The Islamic University of Gaza · The Islamic University of Gaza Deanship of Postgraduate Studies...
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Transcript of The Islamic University of Gaza · The Islamic University of Gaza Deanship of Postgraduate Studies...
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The Islamic University of Gaza
Deanship of Postgraduate Studies
Biological Sciences Master Program
Assessment of serum Vitamin D in Type 1 Diabetic
Patients from Gaza Strip
BY:
Inass Mohammad Fathy Elhamalawi
B.Sc. Medical Technology
Supervisor:
Prof. Dr. Maged M. Yassin
Professor of Physiology
Faculty of Medicine
The Islamic University of Gaza
Submitted in Partial Fulfillment to the Requirements for the Master Degree of
Biological Sciences / Medical Technology
2015 – 1436
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Declaration
I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material 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 or other institute, except where due
acknowledgement has been made in the text.
Signature Name Date
Inass Inass M.F Elhamalawi August, 2015
Copy Right
All rights reserved: No part of this work can be copied, translated or stored in any
retrieval system, without prior permission of the author.
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Dedication
I dedicate this work to:
My beloved parents who have always supporting me
My brothers and sisters who helped me to accomplish this thesis
The dearest to me; my daughter
Rawand and my sons Mohammed, Hazem and Ahmed
All researchers who are working to improve the quality of life
Dedication is almost expressed to the Palestinian people who have
suffered and will be struggling with the persistence to
have a free Palestine.
Inass M.F.Elhamalawi
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Acknowledgment
I would like to express my deepest gratitude and appreciation to my supervisor Prof.
Dr Maged M. Yassin, Professor of Physiology, Faculty of Medicine, The Islamic
University of Gaza for his planning and initiating of this work and for his continuous
support, encouragement and kind of supervision that leads to the emergence of this
work in its current form.
Special thanks for the dearest persons to me my mother, my father and my
beloved brothers and sisters Rami, Mahmood, Hany, Yosri, Heba and Shymaa for
their support and encouragements.
I would like to thank the staff of diabetic Units at El-shifa Hospital, Al-Aqsa Hospital,
Paletinian Medical Relief Center and Al-Nusairat Clinic for their facilitation and
helping me in samples collection .
Special thanks to Basem Medical Laboratory for helping me in biochemical
analysis.
My special thanks to Mr. Abdul Rahman Hamad for his help in statistical analysis.
Also I cannot forget Mrs Nisreen Rayan, Mr Mohammed Yaseen, Mr Hisham
AL-Ashkar, Mr Haytham Weshah, Mr Ayman Qandeel and Mr Alaa jibrel for
their helping.
At the end, I am very grateful to every person who participated and helped me to
complete this study.
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Assessment of serum Vitamin D in Type 1 Diabetic
Patients from Gaza strip
Abstract
Background: Type 1 diabetes usually strikes children and young adults. Although
vitamin D deficiency has been recently linked to diabetes, biochemical tests are
restricted to traditional monitoring of glucose. Therefore, introducing vitamin D test
in Gaza hospitals and its supplementation may help in the management of the disease.
Objective: To assess serum vitamin D level in type 1 diabetic patients from Gaza
Strip
Materials and methods: This case-control study comprised 44 type 1 diabetic
patients (22 males and 22 females) and 44 healthy controls (22 males and 22 females).
Questionnaire interview was applied. Body mass index was determined. Serum
vitamin D, glucose, insulin, cholesterol, triglycerides, high density lipoprotein
cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), Alkaline
phosphatase, calcium and phosphorus were determined. Blood glycated hemoglobin
(HbA1c) was measured. Data were computer analyzed using SPSS version 18.0.
Results: Type 1 diabetes mellitus was more frequent among individuals with family
history of the disease (P
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mg/dl versus 152.9±30.7, 94.0±51.3 and 56.5±24.6, P=0.000, respectively). Serum
alkaline phosphatase (ALP) was also significantly increased in cases (164.2±129.6
versus 116.6±42.5 U/L, P=0.023). Serum calcium was significantly lower in cases
compared to controls (9.04±0.41 versus 9.38±0.56 mg/dl, P=0.024). Vitamin D levels
were found to be lower in individuals who were not doing physical activity (P=0.010).
Serum vitamin D levels showed significant negative correlations with HbA1c (r=-
0.258, P=0.015), insulin (r=-0.257, P=0.016) and LDL-C (r=-0.281, P=0.008), and
significant positive correlation with calcium (r=0.251, P=0.018).
Conclusions: Serum vitamin D was significantly lower in type 1 diabetic patients
compared to controls. Serum vitamin D levels showed significant negative
correlations with HbA1c, insulin and LDL-C, and significant positive correlations
with calcium.
Keywords: Type 1 diabetic patients, Serum vitamin D, Gaza strip.
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االولالدم لمرضى السكري من النوع مصلمستوى فيتامين د فى تقييم
فى
قطاع غزة
ملخص الدراسة
ن أ او قد وجدد مدر ر. طفال و الشبابمراض التي تصيب األمن األول من النوع األ السكري يعد مرض: المقدمة
مقتصدر ال تداال تبدارا الييويدا الكيمياةيدا ان اال بما و, ارتباط وثيق بمرض السكري مرتبط دنقص فيتامين
و امدداد فيتدامين د فدي مستشدفيا دا لدلل،, فدد دال ا تبدار .في الدم يص التقليدي لمستوى الجلوكوزفعلى ال
.لمرضامتابعا و عمليا التيكمقد يساعد في المرضى بفيتامين د
.ول فى قطاع ا أللدى مرضى السكري من النوع ا : تقييم مستوى فيتامين دالهدف
44 علدى تيتدوي المرضديا المجموعا )ضابطا مجموعا - مرضيا مجموعا ) الدراسا : منھجالطرق واالدوات
علدى اليصول تم وقد ,امرأ ( 22 -رجل 22 األصياء ) من شخصا), 44 امرأ 22 -رجل 22 مريض سكر)
قيدا مسدتوى فيتدامين , حساب مرشر كتلدا الجسدموتم ,واألصياء الشخصيا للمرضى المقابلا الل من النتاةج
الكثافدا عدالي الددوني البدروتين الثالثيدا, الددوون ,الكوليسدترول انسدولين, د, مسدتوى الجلوكدوز, مسدتوى األ
مخداون السدكر. قيدا و والفوسدفور, الكالسديومانايم الفوسفاتيا القلوي, , الكثافا الدوني منخفض البروتين,
. SPSS-18 اإلحصاةي البرنامج باستخدام عليھا اليصول تم التي والنتاةج البيانا تيليل وتم
كمدا أهھدر للمدرض أكثر شيوعا بين األفراد اللين لديھم تداري عداةلي االول : مرض السكر من النوع النتائج
باسدتمرار و اكثدر مدن ثلدث فيدص مسدتوى الجلوكدوز لدديھم يتدابعوالدم الياال المرضيا ثلثحوالي النتاةج بأن
أ ر بااقر كما أظهرت النتائج بأن أكثرر مرن ن رل الترالت المر ر حميا لاةيا ايتبعو لم الياال المرضيا
الوحيدد كمدا أهھدر الدراسدا بدأن التعقيددا سناات من عمررم الترال 7مرض السكري منذ أقل اا سااي ب
كدان مدرتين باألنسولينط عدد مرا اليقن النتاةج ان متوس كلل، بينت الناتجا عن المرض وي اعتالل الشبكيا.
في الياال أقل بكثير مقارنا مد الودوابط وودلا كاندت د مستوى فيتامينيوميا لدى ثلثي المرضى. كان متوسط
بشدكل مليدوه فدي األنسدولينذا داللا إحصاةيا, وزاد مستويا نسبا مخداون السدكر والجلوكدوز فدي الددم و
والبروتين الدوني منخفض الدوون الثالثياالكوليسترول و المستويا من وكانتالياال بالمقارنا م الووابط,
أنشدطا وكاندت, وكاندت ودلا النتيجدا ذا داللدا إحصداةيا, مقارندا مد الودوابط اليداال أعلى بكثير فدي الكثافا
فدي أقدل مدن ذلد، بكثيدر الكالسديوم كدان, مقارندا مد الودوابط يداال الأعلى بكثيدر فدي الفوسفاتيا القلويإنايم
داللددا احصدداةيا . كمددا انخفددض مسددتوى فيتددامين د لدددى االفددراد الددلين ال وووددلا ذ مقارنددا مدد الوددوابط اليدداال
يمارسون االنشطا الرياضيا.
