Years

Blood glucose check

Diagnostic age:

Yes (Regular)

Years

No (Sometime)

Type of medication
Diet

Tablet

Insulin

All

Complications
Retinopathy

Yes

No

Cardiovascular diseases

Yes

No

Neuropathy

Yes

No

Other

Yes

No

108

Annex F
Control individuals questionnaire.
Questionnaire No

Personal data
Name:

Phone No:

Age:
Weight:

Years
Kg

Sex: Male

Height:

cm

Female
Kg/m2

BMI:

Family history of diabetes

Yes

No

Smoking

Yes

No

Retinopathy

Yes

No

Cardiovascular diseases

Yes

No

Neuropathy

Yes

No

Other

Yes

No

Clinical data

109