UNIVERSITAS INDONESIA

LEVEL OF SERUM VITAMIN D IN ALLERGIC RHINITIS

PATIENTS COMPARED TO HEALTHY CONTROLS IN
RUMAH SAKIT CIPTO MANGUNKUSUMO, JAKARTA

RESEARCH REPORT

JONATHAN DARELL WIDJAJA

1206230025
 2

FAKULTAS KEDOKTERAN
PROGRAM STUDI PENDIDIKAN DOKTER
JAKARTA
JULY 2015

UNIVERSITAS INDONESIA

LEVEL OF SERUM VITAMIN D IN ALLERGIC RHINITIS PATIENTS

COMPARED TO HEALTHY CONTROLS IN RUMAH SAKIT CIPTO
MANGUNKUSUMO, JAKARTA

RESEARCH REPORT
A mandatory final project report presented to Universitas Indonesia to obtain
Bachelor of Medicine

JONATHAN DARELL WIDJAJA

1206230025
 3

FAKULTAS KEDOKTERAN
INTERNATIONAL CLASS PROGRAM
JAKARTA
JULY 2015
STATEMENT OF ORIGINALITY

I hereby certify that the material presented in this research project report (undergraduate) is my

original work and that all the resources, whether cited or referred to, has been stated correctly.

Name : Jonathan Darell Widjaja

NPM : 1206230025

Signature :

Date : 2 July 2015

 4

STATEMENT OF CERTIFICATION

This Project Research is submitted by:

Name : Jonathan Darell Widjaja

NPM : 1206230025
Study Program : General Medicine
Title : Level of serum vitamin D in allergic rhinitis patients compared to healthy
controls in Rumah Sakit Cipto Mangunkusumo, Jakarta

Has been successfully defended in front of the Board of Examiners and was accepted as part of the
prerequisites to obtain a Bachelor Degree in Medicine for the General Medicine Study Program,
Faculty of Medicine, Universitas Indonesia.

BOARD OF EXAMINER

Supervisor : dr. Dewi Wulandari, SpPK ( )

Examiner I : dr. Dewi Wulandari, SpPK ( )

Examiner II : dr. Isabella Kurnia Liem, M.Biomed., PA., Ph.D ( )

Endorsed in : Jakarta
Date : 2 July 2015
 5

ACKNOWLEDGEMENT

Author expressed the utmost gratitude to the Almighty God for all His blessings that that led this
research to reach its completion. This research was conducted as a prerequisite to graduate as
Bachelor of Medicine from Faculty of Medicine Universitas Indonesia. The author also would like
to appreciate the following people who have helped through the process and completion of this
research:
1. dr. Dewi Wulandari, SpPK as the supervisor of this research project. Without her guidance and
teaching this research would not reach this far.
2. dr. Retno Asti Werdhani, M.Epid, where the author consulted the statistic for this research.
3. The author research group, through their support and persistence the author were able to
traverse all the obstacle that emerge during the research.
4. My friends, colleagues and the medical students from the Lembaga Pengkajian dan Penelitian
(LPP) organization that has supported and guide author throughout the research development.
5. The authors family that has given an endless support throughout this project
.

Jakarta, 2 July 2015

The Author
 6

DECLARATION PAGE OF RESEARCH PAPER PUBLICATION APPROVAL FOR

ACADEMIC PURPOSES

As the civitas academic of Universitas Indonesia, I whose signature as depicted below:

Name : Jonathan Darell Widjaja
NPM : 1206230025
Study Program : General Medicine
Faculty : Medicine
Type of work : Research Report

for the further development of science, hereby agree to grant Universitas Indonesia a Non-Exclusive
Royalty-Free Right to my scientific work, with the title:

"LEVEL OF SERUM VITAMIN D IN ALLERGIC RHINITIS PATIENTS COMPARED TO

HEALTHY CONTROLS IN RUMAH SAKIT CIPTO MANGUNKUSUMO, JAKARTA

along with its supporting equipment (if any). With this Non-Exclusive Royalty-Free Right,
Universitas Indonesia has the right to store, convert to other media, manage in a database, maintain
and publish this Final year Project as long as it states my name as the Author.

I hereby affirm that I have made the above statement truthfully and under my own volition.
Prepared in : Jakarta
Date : 2 July 2015
Declared by,

Jonathan Darell Widjaja

 ABSTRAK

Nama : Jonathan Darell Widjaja

Program Studi : Pendidikan Dokter
Judul : Perbandingan kadar serum vitamin D pada pasien alergi
rinitis dengan kontrol sehat di Rumah Sakit Cipto
Mangunkusumo, Jakarta

Latar Belakang: Vitamin D telah diteliti terhadap fungsinya dalam system imun
karena terdapat reseptor vitamin D pada sel-sel imun. Karena penemuan baru ini
vitamin D dapat dihubungkan perannya dalam patofisiologi rinitis alergi. Namun,
data mengenai status vitamin D pada individu sehat maupun pada pasien yang
menderita alergi rinitis di Indonesia sangatlah terbatas. Berdasarkan alasan
tersebut riset ini dilakukan, untuk mengetahui status vitamin D pada kelompok
individu sehat dan membandingkannya dengan kelompok rinitis alergi.
Metode: Riset ini menggunakan metode observasi seleksi silang. Terdapat 22
subjek dalam riset ini yang didiagnosis dengan rhinitis alergi. Pendeteksian
25(OH)D dari serum pasien dilakukan dengan menggunakan teknik
Electrochemiluminescence Binding Assay (ECLIA). Data tersebut akan dianalisa
lebih lanjut menggunakan software IBM SPSS versi 22.
Hasil: Hasil vitamin D pada pasien sehat dibandingkan pasien yang menderita
rhinitis alergi adalah 12.710.3 ng/mL terhadap 15.18.1 ng/mL. Tidak ditemukan
perbedaan tingkat vitamin D yang signifikan antara kelompok jenis kelamin pada
pasien rinitis alergi (Independent Sample T-test p = 0.62). Tidak terdapat
perbedaan yang signifikan pada pasien alergi rinitis dengan kadar Interleukin-5
dan hitung eosinophil yang berbeda (kelompok IL-5: one-way ANOVA: p= 0.897;
kelompok eosinofil: One Way ANOVA: p = 0.752)
Kesimpulan: Tidak terdapat perbedaan yang signifikan pada kadar vitamin D
pasien dengan alergi rhinitis dibandingkan dengan pada pasien normal. Tidak
terdapat perbedaan signifikan pada kelompok yang memiliki kadar IL-5, hitung
eosinofil maupun jenis kelamin yang berbeda

