Keywords
visfatin - biomarker - gingivitis - periodontitis - saliva - diagnostic marker
Introduction
A biomarker is “a substance that is measured objectively and evaluated as an indicator
of normal biologic or pathologic process or pharmacologic responses to a therapeutic
intervention.”[1] Biomarkers, whether produced by normal healthy individuals or by individuals affected
by any specific systemic diseases, are telltale molecules that could be used to monitor
underlying health status, disease onset, and treatment response. Saliva, when considered
as a biomarker is an important physiologic fluid, and is rapidly gaining popularity
as a diagnostic tool. Saliva contains both host-derived and microbial-derived factors,
including several enzymes that degrade proteins, proteoglycans, lipids, and carbohydrates
and also enzymes in saliva can originate from cells in salivary glands, microorganisms,
epithelial cells, and neutrophils. Saliva, as a mirror of oral and systemic health,
is a valuable source for clinically relevant information because it contains biomarkers
specific for the unique physiologic aspects of periodontal diseases.[2]
Adipose tissue is composed mostly of adipocytes that produce a variety of cytokines
and inflammatory molecules, commonly referred to as adipocytokines such as adiponectin,
leptin, visfatin, interleukin-6 (IL-6), monocyte chemoattractant protein-1, resistin,
tumor necrosis factor-α (TNF-α), and vaspin that regulate different inflammatory processes.
These factors influence insulin resistance and are thought to play a role in inflammation,
and immune responses and are involved in the pathophysiology of periodontitis.[3]
Visfatin is an adipokine, also known as Pre B cell colony enhancing factor, encoded
by the nicotinamide phosphoribosyltransferase (NAMPT) gene (located on 7q22.3, OMIM
ID: 608764). The structural gene part is composed of 11 exons and 10 introns, which
encode 491 amino acids. The protein belongs to the nicotinic acid phosphoribosyltransferase
family and is thought to play an important role in immune response and inflammation.[4]
[5] Visfatin was first reported as a novel adipocytokine secreted preferentially by
visceral fat tissue compared with subcutaneous fat in humans and mice, though subsequent
reports indicated it is expressed in all adipose tissue and has insulin-mimetic properties.[6]
Expression of visfatin is found to be positively regulated in response to microbial
stimulation by B cells, T cells, monocytes, macrophages, and neutrophils.[7]
[8]
[9] It has been reported that the expression of visfatin is upregulated in a variety
of acute and chronic inflammatory diseases such as rheumatoid arthritis,[10] sepsis,[11] acute lung injury,[12] inflammatory bowel disease,[13] diabetes mellitus,[14] aging,[15] metabolic syndrome, and obesity,[16] nonalcoholic fatty liver disease,[17] where there is persistence of inflammation by inhibition of neutrophil apoptosis.
Also, it has been shown that visfatin synthesis is regulated by some cytokines such
as IL-1β, TNF-α, IL-6, and by lipopolysaccharides.[11]
[18] In a study, during polyclonal immune responses, visfatin was found to be increased
in lymphocytes and stimulated their proliferation.[19] So, visfatin appears to be a key cytokine involved in chronic inflammatory diseases
and immune responses.
Several studies reported that the plasma visfatin concentration is significantly increased
in type 2 diabetes and/or obese individuals as well as in obese nondiabetic children
compared with lean control children influenced by several factors such as age, gender,
and body mass index (BMI).[20]
[21]
Advances in oral and periodontal disease diagnostic research are moving toward methods
whereby periodontal risk can be identified and quantified by objective measures such
as biomarkers. Thus, the aim of this study was to evaluate the role of visfatin biomarker
in oral diseases like periodontitis.
Materials and Methods
Sample Selection
A total of 60 patients (20–50 years) who visited the Department of Periodontics, The
Oxford Dental College, Bangalore, India, were recruited in the study after satisfying
the inclusion and exclusion criteria. These subjects were divided into three groups
according to the 2017 World Workshop Classification of periodontal diseases and periimplant
diseases and conditions.[22] Group I consisted of 20 systemically healthy subjects with healthy periodontium
(with no attachment loss and probing depth ≤ 3mm), group II consisted of 20 systemically
healthy subjects with generalized moderate gingivitis (with a generalized probing
depth of ≤3mm, no attachment loss and generalized bleeding on probing), group III
consisted of 20 systemically healthy subjects with generalized periodontitis (clinically
and radiographically with generalized moderate periodontitis, with a pocket depth
of 5 to 8mm and with moderate alveolar bone loss and clinical attachment loss of 3
to 4 mm).
