Effect of Non-Surgical Periodontal Therapy on Salivary Leptin Levels in Chronic Periodontitis Patients - A Clinico Biochemical Study

Introduction

A microbial infection-related chronic inflammatory condition called chronic periodontitis arises from an ecological imbalance between the microbial biofilm on the teeth and the host immune inflammatory response, leading to the breakdown of tooth-supporting structures, ultimately causing tooth loss. This destructive process is mediated by the generation of eicosanoids, matrix, and cytokines which in turn activate host cells, inducing significant connective tissue damage and bone breakdown. A 16-kDa peptide hormone called leptin is mostly produced by adipocytes, with modest amounts also being produced by the placenta, stomach epithelium, T cells, osteoblasts, and intralobular ducts of the major salivary glands. Leptin can be considered a connecting link between the neuroendocrine and immune systems.¹

Currently, the major limitations preventing people from recognizing full potential of disease detection and seriously hampering the development of clinical diagnostics are - lack of definitive molecular biomarkers for specific diseases, absence of a simple and low-cost sampling method with minimal discomfort and lack of an accurate, easy to use, portable platform to facilitate early disease detection.² A myriad of biomarkers in serum/plasma, gingival crevicular fluid (GCF), and saliva in individuals with periodontitis have the capacity to offer understanding beyond the conventional clinical and radiological characteristics of the disease process.³

A variety of biomolecules that are either produced locally or derived from the vascular beds in the gingival tissues can be located in saliva, an easily accessible biologic fluid. Hence, saliva has emerged as a potential diagnostic medium in the evaluation of systemic and oral diseases, notably periodontal diseases. However, despite the potential use of serum biomarkers in identifying the existence, risk, progression, and impact of chronic periodontitis, leptin concentrations in patients’ saliva have not been investigated. Hence this investigation was done to evaluate the leptin levels in saliva of chronic periodontitis patients before and after NSPT and in doing so to confirm that salivary leptin levels are biomarkers for periodontal health status.

Materials and Methods

Study design

The current research was conducted after obtaining clearance from the Ethical Committee of Sri Siddhartha Dental College. Written informed consent from all the participants chosen for the investigation was obtained. The investigation was carried out in the Periodontics department, Sri Siddhartha Dental College and Hospital, Agalakote, Tumkur and Laboratory Settings (ACUMEN LABORATORY, Bangalore).

Inclusion and exclusion criteria

A total of twenty-four patients in the age group of 25 to 60 years from Sri Siddhartha Dental College and Hospital were chosen and the subjects were divided into two groups. Twelve healthy patients who had no clinical signs of gingivitis or periodontitis made up Group I - the control group. Twelve patients who were diagnosed with chronic periodontitis and had pockets that measured 4-6 mm deep, with at least 20 teeth present made up Group II - the study group. Two subjects with chronic periodontitis with a body mass index (BMI) less than 30 and in good general health were identified.

Subjects with systemic diseases, smokers and alcoholics, pregnant / lactating mothers, those using or used antibiotics or immune suppressants in the last six months, those who had undergone periodontal therapy (surgical/non-surgical) in the past six months were excluded from the study.

Saliva sample collection

Before the treatment and one month after periodontal treatment, saliva samples were collected using the following method. Unstimulated saliva was collected by asking patients to expectorate into disposable plastic container. To remove cell debris, saliva samples were centrifuged. 0.5 mL of the supernatant was divided into 1.5 mL aliquots after being spun at 4,000 rpm for 10 minutes. A tracking number was assigned to each aliquot, and sent to lab for analysis. Group II patients were evaluated clinically after one month of periodontal therapy to revaluate the previously mentioned periodontal measures.

Clinical examination

Clinical examination was carried out by a single examiner. Periodontal health status was assessed using Plaque index by Silness and Loe (1964), Gingival index by Loe and Silness, Modified sulcular bleeding index (mSBI) by A Mombelli and associates, Probing depth (PD) and Clinical attachment level (CAL) measurements.

Reagents preparation

All reagents were brought to room temperature before use. Then 500 mL of 1x wash buffer was prepared by diluting 20 mL of wash buffer concentrate 30x into deionized or distilled water. Crystals formed in the concentrate were dissolved by mixing gently. 

