Comparative Evaluation of Chemerin and Visfatin Levels in Saliva of Patients with Chronic Periodontitis and Type-2 Diabetes Mellitus: A Clinicobiochemical Study

Introduction

Periodontitis is a chronic inflammatory disease characterized by destruction of hard and soft supporting tissues of the teeth. A large number of inflammatory cytokines are involved in the disease process. The environmental factors, individual differences and genetic predisposition, can influence tissue response and increase the severity of periodontitis.¹

Diabetes mellitus constitutes a heterogeneous group of metabolic diseases in which elevated blood glucose levels lead to disturbances in the metabolism of carbohydrates, fats and proteins.^2,3 Adipose tissue produces several inflammatory cytokines; the most important of which are adipokines. These cytokines affect insulin sensitivity, alter glucose and lipid metabolism, and influence inflammatory responses.⁴

Chemerin is a pro-inflammatory adipokine. Under normal physiological conditions, chemerin remains in an inactive state as prochemerin. However, under inflammatory conditions, it is activated by removal of the C-terminal amino acid by proteolytic enzymes such as cathepsin G. There is evidence for the pro-inflammatory impact of chemerin as chemerin volumes have been shown to positively correlate with recognized markers of inflammation such as TNF α, IL6.^4,5

Visfatin, also known as preB cell colony enhancing factor and nicotinamide phosphoribosyl transferase, was originally described by Samal et al. Other than adipose tissue, this protein is synthesized by a variety of cell and tissue types, including lymphocytes, bone marrow, liver cells, skeletal muscle, trophoblasts, and fetal membranes. It has been suggested that visfatin can induce production of IL1β, TNFα and IL6 during infection and inflammation. Studies have shown that visfatin serum/plasma levels are increased in a number of inflammatory conditions. Visfatin levels in both GCF and serum have been shown to progressively increase as periodontitis progresses. Visfatin has been reported to be identifiable and measurable in saliva.⁶ With this in mind, chemerin and visfatin levels can be considered as potential pro-inflammatory biomarkers for diabetes and periodontitis.

Saliva is a clinically informative biological fluid (biofluid) useful for novel prognostic, laboratory, or clinical diagnostic approaches and monitoring of patients with oral and systemic diseases. Saliva is easy to collect, store and is ideal for early disease detection as it contains specific soluble biological markers (biomarkers).⁷

The main objective was to detect the biomarkers chemerin and visfatin in the saliva of patients with chronic periodontitis and Diabetes mellitus 2 (T2DM). The secondary objective was to determine whether levels of these biomarkers correlate with the severity of periodontitis in patients with and without T2DM.

Materials and Methods

A total of 80 subjects in the age range of 35-75 years were recruited from the out patients visiting the Outpatient Department between December 2019 - September 2021.

The study protocol was approved by the Ethics Committee and the study was performed in accordance with the code of ethics of the World Medical Association according to the Helsinki Declaration of 1975, as revised in 2013.⁸ The study was clearly explained and the informed consent was obtained from the subjects.

A total of 80 subjects were recruited for the study and grouped into Group I - 20 subjects without chronic periodontitis or T2DM (healthy individuals), Group II - 20 subjects with chronic periodontitis and without T2DM, Group III - 20 subjects with T2DM and without chronic periodontitis and Group IV - 20 subjects with T2DM and chronic periodontitis.

Patients with chronic periodontitis were diagnosed based on the classification of 1999 World Workshop of Periodontology by American Academy of Periodontology (attachment loss ≥5 mm at more than 30% of the sites in patients aged ≥35 years and who showed radiographic evidence of bone loss).⁹ The diagnosis of patients with T2DM was based on the criteria given by the World Health Organization.¹⁰ Both well controlled and poorly controlled diabetic patients were included.