Kata kunci: vitamin D, Rinitis Alergi, Indonesia

7 Universitas Indonesia
 ABSTRACT

Name : Jonathan Darell Widjaja

Study Program : General Medicine
Title : Level of serum vitamin D in allergic rhinitis patients
compared to healthy controls in Rumah Sakit Cipto
Mangunkusumo, Jakarta

Background: Vitamin D role in immune system have been investigated due to the
presence of VDR on immune cells. Based on this information deficient level of
vitamin D could affect the progression of allergic rhinitis. Unfortunately the data
regarding vitamin D status in the normal population and allergic rhinitis patient
were very limited in Indonesia. This research was done to provide illustration
regarding the status of vitamin D in healthy and allergic rhinitis patients in Jakarta
and also to investigate the factor that might affect the level of vitamin D in
allergic rhinitis.
Methods: This research was an observational cross sectional research. There were
22 subjects used during this research all diagnosed with moderate-severe allergic
rhinitis. The study used the Electrochemiluminescence Immunoassay (ECLIA)
technique. The data then were analyzed with IBM SPSS statistic version 22
Results: The difference between the mean vitamin D of patients suffering from
allergic rhinitis with healthy controls (12.710.3 ng/mL to 15.18.1 ng/mL).
There was no significant diference in mean vitamin D between the gender groups
(Independent Sample T-test p= 0.62). There were no statistical difference between
the vitamin D level in patient with different eosinophil count and IL-5 level (IL-5
group: one-way ANOVA: p= 0.897; eosinophil group: One Way ANOVA: p =
0.752).
Conclusion: The mean level of vitamin D in allergic rhinitis patients compared to
healthy controls showed no significant difference. Comparison studies about level
of vitamin D between groups with different gender, IL-5 and eosinophil count
showed no significant difference

Keywords: Vitamin D, Allergic Rhinitis, Indonesia .

8 Universitas Indonesia
 TABLE OF CONTENT

TITLE PAGE..........................................................................................................ii
STATEMENT OF ORIGINALITY.....................................................................iii
ENDORSEMENT PAGE......................................................................................iv
ACKNOWLEDGEMENT.....................................................................................v
DECLARATION PAGE.......................................................................................vi
ABSTRAK............................................................................................................vii
ABSTRACT.........................................................................................................viii
TABLE OF CONTENT........................................................................................ix
LIST OF TABLES.................................................................................................xi
1. INTRODUCTION............................................................................................1
1.1 Background..................................................................................................1
1.2 Problem Identification..................................................................................2
1.3 Research Question........................................................................................2
1.4 Research Objective.......................................................................................2
1.4.1 General Objective......................................................................................2
1.4.2 Specific Objective.....................................................................................2
1.5 Hypothesis....................................................................................................3
1.6 Benefits of the Research...............................................................................3
1.5.2 Benefit for Society....................................................................................3
1.5.2 Benefit for Institution................................................................................3
1.5.3 Benefit for Researcher...............................................................................3
2. LITERATURE REVIEW................................................................................4
2.1 Vitamin D.....................................................................................................4
2.1.1 Metabolism of Vitamin D..........................................................................6
2.1.2 Storage of Vitamin D.................................................................................7
2.1.3 Factor Affecting Vitamin D Level.............................................................7
2.2 Vitamin D and Immunity..............................................................................9
2.2.1 Innate Immunity......................................................................................10
2.2.2 Adaptive Immunity..................................................................................11
2.3 Allergic Rhinitis.........................................................................................12
2.3.1 Pathophysiology of Allergic Rhinitis......................................................13
2.3.2 Vitamin D and Allergic Rhinitis..............................................................10
2.4 Theoretical Framework..............................................................................12
2.5 Conceptual Framework..............................................................................13

3. METHODS.....................................................................................................14
3.1 Research Design.........................................................................................14
3.2 Time and Location of Study.......................................................................14
3.3 Data Sources...............................................................................................14
3.4 Experimental Subjects................................................................................14
3.4.1 Inclusion Criteria.....................................................................................15
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 3.4.2 Exclusion Criteria....................................................................................15
3.5 Sample Frame.............................................................................................15
3.5.1 Sample Size.............................................................................................15
3.6 Research Method........................................................................................18
3.6.1 Variable Identification.............................................................................18
3.6.2 Sample Gathering....................................................................................18
3.6.3 Materials & Equipment...........................................................................18
3.6.4 Sample Processing..................................................................................18
3.7 Data Analysis.............................................................................................19
3.8 Research Framework..................................................................................19
3.9 Operational Definition...............................................................................20

4. RESULT..........................................................................................................21
4.1 Descriptive Test Result...............................................................................21
4.2 Parametric Test Result................................................................................22

5. DISCUSSION.................................................................................................24

6. CONCLUSION AND RECOMMENDATION............................................26

References.............................................................................................................27
Appendix...............................................................................................................29

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 LIST OF TABLES

Table 1 Gender Distribution ..................................................................................21

Table 2 Group by Age ..........................................................................................21

Table 3 Comparison of vitamin D in Healthy and Allergic Rhinitis Patients........22

Table 4 Vitamin D Comparison in Gender Group.................................................22

Table 5 Comparison of Vitamin D based on Eosinophil Count.............................23

Table 6 Comparison of Vitamin D based on IL-5..................................................23

CHAPTER 1
INTRODUCTION

1.1 Background
Vitamin D is a microvitamin that are well known for its functions in calcium
homeostasis.1,2,3 However, new role of vitamin D in the immune system was
found.2,3 It is investigated that vitamin D works towards both the innate immune
system and the adaptive system due to the presence of vitamin D receptor in
immune cells such as macrophage, dendritic cells and also in the T
lymphocytes.2,3,4,5

To elicit its effect, vitamin D needs to be at a certain amount that are reflected by
its inactive form which are the 25-hydroxyvitamin D3.1 This form are distributed
throughout the body and if the level falls below 30ng/ml then vitamin d deficiency
manifestation may occur.1 To avoid these symptoms, a proper level of vitamin D
needs to be maintained.1 The level of vitamin D are majorly influenced by the
11 Universitas Indonesia
exposure to ultraviolet B (UVB) from sunlight that can induce synthesize of
vitamin D by breaking down the precursor of vitamin D.

Regardless of that fact, there were several cases of vitamin D insufficiency even
in a tropical country that supposed to receive an adequate sun exposure. 6,7 A
research in Indonesia conducted by Setiati S about the status of vitamin D in
elderly woman living in Jakarta and Bekasi showed that 35.1% from 75 subject
have vitamin D deficiency.8 This was a very concerning issue as the data for other
population in Indonesia were very limited and the risk of deficiency was still
shown in tropical country.

