Diabetes Mellitus

Inflammation is a biological response of vascular tissues to irritants such as microbial or their products irritants.1 Chronic inflammatory state such as obesity and metabolic syndrome are characterised by accumulation of macrophages in adipose cells and secretion of various inflammatory mediators. Several novel inflammatory markers have been suggested to integrate metabolic and inflammatory signals.2
A recently identified adipokine chemerin, highly secreted in liver and adipocytes is associated with adiposity, insulin resistance, metabolic syndrome risk factors, and degree of non-alcoholic fatty liver.3,4 Chemerin is a chemoattractant for immature dendritic cells and macrophages.5,6 It is activated by serine protease activity.7 It also induces adhesion of macrophages to extracellular matrix proteins at the site of inflammation.8 Various studies have associated chemerin with several inflammatory markers in obesity and type-2 DM.9,10 Chemerin is considered a candidate in linking inflammation to metabolic disorders such as type-2 DM.2
Diabetes mellitus is characterised by abnormally high levels of glucose in the blood11, brought about either by beta cell dysfunction in pancreas, increased resistance to insulin in liver and muscles cells or a combination of both.12 Diabetes is now viewed an inflammatory condition and its development is preceded by a low grade systemic inflammation13, with elevated plasma concentrations of pro-inflammatory mediators ' markers.14
Diabetes has been found to be an important host risk factor in periodontal diseases in large epidemiological studies.15,16 Loe17 described periodontitis as the 6th complication of diabetes.17
Chronic periodontitis (gum disease) is a chronic inflammatory disease characterized by inflamed gingiva, bleeding on probing, alveolar bone and attachment loss between the tooth and its supporting structure.18 Imbalance in the anabolic and catabolic processes and increased secretion of inflammatory mediators leads to destruction of surrounding structure of the tooth.12,18
GCF is closely approximated with the periodontal tissue 19, and has been used as a source for detection of various inflammatory markers in type 2 DM and CP.20,21
Tear fluid is an aqueous layer on the ocular surface22,23, consists of over 20 components24, including, various inflammatory and anti-inflammatory markers.25-27 Tear fluid has been used as a diagnostic tool for detection of glucose in subjects with diabetes.28-29
Till date, no study has reported human chemerin levels in GCF and tear fluid in CP subjects with and without type 2 DM. In this context, this clinico-biochemical study was designed to assess the levels of human chemerin in GCF and tear fluid and its in CP subjects with and without type 2 DM.

MATERIAL AND METHODS:
This was a 3 months duration cross-sectional study performed from November 2013 to February 2014. The study group consisted of 40 subjects age and sex-balanced individuals (19 males and 21 females, aged 25 to 45 years). Subjects reporting to the Department of Periodontics, Government Dental College and Research Institute (GDCRI), Bangalore, India were included in the study with least 20 natural teeth diagnosis of CP based on clinical parameters like probing depth (PD), clinical attachment level (CAL)30, gingival index (GI)31, Body mass index (BMI) in range of 18.5-22.9 kg/m2 and waist circumference<90cm (men) and <80cm (women) (WHO, 2004)32; subjects should not have received periodontal therapy within preceding six months; Well-controlled type 2 diabetic patients classified based on criteria given by American Diabetic Association (ADA) in 2012, fasting plasma glucose (FPG) and glycated haemoglobin, hemoglobin A1c (HbA1c) levels.33
Exclusion criteria was presence of systemic disease (e.g., cardiovascular disease, rheumatoid arthritis), Clinical examination and routine laboratory testing was performed to determine history of any infection, systemic antibiotics in the last 6 months or any other medications affecting the periodontal status or subjects with the use of contact lenses.
Written informed consent was obtained from all the subjects participating in the study. The Institutional Ethical Committee and Review Board, GDCRI approved the ethical clearance for the study.
Subject Grouping:
Participants were categorized into three groups. Group 1 (healthy) consisted of 10 individuals with clinically healthy periodontium, GI = 0 (absence of clinical inflammation), PD <3 mm, and CAL = 0, with no evidence of bone loss on radiographs, HbA1c < 6.5%, FPG < 126 mg/dl. Group 2 (CP without type 2 DM) consisted of 15 individuals who had signs of clinical inflammation, GI >1, PD >5 mm, and CAL >3 mm, with radiographic evidence of bone loss, HbA1c <6.5%, FPG < 126 mg/dl. Group 3 (type 2 DM among individuals with CP) consisted of 15 individuals who had signs of clinical inflammation, GI >1, PD >5 mm, CAL >3 mm, and HbA1c <7% with radiographic evidence of bone loss. Only well controlled (HbA1c <7%) type 2 DM individuals were selected based on American Diabetes Association's criteria for diagnosis of diabetes.33