Smokers, alcoholics, obese individuals, pregnant and lactating women and those taking oral contraceptive drugs, presence of an active infection other than periodontitis, intake of antibiotics/corticosteroids and/ or non-steroidal anti-inflammatory drugs during the previous three months, those who received professional periodontal treatment during the past six months, subjects with periapical pathology, orthodontic appliances and multiple systemic complications of diabetes mellitus were excluded from the study.

The demographics and baseline clinical characteristics were collected from patients. Periodontal examination included: clinical measurement of Gingival index (GI) by Loe and Silness (1963), Plaque index by Silness and Loe (1964),¹¹ Bleeding on probing by Ainamo and Bay (1975),¹² Probing pocket depth (PPD) and clinical attachment level (CAL). A single, examiner completed full mouth assessment of periodontal conditions, except for third molars, using William’s graduated manual probe (Hu- Friedy, Chicago, IL) and recorded the information.

Sampling was performed in the morning around 9 am to avoid any variations due to circadian rhythm. Whole human saliva was collected from the seated subjects as it flows into the anterior floor of the mouth over 10-minute period and collected into sterile polypropylene container by passive spitting method.

Saliva samples was frozen immediately at -80°C until analysis at which point the samples were thawed and cleared by centrifugation at 3000 rpm for 20 minutes. Saliva sample was assayed using commercially available ELISA kits (enzyme linked immunosorbent assay) to determine the level of chemerin (BIOASSAY) and visfatin (BIOASSAY) according to the manufacturer’s recommendations (Figure 1).

Statistical Package for Social Sciences [SPSS] for Windows, Version 22.0. (Released 2013. Armonk, NY: IBM Corp) was used for statistical analyses. Descriptive statistical analysis was carried out in our study. It included expression of the study variables with categorical data in terms of number & percentage, and in mean & SD for continuous data. Inferential statistics used in the study were: Chi Square test was used to compare the difference between gender and bleeding on probing between different study groups. Kruskal-Wallis test followed by Mann-Whitney’s post hoc analysis was used to compare the age, mean values of different clinical, diagnostic parameters, chemerin and visfatin biomarkers between the four study groups. Spearman’s Correlation test was used to estimate the relationship between diagnostic, periodontal parameters, chemerin and visfatin levels in different study groups. Stepwise multiple linear regression analysis was performed to predict the salivary chemerin and visfatin levels using clinical & diagnostic parameters in each study group. The level of significance [p-value] was set at p <0.05.

Results

In this study, chemerin and visfatin were detected in all the saliva samples. The concentration differed in all the groups as shown in Table 1. 

Table 2 infers that the clinical parameters such as PI, GI, PPD and CAL were highest in group 4 followed by group 2, group 3 and group 1. Group 1 showed considerably reduced scores of clinical parameters.

The salivary levels of chemerin and visfatin were compared between four groups (Table 3 & 4). The chemerin and visfatin levels ranged from 0.75 ng/mL - 2.87 ng/mL and 22.08 ng/mL -36.17 ng/mL, respectively. Comparison showed that the concentration of salivary chemerin was least in healthy group and highest in CP (2.87±0.86) and T2DM groups (2.01±0.29). The maximum levels of visfatin were seen in CP ( 36.17±7.99) followed by T2DM (31.19±9.31) groups.

Chemerin showed a weak positive correlation with age and moderate positive correlation with PI, GI, FBS, and HbA1c. A strong positive correlation was seen with PPD, CAL, visfatin in chronic periodontitis + T2DM subjects. A statistically significant correlation was seen with PI, GI, FBS, PPD, CAL, HbA1c and visfatin.

This infers that with an increase in the periodontal parameters like PI, GI, PPD and CAL, chemerin also increases suggesting a positive correlation of chemerin and chronic periodontitis.

Overall, visfatin was shown to have statistically significant correlation with the parameters like age, GI, PI, PPD, CAL, and chemerin.

This infers that with an increase in the periodontal parameters like PI, GI, PPD and CAL, visfatin levels also increase suggesting a positive correlation of visfatin levels and chronic periodontitis.