As mentioned before that vitamin D has an immunomodulatory effect and this

effect could affect disease progression such as allergic rhinitis. 9 According to a
Greiner et al.10 the prevalence of allergic rhinitis are growing larger, there are
around 400 million people suffering from allergic rhinitis in the world. The study
also involved a group of 13 years old children where it was revealed that 24%
subject that were included in the study were suffering from allergic rhinitis and
even worse number (44%) were seen in children whose parents had history of
allergic disease.10 Based on this fact the assessment of vitamin D are considered to
be essential as the function could be use to help relieve allergic rhinitis symptoms.
However, the data to support the importance of vitamin D and even the status of
vitamin D in allergic rhinitis were very limited especially in Indonesia. That is
why this research aimed to compare the serum vitamin D level and compare it to
the healthy population.

1.2 Problem Identification

Data regarding level of vitamin D in healthy individual and allergic rhinitis
patients are very limited in Indonesia
The need of supporting data that can show that vitamin D assessment can
be beneficial for patients with allergic rhinitis

12 Universitas Indonesia
1.1.3 Research Question
Is there any association between presence of allergic rhinitis with the level
of serum vitamin D?
Is there any association between the patients characteristics such as
gender with the level of vitamin D?
Is there any difference in mean vitamin D level based on the allergic
rhinitis patients eosinophil count and also patients Interleukin-5 level?

1.4 Research Objective

1.4.1 General Objective
Investigate factor that might affect the level of vitamin D in
allergic rhinitis patients
1.4.2 Specific Objective
Compare the level of serum vitamin D between patients suffering
from allergic rhinitis towards the level of vitamin D in healthy
individuals in Rumah Sakit Cipto Mangunkusumo, Jakarta
Compare the vitamin D levels in allergic rhinitis patients between
gender
Compare the serum vitamin D in allergic rhinitis patients based on
their eosinophil count and Interleukin-5 level

1.5 Hypothesis
The level of vitamin D in allergic rhinitis patients will be lower
compared to healthy control
The level of vitamin D will be lower in patient with higher interleukin-
5 level and higher eosinophil count
The level of vitamin D between male and female will not show
significant difference

1.6 Benefits of the Research

1.6.1 Benefit to Society

To increase knowledge about the importance of vitamin D

especially in the immune system

13 Universitas Indonesia
 Increase knowledge regarding allergic rhinitis and nutritional
diet that can be used by allergic rhinitis patient
1.6.2 Benefits for Institution

To provide a resource data regarding the epidemiology of

vitamin D in allergic rhinitis patient
1.6.3 Benefit for Researchers
Gain information regarding vitamin D and its function
especially in the immune system
CHAPTER 2
LITERATURE REVIEW

2.1 Vitamin D

According to Pittas et al.11 vitamin D is not classified as a vitamin due to the

several molecular features they can also be grouped as a steroid hormone. The
intake of this vitamins does not come primarily from food consumption. Instead, it
is simply taken from the metabolism of a cholesterol precursor, the 7-
dehydrocholesterol by the radiation of UVB from the sunrays through the
skin.1,9,12,13,14 Even after the change of the precursor to the advance form the
vitamin is not ready to be used yet. These inactive forms are usually called as the
pre-vitamin where it needs to be further metabolized by our body.1,9,12,14

2.1.1 Metabolism of Vitamin D

The 7-dehydrocholesterol that are mentioned before are changed into a pre-
vitamin called as the pre-vitamin D3 that will be changed to vitamin D3 through a
thermal process.1,9,12,14, The thermal processes can only be induced by an UV light
with a certain wavelength (270-290nm), according to the data there are several
factors that affect the synthesis of the vitamin D3 such as: latitude, changes in
season and also the time of day the subject is exposed to the sunlight. 7 The
location and season affect the intake of UVB not only by the intensity of the
14 Universitas Indonesia
radiation the sun emits but also when a country is closer to the equator then it will
affect their behavior regarding how they dress and if they use anything to avoid
the intense heat and sunlight (e.g using a sunscreen). 7 Another essential factor that
need to be taken into consideration is the presence of the precursor for D3 which
is the 7-dehydrocholestrol that bound by the age of the subject, whereas the
subject gets older then there will be less and less presence of 7-DHC. As people
get older the activity that they mostly due related to household activity meaning
less exposure to the sun and this will affect the production of D3. Although that
being said according to the Holicks rule the exposure about part of the body
with minimal eryhemal dose (MED) or the threshold dose where the subject
will feel sunburn sensation will be equal to 1000 IU of oral vitamin D3 and if the
patient is exposed according to this formula in about 2 or 3 times a week that they
will gain sufficient amount of vitamin D3. 7,14 From a research that conducted in a
tropical country such as Indonesia the intensity of UVB peaked at 11 a.m to 1pm
where it is equal to around 2MED/hour or around 8000 IU of vitamin D3. 7

Alongside of the pre-vitamin D3 there are another type of previtamin which the
pre-vitamin D2 that are obtained from diet.1,13 The pre-vitamin D2 is a plan
product, to be precise it is derived from the irradiation of the plant sterol that is
why after being metabolized this vitamin D2 will become the ergocalciferol. The
inactive form of vitamin D will be catalyzed into 25(OH) D form by using the
CYP27A1, CYP2R1 and other hydroxylation enzyme in the liver. 1,13 Due to the
abundant amount of 25(OH)D then it is mostly used as a functional determinant of
vitamin D status, the 25(OH)D form is also considered as a prohormone due to the
ability to change into the active form of vitamin D which is 1,25
dihydroxyvitamin D (1,25(OH)2D;calcitriol). This calcitriol will works in two
manners: the autocrine and paracrine. The 25(OH)D-1--hydroxylase enzyme that
are produced through the CYP27B1 pathway will activate the 25(OH)D inside the
kidney into its active calcitriol that will circulate and produce it effect by
endocrine manner.1,13 The 1,25(OH)2D will work as a high affinity ligand in the

15 Universitas Indonesia
vitamin D receptor (VDR) an it will produce response towards several organ
targets. The most know target is located at the intestinal mucosa where it will
affect the calcium and phosphate regulation.1,9,11

2.1.2 Storage of Vitamin D

The human body monitors Vitamin D usages carefully. There are several storing
place to give adequate supply of vitamin D towards the body, one of the storing
place is the fat where it can be breakdown by the liver and it is maintained with a
feedback system. 1,13 The vitamin D also believed to play a role in the metabolism
of calcium. When there is a decrease in the free circulating calcium amount then
there will be a hormone called as the parathyroid hormone that will give signal to
the kidney to increase its tubular reabsorption of the calcium mineral and thus will
cause a surplus in the production of 1,25(OH) 2D and when this vitamin circulate
and bind to the receptors in the small intestine it will increase the efficiency of
intestinal calcium absorption.1,13

2.1.3 Factor Affecting Vitamin D Level

If the level of serum 25(OH)D is below certain limits (<20ng/mL) then the subject
is classified as having vitamin D deficiency.14,15, To maintain these normal limit we
need to take around 2000 IU/day of vitamin D. The level of the serum itself are
also affected by the subjects weight, height and even physical activity. According
to a research conducted by Jacob et al.16 demonstrated the patient with a high body
mass index and low physical activity will tend to have lower level of serum
25(OH)D compared to ones with lower body mass and more active.

