Estimation and Correlation of Salivary and Serum Omentin-1 in Chronic Periodontitis: An Interventional Study

Introduction

Periodontitis is a polymicrobial disease of inflammatory origin and plaque biofilm is the primary cause of periodontitis. Because of the microbial dysbiosis within the biofilm, an inflammatory   condition   develops that is persistent, damaging, and non-resolving. The biofilm poses a microbial challenge that causes tissue damage (such as alveolar bone resorption, pocketing, and periodontal ligament destruction) that is mainly brought on by the inflammatory response of the host.¹ Inflammatory mediators, such as the pro-inflammatory cytokines like Interleukin-1 (IL-1), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Prostaglandin-E2 (PGE2), are released by the host in reaction to these infectious pathogens, and these mediators exacerbate the periodontal destruction.² Environmental variables, systemic conditions like diabetes mellitus, and genetic susceptibility also affect periodontal destruction along with the host immune- inflammatory reaction in the periodontal tissues.³

Adipose tissue releases numerous inflammatory substances known as ‘adipokines’, which include cytokines, hormones, and different proteins.⁴ These molecular species are thought to have an effect on inflammation   and   immune   responses   in   addition to affecting insulin resistance.⁵ The influence of adipokines in the pathogenetic processes interlinking it to periodontal disease (PD), are not well elucidated.

Omentin-1, also referred as Intelectin-1, is a new adipocytokine having 313 amino acids that has only recently been discovered, which is released in visceral adipose tissue in addition to mesothelial cells, vascular cells, plasma, etc.⁶ Of the homologous isoforms in humans, Omentin-1 is primarily found in human blood circulation.⁷ Omentin is known to exhibit anti- inflammatory, anti-diabetic and anti-atherogenic features.⁸ This anti-inflammatory adipokine has paracrine and endocrine effects and plays a significant role in glucose metabolism.⁷ Omentin-1 has been associated with inflammatory conditions and also periodontitis. Gingival Crevicular fluid (GCF) Omentin-1 levels have been down-regulated by inflammatory conditions like periodontitis.^9,10 Scaling and root planing (SRP) reduces the oral bacterial load in the oral cavity. Few studies so far have assessed the salivary and serum levels of Omentin-1 in periodontitis. Therefore, this research was carried out to measure and compare serum and salivary Omentin-1 levels in chronic periodontitis patients before and after SRP.

Materials and Methods

The sample size was calculated using G Power software version 3.1.9.2. The total sample size estimated was 35 samples when the effect size to be measured (d) was set at 80%, the power of the study was set at 80%, the confidence interval was set at 95%, and the margin of error was set at 5%.

For the enrollment of thirty-five participants, patients in the age range of 35 and 60 years visiting the Department of Periodontology and Oral Implantology at the Vydehi Institute of Dental Science and Research Center in Bangalore were screened. The research included 35 systemically healthy subjects with clinically diagnosed chronic periodontitis according to the American Academy of Periodontology (1999) classification, with clinical attachment ≥ 3mm in 30% of the sites and probing pocket depth ≥ 5 mm with at least six teeth. The research excluded patients with any active intraoral lesions, any other systemic diseases, pregnant or lactating patients, patients with a current or previous history of smoking, patients with a history of periodontal therapy within the previous six months, or patients who took antibiotics or anti-inflammatory drugs in the last three months. Ethical clearance from the Institutional Ethics Committee was obtained. All the participants provided their written, informed consent.

Clinical protocol

Saliva sample: For the research, passive drool sampling was used to collect 5 mL of whole, unstimulated saliva from each participant between the hours of 9:00 am and 10:00 am. Collected samples were spun at 5000 rpm for 10 minutes in a centrifuge. As soon as possible, supernatant was moved to a sterile plastic vial and kept at -80^o C.

Serum sample: 5 mL blood was drawn from the antecubital fossa by venipuncture using a 5 mL syringe with a 20-gauge needle and incubated at room temperature for 30-45 minutes to allow clotting. Serum was separated by centrifugation at 5000 rpm for 10 minutes. The extracted serum was immediately moved to a sterile plastic vial and stored at -80^o C until assay.

Post sample collection (Figure 1), all the clinical parameters i.e. Plaque index (PI), Gingival index (GI), Percentage of bleeding sites (BOP), Probing Pocket Depth (PPD) and Clinical Attachment Level (CAL) were recorded at baseline and at three months. Following the collection of baseline samples, all patients received oral hygiene instructions (OHI) and periodontal therapy in the form of scaling and root planing (SRP). Patients' saliva and blood samples were taken at baseline and three months after SRP. Enzyme-linked immunosorbent assay (ELISA) method was used to measure levels of Omentin-1.