Discussion

Periodontal disease is considered as the most important and widespread pathologies worldwide and represents one of the most remote causes of low-grade systemic inflammation. Diabetes mellitus represents a heterogeneous group of metabolic diseases in which elevated blood glucose levels lead to impaired lipid and protein metabolism. Adipokines are soluble proteins secreted by adipocytes that bind to receptors on target cells and initiate intercellular signaling cascades that lead to phenotypic changes in the cell through altered gene expression and regulation.

Chemerin is a new adipokine secreted by the liver and adipocytes and activated by serine proteases of the hemostatic or inflammatory cascades by cleaving the carboxyl-terminal peptide of the molecule, releasing its chemotactic potential and causing the recruitment of CMKLR1-positive cells, such as immature dendritic cells and macrophages. The main function of chemerin in inflammation is its chemotactic role. In addition, recent evidence indicates that inflammatory cytokines may play a role in the release of chemerin from adipose tissue.

Visfatin is also known as nicotinamide phosphoribosyl transferase (NAMPT), an enzyme that inhibits the biosynthesis of nicotinamide adenine dinucleotide. It is a multipotent mediator, functioning as a growth factor, cytokine, enzyme with a role in energy metabolism, and pro-inflammatory mediator. It is preferentially secreted by visceral adipose tissue and can also be produced by neutrophils and macrophages. It also triggers the synthesis of prostaglandin E2 and stimulates IL6, IL1β, TNF α in inflammatory conditions.

Therefore, our study was performed to determine the baseline levels of two biomarkers (chemerin and visfatin) associated with chronic inflammation in saliva and to assess their concentrations in patients with and without chronic periodontitis and type 2 diabetes mellitus. The highest levels of chemerin and visfatin were found in type 2 diabetics with chronic periodontitis (group IV), followed by only patients with chronic periodontitis (group II), only patients with diabetes mellitus (group III), and the healthy group (group I).

Our study was in accordance with the study done by Ozcan et al¹³ and Jentsch et al¹⁴ who assessed chemerin levels during periodontal inflammation in saliva and gingival crevicular fluid, respectively. Both found that salivary chemerin levels were notably greater among the periodontitis group than the healthy and gingivitis groups. Similar feature was observed in our study, with increased chemerin levels in chronic periodontitis group than the healthy group.

Study by Abolfazli N et al.,¹⁵ inferred significant decrease in mean salivary and serum levels of visfatin after non-surgical periodontal treatment (p <0.05). Also, significant decrease in the clinical parameters of PI, GI, PPD and CAL following non-surgical periodontal treatment was observed, indicating a decrease in the inflammatory load of disease condition. Our study was different from this study, where salivary visfatin levels were compared in four different groups.

Thus, visfatin can be considered as a possible marker in the inflammatory activity of periodontal disease.

To the best of our knowledge, we believe that this is the first study to evaluate the relationship between chronic periodontitis and diabetes in relation to the salivary levels of chemerin and visfatin in a random Indian population sample.

The main strength of the present study can be owed to the fact that the two confounding factors namely obesity and smoking were eliminated and the subjects with any other systemic disorders were also not included in the study.

There were few limitations associated with the present study. First, the study had a small sample size. Larger sample is required to determine the value of salivary chemerin and visfatin necessary for evaluating diabetes control and complications. Second, the concentrations of the chemerin and visfatin signifies the limitations at baseline and after periodontal therapy to analyze the actual involvement of these biomarkers in the pathogenesis of periodontitis and T2DM. Third, the present study could not completely exclude the possible influence of oral antidiabetic drugs in salivary chemerin and visfatin levels.

Conclusion

Within the limitations of the present study, it can be concluded that saliva of patients with chronic periodontitis and T2DM expressed higher levels of chemerin and visfatin than those without T2DM. The present study showed up regulation of chemerin and visfatin in the saliva of subjects with chronic periodontitis with/without well controlled type 2 diabetes mellitus and poorly controlled type 2 diabetes mellitus, when compared to healthy subjects.

Conflict of Interest

None