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1]

Original Article

Relationship between Salivary Calprotectin Levels and Recurrent

Aphthous Stomatitis: A Preliminary Study
M Koray, B Atalay, S Akgul1, FS Oguz1, G Mumcu2, A Saruhanoglu3

Faculty of Dentistry, Aim: Recurrent aphthous stomatitis (RAS) is an inflammatory condition of the oral

Abstract
Department of Oral and
Maxillofacial Surgery,
mucosa. The etiology of RAS remains unclear. Calprotectin is a major cytoplasmic
Istanbul University, Capa, protein contained in granulocytes, monocytes/macrophages and epithelial cells,
Fatih, Istanbul, 1Istanbul and its level is increased body fluids in some inflammatory diseases. The aim is
Faculty of Medicine, to determine the relationship between salivary calprotectin and RAS. Material
Department of Medical and Methods: In the cross-sectional study, 67 patients with active lesions of RAS
Biology, Istanbul University, (F/M: 43/24, mean age: 30.27 ± 9.14 years) and 42 healthy controls (HC, F/M:
Capa, Fatih, Istanbul,
2
Faculty of Health Sciences,
30/12, 30.54 ± 9.49 years) were included. Calprotectin levels were evaluated in
Department of Health unstimulated whole saliva samples by using the ELISA method in both groups.
Management, Marmara Results: Salivary calprotectin levels were significantly higher in RAS group (23.72
University, Maltepe, Istanbul, ± 4.28 mg/L) compared to the HC group (21.59 ± 4.27 mg/L) (P = 0.013). No
3
Faculty of Dentistry, significant relationship was found between calprotectin levels and age or gender
Department of Oral and in both groups (P >0.05).Conclusion: RAS is a very common inflammatory
Maxillofacial Surgery,
Istanbul University, Capa,
ulcerative condition of the oral cavity and its etiology is uncertain. Regarded as an
Fatih, Istanbul, Turkey inflammatory mechanism, releasing a high level of calprotectin in saliva has been
suggested that it may play a role in pathogenesis of RAS.

Date of Acceptance: Key words: Calprotectin, Protein, Recurrent Aphthous Stomatitis, S100A8/
02-Mar-2017 S100A9, Saliva

Introduction 14), or cystic fibrosis antigen.[9,10] It is a complex of

R ecurrent aphthous stomatitis (RAS) is an
inflammatory condition of the oral mucosa.[1-3]
The etiology of RAS remains unclear.[4,5] The
histhopathological changes in the pre-ulcerative stage
include infiltration of the epithelium by mononuclear
cells, development of edema, followed by keratinocyte
vacuolisation and localized vasculitis causing localized
swelling that ulcerates and is infiltrated by neutrophils,
lymphocytes, and plasma cells before there is healing
and the regeneration of the epithelium.[4] Moreover, the
salivary defense system such as the enzyme superoxide
dismutase participates in the inflammatory response in
RAS.[6]
Calprotectin is a major cytoplasmic protein contained
in granulocytes, monocytes/macrophages, and epithelial
cells[7,8] and is also known as L1 antigen, macrophage
migration inhibitory, factor-related protein (MRP8 and
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S100A8 and S100A9 proteins, and its level is increased
body fluids in some inflammatory diseases.[11,12]
Calprotectin has antimicrobial properties and plays an
important role in the innate immune system. This protein
can be found in feces, various tissues, fluids, as well as
in gingival cervical fluid and saliva.[13,14]
Calprotectin has been found in infiltrating macrophages
during inflammatory reactions.[15] Macrophages are likely
to participate in every stage of inflammatory process.[16]
Immunological aberrations involving both cell-mediated
and humoral immunity have been reported in RAS
studies.[16,17] Proteins that take place in the non-specific
acute-phase response were studied in RAS etiology.[18,19]
Address for correspondence: Assoc. Prof. M Koray,
Department of Oral and Maxillofacial Surgery, Faculty of
Dentistry, Istanbul University, Capa Fatih, Istanbul, Turkey.
Email: mkoray@istanbul.edu.tr
This is an open access article distributed under the terms of the Creative Commons
Attribution-Non Commercial-Share Alike 3.0 License, which allows others to remix,
tweak, and build upon the work non-commercially, as long as the author is credited
and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

DOI: 10.4103/njcp.njcp_23_17
How to cite this article: Koray M, Atalay B, Akgul S, Oguz FS, Mumcu G,
Saruhanoglu A. Relationship between salivary calprotectin levels and
PMID: ******* recurrent aphthous stomatitis: A preliminary study. Niger J Clin Pract
2018;21:271-5.

