Professional Documents
Culture Documents
By
Raffat M. El Telbani
Supervisor
Prof. Dr. Baker M. Zabut
2013
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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:
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: 95 ) (
95 ) ( .
.
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:
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: %51.6
. .
.
.
,
V
.
, .
.
.
.
.
. 30/
ROC 0.719 0.624 300
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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
XIV
XVI
Abbreviations .
XVII
Introduction ..
Chapter 2
10
10
12
13
14
15
16
Chapter 1
16
17
19
2.5.3.1 Hyperglycemia .
19
19
20
20
21
21
2.6 Cystatin C ..
24
27
31
32
32
32
32
33
33
33
33
34
34
35
36
37
38
39
40
41
42
Chapter 3
XI
43
44
45
45
Chapter 4
Results .......
46
47
4.1.1 Distribution of the study population by governorates of the Gaza Strip ...
47
47
48
49
50
50
51
51
53
53
54
54
54
55
4.3.3 Serum urea and creatinine among controls and diabetic patients
55
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
58
59
59
XII
60
60
61
61
62
62
63
4.7.5 Urine albumin, urine creatinine and eGFR among diabetic groups ..
64
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
68
69
4.9 Receiver operating characteristic curve analysis for the diagnostic accuracy
..of cystatin C and creatinine ...
70
Chapter 5
Discussion ..
72
Chapter 6
80
81
82
83
Chapter 7
References .
84
Appendices .....
103
XIII
List of tables
Table
Title
Page
Table 2-1
Table 2-2
22
Table 2-3
23
Table 2-4
26
Table 4-1
49
Table 4-2
50
Table 4-3
51
Table 4-4
53
Table 4-5
54
Table 4-6
55
Table 4-7
55
Table 4-8
56
Table 4-9
57
Table 4-10
58
Table 4-11
59
Table 4-12
59
XIV
Table 4-13
60
Table 4-14
61
Table 4-15
62
Table 4-16
62
Table 4-17
63
Table 4-18
64
Table 4-19
65
Table 4-20
66
Table 4-21
67
Table 4-22
68
Table 4-23
69
XV
List of figures
Figure
Title
Page
Figure 2-1
11
Figure 2-2
17
Figure 2-3
23
Figure 2-4
25
Figure 4-1
47
Figure 4-2
Figure 4-3
48
Figure 4-4
49
Figure 4-5
Figure 4-6
52
Figure 4-7
52
Figure 4-8
53
Figure 4-9
54
Figure 4-10
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
ADA
AER
AGEs
Ang II
: Angiotensin II
BMI
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
ESRD
ET-1
: Endothelin-1
G3P
: Glycerol-3-phosphate
GDM
GFR
GK
: Glycerol kinase
GLDH
: Glutamate dehydrogenase
GOD
: Glucose oxidase
GPO
: Glycerol-3- phosphateoxidase
HbA1c
: Glycated hemoglobin A 1 c
HDL
HHS
XVII
HNF
IDDM
IDF
IFG
IGT
IPF
LA
: Lactic acidosis
LDL
LPL
: Lipoprotein lipase
MDRD
MODY
MOH
: Ministry of health
NIDDM
NKF
NO
: Nitric oxide
NPDR
NuroD1
: Neurogenic differentiation 1
OHA
PDR
PEG
: Polyethylenglycol
PKC
: Protein kinase C
POD
: Peroxidase
PVD
ROC
ROS
T1DM
T2DM
UAE
UK
: United Kingdom
US
: United states
UTI
VLDL
WHO
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
To find out the relationship between serum cystatin C and the other studied
parameters.
Chapter 2
Literature Review
Chapter 2
Literature Review
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].
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].
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.
1.
2.
3.
4.
5.
6.
7.
8.
Others
8.
Acromegaly
Cushings syndrome
Glucagonoma
Pheochromocytoma
Hyperthyroidism
Somatostatinoma
Aldosteronoma
Others
1.
2.
3.
4.
5.
1.
2.
3.
4.
5.
Vacor
Pentamidine
Nicotinic acid
Glucocorticoids
Thyroid hormone
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
Infections
1.
Down syndrome
1.
2.
3.
2.
3.
4.
Klinefelter syndrome
Turner syndrome
Wolfram syndrome
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].