Inclusion and Exclusion Criteria
Patients with at least 20 natural teeth with an age group of 25 to 50 years, no systemic
disease with BMI of 18.5 to 29.9 kg/m2 were included in the study. Patients with a history of periodontal therapy during
the previous 6 months, any systemic disorders that would influence the course of periodontal
disease or treatment; patients using glucocorticoids, bisphosphonates, antibiotics,
and immunosuppressant medication during the preceding 6 months, menstruating, pregnant,
and lactating women; patients undergoing orthodontic therapy; patients with oral mucosal
inflammatory conditions; and obese patients with BMI ≥30kg/m2 were excluded from the study.
The study protocol was approved by the Institutional Ethics Committee (No. 433/2015–16)
of The Oxford Dental College. All subjects were given a detailed verbal and written
description of the study and, written informed consent was obtained from all patients
prior to the commencement of the study.
Clinical Examination
Patients were selected for each group after a brief and precise case history recording
that included patient's chief complaint, medical and dental history, clinical examination
and radiographic examination. All the clinical measurements gingival index,[23] plaque index,[24] probing pocket depth,[25] and clinical attachment levels[26] were performed by a single operator using sterile mouth mirror and UNC-15 periodontal
probe. Orthopantomographs were taken to confirm the bone loss, and BMI[27] was recorded. Clinical parameters were recorded 1 day before saliva sample collection
to avoid stimulation of the sample and its contamination with blood.
Method of Salivary Sample Collection
Unstimulated whole saliva was collected according to Navazesh[28] method on the second day after baseline measurement. According to this method, the
patient was advised to avoid food and beverages at least 1 hour before the test session
following which the patients were asked to rinse the mouth with water to remove food
residues. The patient was then asked to relax for 5 minutes to avoid sample dilution
before collection. During sample collection, patients were instructed to minimize
movements of the mouth, lean their head forward, and get the Eppendorf tubes close
to their mouth that is slightly open, to allow saliva to drain into the tube. Following
sample collection, the samples were refrigerated immediately and were transported
in an icebox for storage at or below –80°C, for further analysis of visfatin levels
using an enzyme-linked immunosorbent assay (ELISA) kit.
Biomarker Analysis
The concentration of visfatin from saliva was determined with the human visfatin ELISA
Kit (Ray Biotech Life, Georgia, USA) according to the manufacturer's instructions
using an ELISA. It is based on the principle that the microplate in the kit is precoated
with antirabbit secondary antibody. After a blocking step and incubation of the plate
with antivisfatin antibody, both biotinylated visfatin peptide and peptide standard
or targeted peptide in samples interact competitively with the visfatin antibody.
Uncompleted (bound) biotinylated visfatin peptide then interacts with streptavidin-horseradish
peroxidase (SAHRP), which catalyzes a color development reaction. The intensity of
colorimetric signal is directly proportional to the amount of biotinylated peptide-SAHRP
complex and inversely proportional to the amount of visfatin peptide in the standard
or samples. This is due to the competitive binding to visfatin antibody between biotinylated
visfatin peptide and peptides in standard or samples. A standard curve of known concentration
of visfatin peptide can be established, and the concentrations of visfatin peptide
in the samples were calculated accordingly. The results of the visfatin assay were
expressed as ng/ml for concentrations.
Statistical Analysis
Statistical analysis was performed using commercially available software (SPSS 22.0,
SPSS Inc., Chicago, Illinois, United States). Power analysis was based on the supposition
that a mean difference of 0.5 mm in PD should be detected at a significance level
of 0.05, and the desired study power of at least 80%. One-way analysis of variance
test followed by Tukey's post hoc analysis was used to compare the mean values of
various clinical parameters and salivary visfatin levels between three groups. Pearson
correlation to assess the relationship of periodontal parameters, BMI, and waist to
hip ratio with salivary visfatin levels during pretreatment and post-treatment periods
in different study groups. The level of significance was set at p < 0.05.
Results
In this study, we have evaluated salivary visfatin concentrations in periodontally
healthy, generalized moderate gingivitis, and periodontitis subjects. All the groups
were matched in terms of age (p = 0.005) and gender (p = 0.48). Visfatin was detected in all samples. The levels were highest for periodontitis
group (38.22 ± 3.38 ng/mL) followed by the gingivitis group (26.66 ± 2.24 ng/mL) and
periodontally healthy subjects (25.60 ± 2.19 ng/mL). Also, the visfatin levels in
both normal weight (33.14 ng/mL) and overweight (34.18 ng/mL) subjects were almost
equivocal which implies that visfatin levels increase during periodontal inflammation
irrespective of the BMI.