Another factor that affect the concentration of 25(OH)D is the presence of a

particular gene such as the vitamin D binding protein (DBP or GC) where if there
is a polymorphism toward the GC protein will elicit a unusual reaction that
results in higher than normal concentration of vitamin D when given in the
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supplement form.16 Vitamin D level are also affected by drugs usage such as
corticosteroid. The pathway on how glucocorticoid decrease the level of vitamin
D are not completely described, but it is believed that the glucocorticoid function
will cause increased bone resorbtion, decrease bone formation and also increase
urinary calcium excretion. Diseases such as cancer, cardiovascular disease and
also diabetes are spotted in patients with low vitamin D. 16 Level of vitamin D are
also correlated with the immunity level of the patient.10,16

2.2 Vitamin D and Immunity

2.2.1 Innate Immunity
The immune system composed of two component: the innate and adaptive
immunity. The innate arranged by the physical barrier and the complement system
which are boosted by the presence of vitamin D.10 It is said that if the vitamin D
level reach 30ng/mL it will give a protective function towards chronic diseases. A
research conducted in the 1903 by Nobel prize winner Niels Finsen, the scientist
treat a patient suffering Lupus Vulgaris by exposing them light ray. The
mechanism of the action is due to the activation of monocyte CYP27B1 that
provide the intracrine killing of M.tuberculosis. When the tuberculosis bacteria
attacks the host will identify the presence of the gram positive bacteria by the
activation of the Toll like receptor 2/1 and thus increasing the availability of
CYP27B1 and VDR in the macrophages. Furthermore the expression of
CYP27B1 will induce a localized synthesis of 1,25(OH)2D that will involve in
further process regarding the cathelicidin gene.10,13 The cathelicidin activity are
not fully understood but it plays a role in enhancing the intracellular killing of
bacteria and also wound repair via a growth factor.10,13

Additional data also has been gathered regarding the action of vitamin D in
immunity. Vitamin D are able to stimulate the expression of monocyte LL-37 that
also have a significance role in killing bacteria.10,13 The 1,25(OH) 2D are also
capable of promoting a good bactericidal environment. It will increase the

17 Universitas Indonesia
autophagy effectiveness that are used by the monocyte. 1,25(OH) 2D will also
increase the potency for the cell to recognize the pathogen by increasing the
presence of NOD2, a pathogen recognition receptor. The NOD2 will recognize
these gram bacteria by identifying the presence of ligand muramyl dipeptide.
Then the NOD2 give signals to the NF-kB where it will lead to betha-defensin 2
(DEFB4) production which plays a role as antibacterial protein.10,13

Specific to the innate immunity vitamin D will give a tolerogenicity towards the
antigen presenting cells such as dendritic cells and the macrophages, meaning that
it will increase the production and also the function of T regulatory cells that
works to increase the immune system tolerance. So the effect of 1,25(OH) 2D
towards the dendritic cell can be considered as a protective mechanism towards
autoimmune disease since it will prevent the exaggeration response of normal
body immunity.10,13

2.2.2 Adaptive Immunity

The 1,25(OH) 2D also works to decrease the production of T-helper 1 (Th1) cells
that works as proinflammatory cytokines and also the vitamin will interfere with
the action of products of the Th1 such as the Interferon where it should induce
the activation of CYP27B1, but under the influence of the 1,25(OH) 2D they will
express it towards the CYP 24A1 activity. This will give a colliding effect with the
IL-4 that works with the same pathway of interferon , giving a decrease effect
towards the TLR 2/1- induced expression of CYP27B1.2,3,4,10

Vitamin D produces several effect towards the adaptive immunity exclusively.

The evidence of the theory is the presence of the vitamin D receptor on the surface
of T-cells and B-cells. The T cell phenotype, the Th1 cells will greatly reduced by
the presence of 1,25(OH) 2D and through several studies it is proven that the Th1
cells will shift into the Th2 cell which give rise to humoral immunity. Another T
cells agent that are affected are the Th17. The function of this type of cell is to

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give immune response towards a specific pathogen, unfortunately they are also
considered to give inflammatory tissue damage, and by the presence of1,25(OH)
2D it will suppress the production of Th17. 2,3,4,17

The 1,25(OH)2D also has a remarkable effect when they are attached to the
vitamin D receptor located on the B-cells, they are able to reduce cell proliferation
as well as the Ig production which usually cause inflammation process. The
effects are also considered as a protective mechanism towards the development of
autoimmune disorder. A studies suggest the involvement of 1,25(OH) 2D in
disease like diabetes type I. 2,3,4,13,17

2.3. Allergic Rhinitis

Allergic rhinitis or also known as hay fever is a condition that are induced by type
I hypersensitivity, the condition composed of several features such as nasal
discharge followed by sneezing and itchy sensation also it is usually accompanied
by conjunctivitis (conjunctival discharge). The patient will elicit the
hypersensitivity if they are in contact with pollens from ragweed, grasses trees
and also if they inhaled microorganism such as fungal spores, house dust mites
feces and also feathers. Although the systemic symptoms might been exhibited
but in allergic rhinitis there are no presence of fever. The allergic rhinitis are
differentiated into two types the perennial type where the patient will suffers the
rhinitis throughout the year and the second type is the seasonal times and as it
names suggest the patient will only develop symptoms during several time where
there are high level of allergen and usually the course are more severe compared
to the perennial type.18

According to classification by Allergic Rhinitis and its Impact on Asthma (ARIA),

the severity of allergic rhinitis are measured by the symptoms. The disease are
called as mild when the patient are able to do daily activities without any
limitation. If the patients quality of life is disturbed then the allergic rhinitis could
19 Universitas Indonesia
be classified as moderate/severe allergic rhinitis where the disease usually
progress to more than 4 days/week.19

