Evaluation of Level of TNF-α among Obese Patients with and without Chronic Periodontitis.

Aim: The purpose of this case-control study was to evaluate the role of TNF-α in gingival crevicular fluid among obese women with chronic periodontitis. Materials and
Methods: A total of 60 subjects were randomly selected for the study. The study population was further divided into three groups (Group 1, Obese women without periodontitis; Group 2, Non-obese women without periodontitis; Group 3, Obese with chronic periodontitis). Gingival crevicular fluid samples were taken for assessment based on clinical parameters like probing depth, bleeding on probing, and BMI.
Results: There was significant increase in the level of TNF-α in gingival crevicular fluid from obese patients with chronic periodontitis and a positive correlation was found between TNF-α levels and values of the plaque index, OHI -I and BMI.
Conclusion: Based on these results, it can be concluded that GCF level of TNF-α was significantly higher in obese patients with and without chronic periodontitis, and highest in obese patients with chronic periodontitis group. Copyright:

INTRODUCTION

Obesity is recognized as a chronic disease with a multifactorial etiology. Genetic, environmental factors, socioeconomic and behavioral influences lead to excess caloric intake and decreased physical activity, which leads to energy imbalance. Energy imbalance leads to storage of excess energy in adipocytes, which exhibit both hypertrophy and hyperplasia that leads to intracellular abnormalities of adipocyte function, particularly endoplasmic reticulum and mitochondrial stress. The abnormalities of adipocyte function leads to consequences like adipocyte insulin resistance, production of adipokines, free fatty acids, inflammatory mediators and promotion of systemic dysfunction that leads to clinical manifestations and sequelae of obesity. The adipose tissue–derived adipokines are TNF-α, IL-6, IL-1β, leptin, adiponectin, resistin, acylation-stimulating protein, SAA3, α 1 acid glycoprotein, pentraxin, IL-1 receptor antagonist, and macrophage migration inhibitor factor.[1] Among cytokines, TNF-α is considered one of the main cytokines related to inflammation and immune processes, and operates in various parts of the body. It was discovered by Carswell et al. in 1975.[2] The present study is done to evaluate the role of TNF-α among obese women with and without chronic periodontitis.

SUBJECTS AND METHODS

Examination included 60 female patients, aged 20–45 years. The study population was further divided into 20 obese patients without periodontitis, 20 non-obese women without periodontitis, and 20 obese patients with periodontitis. All subjects were systemically healthy. Women under hormonal therapy, who were pregnant, lactating, patients who had periodontal therapy done six months prior to the study, patients under any medications within three months prior to this study, patients with lesser than 20 permanent teeth, and teeth with fixed or removable prosthetics were excluded from this study. Alcoholics, paan chewers, and drug addicts were also excluded from this study. Obese individuals with BMI greater than 25 kg/m2 were included in this study.

The study protocol was explained to the participants; each subject completed a detailed medical questionnaire and received a complete periodontal examination, which included oral hygiene index (simplified),[3] plaque index (PI),[4] CPITN,[5] probing depth (PD), and clinical attachment level. Ethical clearance was obtained and written informed consent was also obtained from each patient.

BMI is calculated using the formula

The study population was divided into obese and non obese group based on Table 1.

Table 1

Classification and definition of overweight and obesity based on expert panel, 1998[6]

Classification	BMI kg/m2
Underweight	<18.5
Normal	18.5-24.9
Overweight	25-29.9
Obesity Class - I	30-34.9
Obesity Class - II	35-39.9
Obesity Class - III	40+

The study population was divided into:

Group 1 - Obese women without periodontitis: Absence of bleeding on probing, probing depth ≤3 mm and no evidence of clinical attachment loss with BMI ≥25.

Group 2 - Non-obese women without periodontitis: Absence of bleeding on probing, probing depth ≤3 mm and no evidence of clinical attachment loss with BMI ≥ between 18.5–24.9.

Group 3 - Obese with chronic periodontitis: Presence of bleeding on probing, probing depth ≥5 mm, CPITN index >3 and BMI ≥25.

Gingival crevicular fluid (GCF) sampling

After isolation and preparation of the concerned tooth, calibrated microcapillary tubes were used to collect GCF sample from the site with greatest probing pocket depth as per the recommendation of Sueda et al. 1969.[7] A standardized volume of 2–3 microlitre of crevicular fluid was collected by placing the tip of the pipettes extracrevicularly. The sampling time was 5–15 mins. Samples of GCF contaminated by blood or saliva were discarded. GCF samples were then transferred into Eppendorf tube. A total of 60 GCF samples collected were stored at -80°C. The samples were then assayed for TNF-α concentration by using the human TNF-α ELISA kit.

Determination of TNF-α levels

The TNF-α level was evaluated using Diaclone TNF-α ELISA kit (Gen-Probe diagnostic, Cat no. 950.090.096) which is a solid phase sandwich ELISA kit. The detection limit of TNF-α obtained after preparing the standard curve according to the manufacturer's instruction as follows: TNF-α (concentration ranging from 25 to 800 pg/mL) and the TNF-α level in each group was measured according to the manufacturer's instructions.