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].
11
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
neuropathy
can
be
classified
as
generalized
symmetrical
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].
15
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].
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].
17
18
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].
20
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].
30
30-299
300
Timed collection
(g/min)
30
30-299
300
20
20-199
200
22
Description
GFR (ml/min/1.73m2)
90
60-80
30-59
15-29
Kidney failure
15 or dialysis
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):
-1.154
age (years)
-0.203
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
25
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
ml/min/1.73 m
Diabetic patients
ml/min/1.73 m2
Children
ml/min/1.73 m2
ml/min/1.73 m2
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].
27
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
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
Diabetics who have high blood pressure: more than 130/80 mmHg.
B. Controls
Subjects who have high blood pressure: more than 130/80 mmHg.
32
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).
33
34
(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
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
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
Value
Parameter
Value
Calibrator 2 (mg/l)
0.50
10
180
Calibrator 3 (mg/l)
1.50
505
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
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
POD
Quinoneimine + 4 H2O
Reagents
Reagent
Component
Concentration
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
Value
Parameter
Value
505
Calibrator type
linear
10
2.5
Calibrator 1 (mg/dl)
0.0
Assay point 1
35
250
Calibrator 2 (mg/dl)
100
37
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 NH4+ + 2 HCO3-
GLDH
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
Value
Parameter
Value
340
Calibrator type
linear
10
2.5
Calibrator 1 (mg/dl)
0.0
Assay point 1
21
200
Calibrator 2 (mg/dl)
50
Assay point 2
24
50
38
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
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
Value
Parameter
Value
15
Calibrator type
linear
10
505
Calibrator 1 (mg/dl)
0.0
Assay point 1
20
15
Calibrator 2 (mg/dl)
Assay point 2
26
200
Assay point 3
10
50
39
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
CHOD
Cholesterol-3-one + H2O2
POD
Reagents
Reagent
Component
Concentration
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
Value
Parameter
Value
505
Calibrator type
linear
10
2.5
Calibrator 1 (mg/dl)
0.0
Assay point 1
35
250
Calibrator 2 (mg/dl)
200
40
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
GPO
POD
Quinoneimine + HCl + 4 H
Reagents
Reagent
Reagent
Component
Concentration
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
Value
Parameter
Value
505
Calibrator type
linear
10
2.5
Calibrator 1 (mg/dl)
0.0
Assay point 1
35
250
Calibrator 2 (mg/dl)
200
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)
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
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
Value
Parameter
Value
12
Calibrator 2 (mg/l)
11.4
10
250
Calibrator 3 (mg/l)
20.9
415
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
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)
45
Chapter 4
Results
46
Chapter 4
Results
Figure 4-1 Distribution of the study population by governorates of the Gaza Strip
47
48
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
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
50
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].
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.
52
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.
53
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
54
Statistics
t
PValue
-0.008
0.994
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
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
55
Statistics
t
PValue
3.571
0.000
2.299
0.023
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
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
58
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
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
59
X2
P-Value
1.669
0.434
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.
60
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
concentration
increased
significantly
from
normoalbuminuric
to
61
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.
mean
levels
of
glucose
in
normoalbuminuric,
microalbuminuric
and
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.
62
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
HDL,
and
LDL
among
diabetic
groups
(normoalbuminuric,
and
232.6275.70
mg/dl
for
triglycerides
and
106.4544.42,
63
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
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
1.753
0.179
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
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
Table 4-21 Correlation of serum cystatin C with serum glucose, urea and creatinine
Cystatin C (mg/l)
Serum
Parameters
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
Lipid
Profile
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
Table 4-23 Correlation of serum cystatin C with serum urine albumin, ACR, urine
creatinine, and eGFR
Cystatin C (mg/l)
Urine
Parameters
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
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,
73
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
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.
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
6.1 Conclusions
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.
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.
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.
81
6.2 Recommendations
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.
82
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.
83
Chapter 7
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84
Chapter 7
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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:
Yes
No
Smoking
Yes
No
Duration of DM:
Years
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:
Yes
No
Smoking
Yes
No
Retinopathy
Yes
No
Cardiovascular diseases
Yes
No
Neuropathy
Yes
No
Other
Yes
No
Clinical data
109