Discussion
The presence of visfatin/NAMPT in white blood cells and tissue-bound macrophage suggests
an important role in the regulation of immune and defense functions.[29] Pradeep et al reported increased levels of visfatin in gingival crevicular fluid
(GCF) in patients with periodontitis.[30]
In the present study, the mean age, for the group I, group II, and group III, was
27.10 ± 3.92, 33.65 ± 4.90, and 35.90 ± 8.89 years, respectively. The influence of
age on the visfatin concentration was minimized by selecting the subjects within the
specified age group (25–45 years) since age is a known risk factor for both periodontitis
and visfatin expression.[31]
In the present study, the mean baseline salivary visfatin levels in group II and group
III were 26.66 ± 2.24 and 38.22 ± 3.38 ng/mL ([Table 1], [Fig. 1]). Similar observations were made by Pradeep et al,[30] where visfatin levels in serum and GCF increased proportionately with the severity
of the disease. These changes were seen since visfatin is actively secreted by predominant
cells involved in periodontal disease activity and variability in visfatin concentrations
within patients of each group could be attributed to the role of visfatin in different
stages of a disease process. Also, Tabari et al[32] speculated that changes in the microbial composition and the ongoing inflammatory
process in the pocket environment have a close relationship with the visfatin levels
in saliva.
Fig. 1 Comparison of salivary visfatin levels in group I, II, and III.
Table 1
Comparison of salivary visfatin levels in group I, II, and III
|
Group I
|
Group II
|
Group III
|
p-Value
|
Intergroup comparison
|
|
Mean
|
SD
|
Mean
|
SD
|
Mean
|
SD
|
|
25.60
|
2.19
|
26.66
|
2.24
|
38.22
|
3.38
|
<0.001[a]
|
Group I vs. III (p < 0.001**)
Group II vs. I (p = 0.59)
Group III vs. II (p < 0.001**)
|
Abbreviation: SD, standard deviation.
a
p < 0.001 highly significant.
The mean levels of salivary visfatin in periodontitis subjects were 38.32 ± 3.83 ng/mL
that are in accordance with the study done by Nokhbehsaim et al,[33] who demonstrated that visfatin stimulates the production of C–C motif chemokine
ligand 2 and matrix metalloproteinase-1 in the periodontal ligament cells and thus
can lead to inflammation of periodontium and destruction of connective tissue. This
production of visfatin in the periodontal ligament cells could be induced by periodontal
pathogens, Porphyromonas
gingivalis and Fusobacterium
nucleatum, and proinflammatory cytokine, IL-1β. Hence, the microbial and inflammatory signals
can use visfatin for its destructive effects on the periodontium. In addition, visfatin
can result in the production of proinflammatory and matrix-destructing cytokines,
and therefore, interfere with the regenerative capacity of periodontal ligament cells.[34]
In the previous studies, the correlation between obesity and visfatin levels is confirmed,[33] showing that visfatin levels were higher in obese individuals as compared with normal-weight
controls. In this study, we tried to find the correlation between salivary visfatin
levels and normal and overweight patients (based on BMI) that was not compared in
the previous studies to date. The results we got were surprising. Visfatin levels
in the normal weight patients were 3.31482 ng/mL, and the visfatin levels in the overweight
patients were 3.1487ng/mL. This shows that at baseline, visfatin levels increase irrespective
of whether the patient is normal weight or overweight. These values were statistically
significant (p < 0.001) ([Table 2], [Fig. 2]). This negative correlation between BMI and salivary visfatin levels may be because
of (i) ethnic heterogeneity may affect visfatin level or visfatin sensitivity and
(ii) genetic association analysis found that single-nucleotide polymorphism locus
and two other loci located at the intron region of the visfatin gene were associated
with lipid metabolism, which indicates that this gene may account for some variation
in the concentrations of visfatin, which is in accordance with the study done by Jian
et al.[35]
Fig. 2 Mean visfatin levels in normal and overweight subjects in all three groups.
Table 2
Relationship between BMI (kg/m2) and salivary visfatin levels
|
BMI category
|
Mean
|
Sample (n)
|
SD
|
SE mean
|
Significance
|
|
Normal weight
|
33.148
|
33
|
6.7098772
|
1.3991061
|
p < 0.001
|
|
Overweight
|
31.487
|
27
|
6.2788810
|
1.5228523
|
Abbreviations: BMI, body mass index; SD, standard deviation; SE, standard error.
This study has a few limitations. First, the study sample size could have been larger
and second, the study could have included obese patients and could have been done
for a longer duration. Therefore, further multicenter longitudinal studies have to
be conducted to critically evaluate the results of the current study, with a larger
sample population.
Despite these limitations, our study confirmed that salivary levels of visfatin were
increased in the order of disease severity; least in periodontally healthy, followed
by gingivitis and most in periodontitis subjects. Hence, visfatin can be used as a
diagnostic marker in periodontal disease.
Conclusion
The results of this study suggest that salivary visfatin concentrations were higher
in patients with gingivitis and periodontitis compared with periodontally healthy
individuals. Therefore, salivary visfatin is a potential inflammatory biomarker and
acts as a mediator in the pathogenesis of periodontal disease and may be considered
as a diagnostic biomarker of oral diseases like periodontitis.