Clinical evaluation of subjects:
Group allocations and sample site selections in each subject was performed by the chief coordinator (ARP). A thorough case history record was taken for each subject. Each subject then went through a full-mouth periodontal probing and charting and body mass index (BMI) charting procedure according to the World Health Organization guidelines.32 A calibrated examiner (KP) did the clinical evaluation measuring the clinical parameters including PD, CAL, GI using a periodontal probe (Universal gracey curette #4R/4L, Hu-friedy). Radiographic bone loss was recorded dichotomously (presence or absence) to differentiate between healthy and CP subjects. Radiographic assessment was standardized. Full mouth intra-oral periapical radiographs were obtained from all subjects using individually customized bite blocks and paralleling angle technique (long cone technique) and developed under guided, standard conditions. The same examiner (KP) did the radiographic evaluation and collected the GCF samples.

Site selection and GCF collection:
GCF sample collection was done from most inflammed sites with the highest scores. In group 2 and group 3 subjects, the sites showing signs of inflammation and greatest CAL with radiographic of bone loss, were selected for sampling. One site selected/ subject was used for the analysis of human chemerin molecule. In the healthy group sampling was done from the mesio-buccal region of the maxillary left first molar, in the absence of which the right first molar was sampled to standardize site selection and obtain adequate fluid volume. Sterile cotton rolls was used to clean, isolate the selected site and the supragingival plaque was removed gently using a gracey curette so as to avoid contamination of the paper strips (Periopaper, Ora Flow Inc). The paper strips were placed gently at the entrance of the gingival sulcus/ crevice until the light resistance was felt34, without any trauma, and left in place for 60 seconds. The absorbed GCF volume of each strip was determined by electronic impedance (Periotron 8000, ProFlow Inc). Samples that were suspected to be contaminated with blood and saliva were excluded from the study to avoid any kind of bias in sample collection. After collection of the gingival fluid, the two periopaper strips / site that absorbed GCF from each subject were pooled and were immediately transferred in microcentrifuge tubes (premarked with the biomarker name) containing 400??l of phosphate buffer saline and stored frozen at -70??C for subsequent analysis. Periodontal treatment (Scaling and root planing) was performed for CP subjects at the same appointment after GCF collection by the operator (KN).

Tear fluid collection:
Minimally stimulated tear fluid was collected from inferior tear meniscus of each eye using a 0.5??L glass capillary micropipette (Sigma-Aldrich Co) . The tear samples from both eyes (1 ??L) total were eluted into one tube containing 9 ??L of assay buffer for a final dilution of 1:10, centrifuged for two mins and immediately transported in an insulated coolers to a minus -70 oC freezer where they remained frozen until they were used for immunoassay.35
Human Chemerin analysis:
The samples were assayed for chemerin using enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer's instructions. The elute prepared from homogenization of GCF and tear fluid samples for 30 secs and centrifugation for 5 minutes at 1,500 g was used for ELISA estimation from GCF and tear fluid samples.
Each sample was assayed using commercially available ELISA kit (Human Chemerin Elisa kit, ABO Swiss Bio Ltd) in accordance with the manufacturers' instructions. A Microplate reader was used to observe the colour development, a stop solution was added when an optimum optical density was reached which was read at 450 nm. The total chemerin was determined in nanograms (ng), and the calculation of the concentration in each sample was performed by dividing the amount of chemerin by the volume of sample (ng/ml).

STATISTICAL ANALYSIS:
Power calculations were performed before the study was initiated and the sample size was selected based on the previous study.20 Sample size and grouping was based on the power of the study and the confidence interval of 95% (p<0.05). Statistical analysis was done using a software program (SPSS statistical software, SPSS version 16.0). Analysis of Variance (ANOVA) was carried out for a comparison of GCF and Tear fluid Chemerin levels between the groups. Using Pearson's correlation coefficient, the relationship between Chemerin concentration and the clinical parameters were analyzed. The intra-group correlation of tear fluid and GCF concentrations of Chemerin was also performed using the pearson's correlation coefficient. Pair-wise comparisons between groups for Chemerin concentrations in GCF and tear fluid were carried out by Scheff's test. P values <0.05 were considered statistically significant. The mean intra-examiner standard deviation of differences in repeated PD measurements and CAL measurements obtained using single passes of measurements with a UNC- 15 probe (correlation coefficients between duplicate measurements; r = 0.95).