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نسدولين, و البدروتين الددوني األ السدكر,مخداون كدل مدن كدان لدا ارتبداط سدلبي مد د الدراسا ان فيتامين ھر كما أه
الكالسيوم.كما وأثبتت الدراسا ان فيتامين د لا ارتباط ايجابي م منخفض الكثافا
ولدوح ان ونداع عالقدا ,السدكر مدن الندوع االول مرضدى لدى انخفاض في مستوى فيتامين د وجدياالستنتاج:
نسدولين, و البدروتين الددوني مدنخفض الكثافدا وعالقدااأل السكر,مخاون من كلم سلبيا بين مستويا فيتامين د
الكالسيوم.ايجابيا م
. ا قطاع ,فيتامين د , سكري النمط االول : المفتاحية الكلمات
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Table of contents
Page Contents
I Declaration
II Dedication
III Acknowledgement
IV Abstract (English)
VI Abstract (Arabic)
VIII Table of Contents
XII List of tables
XIII List of figures
XIV List of Appendices
Chapter 1: Introduction
1 Overview 1.1
2 General objective 1.2
2 Specific objectives 1.3
Chapter 2: Literature Review
3 Diabetes mellitus 2.1
3 Definition of diabetes mellitus 2.1.1
3 Common types of diabetes mellitus 2.1.2
3 Type 1 diabetes (insulin-depended diabetes mellitus) 2.1.2.1
3 Type 2 diabetes (Non-insulin dependent diabetes mellitus) 2.1.2.2
4 Gestational diabetes 2.1.2.3
4 Type 1 diabetes mellitus 2.2
4 Definition and etiology 2.2.1
5 Prevalence and mortality rate of type 1 diabetes 2.2.2
5 Carbohydrates metabolism in type 1 diabetes 2.2.3
6 Lipid metabolism in type 1 diabetes 2.2.4
7 Vitamin D 2.3
7 Definition and structure 2.3.1
7 Sources of Vitamin D 2.3.2
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7 Dietary sources of vitamin D (exogenous vitamin D) 2.3.2.1
8 Photosynthesis of the skin (endogenous vitamin D) 2.3.2.2
9 Mechanism of action of vitamin D 2.3.3
9 Roles of vitamin D 2.3.4
10 Bone health and calcium absorption 2.3.4.1
01 Vitamin D and autoimmune diseases 2.3.4.2
11 Vitamin D and type 2 Diabetes 2.3.4.3
11 Vitamin D and cardiovascular disease 2.3.4.4
01 Vitamin D and cancer 2.3.4.5
01 Related studies 2.4
Chapter 3: Materials and Methods
01 Study design 3.1
01 Study population 3.2
01 Sample size and sampling 3.3
07 Exclusion criteria 3.4
07 Ethical Consideration 3.5
07 Limitation of the study 3.6
07 Data collection 3.7
07 Questionnaire interview 3.7.1
08 Body mass index 3.7.2
08 Specimen collection and processing 3.7.3
08 Biochemical analysis 3.8
08 Determination of serum vitamin D 3.8.1
10 Determination of glycated hemoglobin in whole blood 3.8.2
13 Determination of serum glucose 3.8.3
15 Determination of serum insulin 3.8.4
11 Determination of serum cholesterol 3.8.5
18 Determination of serum triglycerides 3.8.6
31 Determination of serum high density lipoprotein (HDL-C) 3.8.7
31 Determination of serum low density lipoproteins (LDL-C) 3.8.8
31 Determination of serum alkaline phosphates 3.8.9
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33 Determination of serum calcium 3.8.10
31 Determination of serum phosphorus 3.8.11
38 Statistical analysis 3.9
Chapter 4: Results
39 Personal profile of the study population 4.1
31 Socioeconomic data of the study population 4.2
30 Frequent testing of blood glucose level, diet and physical activity among
the study population
4.3
31 Duration of diabetes and self-reported complications among cases 4.4
33 Insulin therapy among cases 4.5
33 Body mass index of the study population 4.6
33 Serum vitamin D levels of the study population 4.7
35 Categories of serum vitamin D levels of the study population 4.8
31 Whole blood HbA1c, serum glucose, and insulin level of the study
Population
4.9
37 Lipid profile of the study population 4.10
38 Alkaline phosphatase enzyme activity of the study population 4.11
39 Serum calcium and phosphorus of the study population 4.12
39 Relations of vitamin D 4.13
39 Vitamin D level in relation to family history of diabetes, diet and physical
Activity
4.13.1
51 Vitamin D level in relation to whole blood HbA1c, serum glucose and
iinsulin levels of the study population
4.13.2
51 Vitamin D levels in relation to lipid profile of the study population 4.13.3
53 Vitamin D levels in relation to ALP of the study population 4.13.4
55 Vitamin D levels in relation to calcium and phosphorus concentrations of
the study population
4.13.5
Chapter 5:Discussion
51 Socioeconomic data of the study population 5.1
51 Frequent testing of blood glucose, diet and physical activity of the study
population
5.2
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57 Duration of diabetes and self-reported complications among cases
5.3
57 Insulin therapy among cases 5.4
58 Serum vitamin D levels of the study population 5.5
59 Whole blood HbA1c, serum glucose, and insulin level of the study
population
5.6
11 Vitamin D in relation to Lipid profile of the study population 5.7
10 Alkaline phosphatase enzyme activity of the study population 5.8
10 Serum calcium and phosphorus of the study population 5.9
Chapter 6:Conclusions and Recommendations
62 Conclusions 6.1
63 Recommendations 6.2
17 CHAPTER 7:REFERENCES
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Page List of tables 40 Personal profile of the study population
Table 4.1
41 Socioeconomic data of the study population
Table 4.2
42 Frequent testing of blood glucose level, diet and physical activity Table 4.3
42 Duration of diabetes and self-reportd complications among cases Table 4.4
33 Insulin injection among cases Table 4.5
44 Body mass index of the study population Table 4.6
33 Serum vitamin D levels of the study population Table 4.7
45 Different categories of serum vitamin D levels of the study population Table 4.8
37 Whole blood glycated hemoglobin (HbA1c), serum glucose and insulin
levels of the study population
Table 4.9
38 Lipid profile of the study population Table4.10
38 Serum alkaline phosphatase activity (ALP) of the study population Table 4.11
39 Serum calcium and phosphorus concentration of the study population Table 4.12
51 Vitamin D levels in relation to family history of diabetes, diet and
physical activity among the study population
Table 4.13
51 Vitamin D levels in relation to whole blood HbA1c,serum glucose and
insulin levels of the study population
Table 4.14
51 Vitamin D levels in relation to lipid profile of the study population Table 4.15
53 Vitamin D levels in relation to (ALP) of the study population Table 4.16
55 vitamin D level in relation to calcium and phosphorus levels of the
study population .
Table 4.17
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Page List of figures
7 Structure of vitamin D Figure 2.1
8 Biosynthesis of vitamin D Figure 2.2
9 Action of vitamin D Figure 2.3
45 Serum vitamin D levels in cases and controls Figure 4.1
31 Different categories of serum vitamin D levels of cases and controls Figure 4.2
51 Serum vitamin D levels in relation to whole blood glycated
hemoglobin HbA1c, serum glucose and insulin levels of the study
population
Figure 4.3
53 Serum vitamin D level in relation to lipid profile of the study
population
Figure 4.4
53 Serum vitamin D level in relation to alkaline phosphatase (ALP)
activity of the study population
Figure 4.5
55 Serum vitamin D level in relation to calcium and phosphorus
concentration of the study population .
Figure 4.6
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List of Appendices
Page Description Appendix
64 Ministry of Health permission letter Annex1
65 Interview questionnaire Annex2
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Chapter 1
Introduction
1.1 Overview
Diabetes mellitus is a condition in which a person has high blood sugar, either
because the body does not produce enough insulin (insulin deficiency), or because
cells do not respond to the insulin that is produced (insulin resistance). This high
blood sugar produces the classical symptoms of diabetes mellitus including polyuria,
polydipsia and polyphagia (American Diabetes Association, ADA, 2010).
Two major types of diabetes were identified; type 1 and type 2. Lack of or
severe reduction in insulin secretion due to autoimmune or viral destructions of β
cells is responsible for type 1 diabetes, which accounts for 5-10% of diabetic patients.
Patients with type 1 diabetes usually required insulin injection (Belle et al., 2011).
The more prevalent form, type 2 diabetes, accounts for more than 90% of cases. Type
2 diabetes usually begins as insulin resistance, a disorder in which the cells do not use
insulin properly. Patients with type 2 diabetes usually required oral hypoglycemic
tablets (ADA, 2013).
Vitamin D is a fat-soluble vitamin that plays an essential role in calcium
homeostasis and the maintenance of normal function in multiple tissues. Humans
obtain vitamin D either directly from the diet or through exposure to solar ultraviolet
B radiation (Holick, 2011 and Kannan and Lim, 2014). In addition to its well-
recognized effects on skeletal health, vitamin D has suggested to have a potential role
in other disease states and health conditions including autoimmune disorders such as
type1 diabetes, cardiovascular disease, type 2 diabetes and cancer (Drake et al.,
2010; Dalgard et al., 2011; Assy et al., 2012; Joergensen et al., 2012 and Grober
et al., 2013).
Numerous global studies have found that patients with type 1 diabetes are
significantly more likely to have a lower serum vitamin D concentration compared
with those without diabetes (Assy et al., 2012 ; Dong et al., 2013 and Daghri et al.,
2014). In this regard, vitamin D supplementation has a protective effect against type 1
diabetes (Zipitis and Akobeng, 2008). In Gaza strip, studies on vitamin D are limited
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and restricted to rickets to investigate its status in nutritional rickets (Yassin and
Lubbad, 2010). Recently, two studies investigate vitamin D status in type 2 diabetes
and CAD (El-henawe, 2014 and Masoud, 2014). However, No previous study
linked vitamin D with type 1 diabetes mellitus. Therefore, the present study is the first
to assess serum vitamin D level in type 1 diabetic patients from Gaza strip.
1.2 General objective
The general objective of the present study is to asses serum vitamin D level in type 1
diabetic patients from Gaza strip .
1.3 Specific objectives
1. To determine vitamin D level in cases compared with controls.
2. To estimate serum glucose and insulin as well as blood HbA1C in cases and
controls.
3. To measure lipid profile including cholesterol, triglycerides, high density
lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-
C) in cases compared to controls.
4. To evaluate serum alkaline phosphatase activity, calcium and phosphorus in
cases compared with controls.
5. To verify the relationship between vitamin D and the studied parameters in type
1 diabetic patients.