© 2018 Nigerian Journal of Clinical Practice | Published by Wolters Kluwer - Medknow 271
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Koray, et al.: Salivary Calprotectin in Recurrent Aphthous Somatitis
Although calprotectin can be found in the oral
environment and appears to have regulatory functions
in the inflammatory process, there are no clinical data
regarding the relationship between calprotectin levels and
RAS. We aim to determine the relationship between RAS
and the calprotectin, which is a highly immunogenetic
protein and secreted by non-specific immune system
cells.
Material and Methods
Patient selection
The study was designed as a cross-sectional study. The
study group consisted of 67 patients with active lesions
of RAS (F/M: 43/24, mean age: 30.27 ± 9.14 years) and
42 healthy controls (HC) who had no RAS history in all
life (F/M: 30/12, 30.54 ± 9.49 years. The study group
was collected out of 103 RAS patients from a group of
patients who were referred to the Department of Oral and
Maxillofacial Surgery with the complaint of aphthous
ulcers between March 2013 and March 2014. The RAS
diagnosis was made based on medical history and clinical
examination. Patients having at least one aphthous
ulcer, defined as round or oval ulcers with a gray-white
pseudomembrane and an erythematous halo less than
5  mm in diameter, were considered as RAS positive.[4]
HC comprised of patients with no history of RAS and
was matched with study group, which comprised of
patients who were referred to our clinic for dental check-
ups, in terms of age and gender.
The exclusion criteria included patients/subjects with
any systemic disorders that were associated with RAS,
any pharmacological therapies and/or drugs containing
iron or vitamins, patients with Behcet’s disease, coeliac
disease, and other gastrointestinal or dermatological
diseases in which RAS was a part of their clinical
manifestation. And also patients/subjects with any
inflammatory diseases that may affect the calprotectin
levels were excluded from the present study. All patients/
subjects were examined in detail by an internal medicine
specialist before being included in the present study. The
participants who were < 18-year old and patients with
the presence of inflammatory lesions on oral mucosa, and
periodontitis and/or gingivitis were also excluded from
the study after detailed intraoral examination. In total, 36
patients were excluded from the study according to these
exclusion criteria.
All patients and subjects were evaluated by the
same specialist (BA) at the Department of Oral and
Maxillofacial Surgery, Faculty of Dentistry, Istanbul
University. The Ethics Committee of Istanbul University,
Istanbul Medicine Faculty approved the study protocol
and the principles of the Helsinki Declaration were
272 Nigerian Journal of Clinical Practice  ¦  Volume 21  ¦  Issue 3  ¦  March 2018
followed (Project No: 203/107). Each subject signed a
detailed informed consent form.
Collection of unstimulated whole saliva
Patients and subjects were requested not to eat and drink
90 min before saliva collection. Smoking, chewing gum,
and intake of coffee were also prohibited during this
time. The subjects were advised to rinse his/her mouth
several times with deionized (distilled) water and then to
relax for 5 min. The subjects were asked to collect any
remaining saliva in his/her mouth and spit it into the test
tube. Unstimulated whole saliva samples were collected
in RAS patients and HC within 15 min (between 9.00
a.m. and 12.00 p.m.) considering the circadian rhythm of
saliva by using a sterile plastic container (MedSanTek®
Istanbul, Turkey). All samples were frozen at -20 °C
before the ELISA analysis. After the collecting saliva
from the participants, the treatment regimes were given
for all RAS patients.
Determination of salivary calprotectin levels
Calprotectin levels in saliva were determined by ELISA
according to the manufacturer’s protocol (Peninsula
Laboratories, LLC, CA, USA). RAS and HC samples
were diluted in the ratio of 1:100. The 1000 ng/mL stock
solution of calprotectin was diluted (200 ng/mL, 80 ng/
mL, and 20 ng/mL). The diluted samples or calprotectin
standards with peroxidase conjugated detection antibody
were added to the antibody-coated wells and incubated
overnight (15–17 h) at 4–8  °C. After incubation, they
were washed six times. The substrates were added to