Assay procedure

The strips were inserted in the frame after determining the numbers required and the unused strips were stored at 2 to 8 ̊C. All the reagents were brought to room temperature before use after being properly prepared, including the standard solutions and samples. 50 μL standard solutions were added to standard wells. 40 μL samples were added to sample wells and then 10 μL of anti-LEP antibody were added to sample wells. Following this, 50 μL streptavidin-HRP was added to sample wells (Not blank control wells). Once well combined, a sealant was applied to the plates and were then incubated for 60 minutes at 37°C. A wash buffer was used to wash the plates five times after the sealer was removed. For each wash, the plates were soaked with at least 0.35 mL wash buffer for 30 seconds to 1 minute. Then each plate was blotted with paper towels or other absorbent material. 50 μL each of substrate solution A and substrate solution B were added respectively to each well and kept for incubation, covered with a new sealer for 10 minutes at 37 ̊C in the dark. 50 μL stop solutions were added to each well and the colour change from blue to yellow was noted immediately. Within 30 minutes after adding the stop solution, the optical density of each well was determined using a microplate reader set to 450 nm.

Statistical analysis

The mean value and standard deviation was compared using student t test and pre-treatment and post-treatment measurements were compared using student paired t-test. Measurements were statistically analysed using software SPSS version 16. p value ≤0.05 was considered as significant.

Results

This was an in vivo interventional study conducted to assess salivary leptin levels in individuals with chronic periodontitis after NSPT. Subjects with normal BMI were clinically examined and divided into Group-I (healthy subjects) and Group-II (chronic periodontitis patients). Clinical parameters such as clinical attachment level, plaque index, gingival index, modified sulcular bleeding index, probing depth were recorded. Salivary leptin was obtained to assess the leptin levels using an enzyme before and one month after periodontal therapy.

A comparison of salivary leptin levels between Group I (healthy subjects) and Group II (chronic periodontitis patients) is shown in Table 1. Table 2 shows the comparison of clinical parameters between Group I (healthy subjects) and Group II (chronic periodontitis patients). Descriptive statistics of the periodontal variables, including clinical attachment level, probing depth (PD), modified sulcular bleeding index (mSBI), plaque index (PI), gingival index (GI), at baseline in Group I (healthy subjects) and Group II (patients with chronic periodontitis) is displayed in Table 2. PI, GI, mSBI, PD, and CAL mean values were different between Group I (healthy participants) and Group II (patients with chronic periodontitis) at baseline, and these differences were highly significant in terms of statistics (p >0.0001). Every sample in each group was positive for the leptin assay.

Lipid-based leptin levels in Group II patients with chronic periodontitis before and one month after treatment was assessed. The mean value of salivary leptin levels before treatment was 21.00±8.83 ng/mL and one month after the treatment was 24.58±8.50 ng/ mL. The mean difference was 3.57±3.53 which was not statistically significant (p <0.05).

Table 3 shows the comparison of values of clinical attachment loss, modified sulcular bleeding index (mSBI), probing depth (PD), plaque index (PI), gingival index (GI), pre-treatment and one month after treatment in Group II (patients with chronic periodontitis). From baseline to one month after therapy, PI, GI, mSBI, PD, and CAL values dropped dramatically, and the variations in their averages were statistically highly significant (p >0.0001).

Discussion

All the diagnostic procedures for the periodontal diseases should focus on providing essential data to insightful clinicians pertaining to the type, location and severity of periodontal disease. These results form the basis for the early detection and determination of holistic treatment plan and can serve as valuable tools during all the phases of treatment. The future of diagnostic research in periodontal and oral diseases is driving rapidly towards systems whereby periodontal biomarkers are relevant to detect the severity of periodontal diseases early. Studies by Zhong et al., ⁴ and many others comparing individuals with periodontitis to healthy controls have reported elevated levels of biomarkers to the inflammatory response.