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Chapter 2
Literature Review
2.1 Diabetes mellitus
2.1.1 Definition of diabetes mellitus
Diabetes mellitus is a chronic disease that affects the lives of millions around the
world (International Diabetes Federation, IDF, 2006). Diabetes mellitus is defined
as diabetes treated by diet alone or by diet combined with oral hypoglycemic agents
or as treatment with insulin (Tanriverd, 2011). It is a metabolic disorder
characterized by chronic hyperglycemia due to disturbances of carbohydrate, fat
metabolism that are associated with absolute or relative deficiencies in insulin
secretion, insulin action or both. Diabetes mellitus possess a major and growing health
and socioeconomic burden on society, that affects over 177 million people worldwide
and this figure is likely to be more than double by the year 2030 (World Health
Organization, WHO, 2003).
2.1.2 Common types of diabetes
2.1.2.1 Type 1 diabetes (Insulin - depended diabetes mellitus)
Type 1 diabetes develops when the body's immune system destroys pancreatic β cells
resulting in failure of insulin production. This form of diabetes usually strikes
children and young adults, although disease onset can occur at any age. Type 1
diabetes accounts for 5-10% of all diagnosed cases of diabetes (Olefsky, 2001;
Achenbach, 2005 and Belle, 2011).
2.1.2.2 Type 2 diabetes (Non insulin-dependent diabetes mellitus)
Type 2 diabetes results from insulin resistance, a condition in which the body fails to
properly use insulin, combined with relative insulin deficiency (Cnop, 2008). This
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form of diabetes accounts for about 90-95% of all diagnosed cases of diabetes. Type 2
diabetes is associated with older age, obesity, history of gestational diabetes, impaired
glucose metabolism, physical inactivity, and race/ethnicity (Mohan et al., 2007;
Hussain et al., 2010 and Albakr et al., 2013).
2.1.2.3 Gestational diabetes
Gestational diabetes mellitus has been defined as any degree of glucose intolerance
with onset or first recognition during pregnancy. Although most cases resolve with
delivery, the definition applied whether or not the condition persisted after pregnancy
(ADA, 2012). The risk for developing type 2 diabetes within the first decade
following pregnancy in gestational diabetes cases ranges between 35% and 60%
(Seniuk et al., 2009). Similarly, children of women with gestational diabetes are
known to be at risk for obesity and diabetes mellitus in their later life (Bánhidy et al.,
2011).
2.2 Type1 diabetes mellitus
2.2.1 Definition and etiology
Type 1 diabetes develops from a cellular-mediated autoimmune destruction of
pancreatic β cells resulting in insulin deficiency. The immune system incorrectly
manufactures antibodies and inflammatory cells that are directed against and cause
damage to patients' own body tissues. It is believed that the predisposition to develop
these abnormal antibodies in type1 diabetes mellitus is, in part, genetically inherited,
though the details are not fully understood (William et al., 2002; Kantarova and
Buc, 2007; Bluestone et al., 2010 and Zhao et al., 2012). Exposure to certain viral
infections or other environmental toxins may serve to trigger abnormal antibody
responses that cause damage to the pancreatic cells where insulin is made. These
antibodies can be measured in the majority of patients, and may help determine which
individuals are at risk for developing type1 diabetes (Hyoty, 2002; Achenbach et al.,
2005 and Aljabri et al., 2013). In type 1 diabetes, the rate of β-cell destruction is
quite variable, being rapid in some individuals (mainly infants and children) and slow
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in others (mainly adults). Some patients, particularly children and adolescents, may
present with ketoacidosis as the first manifestation of the disease. Others have modest
fasting hyperglycemia that can rapidly change to severe hyperglycemia and/or
ketoacidosis in the presence of infection or other stress (ADA, 2005 and Belle et al.,
2011).
2.2.2 Prevalence and mortality rate of type 1 diabetes
Over 17 new cases of type 1 diabetes were identified per 100 000 children worldwide.
The prevelence rate is particularly high in Finland, Sweden and Norway, with over 25
new cases detected every year per 100 000 children. In Mexico and Korea, the rate is
less than five new cases per 100 000 children. While type 1 diabetes currently
accounts for only 10-15% of all diabetes cases, there is evidence that incidence rates
are rising strongly in some countries. Between 2005 and 2020, new cases of type 1
diabetes for children are expected to double in Europe (Patterson et al., 2009). In
2011, there were almost 660 000 diabetes-related deaths worldwide, and the 2010
global burden of disease study showed that diabetes was the ninth leading cause of
death in the world (International Diabetes Federation, IDF, 2011). In Palestine,
there is under diagnosis and under reporting of diabetes milletus. This is due to lack of
proper hospital and clinic recording system (Ministry of Health, MOH, 2005). In
2011, the total number of new reported cases of diabetes mellitus in West Bank was
3984 with incidence rate 154.4 per 100,000 of population (MOH, 2012). The
mortality rate of diabetes mellitus among Palestinians constituted 5.9 per 100,000
population in the year 2009 (MOH, 2009), and this figure raised to 8.5 per 100,000
population in the year 2010 (MOH, 2010).
2.2.3 Carbohydrate metabolism in type 1 diabetes
Uncontrolled type 1 diabetes leads to increased hepatic glucose output. First, liver
glycogen stores are mobilized then hepatic gluconeogenesis is used to produce
glucose. Insulin deficiency also impairs non hepatic tissue utilization of glucose. In
particular in adipose tissue and skeletal muscle, insulin stimulates glucose uptake.
This is accomplished by insulin mediated movement of glucose transporters proteins
to the plasma membrane of these tissues. Reduced glucose uptake by peripheral
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tissues in turn leads to a reduced rate of glucose metabolism. In addition, the level of
hepatic glucokinase is regulated by insulin. Therefore, a reduced rate of glucose
phosphorylation in hepatocytes leads to increased delivery to the blood. The
combination of increased hepatic glucose production and reduced peripheral tissues
metabolism leads to elevated plasma glucose levels (Ozougwu et al., 2013). When
the capacity of the kidneys to absorb glucose is surpressed, glucosuria ensues.
Glucose is an osmotic diuretic and an increase in renal loss of glucose is accompanied
by loss of water and electrolyte. The result of the loss of water leads to the activation
of the thirst mechanism (polydipsia). The negative caloric balance, which results from
the glucosuria and tissue catabolism leads to an increase in appetite and food intake
that is polyphagia (Raju and Raju, 2010).
2.2.4 Lipid metabolism in type 1 diabetes
In uncontrolled type 1 diabetes where insulin is lacked there is a rapid mobilization of
triglycerides leading to increased levels of plasma free fatty acids. The free fatty acids
are taken up by numerous tissue (except the brain) and metabolized to provide energy.
In the absence of insulin, malonyl COA levels fall, and transport of fatty acyl-COA
into the mitochondria increases. Mitochondrial oxidation of fatty acids generates
acetyl COA that can be further oxidized in the tricarboxylic acid cycle (TCA cycle).
However, in heaptocytes the majority of the acetyl COA is not oxidized by the TCA
cycle but is metabolized into the ketone bodies (acetoacetate and β-hydroxybutyrate).
These ketone bodies are used for energy production by the brain, heart and skeletal
muscle. In type 1 diabetes, the increased availability of free fatty acids and ketone
bodies exacerbates the reduced utilization of glucose, furthering the ensuing
hyperglycaemia. Production of ketone bodies in excess of the body’s ability to utilize
them leads to ketoacidosis (Vergès, 2011). A spontaneous breakdown product of
acetoacetate is the acetone that is exhaled by the lungs, which gives a distinctive odor
to the breath. Normally, plasma triglycerides are acted upon by lipoprotein lipase that
requires insulin. lipoprotein lipase is a membrane bound enzyme on the surface of the
endothelial cells lining the vessels, which allows fatty acids to be taken from
circulating triglycerides for storage in adipocytes. Therefore, the absence of insulin
results in hypertriglyceridemia. (Raju and Raju, 2010).
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2.3 Vitamin D
2.3.1 Definition and structure
Vitamin D is a seco-steroid hormone and it is critically important for the
development, growth and maintenance of a healthy skeleton from birth until death
(Andersen et al., 2008). Vitamin D has other roles in human health; it can play a role
in decreasing the risk of many chronic illnesses, including cardiovascular disease,
diabetes, autoimmune diseases, infectious diseases, and cancer (Zhang and
Naughton, 2010 and Grober et al ., 2013). The molecular structure of vitamin D is
closely allied to that of classic steroid hormones in that it has the same root
cyclopentanoperhydrophenanthrene ring structure (Figure 2.1, Wang, 2013).
Figure 2.1. Structure of vitamin D (Wang , 2013).
2.3.2 Sources of vitamin D
Vitamin D is obtained either directly from the diet or by means of photosynthesis in
the skin.
2.3.2.1 Dietary sources of vitamin D (exogenous vitamin D)
Fish, liver oils, fatty saltwater fish, dietary products and eggs all contain vitamin D. It
is also found in butter, cod liver oil, dandelion green, egg yolks, halibut, liver, milk,
oatmeal, salmon, sardines, sweat potatoes, tuna and vegetables (Balch, 2001;
Palomer et al., 2008 and Holick, 2011).
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8
2.3.2.2 Photosynthesis in the skin (endogenous vitamin D)
The solar ultra violet B radiation penetrates the skin to convert 7-hydrocholestorol to
previtamin D3, which is thermodynamically unstable and undergoes thermally
induced conversion to vitamin D3 (Figure 2.2). Whatever the source, vitamin D3 must
be hydroxylated twice to produce the biologically active form. Thus, the first
hydroxylation process takes place in the liver and forms 25-hydroxyvitamin D3 and is
catalysed by vitamin D-25-hydroxylase. The second hydroxylation step, which
produces the final active metabolite of vitamin D3 (1,25(OH)2D3), is mediated by 25-
hydroxyvitamin D3 1-hydroxylase and occurs predominantly in the kidney. Then,
1,25(OH)2D3 is released into the circulation where it binds to vitamin D-binding
protein until it reaches its target tissue by means of the vitamin D receptors (Chagas
et al., 2012 and Kannan and Lim, 2014).