Assay preparation

After preparing the standard curve according to manufacturer instruction, Add 100 ml of each, Sample, Standard, Control and zero (appropriate standard diluent) in duplicate to appropriate number of wells. Add 50 ml of diluted biotinylated anti TNF-α to all wells. Cover with a plastic plate and incubate at a temperature range of about (18 to 25°C) for three hour(s). After three hours, remove the cover and wash the plates three times; then add 100 μl of Streptavidin-HRP solution into all wells, cover with a plastic plate, and incubate again at temperature range of about (18 to 25°C) for 30 minutes. After 30 minutes, again remove the plastic plate cover and wash the plates three times. Add 100 μl of ready-to-use TMB Substrate Solution into all wells and incubate in the dark for 12–15 minutes at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil. Add 100 μl of H2SO4 (Stop Reagent) into all wells and immediately read the absorbance value of each well on a spectrophotometer using 450 nm as the primary wavelength and, optionally, 620 nm as the reference wave length. Incubation time of the substrate solution is usually determined by the ELISA reader performance. Many ELISA readers only record absorbance of up to 2.0 O.D. Therefore the color development within individual microwells must be observed and the substrate reaction stopped before positive wells are no longer within recordable range.

The levels of TNF-α in the samples were estimated using the standard curve. TNF-α concentration obtained in pg/mL.

RESULTS

The data was collected for various parameters like age, oral hygiene index (simplified), plaque index, probing depth, and BMI. The data was analyzed statistically. The obtained data was presented as mean and standard deviations. Data analysis was performed by using SPSS as software for statistics. Mean values were compared among different study groups by using one-way ANOVA followed by Tukey's HSD test. For comparison between groups, Kruskal–Wallis test was used and group comparison was done using Pearson correlation or Kendall Tau-b test.

The TNF-α levels in Group 1 ranged between 184.388 pg/mL - 1648.94 pg/mL, 146.8354 pg/mL - 817.28 pg/mL in Group 2 and in Group 3 between 227.84 pg/mL - 1335 pg/mL The mean value of PI, OHI–S, GI and TNF-α concentration are shown in Table 2 and comparison of TNF – α levels with other variables are shown in Table 3

Table 2

Represents the comparison of mean values of clinical parameters between the study groups

Variables	Group 1	Group 2	Group 3
OHI	1.73±0.44	0.99±0.69	3.72±0.791
PI	0.63±0.144	0.30±0.20	1.536±0.31
BMI	33.51±3.48	22.85±2.42	35.52±3.977
TNF-α	533.517±376.11	399.20±179.27	650.79±357.21

Table 3

Pearson correlation analysis of TNF-α with other parameters

Variables	Correlation coefficient (‘r’) P
	Group 1		Group 2		Group 3
OHI	0.137	0.565 (NS)	-0.005	0.985 (NS)	-0.202	0.394 (NS)
PI	-0.164	0.490 (NS)	-0.217	0.357 (NS)	0.041	0.864 (NS)
BMI	0.254	0.279 (NS)	-0.351	0.219 (NS)	0.015	0.95 (NS)

DISCUSSION

Obesity is considered as the result of a positive energy balance in conditions of energy excess. It has become a challenging health problem in recent years. Adipose tissue is known to secrete a variety of bioactive peptides like adipokines. Cytokines (TNF-α, IL-6, PAI-1) produced by the adipokines are the link between obesity and obesity-related complications. The present study is done to evaluate the role of TNF-α among obese women with and without chronic periodontitis.

The results of the present study shows that TNF-α levels are increased in both the groups, Group 1 (533.51 pg/mL) and Group 3 (650.79 pg/mL) when compared to Group 2 (399.20 pg/mL), but significantly elevated in Group 3 (650.79 ± 357.21 pg/mL) individuals when compared with the other groups, and the results were similar with studies done by JN Fain et al. 2004,[8] Lundin et al. 2004,[9] Wang Y, et al. 2003,[10] Suruchi Khanna et al. 2010,[11] all of whom stated that obesity and periodontitis results in elevated TNF-α level.

The possible explanation is that the cytokine TNF-α negatively affects the local immunity in the periodontal tissue which might be the possible link whereby obesity acts as a risk factor for periodontal disease, as reported by Al-Zahrani et al. 2003,[12] Wood et al. 2003,[13] Saito et al. 2001.[14]

On comparing the mean PI values in the present study, Group 1 patients had a higher plaque score of 0.63 ± 0.144 when compared to Group 2 0.30 ± 0.20, and the mean TNF-α levels were 533.517 ± 376.11 pg/ml and 399.20 ± 179.27 pg/ml for Group 1 and Group 2, respectively. From this observation, it is understood that even in the absence of periodontitis, the presence of higher plaque values, especially in obese patients, has led to higher TNF -α levels. Even on comparison of the values of OHI–S between groups, obesity without periodontitis group (Group 1, 1.73 ± 0.44) had higher value than non-obese without periodontitis group (Group 2, 0.99 ± 0.69).

This shows the definitive influence of obesity on TNF-α levels, especially in the presence of plaque, which is in accordance with the study done by Lundin M, et al. 2004[9] who stated that patients with higher plaque value and obesity had increased TNF-α levels.

The positive association found between obesity and GCF TNF-α level in this study, as shown in Table 2, could explain the fact that it is the first step in the cellular and molecular chain of events which could lead to the development of periodontal disease, as TNF-α plays an essential role in soft tissues (interleukins and matrix – metalloproteinase activation) and alveolar bone destruction.[15]

CONCLUSION

Within the limits of this study, it can be concluded that GCF level of TNF-α was significantly higher in obese patients with and without chronic periodontitis, and highest in obese patients with chronic periodontitis group. Therefore, it is plausible that low-grade inflammation produced by the excess adipose tissue over a long period of time influences the development of periodontal disease by stimulating the inflammatory cascade.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.