2.3.1 Pathophysiology of Allergic Rhinitis

The pathophysiology of the disease is developed in two settings: the first reaction
happens in the immediate phase allergic reaction that happen in the first hour of
contact with the allergen, the second phase is called the late phase reaction where
it remains for two until four hours and usually it can even reach 24-48 hours. The
mechanism of sensitization are also similar to those in asthma cases. It started by
the activation of antigen presenting cell mainly the monocyte and basophil that
will recognize the allergen and produce the antigen that will stimulate the release
of cytokines such as IL1 which cause the production of Th1 and Th2 that will
release more cytokines such as IL3, IL4, IL 5 and also IL 13. Type 1
hypersensitivity are characterized by the activation of IgE which is the result of
the binding of IL 4 and IL 13 to the lymphocyte B and the release of
Immunoglobulin E will also cause the degranulation of the mast cell and basophil
where there wil be a release of inflamamtory mediator such as histamine,
prostaglandine and leukotrienes in the nasal mucosa.18,20

The release of histamine will stimulate the viridanus nerve that will produce the
itching sensation that are detected by the brain and the response that will be
produced is the sneezing reaction. The increase production of mucus are due to
the effect of histamine to the goblet cell. The stuffy nose that are experienced by
the allergic rhinitis patient are mainly caused by the vasodilatation that are
provided by the histamine and prostaglandin. Another cell that holds a major role
in the pathophysiology of AR is the eosinophil which activated by the type 2
cytokine originated from IL 3, granulocyte macrophage colony stimulating factor
and IL 5 in which their number are increased during the acute reaction phase. So

20 Universitas Indonesia
during the acute attack we can find inflammatory cell such as neutrophil, basophil,
eosinophil, mast cell and lymphocyte. 18,20

2.3.2 Vitamin D and Allergic Rhinitis

The effect of vitamin D towards innate and adaptive immunity explains why there
are more severe reaction in patient with low level of vitamin D. The innate system
which are compromised by Toll-like receptor are blocked in the presence of
vitamin D causing the decrease of monocyte activation that leads less severe
inflammation, vitamin D also serves as a supplements towards our physical barrier
such as our epithelial cell in skin and lungs making them able to resist the
extrinsic allergen.20,21 However, the vitamin D also acts to inhibit the innate
immune system, where it will decrease the expression of MHC class II molecule
that will cause inhibition of the differentiation, maturation and even the effect of
dendritic cell to the immune system which is pathogen recognition. The dendritic
cell will remains immature and it will promote T-cell tolerance compared to the
mature one that will increase the nave T cells.21

Vitamin D role in the adaptive immunity is that vitamin D will affect the T-
regulatory cell so there will be more upregulatory gene to express the Th2 cell
compared to Th1 cell. This event will cause a decrease in Th1 product which is
interferon gamma, IL-2, IL-12 and tumor necrosis factor alpha, the decrease level
of this product will cause an increase level of increase of interleukin 10 level that
makes a less sensitive T regulatory cells. The IL-10 level also indicate the severity
of skin inflammation where if there are high level of IL-10 the inflammation
process will be less noticeable. The increase polarization towards Th2 also cause
an elevated number of its prodct such as IL4, IL5 and IL13 which plays a major
role during parasitic infection due to the activation of eosinophil, these cytokines
also could be seen during the regulation of atopy in asthma. A study conducted in
a mice model also show that there are less migration of eosinophils towards the
lung during administration of vitamin D.22
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2.4 Theoretical Framework

2.5 Conceptual Framework

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 Vitamin D Function
Decrease expression of
MHC class II
Vitamin D
Level Suppress the
maturation of
monocyte
Blocks TLR
Promote Th1 to Th2
Shift
Reduc
Allergic
Sensitizati
Rhinitis
on

DECREAS
Degranulatio
n
Stimulation
of CD4 T
Cell
Release of
Inflammatory
Corticostero Mediators
id Predominantly
Treatment Eosinophil

23 Universitas Indonesia
 CHAPTER 3
METHODS

3.1 Research Design

The research was an observational study more specifically a cross-sectional
study. This study compared difference between level of 25(OH)D in healthy
subjects with allergic rhinitis patient and also compared 25(OH)D level between
gender, IL-5 level and eosinophil count.
The dependent variable was 25(OH)D level measured in ng/mL and it was
recorded as numerical variable. The independent variables were the patients
serum, patients gender, and patients IL-5 and eosinophil count that were
analyzed as categorical variable.

3.2 Time and Location of Study

The research was held for two months, starting from May to June 2015. The
specimen taking and analysis were done in the Department of Clinical Pathology
in Rumah Sakit Cipto Mangunkusumo, Jakarta

3.3 Data Sources

The patients serum as primary data that were stored in Department of Clinical
Pathology at Rumah Sakit Cipto Mangunkusumo, Jakarta were used. The
research also utilized secondary data from medical records at Rumah Sakit Cipto
Mangunkusumo

3.4 Experimental Subjects

In this study, the samples were divided into two groups. The first group
constituted the healthy controls serum that was taken during regular medical
check-up and the patients were not affected by any disease or drug.

24 Universitas Indonesia
The second group included serum of patients that were diagnosed with allergic
rhinitis by the doctors in the ENT Department in Rumah Sakit Cipto
Mangunkusumo, Jakarta

The serums were selected based on the following criteria:

3.4.1 Inclusion criteria

Inclusion Criteria for healthy control:

Healthy individual without any clinical symptoms and with normal
hematology, liver function and renal function test
Young age adult (18-59 years old)

Inclusion Criteria for allergic rhinitis group

Serum from patients that suffer from moderate/severe allergic rhinitis
Young age adult (18-59 years old)

3.4.2 Exclusion criteria

Exclusion Criteria for healthy control group
Patients suffering from illness that affect kidney and liver function

Exclusion Criteria for Allergic rhinitis group

Patients suffering from allergic rhinitis that has been treated with
intranasal steroid for a month before the project starts as it will affect
the vitamin D level and also the inflammatory mediators such as
eosinophil

3.5 Sample Frame

3.5.1 Sample size
The sample size for each test group was calculated using the formula for unpaired
categorical-numeric analytic research:
2
( Z + Z ) S
n=2 ( X 1 X 2 )
25 Universitas Indonesia
In which,
n = total sample
Z = deviat baku alfa (1.64)
Z = deviat baku beta (1.28)
S2 = combined standard deviation (obtained from previous similar research)23
X1 X2 = minimum significant difference in mean from previous similar research.
So, the calculation is X1 X2 = 34 ng/ml 19.52 ng/ml)23
For determination of combined standard deviation (S2), the value of difference
between 25(OH)D levels between allergy rhinitis patients and healthy controls
from previous similar research (Modh D, Shah P, Thakkar B, Jain A, Joshi K,
Katarkar A. Role of vitamin D supplementation in allergic rhinitis. Indian Journal
of Allergy, Asthma and Immunology. 2014;28(1):35).