Figure 2.2. Biosynthesis of vitamin D. UVB: Ultra Violet B, 25(OH)D3: 25-
hydroxyvitamin D3, 25-OHase: Vitamin D-25-hydroxylase, 1-OHase: 25-
hydroxyvitamin D3 1-hydroxylase, DBP: Vitamin D-binding protein (Palomer et
al., 2008).
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9
2.3.3 Mechanism of action of vitamin D
Vitamin is bound to vitamin D-binding protein in circulation, crosses the cell
membrane, and binds to vitamin D receptor (Figure 2.3). The conjugated vitamin D
with its receptor forms a heterodimer complex with retinoid X receptor and with other
factors, attaches to vitamin D–responsive elements on deoxyribonucleic acid, and
alters gene expression. It has been estimated that vitamin D regulates more than 200
genes, directly or indirectly, thereby influencing a wide variety of physiological
processes (Vanga et al., 2010; Stivelman and Retnakaran, 2012 and Wang,
2013).
Figure 2.3. Action of vitamin D. DBP: Vitamin D-binding protein, VDR: Vitamin D
receptor, RXR: Retinoid X receptor, RNA: Ribonucleic acid (Vanga et al. 2010).
2.3.4 Roles of vitamin D
Vitamin D has several roles in the body; many of these arise from its action on gene
transcription and expression. In addition to the well-recognized effects of vitamin D
on skeletal health, emerging evidence suggests a potential role for vitamin D in
numerous other disease states and health conditions including autoimmune disease
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11
such as type 1 diabetes, type 2 diabetes, cardiovascular disease and cancer ( Kannan
and Lim, 2014).
2.3.4.1 Bone health and calcium absorption
Vitamin D facilitates calcium absorption in the intestine by influencing the expression
of epithelial calcium channels and thus calcium-binding proteins. This process allows
calcium to be better absorbed from the foods eaten (Holick, 2005). Due to the
increase in absorption of calcium, parathyroid hormone (PTH) levels are better
regulated. When serum calcium levels are low, the parathyroid gland secretes PTH,
which leads to increased production of vitamin D. This further increases absorption of
calcium from the intestine as well as increases reabsorption of calcium by the kidneys.
Increased PTH leads to resorption of calcium from the bone. Leaching calcium out of
the matrix of bone leads to decreased bone strength. If adequate vitamin D is present
before this occurs, PTH levels are likely to be kept low as calcium absorption is
increased (Wacker and Holick, 2013). Another way in which vitamin D works to
increase bone strength is by mediating the incorporation of calcium into the matrix of
bone (Reese, 2006).
2.3.4.2 Vitamin D and autoimmune diseases
Various epidemiological studies suggested associations between vitamin D deficiency
and a higher incidence of autoimmune diseases, such as type 1 diabeties, multiple
sclerosis, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel
disease (Grober et al., 2013 and Sabbagh et al., 2013). Vitamin D receptors are
present in many cell types including various immune cells such as antigen-presenting-
cells, T cells, B cells and monocytes (Prietl et al., 2013). Focusing on type 1 diabetes
mellitus, meta-analysis of data from the case–control studies showed that the risk of
type 1 diabetes was significantly reduced in children who were supplemented with
vitamin D compared to those who were not supplemented (Zipitis and Akobeng,
2008 and Dong et al., 2013). There was also some evidence of a dose–response
effect, with those using higher amounts of vitamin D being at lower risk of
developing type 1 diabetes mellitus (Antico et al., 2012). Retrospective analysis and
observational studies demonstrated high prevalence of vitamin D deficiency in
patients with type 1 diabetes mellitus (Bener et al., 2009, Janner et al., 2010 and
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11
Greer et al., 2012). In addition, a cross-sectional study from Switzerland reported 60
to 84% of type 1 diabetics to be vitamin D deficient (Janner et al., 2010).
2.3.4.3 Vitamin D and type 2 Diabetes
Recent cross-sectional and prospective studies have reported a significant inverse
association between serum vitamin D and the presence of type 2 diabetes (Pittas et
al., 2010; Dalgard et al., 2011; Gonzalez-Molero et al., 2012 and Djalali et al.,
2013). In addition, case-control studies found that patients with type 2 diabetes or
impaired glucose tolerance are significantly more likely to have a lower serum
vitamin D level compared to those without diabetes (Targher et al., 2006 and
Gorham et al., 2012). Furthermore, Tracy and Mazen, (2010) and Afsaneh et al.,
(2013) reported a significant inverse association of serum vitamin D with insulin
resistance. A positive association between vitamin D and β-cell function was also
found (Palomer et al., 2008; Ozfirat and Chowdhury, 2010 and Takiishi et al
2012).
2.3.4.4 Vitamin D and cardiovascular disease
A growing body of evidence suggests a possible association between vitamin D
deficiency and many cardiovascular disorders, including hypertension, peripheral
vascular disease, coronary artery disease and heart failure. Vitamin D receptors are
located on vascular smooth muscle, endothelium, and cardiomyocytes (Wang et al.,
2008). One of the main mechanisms whereby vitamin D appears to decrease
cardiovascular disease risk is its effect on hypertension through the rennin-angiotensin
system (Vaidya and Forman., 2011 and Kienreich et al., 2013). Vitamin D
deficiency is also strongly associated with increased thickness of the intima-media in
carotid arteries (Targher et al., 2006 and Joergensen et al., 2012).
2.3.4.5 Vitamin D and cancer
An inverse association between vitamin D and the incidence of several cancers and
mortality from these cancers has been shown in case-control studies, prospective and
retrospective studies (Freedman et al., 2007; Drake et al., 2010; Manson et al.,
2011 and Robsahm et al., 2013) and especially for cancers of the colon and breast
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12
(Fleet et al., 2012). In addition, vitamin D promotes various apoptotic mechanism and
cell differentiation, and suppresses angiogenesis and tumor invasion and metastasis
(Deeb et al., 2007; Krishnan and Feldman, 2011; Hossein et al., 2013 and Gröber,
2014).
2.4 Related studies
Zipitis and Akobeng (2008) assessed whether vitamin D supplementation in infancy
reduces the risk of type 1 diabetes in later life. The study was a systematic review and
meta-analysis using Medline, Embase, Cinahl, Cochrane Central Register of
Controlled Trials and reference lists of retrieved articles. Meta-analysis of data from
the case-control studies showed that the risk of type 1 diabetes was significantly
reduced in infants who were supplemented with vitamin D compared to those who
were not supplemented (pooled odds ratio 0.71, 95% CI 0.60 to 0.84). The result of
the cohort study was in agreement with that of the meta-analysis. There was also
some evidence of a dose-response effect, with those using higher amounts of vitamin
D being at lower risk of developing type 1 diabetes.
Svoren et al. (2009) conducted a cross-sectional study to assess vitamin D status in
128 youth with type 1 diabetes from the northeastern United States and to examine the
influence of specific patient and disease characteristics on vitamin D status. Less than
25% of type 1 diabetic patients were vitamin D sufficient. Given that individuals with
type 1 diabetes possess multiple risk factors for skeletal fragility, ensuring vitamin D
sufficiency throughout childhood and adolescence in this population seems especially
warranted.
In a prospective cross-sectional study, Janner et al. (2010) measured serum vitamin
D, intact parathyroid hormon, total and ionised calcium, phosphate and alkaline
phosphatase in 129 Swiss children and adolescents with type 1 diabetes. Of the 129
subjects 78 (60.5%) were vitamin D deficient, defined as a vitamin D level below 50
nmol/L. During the winter this number rose to 84.1%. Vitamin D levels showed
marked seasonal fluctuations, whereas there was no correlation with diabetes control.
Despite the high prevalence of vitamin D deficiency, the authors found a low
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13
prevalence of secondary hyperparathyroidism in vitamin D deficient diabetic children
and adolescents ( Dong et al., 2013).
Bin-Abbas et al. (2011) assessed the prevalence of vitamin D deficiency in type 1
diabetic children. The study was prospective cross-sectional included 100 Saudi
children with type 1 diabetes and 100 healthy controls. The mean levels of vitamin D
were significantly lower in the type 1 diabetic children compared to the controls
(36.7±14.3 nmol/l versus 44.8±14.1 nmol/l). In the type 1 diabetic children, 64% were
mildly, 16% were moderately, and 4% were severely vitamin D deficient as compared
with 52% (mildly), 6% (moderately), and 1% (severely) in the control group. Overall,
84% of the type 1 diabetic children, and 59% of the healthy children were vitamin D
deficient. There was no correlation between glycemic control and vitamin D level. In
addition, Devaraj et al. (2011) found that type 1 diabetic patients with and without
microvascular complications from the Diabetes and Pediatric Clinics at University of
California Davis Medical Center were significantly vitamin D deficient compared
with control subjects (P0.05). The association
between vitamin D status groups and HbA1c was significant (P
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14
The prevalence of vitamin D deficiency was assessed in a group of children and
adolescent patients with recent-onset type 1 diabetes mellitus (Ataie-Jafari et al.,
2012). Fifty-three patients with age 8-18 years and duration of type 1 diabetes less
than 8 weeks were recruited. A food frequency questionnaire was used to assess
dietary vitamin D and calcium intake. Sunshine exposure was measured using a
questionnaire to quantify the amount of time children spent in the sun and other sun-
related habits, and a sun index score was generated. Serum vitamin D
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15
years versus 36.7±3.6 years among the controls. Both the type 1 diabetic and healthy
groups had vitamin D deficiency. The mean levels of vitamin D were significantly
lower in the type 1 diabetic adults than in the controls (28.1±1.4 nmol/L versus
33.4±1.6 nmol/L). In the type 1 diabetic adults, 66.7% were mildly, 31.7%
moderately, and 3.3% severely vitamin D deficient as compared with 41.7% (mildly),
31.7% (moderately), and 5% (severely) in the control group. Overall, 100% of the
type 1 diabetic adults and 78% of the healthy children were vitamin D deficient. In
addition, Soliman et al. (2015) found that type 1 diabetic children had significantly
lower serum vitamin D compared to controls.