ELISA protocol

A commercially available kit (Human Omentin-1 kit- GenlisaTM ELISA, Krishgen Biosystems, Mumbai, India) was used to measure the levels of Omentin-1 using the ELISA method. The Omentin-1 concentrations in materials were measured using this kit's double-antibody sandwich ELISA. Microwells were pre-coated with monoclonal antibodies. Standards and 40 μL of diluted saliva or serum samples were added to microwells, where the antibodies adhered to the human Omentin-1 in the sample. Following the addition of 10 μL of biotin-labeled antibody, 50 μL of streptavidin-HRP was pipetted in, and the mixture was incubated for 60 min at 37°C to create a complex. The substrate solution, 3,3′,5,5′-Tetramethylbenzidine (TMB), was introduced to the microwells after washing the microwells. The quantity of human Omentin-1 determines how the colour changes. The addition of 50 μL of stop solution halted colour development. The solution’s colour turned from blue to yellow. The absorbance was measured at 450nm, and the blank well was computed as zero (Figure 2).

Statistical analysis

Statistical Package for Social Sciences [SPSS] for Windows Version 20 (Released 2011. IBM Corp) was used for statistical analysis. Data was entered in the Excel spread sheet. Descriptive statistics of the explanatory and outcome variables were calculated by mean, standard deviation for quantitative variables, proportions and frequency for qualitative variables. Paired t-test was applied to compare the changes in values of different clinical parameters i.e GI, PI, BOP, PPD and salivary and serum Omentin-1 levels from baseline to three months. Pearson's correlation test was applied to correlate the clinical parameters with Omentin -1 saliva and serum levels. Level of significance was fixed at 5%.

The demographic characteristics analysis for age and gender distribution is shown in Figure 3. Gender distribution did not show any statistically significant difference, and mean age was 46.86 ± 4.5 years.

Comparison of clinical parameters (i.e. GI, PI, BOP, PPD and CAL) at baseline and three months post SRP using paired t test demonstrated a statistically significant decrease in all the clinical parameters (p value 0.001).

The levels of Omentin-1 in saliva and serum showed a statistically significant increase from baseline to three months at p value 0.001 (Table 1).

Correlation coefficient range

0.0            - No correlation

0.01 - 0.20 - Very weak correlation

0.21 - 0.40 - Weak correlation

0.41 - 0.60 - Moderate correlation

0.61 - 0.80 - Strong correlation

0.81 - 1.00 - Very strong correlation

The connection between salivary and serum Omentin-1 levels and clinical parameters at baseline and after three months was examined using Pearson's correlation test (Table 2). Salivary Omentin-1 levels and PI, BOP, PPD, and CAL were found to have a very weak negative correlation at baseline, indicating an inverse relationship between them. Additionally, a very modest negative connection between serum Omentin-1 levels and GI, PI, and BOP levels was seen, indicating an inverse relationship between the variables. However, the correlation did not reach statistical significance. Salivary Omentin-1 levels were shown to be inversely correlated with PI, BOP, and PPD at three months, demonstrating a very weak negative connection. Serum Omentin-1 levels and BOP and PPD also showed a very modest negative correlation, indicating an inverse relationship between them. However, the correlation was not statistically significant.

Discussion

Adipokines, which are bioactive paracrine and endocrine substances veiled by adipose tissue regulate insulin sensitivity, energy expenditure and also have a role to play in inflammation and healing.¹¹ While some adipokines, like resistin, are pro-inflammatory in nature, others like leptin and omentin, have anti-inflammatory influence on the tissues. The majority of adipokines aggravate the severity of diseases, while Omentin is a “good” adipokine.¹² Omentin of visceral stromal cells is a crucial molecule that links adipose tissue with other organs and exerts extensive protective effects. It increases adipocytes' sensitivity to insulin. Lower levels of it are found circulating in individuals with Type 2 Diabetes Mellitus (T2DM) and inflammation.¹⁰

Omentin-1 levels in serum and saliva have only been examined in a small number of periodontitis investigations. Omentin-1 was thus investigated in the current research as a probable biomarker for chronic periodontitis.