The 16 kDa leptin is a circulating protein and polypeptide hormone which is derived from adipocytes and has been upgraded to the status of a cytokine in recent years. Leptin is thought to control how the body reacts to diverse inflammatory stimuli by concurrently increasing cytokine synthesis and macrophage phagocytosis, which stimulates the creation of human peripheral blood mononuclear cells and natural killer cells.⁵

Leptin orchestrates various activities like body weight, reproduction, immune function, and angiogenesis and its decisive or crucial function is to regulate the inflammation.⁶ According to the hallmark study done by Karthikeyan & Pradeep, while estimating the leptin levels, obese patients were excluded to avoid bias & only subjects within normal body mass indices were included. When comparing mean BMI in the current study, there was no distinction between Group I (healthy participants) and Group II (patients with chronic periodontitis). This finding matched that of the research conducted by Karthikeyan & Pradeep.⁷ Salivary leptin levels were observed to be lower in Group II than in Group I in the current investigation demonstrating the protective function of leptin in the saliva; however, it was challenging to determine the exact nature of the protective mechanism of leptin. These results are consistent with the findings reported by Purwar et al.,^1,8 and Karam et al. ⁹

Leptin in saliva may have been produced by salivary glands or by the leaching of GCF components from saliva. Leptin receptor expression may have increased during periodontal inflammation as a result of cytopathic changes, increasing quantity of leptin that binds to these receptors and lowering leptin levels in the patient’s saliva. This would explain the decrease in the levels of salivary leptin in chronic periodontitis group.^10,11 After one month following periodontal therapy, there was an elevation of salivary leptin levels in Group II (chronic periodontitis patients) compared to baseline, similar to the findings reported by Karam et al.,¹² and Purwar et al. But the variance in the mean difference was statistically insignificant which might be due to smaller sample size.

Patients in Group II (chronic periodontitis) treated with NSPT showed elevation in salivary leptin values relative to the baseline indicating that treatment effect may exist. The other two patients showed decrease in salivary leptin levels during periodontal therapy that could have been caused by any additional unidentified medical conditions which were not taken into account when choosing the sample or it could also be explained that the inflammation might not have stopped after treatment. Some of the individual leptin concentrations obtained from saliva samples (4 out of 12) of Group I subjects showed variance from normal range as salivary leptin levels can be altered by many minute factors like age, gender, BMI, allergic conditions, menstrual factors, necrotic conditions, taste perception, and other unknown medical conditions.^7,13-17 More precise criteria for inclusion of subjects needs to be considered for obtaining definite leptin concentrations. Periodontal parameters evaluated a month after periodontal surgical therapy showed significantly improvement (p >0.0001) in the current research which is in concurrence with the findings of Sexton et al.,¹⁸ Haween et al.,⁹ and Purwar et al. ¹⁹

Changes in periodontal characteristics could be attributed to a decrease in periodontal inflammation achieved through NSPT. In this study, leptin levels were determined for evaluating periodontal therapy, the state of periodontal health and its efficacy. The findings demonstrated that levels of salivary leptin in individuals with periodontitis possessed lower levels of salivary leptin than the healthy individuals. Additionally, NSPT increased salivary leptin levels in patients with chronic periodontitis, while also improving clinical periodontal parameters. These outcomes are in concurrence with the study conducted by Purwar et al. ⁸ More studies involving interventions with larger sample size with precise inclusion and exclusion criteria are needed to confirm the results obtained. Combining these findings, it is possible to hypothesise that chronic periodontitis may have lower amounts of salivary leptin due to periodontal inflammation. Periodontal characteristics and leptin concentrations were significantly improved by NSPT.

The sample size in the current investigation was constrained due to careful consideration of the inclusion and exclusion criteria. Consequently, long term interventional trials with larger sample size are needed to potentiate the role salivary leptin as a reliable biomarker for assessing the periodontal health status.

Conclusion

Leptin levels in the saliva of chronic periodontitis patients were not severely altered compared to healthy subjects. A notable decrease in salivary leptin levels from the baseline after individuals with chronic periodontitis were treated with non-surgical periodontal therapy was observed and henceforth, salivary leptin level can be considered as a reliable biomarker to measure the efficiency of periodontal treatment.

Conflict of interest

None