Thus, to determine the combined standard deviation (S2), the formula below was
used:

(
2
s 12 x ( n1 1 ) + s22 x ( n2 1) )
S =
n1 +n22

In which,
S2 = combined standard deviation
s1 = standard deviation of allergic rhinitis patients from previous research (7.435
ng/ml)23
n1 = sample size of allergic rhinitis patients from previous research (23)
s2 = standard deviation of healthy controls from previous research (15.65 ng/ml)23
n2 = sample size of healthy controls from previous research (23)

Hence, the calculation was

S2 =
( 7.352 x ( 23 1 )+ 15.652 x (23 1) )
23+ 232

26 Universitas Indonesia
 ( 1188.495 +5388.295 )
S2 =
44
S2 = 149.4725

Therefore, the calculation of sample size was as follows:

2( Z +Z )2 x S 2
n=
X 1X 22

2( 1.64+1.28)2 x 149.4725
n=
(34.9419.52)2

2(2.92)2 x 31.69
n=
15.422

n = 10.719838672
So the minimum sample size was 11

3.6 Research Method

3.6.1 Variable Identification
Independent: Serum patients from both of the tested group (Allregic
rhinitis and healthy controls), patients gender, eosinophil count and IL-5
level.
Dependent: Level of 25(OH)D (ng/mL)

3.6.2 Sample Gathering

The samples were derived from patients serum that were collected using
standard sample tubes and stored in 2-8C. The patients serum then was
evaluated using the electrochemiluminesence immunoassay (ECLIA)
technique.

3.6.3 Materials & Equipment

27 Universitas Indonesia
 The material that were used during this experiment are the total 25-
Hydroxyvitamin D reagent kit (ref no. 05894913 190) which include as
follows:
1. Pretreatment reagent 1 contain: Dithiothreitol 1 g/L, pH 5.5
2. Pretreatment reagent 2 contain: Sodium hydroxide 55 g/L.
3. Streptavidin-coated microparticles
4. Vitamin D binding protein-BPRu
5. 25-hydroxyvitamin D~biotin

3.6.4 Sample Processing:

The step by step methods for detecting the vitamin D were as follows:
1. First incubation where the sample mixed with the pretreatment
reagent 1 and 2 where it removed the binding between the vitamin
D binding protein with vitamin D (25-OH).
2. Second incubation were done to produce a complex between the
vitamin D (25-OH) with the ruthenylated vitamin D binding
protein
3. During the 3rd incubation the streptavidin-coated microparticles
were added alongside the vitamin D labeled with biotin where it
bind the free ruthenium labeled vitamin D binding protein. The
complex between the ruthenylated vitamin D and the biotinylated
were produced and this complex were then able to react to the
solid phase due to the presence of biotin and streptaividin that
were added previously.
4. Finally the formula were aspirated to the measuring well and the
microparticle were attracted to the surface of electrode. A washing
step by using the Procell/ProCell M were done to remove all the
unbound substances. The electrode then were induced by giving
electricity in certrain voltage that resulted in chemiluminescent
emission that was detected by photomultiplier where the result
were determined via a calibration curve and master curve
provided via the reagent barcode.

28 Universitas Indonesia
 3.7 Data Analysis
For the comparison of the level serum 25(OH)D between the healthy and the
allergic rhinitis group were evaluated by IBM SPSS (Statistical Package for
Social Science) version 22 and the test that were used were the parametric T-
Exclusion
Inclusion Criteria Criteria
Fulfilled
independent test that were used to analyze the difference between two
independent means of the vitamin D in healthy and allergic rhinitis group. The
same test was run to compare groups with gender. The interleukin 5 and
eosinophil count criteria were processed by using the one way ANOVA test.

3.8 Research Framework

Sample Collection

ECLIA

3.9 Operation Definition

1. Serum of Healthy Control
a. Serum taken from healthy patient that have no clinical
symptoms and confirmed by normal result of lab tests such as
hematologic examination, renal function and liver function
b. Recorded as a categorical data

2. Serum of Allergic Rhinitis Patient

a. Serum taken from patient suffering from allergic rhinitis that
have been diagnosed by doctors from the Ear, Nose, Throat
department in Rumah Sakit Cipto Mangungkusumo, Jakarta
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 based on the clinical symptoms with additional IL-5 assessment
and eosinophil count to portray the disease progression
b. Data recorded as categorical data

3. Age
a. Subject age during the serum was taken (18-59 years old)
b. The data were obtained from medical record
c. The data recorded as a numerical data

4. Gender
a. Subject gender were gathered from medical record (male and
female)
b. The data recorded as a nominal data

5. Serum 25(OH)D
a. Result of the processed serum of the two group subjects using
the ECLIA method and the result was vitamin D level of the
subject. The result was in ng/mL
b. Data recorded as numerical data

6. Eosinophil
a. White blood cells that are seen in allergic rhinitis
pathophysiology
b. Taken from the nasal swab of the allergic rhinitis patients
c. The result were counted in:
i. 0 = No eosinophil found
ii. 1 + = 1.1 -5. cell/10 hpf
iii. 2 + = 6-15 cell/10 hpf
iv. 3 + = 16-20 cell/10hpf
v. 4+ = >20 cell/10hpf
d. Data was a categorical data

7. Interleukin 5
a. Cytokines that are produced due to the stimulation of T
regulatory cell that express the Th2
b. Data were counted in pg/mL
c. The data was a numerical data

CHAPTER 4
RESULT

30 Universitas Indonesia
This study was a comparative research where it comprised of 62 test subjects in
which 22 of the subject were patients that came to the Ear, Nose and Throat ward
in Rumah Sakit Cipto Mangunkusumo. The remaining 40 subjects were healthy
controls that have no abnormal finding and were not diagnosed with any illness.
The serum from both populations were taken and stored in the Department of
Clinical Pathology Rumah Sakit Cipto Mangunkusumo Jakarta. The serum were
analyzed for 25(OH)D level by using the Cobas E411 immunoassay analyzer.
The result then were processed statistically by using descriptive and parametric
analysis.