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16
Chapter 3
Materials and Methods
3.1 Study design
The present study is a case control-study. Case-control studies are often used to
identify factors that may contribute to a medical condition by comparing subjects who
have that condition/disease (the "cases") with subjects who do not have the
condition/disease but are otherwise similar (the "controls"). Case-control studies are
quick, widely used, relatively inexpensive to implement, require comparatively fewer
subjects, and allow for multiple exposures or risk factors to be assessed for one
outcome (Mann, 2003 and Song and Chung, 2010).
3.2 Study population
The study population comprised type 1 diabetic patients aged 18-35 years
attending various diabetic Units at El-shifa Hospital, Al-Aqsa Hospital, Palestinian
Medical Relief Center and Al-Nusairat Clinic in Gaza strip. Control group was non-
diabetic apparently healthy persons.
3.3. Sample size and sampling
Non probability accidental sample of type 1 diabetic patients, previously diagnosed
according to the World Health Organization diagnostic criteria for diabetes (WHO,
2006), were selected as cases from Diabetic Units at El-shifa Hospital, Al-Aqsa
Hospital, Palestinian Medical Relief Center and Al-Nusairat Clinic in Gaza strip.
Controls were apparently healthy non diabetic individuals selected from the general
population. Cases and controls were age and gender matched. The sample size
calculations were based on the formula for case-control studies. EPI-INFO statistical
package version 3.5.1 was used with 95% CI, 80% power and 50% proportion as
conservative and OR > 2. The sample size in case of 1:1 ratio of case control was
found to be 41:41. For a no-response expectation, the sample size was increased to 44
patients. The controls also consisted of 44 non diabetic individuals.
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17
3.4 Exclusion criteria
Cases and controls whose aged under 18 years and above 35 years old.
Type 2 diabetic patients.
Pregnant women.
Patients with other chronic diseases.
Patients who take hormone replacement therapy or corticosteroid
therapy.
3.5 Ethical Consideration
An official letter of request was sent from the Palestinian Ministry of Health to
Medical Center Administrations in different Gaza hospitals to facilitate the
conduction of the study (Annex 1). The participants were given a full explanation
about the purpose of the study and assurance about the confidentiality of the
information and the participation was optional.
3.6 Limitation of the study
1. The number of patients who frequently visiting the diabetic clinics was relatively
low.
2. Many patients refused to supply blood sample.
3.7 Data collection
3.7.1 Questionnaire interview
A meeting interview was used for filling in a questionnaire which designed for
matching the study need for both cases and controls (Annex 2). All interviews were
conducted face to face by the researcher herself. During the survey the interviewer
explained any of the questions that were not clear. The questionnaire was based on
recent studies conducted on diabetes melitus in Gaza Strip with some modifications
(EL-Qreenawy, 2013). Most questions were the yes/no questions which offer a
dichotomous choice (Backestrom and Hursh-Cesar, 2012). The validity of the
questionnaire was tested by six specialists in the fields of endocrinology,
epidemiology, public health and biochemistry. The questionnaire was piloted with 8
patients not included in the study. The questionnaire included questions on the
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18
personal profile of the study population (Age, gender, marital status and education),
socioeconomic data (employment, family income/month and family history of
diabetes), frequent testing of blood glucose level, diet and physical activity, clinical
data (duration of diabetes and self-reported complications) among the study
population and insulin injection among the cases (frequency of insulin injection/day
and insulin dose).
3.7.2 Body mass index
Body mass index was calculated as the ratio of body weight in Kg/height in square
meter. Patients were asked to remove heavy clothes and shoes before measurement of
weight and height. Medical balance (Seca Model 762, Germany) was used for weight
measurement. People with BMI=18.5-24.9 were considered to have normal weight,
people with BMI=25.0-29.9 were classified overweight and people with BMI≥30.0
were considered obese (WHO, 2012).
3.7.3 Specimen collection and processing
Twelve hours fasting overnight venous blood samples were collected from 44 type 1
diabetic patients and 44 healthy non diabetic controls. Blood samples (8 ml each)
were drawn by the researcher herself into vacutainer and plastic tubes from each
control and diabetic patients. About 2 ml blood was placed into ethylene diamine tetra
acetic acid (EDTA) vacutainer tube to perform HbA1c for cases and controls. The
remainder quantity of blood (6 ml) was placed in plastic tube and was left for a while
without anticoagulant to allow blood to clot. Serum samples were obtained by
centrifugation at 3000 rpm for 10 minutes for determination of vitamin D, glucose,
insulin, cholesterol, triglycerides, HDL-C, LDL-C, ALP, calcium and phosphorus.
3.8 Biochemical analysis
3.8.1 Determination of serum vitamin D
25-hydroxy (25-OH) Vitamin D enzyme linked immunoassay (ELISA) is designed by
Calbiotech, Inc for the quantitation of total 25-OH Vitamin D in human serum and
plasma (Bikle, 2010).
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19
Principle
The kit is a solid phase (ELISA), based on the principal of competitive binding. Anti-
Vitamin D antibody coated wells are incubated with Vitamin D standards, controls,
samples, and Vitamin D-Biotin conjugate at room temperature for 90 minutes. During
the incubation, a fixed amount of biotin-labeled vitamin D competes with the
endogenous Vitamin D in the sample, standard, or quality control serum for a fixed
number of binding sites on the anti-Vitamin D antibody. Following a wash step,
bound Vitamin D-Biotin is detected with Streptavidin-HRP. Streptavidin-HRP
conjugate immunologically bound to the well progressively decreases as the
concentration of Vitamin D in the specimen increases. Unbound SA-HRP conjugate is
then removed and the wells are washed. Next, a solution of TMB Reagent is added
and incubated at room temperature for 30 minutes, resulting in the development of
blue color. The color development is stopped with the addition of stop solution, and
the absorbance is measured spectrophotometrically at 450 nm. A standard curve is
obtained by plotting the concentration of the standard versus the absorbance. The
color intensity will be inversely proportional the amount of 25 (OH)D in the sample.
The assay measures both Vitamin D2 and D3. The total assay procedure run time is
2.5 hours.
Composition of reagents
Materials provided 96 Tests
Microwell plate coated with anti-Vitamin D 12x8x1
Vitamin D Standard Set: 7 vials (ready to use) 0.25 ml
Vitamin D Control Set: 2 vials (ready to use) 0.25 ml
Biotinylated 25 (OH)D Reagent: 1 Vial (51X) 0.5 ml
Assay Diluent, 1 bottle 24 ml
Streptavidin-HRP, 1 bottle (ready to use) 23 Ml
Stop Solution, 1 bottle (ready to use) 12 mL
TMB Substrate, 2 bottles (ready to use) 2 x 12 ml
Microplate sealing film 2
Wash Concentrate 20X, 1 bottle 25 ml
Preparation of reagent
Before running the test, prepare the following:
1. Standards and Reagents:
Standards are serum-based solutions and stable when stored at 2-8°C,
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21
protected from light, until the expiration date on the label. Equilibrate the needed
volume of standards and reagents to room temperature before use.
2. 51X Biotin conjugate: Immediately before use, prepare 1X working solution at 1:51
with assay diluent (e.g. Add 0.1ml of the 50x Vitamin D-Biotin conjugate concentrate
to 5ml of assay diluent). Remaining Assay Diluent must be stored at 2-8°C in dark
and tightly capped.
3. Prepare 1X Wash Buffer by adding the contents of the bottle (25 ml, 20X) to 475
ml of distilled or deionized water. Store at room temperature (18-24 °C).
Analytical procedure
All reagents and specimens must be allowed to come to room temperature before use.
All reagents must be GENTLY mixed without foaming. Once the procedure has
started, all steps should be completed without interruption.
1. Dispense 10l of 25(OH)D Standards, controls and samples into each well, as
required.
2. 200l working solution of biotinylated 25 (OH) D reagent, was dispensed into
each well.
3. The contents was carefully mixed in the wells for 20 seconds using a plate shaker at
200-400 RPM (or equivalent motion). the plate was removed from shaker and covered
with the adhesive plate seal making sure there is a complete seal over each well.
4. Incubation #1 –sealed plate was Incubated for 90 minutes at room temperature.
5. the plate seal was carefully removed.
6. the contents was shaked out of the wells into a waste reservoir.
7. Wash # 1- 300l of 1X Wash Buffer was dispensed into each well, and then briskly
the 1X Wash Buffer was shaked out into a waste reservoir. the wells was striked
sharply on absorbent paper to remove residual droplets. It was repeated 2 more times
for a total of 3 washes.
8. 200l of enzyme conjugate (Streptavidin-HRP) was dispensed into each well.
9. Incubation #2 – It was incubated for 30 minutes, at room temperature.
10. Briskly the contents was shaked out of the wells into a waste reservoir.
11. Wash # 2 - 300l of 1X Wash Buffer was dispensed into each well, and then
briskly the 1X Wash Buffer was shaked out into a waste reservoir. the wells was
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21
strike sharply on absorbent paper to remove residual droplets. It was repeated 2 more
times for a total of 3 washes.
12. a multi-channel pipette was used, 200 l of TMB Substrate was dispensed into
each well.
13. Incubation #3 – It was incubated for 30 minutes at room temperature, preferably
in the dark.