each well and incubated for 6–8 min at room temperature.
The reaction was stopped with 1N sulfuric acid. The
absorbance of the solution was measured at 450 nm using
a microplate reader. Calprotectin levels were calculated in
ng/mL. Then, data were converted to mg/L.
Statistical analysis
Data were analyzed by using SPSS 20 software (SPSS
Inc., Chicago, IL, USA). Unpaired t-test was used for the
comparison of salivary calprotectin levels between RAS
and HC. According to gender, salivary calprotectin levels
were also analyzed with unpaired t-test. In addition, the
Pearson correlation test was carried out to determine the
relationship between age and calprotectin levels in both
groups. A P value ≤ 0.05 was considered statistically
significant.
Results
Salivary calprotectin levels were significantly higher in
RAS group (23.72 ± 4.28 mg/L) compared to the HC
group (21.59 ± 4.27 mg/L) (P = 0.013)[Table 1].
No significant difference was seen in regard to gender in
both groups (females; 23
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Koray, et al.: Salivary Calprotectin in Recurrent Aphthous Somatitis
Table 1: Calprotectin levels in the RAS and HC groups
Calprotectin levels (mg/L)
RAS (n = 67) 23.72 ± 4.28
HC (n = 42) 21.59 ± 4.27
* A p value ≤ 0.05 was considered statistically significant.
Table 2: Distribution of calprotectin levels according to
gender (mg/L)
Calprotectin levels (mg/L)
Female Male
RAS (n = 67) 23.84±3.87 23.51±5.09 0.762
HC (n = 42) 22.26±4.16 22.91±4.24 0.135
* A p value ≤ 0.05 was considered statistically significant.
84. ± 3.87 mg/L vs. males; 23.51 ± 5.09 mg/L in RAS;
females; 22.26 ± 4.16 mg/L vs. males; 22.91 ± 4.24
mg/L in HC) (P = 0.762 and P = 0.135, respectively)
[Table 2].
Calprotectin levels were found to be not correlated with
age in the RAS and HC groups (r: -0.06 P = 0.67 and r:
0.17 P = 0.163, respectively).
Discussion
The main purpose of this study was to assess whether
a relationship exists between salivary calprotectin levels
and RAS. Calprotectin levels in unstimulated whole
saliva were found to be high in RAS group (23.72 ± 4.28
mg/L) compared to the HC group (21.59 ± 4.27 mg/L)
in the present study. However, calprotectin levels were
found to be not correlated with age or gender in the RAS
and HC groups.
Calprotectin levels had been detected between 3.2 mg/L
and 40 mg/L in different oral secretions.[20,15] Brun et
al. have found the levels in stimulated whole saliva to
be 23.6 mg/L in Sjogren syndrome. They also showed
that plasma and saliva calprotectin levels in patients
suffering from Sjogren syndrome were higher than those
of the healthy subjects.[20] Eversole et al. determined that
calprotectin levels could increase in some inflammatory
oral mucosal diseases including candidiasis, lichen
planus, herpes virus stomatitis, and leukoplakia. They
suggested that epithelial calprotectin also plays a
prominent role in oral mucosal defense mechanism
against viruses, bacteria, and fungi.[21]
Kido et al. found that calprotectin, a major leukocyte
protein and a marker of many inflammatory diseases,
exists in gingival cervical fluid.[10] Calprotectin levels
in gingival cervical fluid are reported to be a potential
marker of gingival inflammation.[22,12] Fecal calprotectin
levels were used as a diagnostic tool for inflammatory
bowel diseases, ulcerative colitis, and Crohn’s disease.[23]
Nigerian Journal of Clinical Practice  ¦  Volume 21  ¦  Issue 3  ¦  March 2018
In Sjogren syndrome, salivary calprotectin levels seem to
p be correlated with glandular pathology and demonstrate
0.013 the presence of calprotectin in human dental calculus
by using immunohistochemical and immunoblotting
analyses.[20,24,25] Mean levels of calprotectin in parotid
gland and whole saliva are found to be 3.2 mg/L and
22.0 mg/L, respectively.[15] A comparison of our results
with the results of previous studies shows that the
increase in the salivary calprotectin levels detected in the
p present study might be related to the local inflammatory
condition observed in RAS.
In the oral environment, saliva contains antimicrobial
peptides originated from oral epithelium and neutrophils.
Decrease in peptide levels are reported to be a
predisposing factor in pathogenesis of oral ulcers. Since
salivary defence system is a part of the innate immune
system, these peptides in saliva play a significant role
in protecting the oral cavity with their antimicrobial
activities.[26,27]