4.1DescriptiveTestResult

Table1.GenderDistribution
Frequency Percent
Female 10 45.5
Male Table 2. Group by Age
12 54.5
Age Percen
Total Groups Sample t
22 100.0
15-24 9 40.9
25-34 3 13.6
35-44 6 27.3
45-54 4 18.2
4.1.1 Subjects Background Total 22 100.0
From the 22 subjects that were involved in this study, 10 were female patients
(45.5%) and male patients were 12 (54.5%) as shown in table 1. Another
descriptive analysis were run for the age distribution. The distribution was: 40.9%
patients belonged to 15-24 years old group, 13.6% were in the 25-34 years old
group, 27.3% in the 35-44 years old group and finally the oldest group the 45-54
years old only account for 18.2% of the total subjects (Table 4.2). The mean age
of the test subjects was 32.9112.35 years old. Data of each patient occupation
was available, from 22 subjects 1 person had outdoor job and the rest were
indoors.
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 4.2 Parametric Test Result

4.2.2 Comparison of vitamin D in Healthy and Allergic Rhinitis Patients

Table 3. Independent T-test AR vs Healthy

Mean Difference
Mean (s.d) p value
Patient Status (CI 95%)
Healthy (40) 15.2 (8.1) 0.76 2.37 (1.7-2.1)
Allergic Rhinitis
12.7 (10.3)
(22)

Table 3 displays the mean difference between the healthy control and the Allergic
Rhinitis patien. The data showed that mean vitamin D level in allergic rhinitis
patient were lower than in the healthy individuals by 12.710.3 ng/mL to 15.18.1
ng/mL. Due to both of the value of the data were categorized as numerical data
and the samples were both independent, the appropriate test that was used to
process the data was the independent T-Test and because of p = 0.76 statistically
the mean level of vitamin D of the Allergic Rhinitis group compared to the
healthy control group in Indonesia was not statistically significant

4.2.3 Comparison of vitamin D between Gender Groups with Allergic

rhinitis

Table 4. Vitamin D Comparison in Gender Group

Mean Difference
Mean (s.d) p value
Gender Group (CI 95%)
Female (10) 11.53 (1.77) 0.62 1.08 (1.26-1.47)
Male (12) 12.46 (1.86)

To see if there was any association between the gender and vitamin D level,
another descriptive test was run comparing the male and female group amongst
32 Universitas Indonesia
those who were affected by Allergic Rhinitis. The mean result of the both group
were almost in the same level where the male group has 12.461.86 ng/mL whilst
the female 11.531.77 ng/mL. Equal variance was assumed in this test due to the
result of Levenes test (p = 0,399). So statistically from the data provided in Table
4 there was no significance difference between the two means. (p > 0,05)

4.2.4 Comparison of serum vitamin D between eosinophil count groups and

IL5

Table 5. Comparison Vit D Based on

Eosinophil Count
p
n Mean (s.d) value
Eosinophil 0 13 12.25 (8.7) 0.752
Table 6. Comparison Vit D Based on IL5
Count +1 4 8.28 (2.96)
p
+2 4 19.31 (18.61)
n Mean (s.d) value
+3 1
Eosinophil 11.72 1
0.897
Count <15 3 13.28 (11.35)
16-
4
30 11.53 (2.61)
The >30 4 7.7 eosinophil
counts were recorded as 4 group (0, +1, +2, +3 and +4) to analyze if there were
any significant difference between the 4 group the author used the one way
ANOVA test. The result can be seen in Table. 6 showed that there were no
significant mean difference between the groups (p = 0.752). Another analysis was
done to know if there was an association between the IL5 level with vitamin D.
The IL-5 level were grouped into 3 groups (less than 15 pg/mL, 16-29 pg/mL, >30
pg/mL), the analysis was done with one way ANOVA test. The result showed no
significant difference between the 3 groups so the post-hoc test was not
conducted.

33 Universitas Indonesia
 CHAPTER 5
DISCUSSION

The result of this research showed that the mean serum of healthy control in
Jakarta was 15.168.10 ng/mL and can be classified as vitamin D deficiency. This
finding suggest that even with the abundant presence of sunlight, there are still
other factors that contribute to the vitamin D synthesis. A factor that might play a
major role is skin color. The darker the skin means the more melanocyte that will
block the incoming UV light. The publication from Hall et al. 24 suggest that
individuals with high skin pigmentation needs and less sun exposure compared to
those who have lower skin reflectance are in need for larger amount of vitamin D
supplementation. In the research it is also stated the mean level between non-
Hispanic Whites, non-Hispanic Blacks and Mexican-American are 67, 40 and 54
nmol/L.24

Gender variation has also been linked with vitamin D status. It is speculated that
female has a lower vitamin D level compared to male because of lifestyle pattern.
Women in some part of the world used hijab that covers area of the skin and also
some women usually avoid the sun exposure to retain for esthetic reasons by
using sunscreen.25 Study in Iran suggested that vitamin D level was significantly
lower in women than men (26.7 1.9 ng/mL compared to 46.8 1.2 ng/mL),
contrary to the data that were revealed in this study the mean vitamin D in women
compared to men was 11.531.77 to 12.461.86 ng/mL showing no significant
difference between the two groups.25 This result needs to be explored more, since
the data that are associated with lifestyle behavior such as clothing, diet and also
sun exposure pattern were not present. However the data regarding each subject
occupation was present and it was revealed that the mean acquired from the
person who works indoor were lower than those in the outdoor.

Another factor that might affect the vitamin D status are the presence of illness
and disease. Vitamin D function in immune system are postulated because of the
34 Universitas Indonesia
availability of vitamin D receptor that are found in many type cells that mediate
immunes system such as the Th1 and Th2 cells. The presence of illness can
promote the activation of more vitamin D receptor, the stimulaton of CD4+T cell
could elevate the number of VDR expressed by five fold. This shows that
vitamin D deficiency could lead to a more compromised immune system, while
the increase risk of infection and disease that affect the immune system will
increase uptake of vitamin D and if the supply is not enough will lead the
individual to be vitamin D deficient. According to the result of this research there
was no statistical significancy between the presence of allergic rhinitis towards the
mean of vitamin D compared to the healthy control (AR:12.710.3 ng/mL vs
Healthy:15.18.1 ng/mL) so the hypothesis that has been previously postulated
was not accepted. Although the result indicate that the total means were lower
compared to those in healthy controls. A similar study has been conducted
previously in India and the result showed that the difference level of vitamin D
was significant in allergic rhinitis compared to the normal population in the same
region. However, both of the mean vitamin D in the AR group and the normal
group were quite high (AR: 19.527.35 vs normal: 34.9415.65).25

The inflammatory mediator that are usually seen in during allergic rhinitis patient
are also assessed. Theoretically the presence of vitamin D would affect the
number of IL5 and eosinophil available. The binding of vitamin D to the vitamin
D receptor in the T-regulatory lymphocyte will cause elevated expression of T
helper cell 2 which will produce the interleukins (IL3, IL4, IL5 and IL13) that can
activate eosinophils. However an opposing result were shown from this research
where statistically there are no significant difference between the group with
mild, intermediate or high level of eosinophil, the same result are also seen in the
IL5 group. The real mechanism behind the vitamin D function are still missing a
link and there is still more aspect yet to be discovered. From a research conducted
by wjst and Hyppnen finding showed that there were an U shaped relationship
between the level of IgE detected and serum 25(OH)D in patient with asthma

35 Universitas Indonesia
indicated that higher risk of asthma exacerbation might occur in high and low
level of vitamin D.26 This unknown mechanism yields this research to gain more
information.