14. Stop - 50 l of Stop Solution was dispensed into each well to stop the enzymatic
reaction. Carefully plate contents was mixed for 20 - 30 seconds.
15. absorbance was read on ELISA Reader at 450 nm within 10 minutes of adding the
Stop Solution.
Reference values
Deficient (30 ng/dl)
3.8.2 Determination of glycated hemoglobin in whole blood
Glycated hemoglobin was determined by the colorimetric determination of glycated
hemoglobin in whole blood using Stanbio Kit, Texas-USA (Trivelli et al., 1971).
Principle
A preparation of hemolyzed whole blood is mixed with a weakly binding cation
exchange resin. The non-glycosylated hemoglobin (HbA0) binds to the resin, leaving
HbA1c free to be removed by means of a resin separator in the supernate. The percent
of HbA1c is determined by measuring the absorbance values at 415 nm of the HbA1c
fraction and of the total Hemoglobin fraction, calculating the ratio of absorbance's
(R), and comparing this ratio to that of a HbA1c standard carried through the same
procedure. Results are expressed as HbA, but can be converted or derived as HbA1c
by using a conversion factor or when using HbA1c value for the standard.
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22
Reagents
Glycated hemoglobin lon Exchange Resin. Each tube contains 3.0 mL cation exchange
resin 8 mg/dL. pH 6.9
Glycated hemoglobin Lysing Reagent
Contains potassium cyanide 10 mmol/L and surfactants.
Glycated hemoglobin Standard (Lyophilized) (1 vial)
Prepared from packed human erythrocytes.
Procedure
Hemolysate Preparation
1. 500 µl Lysing reagent was pipetted into tubes labeled Standard (S),Unknown (U)
and Control (C).
2. 100 µl of each well-mixed blood sample was pipetted into appropriately labeled
tube and mix.
3. It was allowed to stand for 5 minutes at room temperature (15-30°C) to complete
hemolysis.
Glycated hemoglobin separation and assay
1. resin tubes Standard (S), Unknown (U) and Control (C) was labeled.
2. I00 µl of the prepared hemolysate was pipetted into appropriately labeled resin
tube.
3. a resin separator was positioned in the tube so rubber sleeve is approximately 1-2
cm above liquid level.
4. Tubes mixed on a hematology rocker for 5 minutes. Alternatively tubes may be
mixed by hand if held above the resin.
5. At the end of the 5 minute mixing, resin separator was pushed into tube until resin
is firmly packed in bottom of the 13mm tube.
6. Each supernate was poured directly into separate cuvettes for absorbance
measurements.
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23
7. absorbance (Agly) of Standard was read, Unknown and Control vs. water at 415
nm within 60 minutes.
Total hemoglobin assay
1. 5.0 mL deionized water was pipetted into tubes labeled Standard (S), Unknown (U)
and Control (C).
2. 20 µl of hemolysate was pipetted into appropriately labelled tube.
It was mixed well and transfer to cuvette for absorbance reading.
3. absorbance (Atot) of Standard, Unknown and Control vs. water at 415nm within 60
minutes was read.
Calculation
For each Standard and Unknown calculate the ratio (R) of the glycated hemoglobin
absorbance to the hemoglobin absorbance as follows:
(R) = Agly / Atot
Hemoglobin (%) = (R) Unknown x Hemoglobin Standard (%)
(R) Standard
Results may also be reported as HbA1c when compared to the reference A1c method,
the Stanbio method showed a 98% correlation with an equation of:
Y (A1c value) = 0.838 x (Stanbio value) - 0.732
The value obtained by the Stanbio method may be converted to Calculated A1c value
by use of this formula. For a direct calculated A1c value, the value of the standard
may be changed to 7.6% in lieu of the 10.0% and the results will be A1c values.
3.8.3 Determination of serum glucose
Serum glucose is determined by glucose-oxidase procedure (Trinder, 1969) using
Dialab reagent kits.
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24
Principle
Determination of glucose after enzymatic oxidation by glucose oxidase. The
colorimetric indicator is quinoneimine, which is generated from 4-aminoantipyrine
and phenol by hydrogen peroxide under the catalytic action of peroxidase.
Reagents
Glucose + O2 → Gluconic acid + H2O2
2 H2O2 + 4-Aminoantipyrine + Phenol → Quinoneimine + 4 H2O
Concentration Reagent
250 mmol/l
5 mmol/l
0.5 mmol/l
≥ 15 ku/l
≥ 1 ku/l
Phosphate buffer (pH 7.5)
Phenol
4-Aminoantipyrine
Glucose oxidase (GOD)
Peroxidase (POD)
100 mg/dl Standard
Assay procedure
Wavelength: 500 nm
Optical path: 1cm
Measurement: Against reagent blank.
10 µl of standard (sample or control) was added to 1ml of the reagent and
mixed well.
The mixture was incubated for 10 min at 37 ºC.
The absorbance was measured within 60 min.
GO
D
POD
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25
Calculation
Glucose [mg / dl] = ∆A sample X concentration of standard
Reference value (fasting glucose)
(Palestinian Clinical Laboratory Tests Guide, PCLTG, 2005)
60 – 100 mg/dl Child
70 – 110 mg/dl Adult
3.8.4 Determination of serum insulin
Serum insulin was determined by microparticle enzyme immunoassay (MEIA), using
Abbott IMx Insulin assay, following the instruction manual (Travis, 1980 and
National Committee for Clinical Laboratory Standards, 2001).
Biological principles of the procedure
The IMx insulin assay was used. It is based on the MEIA technology. The IMx insulin
reagents and sample were added to the reaction cell in the following sequence:
1. The probe/electrode assembly delivers the sample, anti-insulin (mouse,
monoclonal) coated microparticles and the assay buffer to the incubation well of
the reaction cell forming an antibody-insulin complex.
2. An aliquot of the reaction mixture containing insulin bound to the anti-insulin
coated microparticles was transferred to the glass fiber matrix.
3. The matrix was washed to remove unbound materials.
4. The anti-insulin: alkaline phosphatase conjugate was dispensed onto the matrix and
binds to the antibody-antigen complex.
5. The matrix was washed to remove unbounded materials.
6. The substrate, 4-methylumbelliferyl phosphate, was added to the matrix and the
fluorescent product was measured by the microparticle enzyme immunoassay
optical assembly.
∆A standard
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26
Reagents
Reagent pack
IMx Insulin Reagent Pack, 100 tests (2A10-20)
1 bottle (7ml) anti-insulin (mouse, monoclonal) coated microparticles in buffer
with protein stabilizers. Preservative: contain sodium azide and antimicrobial
agents.
1bottle (9ml) Anti-Insulin (Mouse, Monoclonal): alkaline phosphatase conjugate in
buffer with protein stabilizers. Minimum concentration: 3μg/ml.
1 bottle (10ml) 4-methylumbelliferyl phosphate, 1.2mM, in buffer.
1 bottle (14ml) assay buffer in calf serum.
Preservative: All of the above mentioned reagents are contain sodium azide and
antimicrobial agents.
Calculation
To convert control ranges to the alternate units, perform the following calculations:
Concentration in μIU/ml = Concentration in μU/ml x 1.0
Concentration in pmol/L = Concentration in μU/ml x 7.175
3.8.5 Determination of serum cholesterol
Enzymatic colorimetric method was used for the quantitative determination of total
cholesterol in serum or plasma, using Diasys Diagnostic Systems, Germany
(Meiattini et al., 1978).
Principle
Determination of cholesterol after enzymatic hydrolysis and oxidation. The
colorimetric indicator is quinoneimine which is generated from 4-aminoantipyrine
and phenol by hydrogen peroxide under the catalytic action of peroxidase.
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27
Reagents
Concentrations are those in the final test mixture.
Cholesterol ester + H2O → cholesterol + fatty acid
Cholesterol + O2 → cholesterol-3-one + H2O2
H2O2 + 4- aminoantipyrine + Phenol → Quinoneimine + 4 H2O
Concentration Reagent
50 mmol/l
5 mmol/l
0.3 mmol/l
≥ 200 u/l
≥ 100 u/l
≥ 3 ku/l
Good’s buffer (pH 6.7)
Phenol
4- Aminoantipyrine
Cholesterol esterase (CHE)
Cholesterol oxidase (CHO)
Peroxidase (POD)
200 mg/dl Standard
Assay procedure
Wavelength: 500 nm
Optical path: 1cm
Temperature: 37 ºC
Measurement: against reagent blank.
Ten µl of standard (sample or control) was added to 1ml of working reagent
and mixed well.
The mixture was incubated for 5 min at 37 ºC.
The absorbance was measured within 60 min.
CHE
CHO
POD
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28
Calculation
Cholesterol (mg/dl) =
Reference value
Child (desirable) < 170 mg/dl
Adult (desirable)
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29
Reagent Concentration
Cood's buffer (pH 7.2)
4-Chlorophenol
ATP
Mg2+
Glycerokinase (GK)
Peroxidase (POD)
Lipoprotein lipase (LPL)
4-Aminoantipyrine
Glycerol-3-phosphate-oxidase (GPO)
50 mmol/l
4 mmol/l
2 mmol/l
15 mmol/l
≥ 0.4 KU/I
≥ 2 KU/I
≥ 2 KU/I
0.5 mmol/l
≥ 0.5 KU/I
Assay Procedure
Wavelength: 500 nm
Optical path: 1 cm
Temperature: 37 ºC
Measurement: Against reagent blank.
Ten µl of standard (sample or control) was added to 1ml of working reagent
and mixed well.
The mixture was incubated for 5 min at 37 ºC.
The absorbance was measured within 60 min.