RESULTS:
Table 1 demonstrates the descriptive statistics (mean + SD) of the study population. The mean Chemerin concentrations both in tear fluid and GCF were highest for Group 3 followed by Group 2 and least in Group 1. ANOVA test showed significant difference in the tear fluid and GCF levels of Chemerin between the three groups test (Table 2). Table 3 demonstrates that tear fluid and GCF levels of Chemerin were positively correlated with all the clinical parameters. The correlation between the levels of these inflammatory markers and clinical parameters was statistically significant (p<0.05) in all the groups.
Table 4 demonstrates statistically significant pair-wise comparisons between groups for Chemerin concentrations in GCF and tear fluid.

DISCUSSION
This study evaluated GCF and tear fluid levels of chemerin in CP with and without association of type 2 DM. The results of the study indicated an increase in the GCF and tear fluid levels of chemerin from health to CP to type 2 DM with CP. The increase in the levels of these inflammatory mediators in conditions of localized inflammation (CP) and systemic condition (DM) leads to the suggestion that chemerin can be considered as potential biomarkers for inflammatory periodontal disease and type 2 diabetes.
Chemerin is a novel adipokine secreted is from liver and adipocytes.3,4 The chemotactic potential is released after activation of chemerin by serine proteases leading to recruitment of CMKLR1-positive cells such immature dendritic cells and macrophages.7 Apart from chemoattractant the macrophage adhesion property by chemerin was demonstrated in a study by flow cytometry and confocal microscopy assays in murine peritoneal exudate cells, which showed that chemerin can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules, proving its role in recruitment and retention of macrophages at the site of inflammation.8
Diabetes is now considered as an inflammatory condition with an elevated levels of pro inflammatory mediators/markers in serum.13,14 The inflammatory response in CP is characterised by the localized production of pro inflammatory mediators such as C-reactive proteins, cytokines (interleukin-1b, interleukin-6, tumor necrosis factor a) and prostanoids (prostaglandin E2).12 As diabetes alters the immunologically active molecule there is an increased level of cytokines seen in the periodontal tissues which accelerates the disease progression providing the scientific basis for increased susceptibility to periodontal disease in subjects with diabetes.12
Recent evidence showed that inflammatory cytokines may play a role in chemerin release from adipose tissue. In an in vitro study both IL-1 beta36 and TNF37 alpha induce chemerin mRNA expression and secretion from adipocytes37, this can be attributed to the rise in serum chemerin levels in diabetes and periodontitis where there is an increase in inflammatory mediators.
Various studies have been carried out to determine the relationship between serum chemerin levels in subjects with type 2 diabetes.2 In a study serum chemerin levels evaluated by ELISA showed significant higher levels in subjects with type 2 DM compared to normal glucose tolerant individuals.38 In another study serum chemerin levels were significantly increased in patients with type 2 diabetes and in patients with type 2 diabetes with ischemic heart disease compared with healthy control subjects.39
Tear fluid, the aqueous layer found on the ocular surface22,23 has been reported to have increased glucose in diabetic subjects28,29 and inflammatory25,26 and anti-inflammatory27 markers in various ocular surface diseases.
Many reports have demonstrated that tear glucose is higher in diabetic subjects than in healthy ones. Quantitative and qualitative assessment on tear fluid of diabatic and non-diabetic individuals shows that tear fluid can be considered as a diagnostic tool in detecting diabetes.28 In another study tear glucose was almost five times higher in diabetic subjects as compared to non-diabetic group.29 Apart from the glucose levels various inflammatory markers have been detected by ELISA technique in subjects with ocular surface disease. Inflammatory markers such as IL-6, IL-17 in keratoconus subjects25, increase in IL-1 in dry eye disease27, presence of Pro-MMP-9 and overexpression of IL-1 beta and IL-6 of conjuctivochalasis26,27 subjects have been detected in tear fluid.
Clinic-biochemical studies done on GCF has revealed the presence of inflammatory biomarkers such as visfatin20, stem cell factor21, in subjects with chronic periodontitis and diabetes.
Numerous studies mentioned above and the fact that GCF and tear fluid contains large amounts of constituents such as proteins, lipids, active biomolecules20-27, suggest that they are derived from the systemic circulation.
To our knowledge this is the first study evaluating and correlating chemerin in GCF and tear fluid in subjects with CP with and without type 2 DM. Further longitudinal studies with larger sample size and before and after treatment levels of chemerin should be carried out to confirm the findings of the study and better understand the role of this inflammatory biomarkers in type 2 DM and CP.

CONCLUSION:
Within the limitations of the study, chemerin can be considered as potential biomarkers of periodontal disease. The results indicate correlation between these two secretory fluids in conditions such as diabetes and periodontitis. These biomarkers can thus be valuable in detecting high risk individuals with periodontitis and systemic diseases like diabetes. Further longitudinal prospective studies must be carried out to affirm these findings and understand the possible role of chemerin in pathogenesis of periodontitis and diabetes.

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