A statistically significant decrease in each clinical parameter from baseline to three months after SRP was observed on the assessment of clinical parameters in the current study. This indicates a reduction in periodontal inflammation following Non- Surgical Periodontal Therapy (NSPT). The decrease in subgingival bacterial load following NSPT in turn leads to a decrease in periodontal inflammation, subsequently leading to improved clinical parameters. There was a statistically significant rise in salivary and serum Omentin-1 levels from baseline to three months after the intervention. Omentin-1 has been connected with inflammatory conditions, periodontitis and diabetes mellitus. Individuals diagnosed with chronic inflammatory bowel disease exhibited lower Omentin-1 levels,¹³ also in synovial fluid of rheumatoid arthritis patients¹⁴ and in T2DM patients.15 Omentin most likely contributes significantly to the etiology of inflammation in these diseases.

Omentin levels were low at baseline in the current investigation, which can be related to inflammation in CP, which changes the host immune-inflammatory equilibrium¹⁶ by producing cytokines that have the ability to reduce Omentin levels. The lower salivary Omentin levels in the study may be because of fluctuation in the release of these molecules under local inflammatory conditions in the periodontium. The results of our study are in agreement with the findings of Doğan et al., where the Gingival Crevicular fluid (GCF) levels of Omentin 1 were decreased at baseline and increased after NSPT.¹⁰ In addition to this, Doğan et al. also showed a reverse correlation between TNF-α levels and Omentin-1 levels in GCF of chronic periodontitis patients.¹⁰ Local release of inflammatory mediators in the periodontium in chronic periodontitis is because of persistent inflammatory conditions. Pro-inflammatory   intermediaries   like IL-6 and TNF-α are common in chronic periodontitis which may have an impact on Omentin 1. This could explain why the Omentin levels were lower in chronic periodontitis. These findings may imply that Omentin-1 levels reveal that chronic inflammation likely plays a significant role in the development of periodontitis.¹⁷

Similar to the reports of Balli U et al.,⁹ Dogan SB et al.,¹⁰, and Dooxa Nongrum et al.,¹⁸ the Omentin-1 levels in saliva and serum were considerably greater after non- surgical periodontal therapy in our study. Omentin-1 levels may be associated with inflammation and adversely affected by it because their levels are lower in individuals with T2DM and chronic inflammatory diseases.^9,10,18 In addition, Omentin-1 levels rose during periodontal treatment, indicating an anti-inflammatory function in periodontitis.⁹

The Pearson’s correlation test determined the association between salivary and serum Omentin-1 levels and clinical parameters exhibited a very weak inverse correlation between salivary Omentin-1 levels and PI, BOP, PPD and CAL at baseline. A very weak inverse correlation was also seen between serum Omentin-1 levels and GI, PI and BOP. At three months, a very weak inverse correlation was found between salivary Omentin-1 and PI, BOP and PPD. A very weak inverse correlation was also seen between serum Omentin-1 levels and BOP, PPD. Only a few other studies have established a link between salivary and serum Omentin-1 levels.

Hypothetical mechanisms for Omentin-1 suggest that it impacts insulin sensitivity and production, which in turn effects adipose, brain, muscle, liver, and other organ cells.¹⁵ Similar to this, a small number of experimental investigations have demonstrated that Omentin-1 activates Adenosine 5-monophosphate (AMP)- activated protein kinase (AMPK) via insulin receptor substrate (IRS) by restricting the rapamycin (mTOR-p70S6K).¹⁹ Also, Omentin regulates the inflammatory process by acting as an anti inflammatory cytokine by impeding the TNF α induced COX2 expression through the inhibition of c Jun N terminal kinase signal via instigation of 5′ adenosine monophosphate activated protein kinase (AMPK)/ endothelial nitrate synthetase (eNOS)/nitric oxide (NO) pathway.²⁰

The present study demonstrated that patients with chronic periodontitis had Omentin-1 detected in theirserum and saliva. There was a significant correlation between clinical parameters and salivary and serum Omentin-1 levels, as well as an increase in Omentin-1 levels after NSPT, according to the findings. The results of the current research demonstrated that Omentin-1 was found in the serum and saliva of chronic periodontitis patients and decreases after NSPT in these individuals. Omentin-1 can be detected in saliva, making it a non-invasive test that may also serve as an indicator for periodontal disease diagnosis, prognosis, and therapeutic response. However, additional studies in molecular research to explore the impact of these adipokines on inflammatory pathways are vital for better comprehension of the pathological mechanisms that link this adipokine to periodontal disease.

Conclusion

In chronic periodontitis, there is a substantial link between salivary and serum Omentin-1 levels. As a result, the Omentin-1 level may be used as an inflammatory marker for periodontal disease assessment and its treatment outcome.

Source(s) of support

Nil

Conflicting Interest

Nil

Acknowledgment

Nil