Calculation
Triglycerides [mg / dl] =
A sample X concentration of standard∆
∆A standard
-
31
Reference value
30 - 150 mg/dl Child (desirable)
40 - 160 mg/dl
35 - 135 mg/dl
Adult (desirable) M
F
3.8.7 Determination of serum high density lipoprotein cholesterol
Liquid high density lipoprotein cholesterol (HDL-C) precipitant was used for the
determination of HDL-C Cholesterol using Diasys Diagnostic Systems, Germany
(Grove, 1979).
Principle
Chylomicrons, VLDL-C and LDL-C were precipitated by adding phosphotungstic
acid and magnesium ions to the sample. Centrifugation leaves only the HDL-C in the
supernatant, their cholesterol content is determined enzymatically using cholesterol
reagent.
Reagents
Concentration Reagent
1.4 mmol/l
8.6 mmol/l
Monoreagent contain: Magnesium chloride
Phosphotungstic acid
200 mg/dl Choesterol standard
Assay procedure
1- Precipitation
Two hundred µl of standard (sample or control) were added to 500 µl of the
precipitation reagent and mixed well.
The mixture was allowed to stand for 15 min at room temperature, and then
centrifuged for 20 min at 4000 rpm.
-
31
2- Cholesterol determination
Wavelength: 500 nm
Optical path: 1cm
Temperature: 37 ºC
Measurement: against reagent blank.
One hundred µl of the supernatant of standard (sample or control) was added
to 1ml of the cholesterol reagent and mixed well.
The mixture was incubated for 5 min at 37 ºC.
The absorbance was measured within 45 min.
Calculation
HDL-C (mg/dl) =
Reference value
3.8.8 Determination of serum low density lipoproteins cholesterol
Serum low density lipoproteins cholesterol (LDL-C) can be calculated using the
empirical relationship of Friedewald (Grove, 1979).
Principle
The ultracentrifugal measurement of LDL-C is time consuming and expensive and
requires special equipment. For this reason, LDL-C is most commonly estimated from
37 – 75 mg/dl Child
35 – 65 mg/dl
35 – 80 mg/dl
Adult: M
F
A sample X concentration of standard∆
∆A standard
-
32
ALP
quantitative measurements of total and HDL-C and plasma triglycerides (TG) using
the empirical relationship of Friedewald.
The Equation
LDL-C = Total Cholesterol - HDL-C - TG/5
3.8.9 Determination of serum alkaline phosphates
Serum alkaline phosphatase (ALP) activity was measured by kinetic photometric test,
according to the international federation of clinical chemistry and laboratory medicine
(Soldin et al., 2007) using DiaSys reagent kit.
Principle
p-nitrophenylphosphate + H2O Phosphate + p-nitrophenol
Reagents
Reagent Components Concentrations
Reagent 1 2-Amino-2-methyl-1-propanol pH 10.4
Magnesium acetate
Zinc sulphate
HEDTA
0.9 mol/l
1.6 mmol/l
0.4 mmol/l
2.0 mmol/l
Reagent 2
Substrate
p-Nitrophenylphosphate 16.0 mmol/l
Substrate start
The reagent and standard are ready to use.
-
33
Sample start
Mix 4 parts of R1 with 1 parts of R2, (e.g. 20 ml R1 + 5 ml R2) = monoreagent.
Stability: 4 weeks at 2-8 o C & 5 days at 15-25
o C.
The monoreagent must be protected from light. The reagent mixture was only
prepared just prior to use.
Procedure
Substrate start
Reagent Blank Sample
Sample - 20μ
Dist. Water 20μ -
Reagent 1 1000μ 1000μ
Mix, incubate for approximately 1 min., then add:
Reagent 2 250μ 250μ
Mix, read absorbance after 1 min. and start stopwatch. Read
absorbance again after 1,2 and 3 min, at wavelength 405 nm.
∆ A/min = [∆ A/min sample] - [∆ A/min blank]
Sample start
Reagent Blank Sample
Sample - 20μ
Dist. Water 20μ -
Monoreagent 1000μ 1000μ
Mix, read absorbance after 1 min. and start stopwatch. Read
absorbance again after 1,2 and 3 min, at wavelength 405 nm.
-
34
Calculation
From absorbance readings calculate ∆A/min and multiply by the
corresponding factor from table below:
∆A/min x factor = ALP activity [U/L]
Substrate start Sample start
405 nm 3433 2757
3.8.10 Determination of serum calcium
Serum calcium was determined by photometric test with cresolphthalein complexone
(Thomas, 1998) using DiaSys reagent kit.
Principle
Cresolphthalein complexone reacts with calcium ions in alkaline medium forming a
red-violet color. Interference by magnesium is eliminated by addition of 8-hydroxy-
quinoline.
Reagents
Reagent Components Concentrations
Reagent 1 Ethanolamine Detergent pH 10.7 600 mmol/L
Reagent 2 2-Cresolphthalein complexone
8-Hydroxyquinoline Hydrochloric
acid pH 1.1
0.06 mmol/L
7 mmol/L
20 mmol/L
Reagent 3 Standard: 10 mg/dL
Preparation and stability of working reagent
Four parts of R1 were mixed with 1 part of R2
-
35
Stability: 3 days at 2-8 oC
Procedure
Wavelength 570 nm, Hg 578 nm (550-590 nm)
Temperature 37°C
Cuvette 1 cm light path
Reading against reagent blank was done
Blank Standard Sample
Working reagent
Distilled water
Standard
Sample
1 ml
20 μ l
-
-
1 ml
-
20 μ l
-
1 ml
-
-
20 μ l
Mixing and reading the optical density (OD) after a 5 minute incubation was done.
The final color is stable for at least 15 minutes.
Calculation
OD Sample
X n
=sample calcium concentration (mg/dl) OD Standard
n = standard calcium concentration
-
36
3.8.11 Determination of serum phosphorus
Serum phosphorus was determined by phosphomolybdate UV end point (Tiez, 1994)
using Amonium Molybdate Diagnostic K.
Principle
Determination of inorganic phosphate was made according to the following reaction:
Phosphorus
Amonium molybdate + Sulforic acid Phosphomolybdic complea
Reagent Components Concentrations
Reagent Sulfuric acid
Amonium molybdate
210 mmol/L
650 mmol/L
Standard Phosphorus 5 mg/dl
Preparation and stability of working reagent
The reagent is ready for use
Procedure:
Wavelength 340 nm
Temperature 37°C
Cuvette 1 cm light path
-
37
Reading against reagent blank was done
Blank Standard Sample
Reagent
Distilled water
Standard
Sample
1 ml
10
-
-
1 ml
-
10
-
1 ml
-
-
10
Mixing and reading the optical density (OD) after a 5 minute incubation was done.
The final color is stable for at least 1 hour.
Calculation
OD Sample
X n =sample phosphorus concentration (mg/dl)
OD Standard
n = standard phosphorus concentration
-
38
3.9 Statistical analysis
Data were computer analyzed using SPSS/ PC (Statistical Package for the Social
Science Inc. Chicago, Illinois USA, version 18.0) statistical package.
Simple distribution of the study variables and the cross tabulation were
applied.
Chi-square (2) was used to identify the significance of the relations,
associations, and interactions among various variables. Yates’s continuity
correction test, 2 (corrected), was used when not more than 20% of the cells had
an expected frequency of less than five and when the expected numbers were
small.
The independent sample t-test procedure was used to compare means of
quantitative variables by the separated cases into two qualitative groups such
as the relationship between cases and controls vitamin D.
Pearson's correlation test was applied.
The results in all the above mentioned procedures were accepted as statistical
significant when the p-value was less than 5% (p
-
39
Chapter 4
Results
4.1 Personal profile of the study population
Table 4.1 summarizes personal profile of the study population. The study included 44
cases (22 males and 22 females) and 44 controls (22 males and 22 females). Age
classification showed that 8 (18.2%) cases and 10 (22.7%) controls were
-
41
Table 4.1 Personal profile of the study population
Personal character Cases (n=44) Controls
(n=44)
Test p- value
No. % No. %
Age (Year)
0.05:not Significant.
4.2 Socioeconomic data of the study population
Table 4.2 provides socioeconomic data of the study population. The employed cases
and controls were 18 (40.9%) and 12 (27.3%) whereas 26 (59.1%) cases and 32
(72.7%) controls were unemployed. The difference between the two groups was not
significant (2=1.821, P=0.177). Regarding family income\month, none significant
difference was also recorded between cases and controls (2 (corrected)
=1.621,
P=0.445). However, family history of diabetes revealed that 36 (81.8%) cases and 20
(45.5%) controls reported that they have family history of diabetes whereas 8 (18.2%)
cases and 24 (54.5%) controls have not (2=12.571, P=0.000), indicating that family
history is associated with type 1 diabetes.
-
41
Table 4.2 Socioeconomic data of the study population
Socioeconomic data Cases (n=44) Controls
(n=44)
2 p- value
No. % No. %
Employment
Yes
No
18
26
40.9
59.1
12
32
27.3
72.7
1.821
0.177
Family income/month (NIS)**
2000
6
9
29
13.6
20.5
65.9
5
4
35
11.4
9.1
79.5
1.621
0.445*
Family history of diabetes
Yes
No
36
8
81.8
18.2
20
24
45.5
54.5
12.571
0.000
Table 4. 1
*P-value of 2 (corrected) test.
** NIS: new Israeli Shekels.
P0.05:not significant.
4.3 Frequent testing of blood glucose level, diet and physical
activity among the study population
Frequent testing of blood glucose level, diet and physical activity among the study
population are presented in Table 4.3. Thirty eight (86.4%) cases and 8 (18.2%)
controls reported that they had frequent testing of blood glucose level whereas 6
(13.6%) cases and 36 (81.8%) had not. The difference between the two groups was
significant (2=40.994, P=0.000). Concerning diet, 27 (61.4%) cases and 10 (22.7%)
controls were on diet whereas their counterparts of 17 (38.6%) and 34 (77.3%) were
not (2=12.512, P=0.000). Although the number of cases 14 (31.8%) doing physical
activity was lower than controls 21 (47.7%), the difference between the two groups
was not significant (2=2.325, P=0.127).