Inflammation is the body’s reaction to invasion by an
infectious agent, antigen challenge, or even just physical
damage. In inflammatory reactions initiated by the
immune system, the ultimate control is exerted by the
antigen itself, in the same way as it controls the immune
response itself. For this reason, the cellular accumulation
at the site of chronic infection or in autoimmune reactions
(where the antigen cannot ultimately be eradicated) is
quite different from that at sites where the antigenic
stimulus is rapidly cleared.[28] Although several studies
reported a relationship in bacterial or viral infections
and RAS, Greenspan et al. concluded that neither cell-
mediated hypersensitivity to streptococcal or viral antigens
nor cross-activity between oral mucosal and streptoccal
antigens is likely to play a role in the pathogenesis of
RAS.[29,30] Ozler and Akoglu found that the neutrophil/
lymphocyte ratio levels in patients with RAS were higher
than the healthy controls. Moreover, the authors reported
a positive correlation between neutrophil/lymphocyte ratio
levels and oral ulcer activity.[3]
The aphthous process is believed to be initiated by the
stimulation of the mucosal keratinocytes by a currently
unknown antigen, leading to T-lymphocyte stimulation and
the liberation of cytokines and various interleukins.[4,31,32]
Calprotectin is produced by cytokines stimulated
kerotinocytes especially in response to combination of
the proinflammatory cytocines.[33] In the initial phase
that precedes the ulcer formation, monocytes and
lymphocytes (mainly of the T type) together with single
mast and plasmatic cells (leukocytes) accumulate under
the basal cell layer. In more advanced stages, polynuclear
leukocytes dominate in the center of the ulcer, wheras
on the lesion border, the abundant mononuclear cell
273
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Koray, et al.: Salivary Calprotectin in Recurrent Aphthous Somatitis
infiltration can be observed.[34,28,35] Calprotectin is a
major cytoplasmic protein contained in granulocytes,
monocytes/macrophages, and epithelial cells.[7,8] Several
factors are known to regulate calprotectin release from
neutrophils and monocytes/macrophages.
Calprotectin is a complex of S100A8 and S100A9
proteins and its level is increased in body fluids in
some inflammatory diseases.[11,12] Hou et al. showed that
stimulating cells with S100A8/9, a repertoire of key
genes found to be up-regulated in pterygium tissue, were
induced in these cells. S100A8/9 may be an upstream
trigger for inflammation in pterygium and any other
disease.[36]
However, the regulatory mechanisms of calprotectin
release by these factors remain unclear.[37] Although
calprotectin could be found in the oral environment and
appears to have regulatory functions in the inflammatory
process, there are no clinical data regarding the
relationship between calprotectin levels and RAS. In the
present study, calprotectin levels were found to be high in
patients with RAS compared to the HC in unstimulated
whole saliva. Calprotectin may play significant roles in
immune response of RAS.
The main shortcoming of this study might be the
lack of blood or fecal samples and after healing data
of RAS. Since RAS is a condition of oral mucosal
inflammation and there are no published data regarding
the relationship between RAS and calprotectin levels in
saliva, the collection of blood samples, a more aggressive
method than saliva collection, was not preferred in the
preliminary study. For further studies, research must
be focused on to the salivary calprotectin levels of
post-treatment stage. In conclusion, with regard to an
inflammatory mechanism, it is suggested that release of a
high level of calprotectin in saliva might play a role as a
biomarker in the pathogenesis of RAS.
Financial support and sponsorship
The present work was supported by the Research Fund
of Istanbul University. Project No. 31343
Conflicts of interest
We affirm that we have no financial affiliation (e.g.,
employment, direct payment, stock holdings, retainers,
consultantship, patent licensing arrangements, or
honoraria) or involvement with any commercial
organization with direct financial interest in the subject
or materials discussed in this manuscript, and no such
arrangements existed in the past three years. Any other
potential conflict of interest is disclosed.
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