This research also suffered from other limitations such as the small sample size
because of the expensive reagents price and also the lack of patients. Thus, it is
recommended in the future to make a nationwide survey of vitamin D hence it can
reveal the status of vitamin D in Indonesia. Another obstacle that this research
needed to overcome were the lack of patients backgrounds due to the fact that the
serum that were taken were stored serum that has limited medical record.
CHAPTER 6
CONCLUSION AND RECOMMENDATION

6.1 Conclusion
1. Statistically the serum 25(OH)D in patient with allergic rhinitis
compared to healthy control in Jakarta had no significant difference
2. The vitamin D level between male and female showed no significant
difference
3. Vitamin D in allergic rhinitis patients with high eosinophil counts
showed no statistical difference compared to lower eosinophil count.
4. There were no significant difference in mean vitamin D between
allergic rhinitis patients with high IL-5 level compared to those with
lower IL-5 level

6.2 Recommendation
1. Future research should be done by using multivariate analysis to include
all the factor that can affect the vitamin D level. The data regarding patient
history of patient lifestyle that might affect the amount of sun exposure
should be assessed as well as subject diet routine should be included.
2. A nation scale research about the prevalence of vitamin D should be done
3. Molecular study by using cytology in patient suffering allergic rhinitis and
given a vitamin D supplementation should be done to see the clear

36 Universitas Indonesia
mechanism about how there is a lower vitamin D level in allergic rhinitis
patient

37 Universitas Indonesia
 REFERENCE

1. Mahan LK, Escott-Stump S, editors. Krauses food & nutrition therapy. 12th ed. St.
Louis: Saunders/Elsevier; 2008. Chapter 3. p74-7
2. Schwalfenberg GK. A review of the critical role of vitamin D in the functioning of the
immune system and the clinical implications of vitamin D deficiency. Mol Nutr Food
Res. 2011 Jan;55(1):96108.
3. Hossein-nezhad A, Holick MF. Vitamin D for health: a global perspective. Mayo Clin
Proc. 2013 Jul;88(7):72055.
4. Adams JS, Hewison M. Update in Vitamin D. J Clin Endocrinol Metab. 2010
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5. Cantorna MT, Zhu Y, Froicu M, Wittke A. Vitamin D status, 1,25-dihydroxyvitamin D3,
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6. Green TJ, Skeaff CM, Rockell JEP, Venn BJ, Lambert A, Todd J, et al. Vitamin D status
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11. Pittas AG, Harris SS, Stark PC, Dawson-Hughes B. The effects of calcium and vitamin
D supplementation on blood glucose and markers of inflammation in nondiabetic
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12. Norman AW. Sunlight, season, skin pigmentation, vitamin D, and 25-hydroxyvitamin D:
integral components of the vitamin D endocrine system. Isr Med Assoc J. 2010
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13. White JH. Vitamin D metabolism and signaling in the immune system. Rev Endocr
Metab Disord. 2012 Mar;13(1):219.7
14. Holick MF. Vitamin D Deficiency. New England Journal of Medicine. 2007 Jul
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15. Rosen CJ. Vitamin D Insufficiency. New England Journal of Medicine. 2011 Jan
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16. Jacobs ET, Martnez ME, Jurutka PW. Vitamin D: Marker or Mechanism of Action?
Cancer Epidemiol Biomarkers Prev. 2011 Apr 1;20(4):58590.

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17. Huang H, Porpodis K, Zarogoulidis P, Domvri K, Giouleka P, Papaiwannou A, et al.
Vitamin D in asthma and future perspectives. Drug Des Devel Ther. 2013 Sep
23;7:100313.
18. Porth C. Essentials of Pathophysiology: Concepts of Altered Health States. Lippincott
Williams & Wilkins; 2011.
19. Bousquet J, Khaltaev N, Cruz A, Denburg J, Fokkens W, Togias A. Allergic rhinitis and
its impac on asthma (AROA) 2008 update (in collaboration with the World Health
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20. Brodie D. Allergic rhinitis: Pathophysiology. Allergy Asthma Proc. 2010;31:370-4
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 APPENDIX
APPENDIX. 1
Healthy Control Data
25(OH)D
Sample
Age Gender (ng/mL)
N1 24 F 8.74
N2 24 F 23.63
N3 26 M 10.57
N4 29 F 24.35
N5 29 F 27.73
N6 38 M 18.00
N7 31 M 9.41
N8 25 M 8.23
N9 26 M 22.59
N10 21 F 24.71
N11 32 M 11.69
N12 30 M 11.71
N13 47 M 25.77
N14 32 F 16.25
N15 47 M 5.93
N16 49 F 12.51
N17 51 M 13.68
N18 28 M 42.69
N19 32 F 13.63
N20 54 M 20.63
D21 32 F 6.32
D22 28 F 7.33
D23 23 M 13.83
D24 34 M 11.26
D25 26 M 10.41
D26 27 M 10.45
D27 24 F 6.09
D28 29 F 15.77
D29 30 M 24.69
D30 25 F 8.85
D31 26 M 7.54
D32 33 M 27.43
D33 25 F 17.90
D34 48 M 12.06
D35 51 F 11.76
D36 52 M 20.83
D37 57 M 14.39
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D38 21 F 7.76
D39 27 F 4.19

APPENDIX. 2
Allergic Rhinitis Data

Sam
Vit D Eosinophi
ple Age Workplace IL5
Level l
No
1 6.69 33 Indoor 1+ 3,9
2 3 40 Indoor 0 3,9
3 11.5 47 Indoor 0 3,9
4 4.21 37 Indoor 2 3,9
5 46.49 54 Outdoor 2+ <3,9
6 35.82 20 Indoor 0 <3,9
7 11.72 18 Indoor 3+ <3,9
8 5.3 25 Indoor 0 <3,9
9 12.46 58 Indoor 0 <3,9
10 5.53 33 Indoor 1+ 10,6
11 12.97 24 Indoor 0 <3,9
12 17.21 23 Indoor <3,9
13 8.97 54 Indoor 0 <3,9
14 7.7 35 Indoor 0 54,5
15 7.5 41 Indoor 0 11,65
16 8.62 18 Indoor 1+ 15,8
17 20.06 37 Indoor 0 <3,9
18 13.83 24 Indoor 0 <3,9
19 12.87 22 Indoor 2+ <3,9
20 3 21 Indoor 0 5,7
21 12.31 38 Indoor 1+ 18,4
22 13.68 22 Indoor 2+ 26,8

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