-
42
Table 4.3 Frequent testing of blood glucose level, diet and physical activity among the
study population
Item Cases (n=44) Controls
(n=44)
2 p- value
No. % No. %
Frequent testing of blood
glucose level
Yes
No
38
6
86.4
13.6
8
36
18.2
81.8
40.994
0.000
Diet
Yes
No
27
17
61.4
38.6
10
34
22.7
77.3
13.477
0.000
Physical activity
Yes
No
14
30
31.8
68.2
21
23
47.7
52.3
2.325
0.127
P0.05:not significant.
4.4 Duration of diabetes and self-reported complications
among cases
Table 4.4 shows duration of diabetes and self-reported complications among cases.
Diabetic patients with diabetes since less than 7 years were 23 (54.7%), whereas those
with diabetic duration of 7-14 years were 11 (26.2%). The rest of patients 8 (19.1%)
had diabetes for more than 14 years. The mean duration of diabetes was found to be
9.1±7.0 years with the range of 1-32 years. The only self-reported complication
among cases was retinopathy 2 (4.5%). However, no complications were reported
among controls.
Table 4.4 Duration of diabetes and self-reported complications among cases (n=44).
Item No. %
Duration of diabetes (Year)
14
23
11
8
54.7
26.2
19.1
Mean duration of diabetes±SD (Year)
Range (min-max)
9.1±7.0
(1-32)
Retinopathy
Yes
No
2
42
4.5
95.5
-
43
4.5 Insulin therapy among cases
Table 4.5 indicates insulin therapy among cases. Six (13.6%), 28 (63.6%) and 10
(22.7%) cases received one, two and three doses of insulin/day, respectively with
mean±SD of 2.1±0.6 UI.cc/ml. The mean insulin dose/day was 49.4±20.5 UI.cc/ml
with range of 8-120.
Table 4.5 Insulin injection among cases
Treatment
Cases
(n=44)
No. %
Insulin injection/day
One dose
Two doses
Three doses
6
28
10
13.6
63.6
22.7
Mean±SD 2.1±0.6
Dose (UI.cc/ml)/day
Mean±SD
Range (min-max)
49.4±20.5
(8-120)
4.6 Body mass index of the study population
Body mass index (BMI) of the study population is illustrated in Table 4.6. The mean
weight of cases was 71.3±16.1 kg compared to 68.8±14.1 kg of controls. The weight
difference between cases and controls was not significant (t=0.754 and P=0.453) with
% difference=3.6 higher in cases. Similarly, there was no significant difference in the
mean height of cases compared to controls (1.66±0.12 versus 1.68±0.10 m, %
difference=1.2, t=0.754 and P=0.453). Therefore, BMI of cases was relatively higher
than that of controls, without significant change (26.0±5.7 versus 24.3±4.2, %
difference=6.8, t=1.596 and P=0.114).
-
44
Table 4.6 Body mass index of the study population.
Anthropometric
measurement
Case (n=44)
mean± SD
Control
(n=44)
mean± SD
%
difference
t P-
value
Weight (kg)*
Range (min-max)
71.3±16.1
(44-120)
68.8±14.1
(45-110)
3.6 0.754 0.453
Height (m)**
Range (min-max)
1.66±0.12
(1.4-1.9)
1.68±0.10
(1.5-1.9)
1.2 0.754 0.453
BMI***
Range (min-max)
26.0±5.7
(17.4-45.0)
24.3±4.2
(17.7-38.3)
6.8 1.596 0.114
*Kg: kilogram, ** m: meter. ***BMI: Body mass index: People with BMI=18.5-24.9 were considered
to have normal weight and people with BMI=25.0-29.9 were classified overweight (WHO, 2012).
P>0.05: not significant.
4.7 Serum vitamin D levels of the study population
Table 4.7 and figure 4.1 show the mean serum vitamin D levels of the study
population. The mean level of vitamin D was significantly lower in cases compared to
controls (34.1±19.1 versus 43.9±16.9 ng/dl, % difference=25.1%, t=2.584 and
P=0.012).
Table 4.7 Serum vitamin D levels of the study population
Parameter Case (n=44)
mean±SD
Control (n=44)
mean±SD
%
difference
t P-
value
Vitamin D (ng/dl)
Range (min-max)
34.1±19.1
(7.2-72)
43.9±16.9
(19-82)
25.1 2.584 0.012
P
-
45
4.8 Categories of serum vitamin D levels of the study
population.
As illustrated in Table 4.8 and figure 4.2 serum vitamin D levels of the study
population were classified into 3 different categories: deficient (30 ng/dl). The number of cases having
vitamin D deficient, insufficient and sufficient were 5 (11.4%), 20 (45.5%) and 19
(43.2%), respectively compared to controls of 0 (0.0%), 13 (29.5%) and 31 (70.5%),
respectively with 2
=6.711 and P=0.035.
Table 4.8 Different categories of serum vitamin D levels of the study population
Vitamin D Case
(n=44)
Control
(n=44)
2 P-
value*
No. % No %
Deficient (30 ng/dl) 19 43.2 31 70.5
*P-value of 2 (corrected) test, P
-
46
4.9 Whole blood HbA1c, serum glucose and insulin levels of
the study population
As indicated in Table 4.9, the mean level of whole blood HbA1c was significantly
higher in cases than that in controls (7.7±1.8 versus 5.9±1.2%, % difference= 26.5,
t=5.863 and P=0.000). Serum glucose level was significantly elevated in cases
compared to controls (212.2±101.2 versus 75.0±14.4 mg/dl, % difference= 95.5,
t=7.097 and P=0.000). In addition, serum insulin level was also significantly higher
in cases versus controls (23.4±16.4 versus 13.0±12.9 lU/ml,% difference= 57.1,
t=3.311 and P=0.001).
0
5
10
15
20
25
30
35
Defficient(30 ng/dl)
5
20 19
0
13
31
Vita
min
D le
ve
l (n
g/d
l)
Figure 4.2 Different categories of serum vitamin D levels of cases (n=44) and controls (n=44)
-
47
Table 4.9 Whole blood glycated hemoglobin (HbA1c), serum glucose and insulin
levels of the study population
Parameter Case
(n=44)
mean±SD
Control
(n=44)
mean±SD
%
difference
t P-value
HbA1c (%)
Range (min-max)
7.7±1.8
(5.4-13.3)
5.9±1.2
(4.3-11.8)
26.5 5.863 0.000
glucose (mg/dl)
Range (min-max)
212.2±101.2
(49-449)
75.0±14.4
(48-123)
95.5 7.097 0.000
Insulin (lU/ml)
Range (min-max)
23.4±16.4
(2.0-91.2)
13.0±12.9
(0.1-70)
57.1 3.311 0.001
P
-
48
Table 4.10 lipid profile of the study population
Lipid profile
(mg/dl)
Case (n=44)
mean±SD
Control
(n=44)
mean±SD
%
difference
t P-
value
Cholesterol
Range (min-max)
197.0±45.6
(133-341)
152.9±30.7
(102-220)
25.2 5.318 0.000
Triglycerides
Range (min-max)
142.1±63.5
(33-306)
94.0±51.3
(37-282)
40.7 3.916 0.000
HDL-C *
Range (min-max)
57.1±23.0
(31-135)
57.7±12.8
(36-105)
-1.1 0.149 0.882
LDL-C**
Range (min-max)
88.5±50.6
(8-229)
56.5±24.6
(12-111)
44.1 3.770 0.000
*HDL-C: High density lipoprotein cholesterol, **LDL-C: Low density lipoprotein cholesterol.
P0.05:not significant.
4.11 Alkaline phosphatase enzyme activity of the study
population
Serum alkaline phosphatase (ALP) activity of the study population is pointed out in
Table 4.11. There was a significant increase in ALP activity in cases compared to
controls (164.2±129.6 versus 116.6±42.5 U/L, % difference=33.9%, t=2.317 and
P=0.023).
Table 4.11 Serum alkaline phosphatase activity (ALP) of the study population
Enzyme Case (n=44)
mean±SD
Control (n=44)
mean±SD
%
difference
t P-
value
ALP (U/L)
Range (min-max)
164.2±129.6
(65-602)
116.6±42.5
(54-267)
33.9 2.317 0.023
P
-
49
4.12 Serum calcium and phosphorus of the study population
Table 4.12 shows serum calcium and phosphorus concentrations of the study
population. Serum calcium was significantly decreased in cases compared to controls
(9.04±0.41 versus 9.38±0.56 mg/dl), recording % difference of 3.7, t=2.293 and
P=0.024. On the other hand, serum phosphorus concentration showed no significant
difference between cases and controls (4.32±1.14 versus 4.21±0.91, % difference=2.6,
t=0.441, P=0.659).
Table 4.12 Serum calcium and phosphorus concentration of the study population
Electrolyte (mg/dl)
Case (n=44)
mean±SD
Control
(n=44)
mean±SD
%
difference
t P-
value
Calcium
Range (min-max)
9.04±0.41
(8.0-10.5)
9.38±0.56
(8.5-10.6)
3.7 2.293 0.024
Phosphorus
Range (min-max)
4.32±1.14
(2.7-6.4)
4.21±0.91
(2.5-6.1)
2.6 0.441 0.659
P0.05: not significant.
4.13 Relations of vitamin D
4.13.1 Vitamin D level in relation to family history of diabetes, diet
and physical activity among the study population
